International Journal of Cytology: A Survey of Cell Biology, Band 26

INTERNATIONAL

CYTOLOGY VOLUME REVIEW 26

Volume 26 Contributors

S. BORST KONHADKECK

A. M. CROWN GILBERTN. ABTEI

TWO ROBERTS

EVALDR. WEBEL LEONARD WEBS

INTERNATIONAL

Cytology review EDITED BY

GH BOURNE

J. F. DANIELLI

Yerkes Regional Primate Research Center Emory University Atlanta, Geórgia

State University of New York Center for Theoretical Biology in Buffalo Buffalo, New York

MITARBEITER K. W. JEON Center for Theoretical Biology State University of New York at Buffalo Buflalo, New York

BAND26

Prepared under the auspices of the International Society for Cell Biology

ACADEMIC PRESS New York and London 1969

COPYRIGHT @ 1969, BY THE ACADEMIC PRESS. INCL. A1.I. RIiSERVI RIGHTS: D. WITHOUT PARI' THIS TOWER MAY BE REPRODUCED IN ANY WAY USING PHOTOSTAT, MICROFILM, RE'I'RIEVAJ. SYSTEM OR OTHERWISE WITHOUT WRITTEN PERMISSION FROM 'JH11 PUBLISHERS.

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l'witeu' K i ~ q d o measurement published by

ACADEMIC

PRESS,

INK. (LONDON) LTD.

Berkeley Square House, Londres W1

LIRRAHY 01: CA'I'ALOG CONGRESS MAP NUMBER: 52-5203

PRIN1F.D IN THE UNITED STATES 01. AMERICA

List of contributors P. BORST, Department of iMediral Enzymologj, Luboiatory University of Am rtejdam, Am iterdam, The Netheilaud KONRADKECK, TUCJ~O?~ Department, Arizoiiu

von

Biochemistry),

Biologii-ul Scieiire~, Uiiiz'es.rjty o j Arizoua,

A. M. KROON, Depurtmetit of Medirul E n z y m o l o g ~ , Labor University of A m iterdam, A m s t e r d ~ ~ n The z , Netherluiidr

von

Biochemistry,

GILBERT N. LING, Department of Molecular Biology, Department of Neurology, Pennsylvania Hospital, Philadelphia, Pa. The Netherluidr

D. W. A. ROBERTS, Rejearr Stutioti, Department of Cmudu Lethbridge, Albestu, Canada

von

Agsii-iltzrre,

EVALDR. WEIBEL, Depnstmeizt of Aiiutomy, VliiverJity of Beru, Besrz, Swifzerland LEONARDWEISS, Departamento de Patologia Experimental, RoJwell Park Memorial Institute, Buffalo, N e w York

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Contents LIST OF STAFF ................................................... .. .. .. .

CONTENT OF THE PREVIOUS VOLUMES ............................................

A new model for the living cell: theory summary and recent experimental evidence supporting it GJLEERT N. LÍNEA Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. Die Membrantheorie. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11. An interesting lead in the search for a better living cell model. . . . . 111. The association induction hypothesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . references . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

10 11 58

The Cellular Periphery LEONARD WEISS Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . lipid bilayers. . . . . . . . . . . . . . . . . . . . . . . . . . ......... III. Other models. . . . . . . . . . . . . . . . . . . . . . . . . . . ...................... IV Cell surface charge. . . . . . . . ......................................... V. Enzyme activity and the cell. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VISA. The Outskirts of Evil ............................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

EU.

64 70 78

91 94 99

Mitochondrial DNA: physicochemical properties, replication and gene function P. BORSTA N DA M. KROON

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Basic composition of mitochondrial DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Closest Neighbor Frequencies of Mitochondrial DNA1. . . . . . . . . . . . . . . . . . . viii

108 109 117

...

VIII

CONTENT

IV. Differences in base composition and base sequence of complementary strands of mitochondrial DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Size and structure of mitochondrial DNA from animal tissue VI. Size and structure of mitochondrial DNA in plants and protozoa. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VISA. The amount of mitochondrial DNA per mitochondrion and per cell. . . . . . viii Mitochondrial DNA replication. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Effects on Ycast mitochondrial DNA of anaerobic GIu and mutagens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. Mitochondrial DNA recombination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI. Renaturation studies with mitochondrial DNA. . . . . . . . . . . . . . . . . . . . . . . . . XI1. Evolution of mitochondrial DNA and the relationship between mitochondrial ............................................... and nuclear DNA rial DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI11. Genetic function of Mitocl ............................................. .............. .... ..... .. XV . references . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

117 118 139 141 145 154 163 165 167 168 179 181

Metabolism of KONRADKECR Enucleated Cells 1 . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I 1. Initiation of the seedless state. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I I I mRNA quantification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV.InRNA Disintegration and Protein Synthesis in Anucleated Cells ........................ V . Type of mRNA degradation. . . . . . . . . . . . . . . . . . . . . . . . . . . . .............VISA. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...................... References . . . . . . . . . . . . . .................................................. .

191 192 196 208 222 225 225

Stereological principles for morphometry in electron microscopy cytology

YOU . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 Basic Stereological Principles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sick . Application of stereological methods to electron microscopic cytology. . . . . IV.An example for the morphometric characterization of organelles: the liver cell V. Cytomorphometric methods in experimental pathology. . . . . . . . . . . . . . . . . . VISA. Problems in the application of stereological methods to anisotropic systems VII.Assessment of the current state and perspectives of future possibilities. . . . . . . . . . references . . . . . . . . . . . . .................................................. .

235 238 261 286 293 294 298 299

CONTENT

Some possible roles of isoenzyme substitutions during cold hardening in plants D. w A. ROBERT I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Effects and Prevention of Ice Formation. . . . . . . . . . 111. The effect of low temperature on proteins. . . IV. Metabolic imbalance. . . . . . . . . . . . . . . . . . . . . . . . V. The isocytic substitution hypothesis. . . . . . . . VISA. Final remarks. . . . . . . . . . . . . . . . . . . . . . . . . references . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

..................................................... ..... ................. ................. ........ . .......... . .....................

AUTORENÍNDICE S U B J F I C T ÍNDICE

303 304 309 313 318

322 323

329 .........................

...................

348

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Contents of Previous Volumes Aspects of Bacteria as Cells and as Organisms - STumT MUDDAND EDWARD Some Historical Characteristics of Cell Biology. DELAMATER OY-ARTHUR HUGHES Ion secretion in J-plants. F. SUTCLIFFE Nuclear Reproduction-C. LEONARDHuS multienzyme sequences in soluble extensions of ExKINS-HENRY R. MAHLER Enzyme capabilities and their relationship The nature and specificity of feulgen with cellular nutrition in animals-GEORGE nuclear reaction-M. A. LESSLER W. KIDDER Quantitative Histochemistry of Phosphate The Application of Lyophilization Rate Techniques-WILLIAM L. DOYLE in Cytology-L. G. E. BELL Alkaline Phosphatase of Nucleo-Enzymatic Processes in the Cell Membrane M. CHBVREMONT E H. FIRKET Penetration-TH. ROSENBERGAND F. Taste and Olfactory Epithelium-A. F. WILBRANDT BARADI AND G. H. BOURNE Bacterial Cytology—K. A. BISSET Growth and differentiation enzymes from the surface of explanted protoplasts and absorbent tissue P. J. GAILLARD by Sugar-R. BROWN Electron microscopy of tissue sections Reproduction of bacteriophage-A. D. A. J. DALTON HERSHEY A redox pump for the biological folding and unfolding of osmotic work and its molecules as the basis of osmotic work in relation to the kinetics of diffusion of free ions R. J. GOLDACRE through E-membranes. J. Nucleocytoplasmic relationships in AmphibCONWAY ian Development-G. FANK-HAUSER A Critical Examination of Current Approaches Structural Agents in Mitosis-M. M. in Histo- and CytochemSWANN istry-DAvID GLICK Quantitative factors affecting staining of nucleus-cytoplasm ratios in acid- and basic-growing tissue sections of Acetdularia-J. HAMdyes-MARCUS SINGER MERLING Sperm behavior in report from ROTHSWorkers Neighborhood Tissue Culture Conference in Cooperstown, New Volume 1

NIÑO

Cytology of the epidermis of mammals and sebaceous glands-wrLLrAM MON-

York-D.

C. HETHERINGTON

AIJTHOR-SUBINDEX

INDICES JECT

TAGNA Volume 3 Electron Microscopic Investigation of Animal Cell Nutrition - CHARITY Tissue Sections - L. H. BRETSCHNEIDER WAYMOUTH Histochemistry of EsterasesKaryometric Studies in Tissue CulturesG. GOMORI OTTO BUCHER AUTHOR INDEX- SUBJECT INDEX The Properties of Urethane Considered in Volume 2 in Relation to Its Action on Mitosis IVORCORNMAN Quantitative Aspects of Nuclear Nucleoproteins-HEWSON SWIFT Composition and Structure of Giant Chromosomes-MAX ALFERT Ascorbic Acid and Its Intracellular Localization, with special reference to How Many Chromosomes Are in Mammalian Somatic Cells?-R. A. Plants BEATTY - J. CHAYEN

XI1

LONTENTOS 01: PREVIOUS VOLUMES

The chemical composition of the bacterial cell wall-C. S. CUMMINS Theories of Enzymatic Adaptation in Microorganisms-J. MANDELSTAM The cytochondria of cardiac and skeletal muscle ~--JOHN W. HARMAN The mitochondria of the neuron-WARREN ANDREW Results of cytophotometry in the study of the content of deoxyribonucleic acid (DNA) in the nucleusR. VENDRELY AND C. VENDRELY Protoplasmic contractility in relation to gel structure: temperature and pressure experiments on cytokinesis and movement of amoebae-DOUGLAS MARSLAND intracellular pH-PETCR C. CALDWELL The activity of enzymes in metabolism and transport in erythrocyte T-cells. A. J. PRANKERD AUTHOR INDEX-SUB J E C T INDEX Volume 4 Uptake and Transfer of Macromolecules by Cells with Special Reference to Growth and Development-A. M. Cytochemical microsurgery-M. J. KOPAC SCHECHTMAN Amebocyte-L. E. WAGGE Cell Secretion: A Study of the Pancreas and Fixation Problems in Cytology, HistolSalivary Glands-L. C. U. JUNQUEIRA ogía e Historhemistry-M. WOLMAN E G. C. HIRSCH BACTERIAL CYTOLOGY — ALFRED MARSHAK The acrosomal reaction — JEAN C . Histochemical DAN of bacteria-R. VENDRELY Cytology of spermatogenesis-VrsHwA Recent studies on plant mitochondriaNATH DAVIDP. HACKETT The ultrastructure of cells like the structure of chloroplasts is revealed by electron microscopy-FRITIor: K. M ~ ~ H L E T H A L E R S. SJOSTRAND Histochemistry of Nucleic Acids-N. B.

The importance of enzymatic studies in isolated cell nuclei-ALEXANDER L. DOUNCE The use of differential centrifugation in the study of tissue enzymes-CHR. DE DUVEAND J. BERTHET Enzymatic aspects of embryonic differentiation-TRYGGVE GUSTAFSON Azo staining methods in enzymatic histochemistry-A. G. EVERSON PEARSE Microscopic Studies in Living Mammals Using ROYG Camera Lucida Methods. WILLIAMS O Mast Cell—G. ASBOE-HANSEN-EDWARD M. DEMPSEY E ALBERTI stretch fabric. LANSING Nerve cell composition examined with new methods - ENOLOFBRA~TGARD AND HOLGER HYDEN

AUTHORS INDEX SUBJECT INDEX KURNICK Structure and Chemistry of NucleoliVolume 6 W. S. VINCENT in goblet cells, particularly of the intes- the antigenic system of Paramecium aurelia-G. H. BEALE lineage of some mammalian species Chromosomal cytology of ascites HARALD MOE Tumors of Rats, with special reference localization of cholinesterases in Neurotus the concept of stem cell muscle junctions-R. COUTEAUX SAJIRO MAKINO Evidence of an active redox pump The structure of the Golgi cation transport apparatus E. J. CONWAY AND PRISCILLA ARTHURW. POLLISTER AUTHOR INDEX - TOPIC INDEX F. POLLISTER Analysis of the Fertilization Process, Volume 5, and A-Egg Activation. Histochemistry of MONROY-Stained Antibodies The Role of the Electron Microscope in the Study of Viruses ALBERT H. COONS-ROBLEY C. WILLIAMS

...

CONTENT OF PREVIOUS VOLUMES

The histochemistry of polysaccharidesARTHURJ. HALE Thyroid dynamic cytology — J. GROSS Recent histochemical results of studies in some avian and mammalian embryos — Jho BORGHESE Carbohydrate metabolism and embryonic determination — R. J. O'CONNOR Enzymatic and metabolic studies in isolated cell nuclei-G. SIEBERT E R.M.S. SMELLIE Recent approaches to the cytochemical study of mammalian tissues - GEORGE EDWARDL. KUFF AND H.HOGEBOOM, WALTERC. SCHNEIDER The kinetics of non-electrolytic penetration in mammals ErythTOCyte-FREDA BOWER AUTHOR INDEX-SUB

INDICES JECT

CUMULATIVE INDEX OF TOPICS

(VOLUMES 1-5) Volume 7 Some Biological Aspects of Experimental Radiology: A Historical Review-F. G. SPEAR The effect of carcinogens, hormones and vitamins on the organ CuhreS-ILSE LASNITZKI Recent advances in the study of the A-kinetochore. FARIA LIME Autoradiographic studies with S35 sulfate D. D. DZIEWIATKOWSKI The Structure of Mammalian Spermatozoa, F A W C E ~ matozoon-DoN The Lymphocyte-0. A. TROWELL Structure and innervation of the lamellar muscle-J. BOWDEN Hypothalamo-neurohypophyseal neurosecretion-J. C. Cell contact SLOPER-PAUL WEISS Ergastoplasma: its history, ultrastructure and biochemistry-FRANCOISE HAGUENAU Anatomy of the renal tubules-JoHANNEs RHODIN Structure and innervation of the inner kidney

XLII

Ohr

Sensory Epithelium-HANS ENGWESKLL Isolation of Living Cells from Animal Tissues-L. M. J. RINALDINI POWER AND JAN

AUTHOR SUBJECT INDEX

INDEX

Volume 8 The structure of the cytoplasm - CHARLES OBERLING. Wall Organization in Plant Cells-R. D. PRESTON Submicroscopic morphology of the synapse-EDUARDO DE ROBERTIS The cell surface of Paramecium-C. F. EHRETAND E. L. POWERS The Mammalian Reticulocyte-LEAH MIRIAM LOWENSTEIN The Physiology of Chromatophores-MILTON FINGERMAN The Fibrous Components of Connective Tissue with Special Reference to Elastic Fiber-DAVID A. HALL Experimental Heterotopic OssificationJ. B. BRIDGES A review of metabolic studies in isolated mammalian nuclei – D. B. ROODYN Trace elements in cell functionBERTL. VALLEE AND FREDERIC L. HOCH Osmotic Properties of Living Cells D. A. T. DICK Movements of Sodium and Potassium in Nerves, Muscles, and Red Blood Cells I. M. GLYNN Pinocytosis-H. HOLTER AUTHOR SUB-TABLE

INDICES JECT

Volume 9 The influence of cultural conditions on bacterial cytology-J. F. WILKINSON AND J. P. DUGUID Organizational Patterns Within Chromosomes - BERWIND P. KAUFMANN, HELEN R. MCDONALD GAY AND MARGARET Enzymatic Processes in Ceh - JAY BOYD BEST Cell Adhesion - LEONARD WEISS Physiological and Pathological Changes

XIV

CONTENT OF PREVIOUS VOLUMES

in Mitochondrial Morphology-CH. ROUILLER The study of drug effects at the G-cytological level. B. WILSON Histochemistry of lipids in oogenesis VISHWANATH Cytoembryology of echinoderms and amphibians-KATsuMA DAN Cytochemistry of non-enzymatic proteins-RONALD R. COWDEN

Histochemistry of Ossification-RoMULO L. CABRINI Cinematography, an indispensable tool for C-cytology. M. POMERAT AUTHOR INDEX SUBJECT

INDEX

Banda 12

Sex chromatin and human chromosomes JOHN L. HAMERTON Chromosomal development in cell populations AUTHORS-INDEX-SUBJECT-INDEX tion-T. C. Hsu Chromosome Structure with Special ReferenceVolume 10 ence to the Role of Metal Ions-DALE M. STEFFENSEN The Chemistry of Schiff's Reagent-FREDElectron Microscopy of Human White ERICK H. KASTEN Blood cells and their stem cells Spontaneous and chemically induced BESSISAND JEAN- PAUL THIERY Chromosomal Breakages-ARuN KUMAR MARCEL In Vivo Implantation as a Technique in SHARMA AND ARCHANASHARMA Skeletal Biology-WILLIAM J. L. FELTS The Ultrastructure of the Nucleus and the Nature and Stability of Nucleocytoplasmic Relationships in the Myelin Nerve-SAUL J. B. FINEAN WISCHNITZER The Mechanics and Mechanism of in vitro fertilization of mammalian eggs C. R. AUSTIN Alter-LswIs WOLPERT Liver growth with special fertilization physiology in fish eggsTOKI-oYAMAMOTO relationship with mammals-F. DOLJANSKI Cytological Studies on the Affinity of AUTHORS-INDEX- SUBJECT-INDEX Carcinogenic Azo Dyes for Cytoplasmic Components of Volume 13-YosHIMI NAGATANI Epidermal Cells in A-Culture. GEDEON The coding hypothesis-MARTYNAs Y t A s Chromosomal reproduction-J. AUTHOR SUBTABLE OF HERBERT MATOLTSY

INDICES JECT

TAYLOR

Sequential genetic action, protein synthesis and cell differentiation-REED A. (VOLUMES 1-9) Flickinger Volume 11 The composition of the mitochondrial membrane in relation to its structure Seand electron microscopic analysis-ERIC G. BALL Function and mechanism of formation-k CLIFF KUROSUMI. JOEL The Fine Structure of Insect Sense Organs Nucleate Metabolism Pathways and ELEANORH. SLIFER Cytology of the E Y ~ A L F R E D Development of anucleated erythrocytes H. A. SCHWEIGER J. COULOMBRE Structures of photoreceptors-J. J. WOL- Some recent developments in the field of transport of alkaline cations-W. WILK EN BRANDT Use of inhibitors in studies of fertilization mechanisms-CHARLES B. Chromosomal aberrations induced by ionizing radiation-H. J. EVANS METZ The Cytochemical Growth Cycle of Protozoan Cells, With Special Reference to the Golgi Apparatus D. M. PRESCOTT CUMULATIVE SUBJECT INDEX

CONTENT V O L I J M E S PREVIOUS

and VrsHwA NATH mitochondrial regeneration from mammalian liver NANCYL. R. BUCHER G. P. DUTTA Collagen formation and fibrogenesis Cell renewal-FELIX BERTALANFFY And with special reference to the role of ascorbic acid CHOSENLAU-BERNARD S. GOULD AUTHOR INDEX- SUBJECT INDEX The behavior of mast cells in the AnaphyBand 14 laxis-IVAN MOTA Inhibition of cell Division: A Critical Lipid Uptake-ROBERT M. WOTTON and Experimental Analysis-SEYMOUR AUTHOR'S INDEX- SUBJECT INDEX GELFANT Electron Microscopy of Plant Protoplasm Volume 16 R. BUVAT Ribosomal Functions Related to Cytophysiology and Cytochemistry of Protein Synthesis-TORE HULTIN Organ de Corti: The Cytochemical The- Physiology and Cytology of Chloroplasty of Audition-J. A. VINNIKOV E Formation and “loss” in EuglenaL. K. TITOVA M. GRENSON Connective Tissue and Serum Proteins Cell Structures and Their Importance in the R. E. MANCINI Ameboid Movement-K. E. WOHLL The biology and chemistry of FARTH-BOTTERMANN cell walls from higher plants, algae and microirradiation and fungi from partial-D cell irradiation. H. NORTHCOTE C. L. SMITH Development of Drug Resistance by Nuclear-Cytoplasmic Interaction with Ionstaphylococci in vitro and in Viuoizing Radiation-M. A. LESSLER MARYBARBER In V i m Studies on the cytological and cytochemical effects of myelinated nerve fibers - CARL CASKEY SPEIDEL Agents involved in various pathological respiratory tissues: structure, histophysical conditions: the effect of viralology, cytodynamics. Part I. Overview of Cigarette Smoke in the Cell and Basic Cytomorphology - FELIX D. Its Nucleic Acid - CEcIm LEUCHTENBERTALANFFY BERGER AND RUDOLF LEUCHTENBERGER AUTHOR INDEX - TOPICS INDEX The Tissue Mast Wall - DOUGLAS E. SMITH Volume 17 Y

AUTHOR-SUB-INDEX

INDICES JECT

The Growth of Plant Cell Walls K. WILSON The Nature of Reproduction and Inheritance of the Lampbrush Chromosome in Trypanosomes: A Critical Review H. G. CALLAN Dealing Mainly with African Species on the Intracellular Transmission of Genetics In Mammalian Host-P. Formation J. WALKER-J. L. SIRLIN Mechanisms of gametic targeting in platelets: electron microscopy studies-J. F. DAVID-FERREIRA Plants-LEONARD MACHLISAND ERIKA The histochemistry of mucopolysaccharRAWITSCHER-KUNKEL migrates-ROBERT C. CURRAN The cellular basis of morphogenesis and development of the sea urchin-T. GUSTAF- Structure of the Respiratory Tissue, Histophysiology, Cytodynamics. Part 11. New Approaches and Interpretations of ApSON Y L. WOLPERT-FELIX D. Plant Tissue Culture in Relation to Developmental Cytology DeBERTALANFFY--CARL R. PARTANEN Adenohypophyseal Cells and Volume 15

xvi Su funcional HERLANT AUTHOR INDEX-SUB

CONTENT OF PREVIOUS VOLUMES

Meaning-MARC JECT INDEX

Phosphorus Metabolism in the K Plant. ROWAN AUTHOR SUBTABLES

INDICES JECT

Banda 20

Banda 18

S. RESPIRATION- The chemical organization of the plasma membrane of animal cells-A. H. MADDY The Structure of Mammalian Egg Subunits of Chloroplast Structure and ROBERT HADEK Quantum Conversion in Photosynthesis Cytoplasmic Inclusions in OogenesisRODERICB. PARK M. D. L. SRIVASTAVA Control of Chloroplast Structure by Light Sorting and Partial Tabulation LFSTI-R PACKI'R E PAUL-ANDRI? SIE of enzymatic studies in subcellular fractions

The Langerhans A cell. AFTER

isolated by D-differential centrifugation. B. ROODYN histochemical localization of enzymatic activities by substrate film methods: ribonucleases, deoxyribonucleases, proteases, amylase and hyaluronidaseR. DAOUSTP cytoplasmic deoxyribonucleic acid. B. GAHANAND J. CHAYEN Malignant transformation of cells in vitro-KATHERINE K. SANFORD Effects of deuterium isotopes on cytologyE. FLAUMENHAFT, S. BOSE, H. L. CRESPI, E J. J. KATZ The use of heavy metal salts as C-electron dyes. RICHARD ZOBELAND MICHAEL BEER AUTHOR'S DIRECTORY

INDEX

Banda 19

GENTHALER

The role of potassium and sodium ions studied in the brain of mammals-H. HILLMAN Triggering of ovulation by intercourse in the rat-CLAUDE ARON, GITTAASCH AND JACQUELINE Roos Cytology and cytophysiology of non-melanophore pigment cells-JOSEPH T. BAGNARA Fine structure and histochemistry of the prostate glands in relation to sex hormones-Davm BRANDES Cerebellar das enzymology LucrE ARVY INDEX AUTHOR SUBSECTION INDEX Volume 21 Histochemistry of Lysosomal P. B GAHAN Physiological Clocks-R. L. BRAHMACHARY

"Metabolic" DNA: A Cytochemical Study H. ROELS The Importance of Sex Chromatin MURRAY L. BARR Some functions of the J-nucleus. M. MITCHISON Synaptic morphology in the normal and degenerative nervous system-E. G. GRAYAND R. W. GUILLERY Neurosecretion-W. BARGMANN Some Aspects of Muscle Recovery E. H. BETZ, H. FIRKET AND REZNIK Gibberellins as P-hormones. W. BRIAN Phototaxis in plants - WOLFGANG MAIN

Ciliary movement and coordination in CihteS-BELA PARDUCA Electromyography: its structural and neural basis-JOHN V. BASMAJIAN Cytochemical studies with acridine orange and the influence of dye contaminants on nucleic acid staining FREDERICK H. KASTEN Experimental cytology of apical cells during phase vegetativa Growth and flowering-A. NOUGAREDE Type and origin of the perisynaptic cells of the T motor end plate. AUTHOR SUBINDEX BY R. SHANTHAVEERAPPA AND G. H. BOURNE

INDICES JECT

CONTENT OF PREVIOUS VOLUMES

xvii

Banda 22

Banda 24

Current techniques in biomedical electron microscopy-SAUL WISCHNITZER The cellular morphology of tissue repair-R. M. H. MCMINN Structural organization and embryonic differentiation-GA JANAN V. SHERBET E M. S. LAKSHMI The dynamics of cell division during the early stages of ovoN cleavage. FAUTREZ-FIRLEFYN AND J. FAUTREZ Lymphopoiesis in Thymus and Other Tissues: Functional Implications-N. B. EVERETT AND RUTH c. TYLER (CAF-

Synchronous Cellular DifferentiationGEORGEM. PADILLA N D IVAN L. CAMERON Mast cells in the nervous system YNGVEOLSON Developmental stages in intermitosis and preparation for mitosis of mammalian cells in VitYO-BLAGOJE A. NEJKOVIC Antimitotic substances-Guy DEYSSON The form and function of the sieve tube: a problem in reconciliation P. E. WEATHERLEY AND R.P.C. JOHN-

FREY)

SOHN

Analysis of antibody staining patterns Structure and organization of myofibrils obtained with striated myofibrils at the neural C junction. COERS and Fluorescence Electron Microscopy The Ecdysial Glands of Arthropods Microscopy-FRANK A. PEPE WILLIAM S. HERMAN Cytology of Intestinal Epithelial Cells Cytokinins in Plants-B. I. SAHAISRIVASPETERG. TAVA TONER liquid junction potentials and their effects of AUTHOR INDEX-SUBJECT INDEX on potential measurements in CUMULATIVE SUBJECT INDEX Systems Biology-P. C. CALDWELL (VOLUMES 1-2 1 ) AUTHOR LIST – LIST OF TOPICS Volume 23

Banda 25

Phenomena similar to transformation in somatic cells-J. M. OLENOV Recent developments in the theory of control and regulation of cellular processes-ROBERT ROSEN Contractile properties of sea urchin egg protein filaments related to cell division-HIKoIcHI SAKAI Electron microscopic morphology of oogenesis-ARNE[email protected]Dynamic aspects of phospholipids during protein secretion-LOWELL E. HOKIN The Golgi apparatus: structure and function-H. W. BEAMSAND R. G. KESSEL The Chromosomal Basis of Sex Determination - KENNETH R. LEWIS AND BERNARD JOHN AUTHOR INDEX - SUB

INDICES JECT

Cytoplasmic control over the nuclear events of cell reproduction-NoET. DE TERRA Coordination of beat rhythm in some M-ciliary systems. A. SLEIGH The importance of structural and functional similarities in bacteria and mitochondria-SYLVAN NASS The effects of steroid hormones on macrophage activity-B. VERNON ROBERTS

The fine structure of malaria parasites ~ ~ A K A. I A RUDZINSKA The growth of liver parenchyma nuclei and their endocrine regulation – RITA CARRIERE Chromosome rectification – SHErDoN WOLFF

xviii

CONTENT OF PREVIOUS VOLUMES

Isoenzymes: Classification, Frequency and Importance - C ~ ~ R. ~ ~SHAW ~s

Protein metabolism in nerve B-cells. DOCTOR OZ

Embryonic LUCIEARVY nephron enzymes

AUTHOR SUBJECT INDEX

Freeze Etching HANs TABLE OF CONTENTS

A New Model for the Living Cell: Summary of Theory and Current Supporting Experimental Evidence GILBERT N. LING Department of Molecular Biology, Department of Neurology, Pennsylvania Hospital, Philadelphia, Pennsylvania

.................................................. .......... ...................................................B The requirements power of the necessary pumps. . . . . C. The physical state of water in the living cell D. Is the cell membrane a universal boundary for intracellular-extracellular traffic of water and all solutes? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. An interesting lead in the search for a better living cell model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. The association induction hypothesis. . . . . . . . . . . . . . . . . . A. The molecular mechanism of solute distribution in living cells: theoretical aspects. . . . . . . . . . . . . . . . . . B. The molecular mechanism for solute distribution in living cells: experimental evidence. . . . . . . . . . . . . . . . C. Responses to fundamental criticisms of ion adsorption in living cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Molecular mechanisms of the integrative function of protoplasm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . references . . . . . . . . . . . . . ..........

1 2 2

5 7

7 10 11 12

36 58

Introduction It is generally accepted that living organisms, although highly complex, can be understood in terms of basic principles derived from studies of the simple inanimate world. A biologist's main task is not so much to derive basic principles - that's part of physics - but to understand how these principles are applied in a single complex system. Therefore, the most logical time to start basic biological research would be when humans gain a complete understanding of the physical world. Then one could be sure that the basis of his argument was correct. Since this was not the case - the study of living matter is as old as the study of the inanimate world - the student of biology must be doubly cautious in accepting assumptions presented as facts. For example, when writing textbooks, what could be is often turned into what is. For this reason, the student must constantly reexamine major assumptions, no matter how popular or venerable, using both current knowledge of the physical sciences and advanced technology.

GILBERT N. LING

Technology unavailable to researchers of earlier days. This review begins with a reassessment of the basic assumptions of an ancient and venerable concept, the membrane theory. After discussing some of the difficulties with this concept, he goes on to summarize the theoretical aspects and experimental evidence for an alternative living cell model.

I. Die Membrantheorie A.

HISTORY

About 90 years ago, H. Pfeffer, impressed by the similarity between the osmotic behavior of living cells and that of an aqueous solution enclosed in a semipermeable membrane, proposed the membrane theory (Pfeffer, 1921). According to this view, the outermost layer of every living cell consists of a membrane, which is a universal barrier that limits the rate of movement of water and all solutes between the interior of the cell and the external environment. A second implicit assumption was that there is no significant interaction between cellular proteins, which constitute 15-25% of cell weight, and cellular water, which constitutes almost all of the remainder (75-85%) (Ling, 1962). Therefore, it was postulated that the water in the cell was essentially the same as in any dilute saline solution. Based on these assumptions, problems of water and solute distribution and maintenance of cell volume can be expressed in terms of a general parameter, meinhrzlm permeability. Thus, the degree of cell dilation in a medium containing a given solute reflects the permeability or impermeability of the cell membrane to that solute (De Vries, 1885; Hamburger, 1889). Much effort was spent over the next six decades to discover the properties of this cell membrane, which allowed him to determine not only the rates of entry and exit of water and solutes into and out of the cell, but also their constant concentrations within the cell. Cell. At the turn of the century, Overton used osmotic methods such as cell swelling to study the relative permeability of many solutes (Overton, 1899, 1907). A parallel between the permeability of many non-electrolytes and their oil/water partition coefficients led him to speculate that the cell membrane consists of a continuous lipid layer. He further suggested that transport of solutes that are not lipid soluble, such as sugars, amino acids, and ions, is promoted by "adenoid" or secretory activity. In 1933, Collander and Batlund confirmed Overton's results by measuring the penetration of solutes into the sap of plant cells. However, they found that water's high permeability was inconsistent with its low oil/water partition coefficient, leading them to postulate the existence of tiny aqueous channels in the lipid membrane that allow water (and other molecules) to pass through. small) (mosaic membrane theory) (Collander and Barluncl, 1933). The discovery, also in the 1930s, that the surface tension of cells is much lower than that at the oil/water interface led to the proposal of a

A NEW MODEL FOR THE LIVING CELL

Protein that covers the lipid layer (Cole, 1932; Davson and Danielli, 1952). Ruhland and Hoffman (1925) first suggested that the cell membrane might be like a sieve, selectively admitting some ions but not others (Ruhland and Hoffman, 1925; see also Mond and Netter, 1930). The sieve theory reached its peak in the version presented in 1941 (Boyle and Conway, 1941): the critical pore size was postulated in such a way that both cationic and anionic permeability could be explained. Small cations, such as the K+ ion and the H+ ion, were considered permeable, while the large cations Na+, Ca++ and Mg++ were not. Small anions like C1- and OH- were permeable, but larger anions like ATP, CrP and hexose phosphate were not. This theory not only provided a unified interpretation for the distribution of all types of ions, but also provided the essential molecular basis for Bernstein's membrane theory of cell electric potential. If one were to accept the small pores of Boyle and Conway as those postulated by Collander and Barlund, one could imagine an internally consistent interpretation for the permeability of almost all solutes and water. However, this state of apparent harmony between membrane theory and experimental facts did not long survive the publication of Boyle and Conway's theory (1941). Around the same time, Heppel (1940) and Steinbach (1940) showed that muscle cells are indeed permeable to Na ions. This was interpreted by most as a refutation of Boyle and Conway's specific theory. In order to explain the constantly low content of Na+ ions in the cell, despite their constant inward diffusion, the corrective hypothesis of the Na+ ion pump was put forward (Dean, 1941; Krogh, 1946; Hodgkin, 1951). However, the reviewer is of the opinion that the results of Heppel and Steinbach are more important than is generally recognized: (1) therefore, an increase in the concentration of Na+ ions from the external environment leads to cell shrinkage; therefore, this ion satisfies the criterion based on membrane theory for an impermeable solute. Evidence that it is permeable reveals such expansion and contraction effects that it cannot be used as a measure of permeability. This invalidates the fundamental assumption associated with the widespread use of osmotic behavior as a measure of membrane permeability. (2) If the Na+ ion enters the cell through water channels (the fact that most Na+ ions that enter muscle cells show no competition supports this; see Fig. 21; Ling and Ochsenfeld, 1965), these channels also they should be wide enough to allow the passage of non-electrolytes such as glycerol, erythritol, etc. These compounds are much more soluble in lipids than in water. Therefore, it cannot be assumed that they enter the cell through the lipid layer and not through the aqueous channels. When entering through aqueous channels, the observed linear correlation between the oil/water partition coefficient and non-electrolyte permeability (Overton, 1907: Collander, 1940) cannot be due to permeation through a lipid membrane. ( 3 ) According to Boyle and Conway's theory, hydrated ions are N a 1 , Ca-1 +-, Mg-1 -1 and

GILBERT N. L I N G

since ATP, CrP and hexose phosphate are all impermeable because they are bigger than pores. However, Na + , Ca + + and Mg + + ions have been shown to be permeable (Ling, 1962). So what prevents the diffusion of ATP, CrP and hexose phosphates? Since, according to membrane theory, these are the impermeable anions in a Donnan equilibrium, their outward diffusion also leads to a collapse of the resting potential (Boyle and Conway, 1941). These are some of the problems posed by demonstrating the permeability of Na+ ions; they cannot be remedied simply by postulating a Na+ pump. Proving that cells are permeable to molecules much larger than Na+ ions poses an even greater challenge: there is increasing evidence that proteins and other macromolecules can move in and out of cells (Avery et al., 1944; Dawson, 1966; McLaren et al., 1960; Zierler, 1958; Ryser, 1968). Dawson (1966) showed that enzymes diffuse from isolated intact chicken muscle. Ryser studied the entry of 131-labelled serum albumin and many other proteins, including poly-L-lysine and poly-D-lysine, and showed that all of these molecules enter cells at a rate generally proportional to their weight. Thus, in its current state, the membrane theory is plagued by internal contradictions. However, there are more fundamental questions that need to be asked regarding the general validity of the membrane theory: (1) Is the resting cell producing energy at a rate sufficient to supply all the necessary "pumps"? (2) Is there really no significant interaction between cellular proteins and cellular water? (3) Is the cell membrane a universal rate-limiting barrier to the movement of water and other solutes between the interior of the cell and its external environment? Using technology not available until recently, the critical experiment provides a mechanism for protein entry into cells based on the concept of pinocytosis; that is, the cell actually phagocytoses droplets from its external environment that contain macromolecules (Bennett, 1956; Holter and Holtzer, 1959). There are a number of difficulties associated with this concept: (1) According to current concepts of pinocytosis, although the phagocytosed macromolecules are inside the cells, they are actually outside the cytoplasm due to a continuous plasma membrane surrounding the vesicle. However, it is difficult to understand how macromolecules can exert some physiological effect on the cell, such as the effect of DNA on capsule formation in Pneumococrus, if the substance does not enter the cell from this vesicle (Avery et al. a., 1944). ). If substances entered cells by a "meltdown" of the plasma membrane, the consequence of pinocytosis would be the creation of a non-discriminatory pathway for the entry of all solutes into the cell. If this is the case, it is difficult to explain why poly-D-lysine enters living cells faster than poly-1.-lysine (Ryser, 1968), as well as the correlation between the rate of entry of non-electrolytes and their water-oil distribution coefficients. (3) Metabolic toxins such as iodoacetate, NaF, 2,4-dinitrophenol, and cyanide, which inhibit phagocytosis, do not inhibit entry of P'-labeled serum albumin into living cells (Ryser, 1968). (5) Finally, how to prevent the cell from absorbing and depositing increasing amounts of foreign proteins in the cell through continual pinocytosis? Should we postulate protein bombs?

A NEW MODEL FOR THE LIVING CELL

Studies have been conducted that have answered these questions. It should be added that some of these experiments, if technically feasible, should have been carried out shortly after Pfeffer presented the membrane theory some 90 years ago. B. THE ENERGY REQUIREMENTS OF

AND

PUMPS REQUIRED

Soon after the postulation of the Na+ pump, no fewer than four sets of experiments (with the exception of the reviewer) were published, all comparing the minimum energy required for the Na+ pump in the sartorius muscles of the toad with the maximum energy available compared (Conway, 1946; Levi and Ussing, 1948; Harris and Burn, 1949; Keynes and Maisel, 1945). Conway (1946) and Levi and Ussing believed that the energy consumption of the cell was too high. However, Conway's Na+ ion efflux data were indirectly derived and may therefore be questionable. Data from Levi and Ussing (1948), Harris and Burn (1949) and Keynes and Maisel (1945) are more or less in agreement and indicate a minimum requirement of around 20% of the total energy production. These numbers were derived from the assumption that all energy from glucose oxidation is converted into a form suitable for consumption by the 100% efficient pump and that the pump itself is also 100% efficient. As neither assumption is likely to be correct, these numbers alone indicate a flaw in the pump design (Ling, 1955). Due to the critical importance of the subject, I revisited the energy question in two ways: (1) The energy consumption of the Naf pump at 0°C. itz microskeletons poisoned with iodoretat atid pnre nitrogelz. Interruption of oxidation and glycolysis does not significantly alter constant levels of Na+ and K+ ions in frog muscle cells (0°C) for up to 7 hours (Ling, 1962, p. 200). During this time, Na+ ion production continues at a rate no slower than that of unintoxicated control muscle (Ling, 1962, p. 198). Without oxidation and glycolysis, muscle cells' energy sources are limited to their pool of ATP and creatine phosphate. By comparing the maximum energy available from the hydrolysis of these compounds with the calculated minimum energy requirement for pumping based on the measured resting potential, intracellular Na+ ion concentration, and Na+ ion efflux rate, I concluded that the minimum energy required is 2 . several times if the Na+ ion efflux value used in the calculation may be overestimated. So, for normal muscle at 0°C, I gave an output rate of 1.76 x 10-11 mol/cm²/sec. (in contrast to the Harris value of 4.7 x moles/cm²/sec, 123). The escape rate of the poisoned muscle was higher (3.9-8.73 x 10-11 mol/cm2/s). Ling, 1962). However, he ruled out

GILBERT N. LING

1500-3500 C/o of the maximum energy available, again assuming an efficiency of 100 u/o (Ling, 1962, p. 211). (2) The energy supply of Nu,+, Ca++ and Mg++ pumps in frog nut reactive cells. When the Na+ ion proved to be permeable, an exception was considered and the Na+ pump was postulated. However, it was not long before it was discovered that no two ions had the same Donnan ratio dividing between the inside and outside of the cell and that all were permeable (Ling, 1955). To explain this phenomenon based on the membrane theory, one must postulate more pumps with the same maximum energy source (already overloaded only for the Na+ pump). Using data available in the literature, I calculated that Na+, Ca++, and Mg++ pumps alone would expend up to 350% of the maximum total energy available from a resting frog muscle (Ling, 1965b). To this must be added pumps to maintain the levels of all other solutes (HCO,-, Ck, amino acids, etc.) that are not distributed according to the thermodynamic equilibrium between the cell and its surroundings.

the first fraction of fasting (15-20 minutes) is attributed to egress from the extracellular space. However, our further investigation of Na+ ion efflux from isolated muscle fibers shows that this fraction cannot be in the extracellular space. Therefore, it takes less than 1 second to remove the adherent solution in this preparation, but these fibers still have a similarly fast fraction (see Fig. 11.4, Ling, 1962) (curves obtained from Horowicz and Hodgkin (Horowicz and Hodgkin, 1957) of fibers Individual muscle measurements were apparently exponential, but the setup used by these authors did not allow for stitches before the first 10 minutes of washout, after which they could take readings at intervals of only about 10 minutes. Under these conditions, the curvature is lost). . All energy balance estimates cited above assume that the flat, slow part of the output curve represents the Na+ ion pump. However, the evidence presented in Section 111, B.4 gives us considerable reason to equate the slow fraction in Na+ ion efflux with the exchange of the adsorbed fraction. If this is the case (for further evidence, see Ling, 1962), the fast fraction actually represents the rate of escape of the free Na + ion into the environment and should be used for the pumping rate (Ling, 1962, ch. 11; Ling, 1966a). Finally, I would like to point out that the method I used to derive the exit rate was chosen to provide a conservative estimate. A small bundle of muscle fibers was immersed in an isotopically labeled solution for a period of time (about 3 minutes). The fabric was then quickly installed in the washer and an effluent curve was obtained for the next 100 to 200 minutes, at which point the curve became exponential. This exponential part of the curve was then extrapolated onto the ordinate to give an (actually very small) estimate of the amount of Na+ ions entering the cell during the 3 min of incubation. Since the total concentration of Na+ ions did not change during this time, the entry and exit rates must have been the same. The pumping rate was then derived from this number.

A NEW MODEL FOR THE LIVING CELL

C. THE PHYSICAL CONDITION OF THE WATER

AND

LIVING CELL

Evidence is accumulating at a rapid pace that cell water is in a different state than water in a dilute saline solution. As this topic will be discussed in detail in the next section, we will just point out here that there are two lines of evidence that point to the above conclusion: (1) the abnormal pattern of water freezing in the living cell (Chambers and Hale, 1932, Rapatz and Luyet, 1958, Ling, 1967~) compared to pure water or water in dilute saline (Ling, 1966b); and (2) abnormal nuclear magnetic resonance (NMR) spectra (Chapman and McLaughlan, 1967; Fritz and Swift, 1967) obtained from water in living cells.

AND

THE CELL MEMBRANE A UNIVERSAL SPEED LIMIT

AND

INTRACELLULAR-EXTRACELLULAR WATER TRAFFIC AND ALL THE SOLUTIONS? Recently, Arbiter has introduced a technique, influx profiling, as a means of determining the rate-limiting step in the movement of water or solutes between the cell and its environment (Ling, 1966a; see Fig. 1). In summary, the absorption fraction of a labeled material t seconds after introducing a cell into a solution containing the isotope is plotted against the square root of t. The profile has specific properties depending on the rate limiting step. Therefore, when the rate-limiting step is at the cell membrane, the curve has a sigmoidal shape (Fig. 1A). On the other hand, if the solute diffuses through the cell, including the cell membrane, at a more or less uniform rate (phase-limited diffusion), the early part of the curve is essentially a straight line (Fig. IB). This technique can be most usefully applied to individual cells. Figure 2 shows the entry profile for tritium hydroxide-labeled water entry into an oocyte from a single frog ovary (Ling et al., 1967). The solid line through the dots has been calculated theoretically to represent confined mass phase diffusion. This shows that the cell membrane is no more resistant to water movement than the cytoplasm. The general diffusion coefficient ranges from one-half to one-third of the diffusion coefficient of tritium hydroxide in a 0.1 N saline solution. This series of experiments disproves one of the fundamental tenets of membrane theory with respect to water. However, water is not the only substance whose movement is not restricted by the membrane. Fenichel and Horowitz showed that the release of many non-electrolytes from frog muscle is also limited by the volume phase (Fig. 3; Fenichel and Horowitz, 1963). This study included many of the same non-electrolytes studied by Overton and Collander. Thus, it appears that the permeability of the "membrane" studied by these authors was, at least in some cases, the permeability of most of the protoplasm. In summary, we can now state that (1) the resting cell does not have enough energy to operate all the necessary membrane pumps

0,8

Mt 0,6

0,6

0,4

METRO,

0,2

me me

COWARDLY. 1. Time of entry of a labeled substance into model systems with rate limiting steps as indicated on each graph. The "influx profiles" are theoretically calm. The ordinate represents the absorbance M of the labeled material at time t as a fraction of the final amount of material in the system (Mw). The abscissa represents the square root of t (Ling, 1966a, with permission from

von

A nova academia do YovR

from Srie?ice.r).

A NEW MODEL FOR THE LIVING CELL

Theory for maintaining the observed asymmetric distribution of solutes; (2) cellular water is not normal, as the membrane theory postulates, and (3) the cell membrane is not a universal limiting barrier to the movement of water and solutes between the cell and its environment. 105

08 /M, mm

Become an influx timeline with the "inversion method". The curve through the points is calculated theoretically on the basis of a simple bulk phase-limited diffusion (Ling et al., 1967, by permission of The Journal of General Phyrirology).

e 0

2000t (seconds)

4000

COWARDLY. 3. Time course of marked thiourea efflux from frog sartorius muscle. Efflux of C14-labeled thiourea was tested by stirring muscles previously equilibrated with thiourea in different portions of unlabelled Ringer's solution, whose activity was then assessed. Curve A is theoretically calculated for membrane-limited diffusion and does not fit the data. Curve B, which fits the data almost perfectly, was calculated theoretically on the basis of limited mass phase diffusion. C is the concentration of labeled thiourea in cells at time t; C, is that at t is equal to 0 (Fenichel and Horowitz, 1767, with permission from Artu Physiologic-a Srundinauira).

GILBERT N. LING

Together, this evidence strongly opposes the diaphragm pump theory. Therefore, we have no choice but to look for a new living cell model to interpret the large body of data already collected and guide future research. 11. An interesting lead in the search for a better living cell model

Figure 4 shows a Starr and Williams electron micrograph of a Congo diphtheroid bacillus flagellum (Starr and Williams, 1952). The dry matter of these flagella is practically pure protein (Weibull, 1960). isolated flagella

COWARDLY. 4. Electron micrographs showing the fine helical structure of flagellar material from Congo diphtheroid bacteria. The structure is that of a left-handed three-strand helix with a diameter of 19 nip and an axial periodicity of 50 m p 100,000 X (Starr and Williams, 1952, by permission of the Journal of Baccleriology).

A NEW MODEL I:O

LIVE CELL

can be reversibly precipitated by ammonium sulfate and behave as homogeneous protein molecules in many respects. There is no membrane covering. The isolated flagellum appears to lack ATPase or any other enzyme. It is directly attached to the protoplasm in a subcellular structure called the basal grain (Weibull, 1960). Despite its structural simplicity, this protein-water system is able to move in a spiral and thus provide the driving force for bacterial mobility (Holwill and Burge, 1963). The bacterial flagellum is a telling example of the basic capabilities of protoplasm, with control and energization taking place in the basal granules beyond the body of the flagellum itself (Astbury, 1951). Thus, this system illustrates the transmission of information and energy over long distances through a protein-water system. According to the association-induction hypothesis, it is this basic ability of organized protein-water-ion systems to undergo reversible changes between metastable equilibrium states that distinguishes this and many other types of protoplasm from the inanimate world. The cell owes its functional coherence and its discontinuity from the external environment not to a lipid membrane but to the unique properties of the protein-water system, just as the naked flagellum, a permanent organelle in an aqueous environment, is functionally coherent. and discontinuous with its surroundings. 111

The association-induction hypothesis

The association-induction hypothesis postulates that the maintenance of the solute distribution pattern reflects the properties of the whole protoplasm (Ling, 1962, 1964b, 196ja,b, see also Butschli, 1894). It is well known that the water content of a living cell is more or less constant. It is also generally accepted that the distribution of water represents an equilibrium state, meaning that the free energy of water inside the cell is equal to the free energy of water outside the cell. Therefore, within a unit time interval, the number of water molecules entering the cell is exactly equal to the number of water molecules leaving the cell. To maintain this constant water level, the cell consumes no energy. The association induction hypothesis states that constant concentrations of all solutes in the living cell also represent steady states, or rather metastable steady states. A stable state of equilibrium is a true state of equilibrium, except that its maintenance is somewhat precarious, as in the case of a narrow block of wood resting on its edge. In the following review, I will specifically address K+ and Na+ ions

GILBERT N. LING

the understanding that the mechanisms involved in their distribution and control are essentially all similar for other solutes.

A. MOLECULAR MECHANISM OF SOLUTE DISTRIBUTION IN LIVING CELLS: THEORETICAL ASPECTS According to the association-induction hypothesis (see also Fischer and Moore, 1908; Troschin, 1958), intracellular solutes exist in two states: (1) solution in cell water and (2) ) Adsorption to cellular proteins. Since the amount of solute in the first state depends on the condition of the water in the cell, an important part of our discussion of solute distribution will be a consideration of the condition of the water in the living cell. Next, we consider the influence of water state on solute distribution, and finally, we deal with the specific sites of solute adsorption. We will first consider the theoretical aspects of these problems, followed by a discussion of the experimental evidence supporting the model. 1. The effect of protein on the condition of water in living beings

cells

Water molecules have a strong permanent dipole moment (1.83 x lo-'* e.s.u.) and high polarizability (1.44 x 10 - 2 4 cm.), i.e. a great tendency to form strong dipoles (Ling, 1962, p. Sixty-five). A simple calculation shows that the electrostatic interaction of water molecules with an electrical charge carried by, say, an ion extends beyond the first layer of water molecules surrounding the ion to several other layers. Polar compounds like titanium dioxide also interact with water. Experimentally, Harkins showed that the heat of desorption of the first layer of water molecules on the surface of titanium dioxide is 6550 cal/mol greater than that of quartz. For the second layer it is higher at 1380 cal/mol; for the upper third layer 220 cal/mol; and for the upper fourth layer 71 cal/mol (Harkins, 1945). Like titanium dioxide, the protein has a large number of polar groups. In muscle cells, the average filament distance between protein molecules is only 16.9 Å, less than the thickness of seven layers of water molecules. All or most of the water in a typical resting cell may be under the polarizing influence of ionic groups and protein hydrogen bonds and exist as polarized multilayers (Ling, 1962, Chapter 2, 1965a; 196613; 19). 6 7~). Furthermore, this polarization must align the water molecules in directions determined by the structure and orientation of the proteins. In this system, the freedom of movement of individual water molecules is more restricted than in normal water, this restriction being more pronounced in the case of rotational motion. The level of restriction gradually decreases with distance from the protein surfaces, as illustrated in the graph in Figure 5, where the length of the curved arrows indicates the rotational degree of freedom.

A NEW MODEL FOR THE LIVING CELL

The entropic exclusion of polyatomic, or indeed polyatomic, solutes from polarized water

In aqueous media, the Na+ ion absorbs at least one layer of water of hydration; that is, water molecules that become highly polarized under the influence of the ion's electrical charge. This hydrated ion behaves as a single entity, that is, it is much more polyatomic. Such a molecule has many types of rotational motion.

COWARDLY. 5 . Schematic representation of the adsorption of water molecules as polarized multilayers on proteins. Upon entering such a system, the hydrated ion shown on the left experiences severe rotational restriction. A simple model of this effect is shown on the right, showing the restricted orientation of small nails in the field of the horseshoe magnet filled with iron filings.

In fact, the rotational entropy, which is a measure of the rotational freedom of the molecule, is responsible for most of the entropy of this ion in an aqueous medium. When a hydrated ion, such as the Na+ ion, is introduced into the cell, where most of the water is multilayer polarized, its rotational movement is restricted in a manner equivalent to losing the disorientation of the small fingernails (Fig. 5) , into which it is inserted the space of a horseshoe magnet filled with iron filings. The result of this rotation constraint is reduced entropy. At equilibrium is the distribution of a solute in a system containing

GILBERT N. LING

Water and a normal aqueous medium are determined by the difference in the standard free energy of the solute in the two media. The AF in turn is the sum of an energy term3 and an entropy term. The energy does not differ much between the two systems. A reduced entropy of the Na + ion in the cell water means a reduced AF and therefore a lower equilibrium concentration of the Na + ion in the polarized water. 3 . O M o l e r z h v Merhanim by Ionic Adsoiptioiz

As early as 1908, Fischer and Moore suggested that selective accumulation of K+ ions could result from adsorption on cellular colloids (Fischer and Moore, 1908). In 1951 and 1952, arbiter suggested that the p- and y-carboxyl groups carried by the side chains of aspartic and glutamic acid could provide anionic sites for the adsorption of K+ and Na+ ions (Ling, 1951, 1952). The hydrated diameter of the K + ion is significantly smaller than that of the hydrated Na + ion. According to Coulomb's law, the electrostatic interaction of the K + ion with negatively charged carboxyl groups would be greater than that of the larger hydrated Na + ion. In theory, taking into account the sharp reduction of the dielectric constant in the immediate vicinity of an ion (the phenomenon of dielectric saturation), it is possible to calculate a selectivity of the K + ion over the Na + ion of the order of 10 to 1. Support of Com based on this hypothesis, the analogy between the recently developed ion exchange resins and the living cell. The selective accumulation of K+ over the Na+ ion in the resin was obtained by introducing anionic groups and fixing them in a three-dimensional network. In the years that followed, more knowledge was gained in the field of ion exchange technology. It has been demonstrated that such resins do not always selectively accumulate K+ ions over Na+ ions. Therefore, resins with strongly acidic groups (lower pKa) prefer the K+ ion over the Na+ ion. However, the opposite is true for resins with weak acid groups (high pKa) (Bregman, 1953). This fact and the theoretical and experimental work by Eisenman, Rudin and Casby on glass electrodes (Eisenman et al., 1957) led to a complete revision of the previous model (Ling, 1960). It was clear that the pK value mainly reflects the electron density of the acid group. To bring this concept into a manageable form, the value c was introduced. This parameter is rigorously defined elsewhere (Ling, 1962, pp. 5-7). For simplicity, a high r value (ie about -1 A , ) corresponds to a high pKa value (eg acetic acid, pK = 4.75). On the other hand, a low c value (ie -5 Å) corresponds to a low pK value (eg trichloroacetic acid, pK < 1.0). 3 More specifically, this term should refer to the enthalpy or heat content H, which is related to the energy U by the relationship H U PV, where P is the pressure and 1.' is the system volume. Since volume changes are small in a liquid system and only changes in H and U are relevant to our discussion, the better known Rnevyy method is used here.

A NEW MODEL FOR 'EL VIVO CB1.L

with the value of r. defined, it is possible to calculate the total interaction energy between a given cation (e.g. K+ or Na+ ion) and an oxyacid group (e.g. a carboxyl group) with a given value c when the cation is of the oxyacid group separated by zero , one, two or three water molecules (see Fig. 6).

configuration 0

Configuration I

Attitude

configuration UI

COWARDLY. 6. The linear model. The shaded cml: on the left in each configuration represents the negatively charged oxygen atom of an oxyacid (eg, carboxyl), and the shaded circle on the right represents its opposite effect. The empty circles represent the water and the different letters indicate the distances used in the calculations. Reprinted with permission from: Gilbert N. Ling "A Physical Theory of the Living State" (Waltham, Massachusetts: Blaisckll Publishing Company, 21 Ginn and Company Division, 1962) p. 61

From these results, the dissociation energies of various alkali metal ions can be determined as a function of the r-value (as well as the polarizability) of the anionic group. Figure 7 shows the results of this calculation. With increasing value of c, the order of anionic group preference for the five alkali metal cations undergoes 11 permutations. At the lowest c value, the order is Cs > Rb > K > Na > Li, while at the highest c value, the order is completely reversed. These theoretically calculated sequential order changes are similar to the sequential order changes observed experimentally by Eisenman (Eisenman, 1961; Fig. 8) in the relative preference of glass electrodes of varying composition to alkali metal ions. An important conclusion that can be drawn from the results of these calculations

sixteen

GILBERT N. LING

is that a small change in the value of c can significantly change the relative preference for an acidic group for the K+ ion over the Na+ ion. If the change in the value of c is large enough, the order of preference can be reversed. This point is revisited in Section 111, D.7.

COWARDLY. 7. The calculated dissociation energy of different cations as a function of the value of c. A polarizability of 0.87 x 10-24 cm3 was assumed for the fixed anionic group. Reprinted with permission from Gilbert N. Ling, "A Physical Theory of the Living State" (Waltham, Massachusetts: Blaisdell Publishing Company, A Division of Ginn and Company, 1962), p. 75

4. Equation for distribution of solcites in living cells according to hypothrys association Figure 9 shows a diagram of a part of a living cell in contact with its external environment. Inside the cell, ions exist in two states, free and adsorbed. The model cell shown has three types of protein sites that adsorb alkali metal cations. Two of these types of sites prefer the K+ ion to the Na+ ion; the Na+ ion is preferred to the K+ ion. Based on this model, intracellular ionic concentrations can be expressed by the following equations: a"a+Illt+y

"a+]:,+

"a+]"

(1)

A NEW MODEL FOR THE LIVING CELL

where [ Na + I in and [ K + I in n are the total intracellular concentrations of Na + ions and K + ions, respectively, a is the percentage of water in the cell. [Na+Iint and [K+],,, are the concentrations of the interstitial ions K+ and Na+, respectively. [N a + ] i d and [K+]: are Na + and K + ions adsorbed on type I sites, "a+]:: and [K+]:' on type I1 sites, etc. intracellular and

kcal/mol

-6918

-4.612

-2306-

0 II .li.-

2.306 -

4612 6,9121

4.612

2 306

- 2 306

AFhak=-RT InKhak=FAE'

COWARDLY. 8. Ionic specificity at ionic glass electrode potentials at neutral pH. Each vertical line of data points corresponds to the observed selectivity properties of a given material (Eisenman, 1961).

Adsorbed ion concentrations are given in units of moles per kilogram of fresh cells. The interstitial ion, on the other hand, is given in moles per liter of cellular water. setting up the equations. (1) and (2) in the most general form:

G I L B E R T N. L I N G

c the Nordics

EU'

111,

=a(K+J,,,,

IK'yl

(4)

eu > 1 ~

,:EU

Here, [N to t and [K+]>l;] refer to the concentrations of Na + and K + ions adsorbed on Lth-type sites. In the case represented in the diagram of figure 9, there are three types of locations in total, that is, N z 3 .

rti. 9. Dynamic illustrations of a living cell. The Stiprlrcl surface represents water-filled space in polarized multilayers.

For. The egicafioiz for the liitevstitlai I o n . Let us first consider the distribution of interstitial ions. According to Henry's law, the ratio of concentrations of a solute distributed in two solvents in equilibrium with each other is constant over a considerable range of concentrations (see also Troschin, 1958). Thus, in the case of the Na+ ion distribution between the cell water and the external aqueous medium, the relationship between the internal and external Na+ ion concentrations is a constant ysa, which is called the partition coefficient. Therefore

where [ N a + ] ,,s is the external concentration of Na+ ions. similar

where [ K + is the external concentration of K + ions and ye is the K + ion equilibrium partition coefficient. rearranging the equations. ( 5 ) and (6) : I N a + I , l , t = ( j ~ , l [Na-‘I..

(7)

A NEW MODEL FOR THE LIVING CELL

y [K+I,rlt = qrc [K+],Y (8) According to these equations, a plot of interstitial Na+ (or K+) ion concentration versus external Na+ (or K + -) ion concentration should form a straight line with a slope equal to qNu (or qK). Furthermore, there is no competition between ions in interstitial water, i. H. cell water contains the same concentration of Na+ ions regardless of whether they are K or not. + ion is also present. 6. The equations for adsorbent I or m. If the concentration of type I adsorption sites [f] shown in Fig. 9 is I and if each site can be occupied by one ion at a time, the total number ( J f Na - 1--ions adsorbed on I-type sites, can be described by the Langmuir adsorption isotherm (Langmuir, 1917) .

where Ki,u and K:( are the adsorption constants for Na+ and K+ ions, respectively, at this type of site in iM-1. Likewise, for adsorption of K+ ions on Type I sites:

F+1º =

Ifl'K":<

[K+l<,x

(10) 1 I?:,, "a+] ('I K":; [ K + ] A graph of [Na+],,,, versus INa-t-],., is a hyperbola (see, for example, Fig. 2G and 30): That is, at low values of "a+- I(,x" the sites are almost empty and there is a proportional increase in adsorbed Na-I- with the increase of "a+] k.x. A Com in sites of higher values are increasingly occupied by [Na-+ ].P The increase in adsorbed Na + ions decreases continuously with a unit increment from I N to + If.X until the total adsorbed Na + ions approaches a value maximum equal to the total number of sites, J [ Equation 3 shows that the adsorbed Na+ ion can be reduced to an insignificant level by increasing the concentration of the concurrent K-t ion. Equation 9 can be written reciprocally:

111

"e +

1a,,

[fl'Qu

+ qi[ K f l e x )

1 „a+

It was

1 (11)

[Florida'

Plotting l/[Na+];,,] as a function of l / l N a + ] , L x with constant [KfJ,,], a straight line is obtained. The intercept on the ordinate (ie, the 1 inch Na+Icr z 0 ) is the reciprocal of the total concentration of Type I sites. The slope of the lines The reciprocal of equation (1 0 ) for is a function of [ K+-.ie , I?:, and the adsorption of K+ ions is

GILBERT N. LING

If Eq. (7) and (9) are substituted in the equation. (3) and equations. (8) and (10) are substituted in the equation. (4) the explicit equations for the total concentrations of Na+ and K+ ions in living cells are obtained.

Zia

Here and gi are the adsorption constants of Na+ and K+ ions at the Lth adsorption site. Equations (13) and (14) are basically similar to an equation first introduced by Troschin for the distribution of sugars in cells (Troschin, 1958). In the following, they are referred to as Troschin's equations.

B. THE MOLECULAR MECHANISM OF DISTRIBUTION OF SOLUTIONS IN LIVING CELLS: EXPERIMENTAL EVIDENCE 1. The physical state of living cells and model systems

The adsorption of polarized multilayer molecules onto solid surfaces is described by a Bradley equation (Bradley, 1936; see also Boer and Zwikker, 1929) Po = K1 K,' log,, K, (15)

PAG

where a is the amount of gas (in this case water) adsorbed at the vapor pressure p; Po is the vapor pressure at saturation. K1, K and K are constants. As shown in Fig. 10, this equation follows the sorption of water on an isolated protein, sheep wool (Ling, 1 9 6 5 ~ ) El . Bull's (1944) full range of experimental data fits the equation. (fifteen). Similar results are obtained for water absorption in collagen (Ling, 1965) and other proteins and macromolecules (Mellon and Hoover, 1950). These experiments indicate that the proteins in zho have the ability to organize the water with which they are in contact into polarized multilayers. You would expect cellular proteins to have the same property, and indeed there are two pieces of evidence that water in cells is in a different state than water in dilute saline.

A NEW MODEL FOR THE LIVING CELL

you Intracellular freezing pattern. Normal water or a dilute saline solution forms tridymite ice in hexagonal patterns when cooled below 0°C (Hallett, 1965). In its general form, normal ice is feather-shaped with branches at regular intervals (Fig. 11). Ice formed in living cells supercooled by seeding with an ice-tipped micropipette has an anomalous structure (Chambers and Hale, 1932; Rapatz and Bradley isotherm

record record ( % / P I +2

FIG. 10. Water vapor sorption on sheep wool plotted from the Bradley multilayer adsorption isotherm. Data of Bull (1944) (Ling, 1966a, courtesy of the Annals of the New York Academy of Sciences).

Lujet, 1958). For example, in skeletal muscle, longitudinal spikes are formed that follow the direction of muscle protein filaments (Chambers and Hale, 1932; Rapatz and Luyet, 1958) (Figs. 1-2). These spines, which have no branches, indicate that the intracellular water is abnormal. That this anomaly is closely related to the structure and orientation of cellular proteins (Ling, 1965a, 1966b, 1967a) is demonstrated by the evidence that when myofilaments twist, the ends also twist (Chambers and Hale, 1932). ii. Nuclear Magnetic Resonator (NMR). The newly developed technique of nuclear magnetic resonance spectroscopy is being used with increasing efficiency to investigate the aggregate state of water in cells. From the amplification of the NMR signal of water in the rabbit sciatic nerve and its dependence on the orientation of the magnetic field, Chapman and McLauchlen concluded that most of this intracellular water is not normal, but is in a normal state, partially oriented (Chapman and McLauchlan). , 1967) (Fig. 13). Fritz and Swift (1967) reached a similar conclusion when working with frog nerves. [To the critique of the above conclusion by Bratton et al. (1965) that only part of the water in muscle cells is subject to restricted rotation, see also Fritz and Swift (1967)1. 2. Sheep wool water ion exclusion,

toad Muscle,

atzd on actomyosin gel Figure 14 shows the ion absorption equilibrium of sheep wool. In the presence of high concentrations of a competing ion, a cumulative labeling graph is displayed

GILBERT N. LING

COWARDLY. 11. Growth of ice crystals in supercooled pure water initiated by insertion of a single crystal at -3.5°C. (Hallett, 1965, by permission of Federation Proceedings).

COWARDLY. 12. Development of a spear of ice in a single muscle fiber at -2. 5" and Luyet, 1c)>8, with permission from Eiodynamicu).

C . (Rapatz

A NEW LIVING CELL MODEL

100 cps

COWARDLY. 13. NMR spectra of water in the rabbit sciatic nerve: (top) with nerve axis parallel to the applied field; (middle) axis perpendicular to field: (lower) nerve axis at about St" to direction of applied field (Chapman and McLauchlan, 1967, with permission from Na/ur.r). 160

-0

I hope that

Off round without lobes b 0 5 round sts without lobes

120

[Rb+l,,

(mm)

COWARDLY. 1.4. Equilibrium of the distribution of labeled Rb+ ions in sheep wool in the presence of different concentrations of competing unlabeled Rb+ ions. Increasing the concentration of unlabeled Rb+ ions from 0 to 0.5 M decreased the concentration of labeled Rb+. Additional increase of 0 . However, 5 to 0.7 M did not further decrease the concentration of labeled Rb+ ions in wool and the distribution curve is linear. Wool contains 30% water. Assuming all labeled Rb+ ions are in the presence of 0.5 or 0.7 M unlabeled Rbtion in this water. the partition coefficient of the labeled Rb+ ion is approximately 0.29.

GILBERT N. LING

The Rb+ ion as a function of the outer Rb+ ion concentration approximates a straight line (bottom line). In the absence of competing ions, there is an additional fraction of Rb+ ions in the wool that is not linearly related to the external concentration of Rb+ ions. In fact, subtracting the bottom curve from the top curve results in a hyperbola. These data show that the accumulation of ions in sheep wool obeys an equation similar to Eq. (13) or (14) (Ling, 1965b). Looking at just the linear portion, we find that the slope of this line is only 0.12, indicating that the Rb+ ion in this 30% protein, 70% water system is only 29% of its concentration in the external absorption solution. Thus, the effect of water polarization in multilayers described above is the partial exclusion of solutes from that water. Figure 15 shows the equilibrium Na+ ion uptake of the living frog Sartorius.

-k

300

5 0 mM 10 0 mM

0, Ln

100

120

C No+lex (mM)

COWARDLY. 15. Equilibrium distribution of Na+ ions in frog sartorius muscle in the presence of varying external concentrations of K+ ions. The external concentrations of K+ ions are 2.5 mM, 5.0 mM, and 10.0 mM. Data were calculated based on 10% extracellular space. The slope of the line passing through the points with the highest concentration of K+ ions is 0.14. Muscle cells contain 78% water. Assuming that all Na+ ions in the cell are in the cell water at the highest concentrations of K+ ions, the equilibrium distribution coefficient of Na+ ions between the cell water and the external environment is 0.18.

muscle (Ling, 1965a,b, 1966b; Troschin, 1958). As in sheep's wool, increasing the concentration of competing K+ ions decreases the concentration of intracellular Na+ ions. But here, too, the intracellular concentration of Na+ ions does not drop to zero with increasing K+ ions. Rather, there is a level at which no further increase in K+ ion concentration will affect Na+ ions in the cell. This remaining fraction (B) of the intracellular Na+ ion concentration is also linearly related to the external Na+ ion concentration with a slope of about 0.14. As in the case of

A NEW MODEL FOR THE LIVING CELL

In sheep wool, at lower concentrations of competing K+ ions, there is an additional fraction of Na+ ions (fraction A) that is approximately hyperbolic relative to the external Na+ ion concentration (see Section 111, D,10). Figure 16 shows the equilibrium distribution of Rb+ ions in a water protein

160

30 -

0,5

[Rb+],,(mM)

COWARDLY. 16. Equilibrium distribution of labeled Rb+ ion on actomyosin gel isolated at neutral (6.7) and acidic (4.3) pH.

(actomyosin) extracted from the living cell (Ling and Ochsenfeld, 1968a). Here the distribution of Rb+ ions follows the same pattern observed in sheep wool and live muscle. In the presence of a strongly competitive cation (H+ ion, pH 4.4), the Rbi ion concentration in the gel is linearly related to the external Rb+ ion concentration (fraction B). At a lower concentration of H+ ions, an additional fraction (A), which shows the typical properties of a Langmuir adsorption isotherm, overlaps fraction B. The actomyosin gel is perfectly homogeneous and contains no anatomical structures, but the concentration of Rb+ ions in the gel Low pH water is only a fraction (50-70%) of the external solution. From these data, we conclude that the water in this gel again has abnormal solubilities for alkali metal ions. The actomyosin gel used in the research shown in Pig. 16 is very dilute (3-576% protein versus 95-97% water). Along with other proteins, actomyosin exists as a much more concentrated gel in living muscle (205% protein versus 80% water). The water ion exclusion property of the isolated actomyosin gel is a compelling reason to believe that the ions of the B fraction of the Na+ ion in frog muscle (Fig. 15) represent the water fraction of the muscle cell and that the water in the muscle

G I L H I X I 'N. LING

The sheep wool-like cells harbor less alkali metal ions than the external aqueous solution. 3. The origin of water extraction

Gel Actomiosiii

Ling and Ochsenfeld studied the temperature exclusion coefficient of Rb+ and other alkali metal ions from water in isolated actomyosin gel (Ling and Ochsenfeld, 196821). From these studies, it was concluded that the exclusion was mainly due to the unfavorable entropy of the hydrated alkali metal ion in the gel water.4

4. The adsorbed fraction of Na+ ions We showed above (Fig. 15) that increasing the concentration of K+ ions to a certain level causes a decrease in the intracellular level of Na+ ions in the sartorius muscle of the frog. This indicates that the K-i- ion competes with an adsorbed fraction of the Na+ ion. There is now considerable independent evidence that such a fraction actually exists: (I) Lewis and Saroff (1957) showed that the Na+ ion binds to isolated actomyosin. (2) Hinke (1959) used an intracellular Na+ ion-sensitive microelectrode to study Na+ ion activity in frog muscle and squid axon and concluded that about two-thirds of the intracellular Na+ ion is bound.6 (3) Cope (1967) recently used NMR techniques to study the Na+ ion in muscle and concluded that about 70% of this ion is complexed. A similar conclusion was reached by Rotunno et al. (1967) on the Na+ ion in frog skin cells. Thus, under normal conditions, the intracellular Na+ ion consists of an adsorbed fraction as well as the interstitial fraction discussed above. 5.

K 4-- 1 ~ nArri/mi/lation

For. Interpietational Accordion

Changing the external concentration of unlabeled K+ ions produces a family of straight lines that converge at the same point as the ordinates. Such a plot is characteristic of a Langmuir adsorption isotherm [Eq. ( 1 1 ) I y in relation to the association-induction hypothesis indicates that the intracellular K + ion is all at 4 N o / r. Added in P w u f : Recently Gary-Bobo and Solomon [Gary-Bobo, C. M. and Solomon, TO . K (1968). /. Gerr. firiol. 52, 8251 published the results of their investigations on the distribution of K+ ions in hemoglobin solutions. K+ ion exclusion from this protein solution was also observed at low pH and these authors attributed this to a Donnan effect. Whether this is a better explanation applicable to our actomyosin liter data (or vice versa) remains to be determined. 5 Nore Addrd/o Test: For comments on the use of ion-sensitive electrodes to analyze ionic activity in living cells, see Ling [Ling, G. (1969). Nature 221, 3861.

A NEW MODEL FOR THE LIVING CELL

the adsorbed state. The concentration of K+ ions in muscle cells (90 mM) is many times higher than in frog plasma (2.5 mM). Therefore, the interstitial K+ ion may represent only a fraction of the concentration in the external solution and therefore less than 1-2 Cjc, which is the limit of experimental error. In

0.I ([K+],,)-'

0,2

0,3

0,4

(Mol/Liter)-'

COWARDLY. 17. Intracellular labeled K+ ion concentration versus externally labeled K+ ion concentration. with which it is in equilibrium, in the presence of 0, 20 and 5.0 mmol/L of unlabeled potassium acetate. The labeled K+ was also in the form of the acetate salt. Each dot represents the marked K+ ion concentration in a single toad sartorius muscle; Lines obtained by the method of least squares. In the lower curve within arc3 from [K+ICS-I = 0 to 0.05 (mmol/liter)-1 and from [K + li , -l = 0 to 0.01 (mmol/kg.)-l , a total of 23 points were determined; they are so close that only a few could be represented. All others override these. Data from two sets of experiments (Ling and Ochsenfeld, 1966, courtesy of The Jourtzal of General Physiology).

With these data we obtain an association constant for the K+ ion of 665 M-1 and a concentration of anionic If] sites equal to 143 mmol per kilogram of fresh cells. Based on the known muscle protein content and the percentage of acidic side chains in these proteins, the maximum number of anionic sites in the cell is 260 mmol/kg. which is more than enough to explain this number of K+ ion adsorption sites (Ling and Ochsenfeld, 1966).I; (yo

Note added iiz test: For additional NMR evidence that most

The K+ ion is in an adsorbed state, see Ling and Cope [Ling, G.N. and Cope, F.W., Srilerce 163, 1335 (1969) 1.

GLBBRT N. LING

B. Alternative Interpretations. you Donna's balance. Although competition is generally considered a feature of the Langmuir adsorption isotherm, it can also result from the fact that macroscopic electroneutrality must be maintained in a Donnan equilibrium (Ling and Ochsenfeld, 1966). However, a Donnan system can be distinguished from a system in which the K+ ion is adsorbed by examining the effect of a second ion on the equilibrium distribution of the labeled K+ ion and comparing the effect of this second ion with that of the K+ ion. Unmarked. . In a Donnan equilibrium, the difference between the two effects depends on the difference in their respective activity coefficients. In Figure 18, we compare the effect of similar concentrations of Cs+ ion and unlabeled K+ ion on equilibrium K+ ion accumulation. The difference observed experimentally is much greater than can be explained by the difference in activity coefficients (less than 2%). ii. The traffic model. According to a more recent version of the membrane theory, most K+ ions enter the cell by associating with certain hypothetical "carrier" molecules, which then transport the K+ ions across the lipid portion of the plasma membrane (Osterhout, 1936; Jacques , 1936; Epstein and Hagen, 1952). Once the carrier ion-K+ complex reaches the inner surface of the cell membrane, the K+ ion dissociates and enters the cell's water. This idea is similar to the activity of the adenoids, or secretaries, postulated by Overton in the early 20th century. The best evidence that can be adduced in favor of such a carrier hypothesis comes from studies of the initial rate of entry of K+ ions. Thus, this rate of entry exhibits saturation (that is, as the external K+ ion increases, the rate of entry per unit increase in external K+ ion concentration steadily decreases until it approaches zero) and competition (that is, ions of similar alkali metals compete with the K+ ion). and reduce the entry fee) (Fig. 19; Ling and Ochsenfeld, 1965). 1;Basic difficulties of the traffic model. (1) Historically, K+ ion entry has always been thought of as free ion diffusion across the cell membrane. Bernstein's original version of the membrane theory (Bernstein, 1902) of the resting potential was based on this thesis. The postulation of the Na+ pump did not imply a revision of this concept. The Hodgkin-KatzGoldman equation [Eq. (16)] was derived based on the fact that the K+ ion migrates across the cell membrane as a positively charged ion under the influence of a constant electric field. When the K+ ion crosses the cell membrane in combination with a carrier, there are two possibilities: either the carrier molecule carries an anionic charge or it is a neutral molecule. The combination of the K+ ion with a negatively charged carrier breaks the basis of the Hodgkin-Katz-Goldman equation: the constant electric field would have no effect on the direction of motion of a neutral complex. On the other hand, if the carrier itself is considered uncharged, the carrier-K+ ion complex would be a monovalent cation. This would overcome the previously mentioned difficulty. Another difficulty now arises, however.

A NEW MODEL FOR THE LIVING CELL

the huge gain in free energy when a charged ion is brought into a lipid phase. To achieve this, the carrier must have a strong affinity for the K+ ion and properties that allow it to remain permanently in the lipid membrane. AND

([CS'I e x )

-' (mmol/liter)-'

COWARDLY. 18. Labeled equilibrium Cs+ ion concentration in muscle cells plotted reciprocally against the external Cs+ ion concentration with which they are in equilibrium. The competing concentrations of K+ ions are 0, 20, and 50 mmolylliters, respectively. Both Cs+ and K+ were in the form of acetates (24°C). Each point represents a single determination in the sartorius muscle of a frog; Lines obtained by the method of least squares. The effect of K+ ion on K+ ion accumulation labeled (dashed lines) in Figure 1 for comparison (Ling and Ochsenfeld, 1966, by permission of The Journal of General Physiology).

GILBERT N. LING

It's not easy to build a molecule with these contradictory properties. Perhaps for this reason, despite the carrier's long history, there is no experimental model that demonstrates the observed behaviors such as satiety and competition. Needless to say, saturation and competition are just evidence that the entry is connected to a limited number of sites. Therefore, the rate of K+ ion entry into an ion exchange resin sheet shows quite similar competition and saturation ability (Ling and Ochsenfeld, 1965) (see also Figure 20). In this case, this resin is nothing more than a three-dimensional matrix carrying anions that does not have supports or membranes. Recently, Ling and Ochsenfeld were able to demonstrate a similar capacity for saturation and competition in the entry rate of the Rb+ ion in the layers of an isolated cytoplasmic water-protein system (actomyosin gel). In such a system, the entry rate is not limited by the surface, but by the bulk phase (Ling and Ochsenfeld, 1968b). These data show that, even in the absence of a cell membrane, ion entry follows the kind of kinetics shown in Fig. 19. (2) Figure 21 shows the inverse of the initial influx of Na+ ions plotted against the inverse of the external concentration of Na+ ions in the presence of different concentrations of K+ ions. Although there is a fraction of the influx of Na+ ions, its rate decreases as the K+ ion concentration increases.

0,yo

0,2

0,3

0,4

0,5

([K+IcJ-', (mmol/liter)-' Figure 19. Inhibitory effect of 2.5 mmol/liter Rb+, Csf, and unlabeled K+ ions on the initial rate of entry of labeled K+ ions into frog sartorius muscles. curve represents rate of K+ ion entry without addition of competing ions Muscles were immersed for 30 minutes at 24 °C... followed by a 10 minute wash at 0 °C Sartorius muscles (Ling and Ochsenfeld, 1965, courtesy of Biophy.ricd Journal).

A NEW MODEL FOR THE LIVING CELL

personal computer

1AC

0. years

0,yo

0,2

e 0,3

([cs+I,,~)-', (mmol/liter)-' Effects of CsCl. KCl, NaCl and LiCl on the initial rate of influx of labeled Cs+ ions into ion exchange resin sheets. Nallilm-1 strips soaked at 5°C for 2 minutes. in an experimental solution containing (approximately) 2.5 mM Tris buffer, pH 7.0, the incoming labeled ion and the ungelled ion (4 mM/liter) as indicated in the figure. The strips were washed in cold distilled water (0°C) for 10 seconds before counting (Ling and Ochsenfeld, 1965, with permission from Biuphyriul J O M W J I ). T'iG. twenty

0 to 30mM; another fraction shown in the top row is unaffected by K+ ion concentrations up to 100 mM. This top line passes through the origin, indicating that there is no saturation (i.e., an infinite number of "adsorption sites") at the input of this fraction. Therefore, there is a fraction of the Na+ ion that does not show competition or saturation and, therefore, may not be able to enter through carriers, but must enter through aqueous channels. If water channels large enough for the hydrated Na+ ion exist, it is difficult to see how they could exclude the smaller hydrated K+ ion.

ANSWER TO

BASIC CRITICISM OF FION (:

A D S O R P I O N IN

LIVING CELLS

There is a body of experimental evidence that appears, at least superficially, to contradict the adsorption model for ion accumulation in living cells. We will first describe this evidence and then discuss some recent advances in our understanding of adsorption that are serving to change this picture.

GILBERT N. LING

$ 3,0 \

0, 0

2.0

PAG

0,yo

([No+],,)

0,2

0,3

0,4

-I (rn mol/litro)

COWARDLY. twenty-one . Effect of different K+ ion concentrations on the initial rate of Na+ ion entry into toad sartorius muscles. Increasing the Kf ion concentration from 30 mmol/liter to 100 mmol/liter has no apparent effect on the rate of Na+ ion influx, whereas a decrease from 30 mmol/liter to 2.5 mmol/liter resulted in an increased influx of causes of Naf ions. . Soak for 15 minutes at 25°C. followed by washing for 10 minutes at 0'. Each point represents the average of three individual determinations (Ling and Ochsenfeld, 1965, by permission of the Biophysical Journal).

(1) Living cells are isotonic with an aqueous solution of about 0.1 M NaCl. This requires that the osmotic activity within the cells be equal to that solution. Since the total ion concentration in the cell is about 0.1 M and the K+ ion makes up most of the cations, it follows that all or most of these K+ ions, as well as the intracellular anions, must be in a free state, as found in 0.1 M NaCl (Hill, 1930). (2) When a pair of electrodes is placed on the surface of an intact nerve or muscle fiber, the total measured resistance is relatively little affected by the distance between the electrodes. This has been interpreted as a result of high membrane resistance and low cytoplasmic resistance (Hermann's central conductor theory (Hermann, 1879). Low cytoplasmic resistance has been interpreted as indicating complete or nearly complete dissociation of the intracellular K+ ion. (3) O the mobility of the Das-labeled K+ ion in squid axons averages 1.5 x 10-5 cm.2/sec. (5 cm.2/sec.) It was concluded that most of the K+ ion within an axon is present as free ions are present (Hodgkin and Keynes, 1953).

A N I X MODEL FOR THE LIVING CELL

(4) Bernstein's membrane theory of cell electric potential (Bernstein, 1902) and its modified version introduced by Hodgkin and Katz (Hodgkin and

Katz, 1949) predicts the correct magnitude of the resting potential. Since this theory is based on the assumption of complete dissociation of the intracellular K+ ion, it follows that most of the intracellular K+ ion must be in the free state. Taken together, this evidence is too compelling to ignore. Unsurprisingly, many cell biologists have thought that the membrane theory is the better option. However, with a more complete understanding of the state of water in living cells and the nature of adsorption, it is possible to resolve the conflict. In the discussion that follows, we will write responses to the above criticisms, point by point. (1) The postulate that most intracellular ions must be free for the cell to be isosmotic with 0.1 M NaCl solution is only valid as long as there is no interaction between cellular water and proteins in the cells, as postulated by the theory of the membrane (see above). There is now overwhelming evidence that most of the water in the cell is adsorbed as polarized multilayers onto proteins. Thus, the decrease in water activity within the cell (expressed as osmotic pressure) is explained by the interaction of water with proteins. With this level of interaction and water activity reduced by the proteins, the fact that the total osmotic pressure does not exceed that of a 0.1 M NaCl solution indicates intracellular ionic adsorption. Arguments (2) and (3) can be answered together. It is known that in nerve and muscle cells the main protein components are longitudinally oriented (Chambers and Kao, 1952; Huxley, 1957). Therefore, a longitudinal alignment of anion sites can be expected, as shown schematically in FIG. In this diagram, the concentric circles around each anion site represent equipotential lines. The closer to the anion site, the lower the potential. As the diagram shows, these lines overlap. Therefore, by looking for a path of minimum energy, a K+ ion adsorbed at one site can move from one site to another following the overlapping low-energy regions. Therefore, the activation energy for this migration is small. Thus, a "driving band" for the K+ ion can be said to be provided by an ordered sequential arrangement of the anion's sites (for more details, see Ling, 1962, ch. 11). On the other hand, the distance between the myofilaments is so great that a greater distance separates the sites of adjacent protein chains. Consequently, a much higher activation energy must be overcome before radial migration of the K+ ion can be achieved. If this hypothesis is correct, we would expect the longitudinal migration of the K+ ion in the cell to be equal to, and perhaps even faster than, the migration of the K+ ion in a dilute saline solution. On the other hand, the radial migration of the K+ ion would be slow. This could explain the central conduction behavior of muscle and nerve cells, as well as the longitudinal mobilities of K+ ions in squid axons. two lines of

GILBERT N. LING

Contingent experimental evidence suggests that this explanation is correct: (a) It has long been known that the migration of K+ ions on the surface of glass with anionic sites is faster than the migration of K+ ions in dilute solution (McBain and Peaker, 1930; Mysels and McBain, 1948; Nielsen et al., 1952). (13) Schwindewolf (1953) and Heckmann (1953) showed that elections

COWARDLY. 2 2 . Schematic representation of an effective "conduction band" for ions adsorbed along a longitudinal array of anionic sites. Along the anion sites are too far apart to produce a conduction band and low conductivity results. The concentric circles represent equipotential lines with lower potentials closer to the anions. The line with arrows indicates a likely path for an adsorbed ion moving along the "conduction band"; it has to overcome relatively low activation energies due to overlapping electric fields. A significantly higher feed energy must be overcome for traverse movement.

The electrical conductivity of a solution containing linear anionic polymers (ie, polyphosphate, thymonucleic acid, or solubilized silk protein) flowing in a long tube is anisotropic. That is, the conductivity is greater in the direction parallel to the solute flow than in the transverse direction. This anisotropic conductivity can also be understood in terms of the interpretation given in Fig. 2-2, considering that the flux produces an alignment effect on long-chain anions. This alignment in living muscle and nerve cells is produced by the pattern of cell growth. (4) There is now a large body of self-consistent data that support part of the Bernstein or Hodgkin-Katz-Goldman equation for cell potential:

+ +

PI< [K+]iii 11, = __ En __-1;

P K t t „a+ P , I K + I,.~ P,,, IN+ ;

liii

+ P,, +

of <.~

(

[C1-] Example 7

ICI-

(sixteen)

where P,,, PN,l,y are the permeability constants K - t - , Na + - and Clk ion "a+ 1,,) and [Cl- I in their intracellular concentrations. R e 1: e I K + I are the Varaday and gas constants, respectively. The dependence of the potential on the absolute temperature T and its logarithmic relationship with the external K +

A NEW MODEL FOR THE LIVING CELL

and Na+ ion concentrations were repeatedly checked (McDonald, 1900; Cowan, 1934; Curtis and Cole, 1942; Ling and Woodbury, 1949; Ling, 1967b, 1962, ch. 10). However, attempts to demonstrate a possible dependence on intracellular concentrations of K+ and Na+ ions have yielded conflicting results. Likewise, injecting highly concentrated saline solutions into squid muscle fibers and axons (Grundfest et al., 1945; Falk and Gerard, 1954) or leaching K+ and Na+ ions from muscle cells does not lead to the predicted changes, the basis of the membrane theory (Tobias, 1950; Koketsu and Kimura, 1960). Furthermore, a change in the external chloride concentration does not lead to a permanent change in potential, as is the case with a change in the external K+ ion concentration (Hodgkin and Horowicz, 1959). Other experiments were performed on living muscle cells and on model systems that played an important role in the development of cell electric potential membrane theories (e.g. glass electrodes, collodion electrodes) (Ling, 1960, 196717, 1962, ch. l o ) led the appraiser to conclude that this potential is not a membrane potential but an interphase potential (i.e., a potential that arises at the interface of the external solution and the solid water-protein charge system; see also Beutner, 1920 ). As such, this potential is determined by the density and nature of the anionic groups on cell surface proteins. Simplified, the equation for the potential is 111

RT = Constante - __ In ( K , ; [ K + I, I;

+ k,,

(N a + I,.,)

(17)

where K and K are the adsorption constants of K + and Na + ions at the surface anion sites. Although this equation has a different basis than the Hodgkin-Katz-Goldman equation, it is formally identical to the part of it that has been verified experimentally. It does not contain terms related to intracellular ion concentrations or external Cl ion concentration whose relationship to potential has not been verified. adsorption constants

K, substitute the permeability constants into the equation. (sixteen). According to the associated induction hypothesis, these constants change during excitation due to a cooperative all-or-nothing change in the r-value of the anion sites (see Fig.

K,; E

7). Thus, the value of r at rest is such that K ,

>> Water,. Consequently, to Eq.

(17)

reduce to (18)

where i l l r is the cell potential at rest. This relationship has been confirmed in various tissues since McDonald (McDonald, 1900) first discovered it.

GILBERTO N. ABADEJO

During excitation, the value of r changes to a value such that KNa > Kg and Eq. (1i) come now

This relationship between the magnitude of the action potential 1 1 ) and ~ ~ log "a+] was discovered and extensively studied by Hodgkin and his collaborators (Hodgkin, 1951; Hodgkin and Katz, 1949). In anticipation of the work presented in the next section, I would like to It should be noted that there are theoretical reasons to believe that a local change in the physical state of water may accompany this change in c-value. Experimental NMR studies by Fritz and Swift (1967) showed a change in water state during nerve depolarization. change, accompanied by an increase in the preference of N over t - i or n of the cell surface sites, would contribute to the generation of the action potential profile. In summary, the fact that there is no constant correlation between the cell potential and the intracellular K+ and Na+ ions indicates that the cell electrical potential cannot be interpreted as evidence for the membrane theory.In fact, this bug adds some very important evidence against it.

D. MOLECULAR MECHANISMS IN THE INTEGRATIVE FUNCTION OF PROTOPLASM In the previous sections we examined the nature of protoplasm in terms of the association-induction hypothesis. We show that the equilibrium properties of the fixed charge protein-water system can explain most of the experimental observations about solute distribution in living cells. However, living protoplasm is very different from sheep's wool or even isolated actomyosin gel. To illustrate this difference, let's look again at the bacterial flagellum. This pure system of protein and water, as found in bacteria, is active, whereas a wool fiber is not. On closer inspection, we find that this activity has the following properties: it is the result of a reversible change between two states (short and long), the change of state is controlled by a signal received at a distance [from the basal grain, box of Astbury (Astbury, 1951)] and stimulates the restoration of the flagellum to its original state, including in the basal granule. In the following discussion, we will examine a theoretical model capable of performing these functions. 1 . Two

Modelo simpleJ

First, let's look at a very simple template. If we were to fasten soft iron nails end to end with pieces of string, as shown in Fig. 23A, they would be distributed in a

A NEW MODEL FOR THE LIVING CELL

random and does not interact with the iron filings scattered around it. When a strong magnet is brought close to the nail at one end, a chain reaction of magnetization takes place, ending with all the nails being magnetized and iron filings being tightly aligned around the nails. We demonstrate the long distance transmission of energy and information possible through the magnetic field.

COWARDLY. 23. Two simple moles demonstrating the transmission of information and energy over distances due to short-range interactions. (A) A chain of soft iron nails, held end to end with pieces of string, is randomly arranged and does not interact with the surrounding iron filings. The approximation of a magnet provokes the propagated alignment of the nails and the interaction with the iron filings. (B) The electrons in a row of insulators are evenly distributed before the approach of the electrified rod R. The approach of the rod displaces the electrons by induction such that the insulator becomes polarized with areas of low electron density and areas of low electron density. high electron density.

Susceptibility to sweet iron. (If we had used wooden nails, for example, nothing would have happened). Of course, this magnetic model has its electrical analogue (Fig. 2 3 B ). When an electrically charged rod is brought close to a row of insulators next to each other, it creates alternating poles of positive and negative charge on the insulators by induction. When removing the electrified rod, the insulators again lose their electrical polarization. Once again, we have the transfer of energy and information made possible by a series of polarizable materials placed close to each other. Depending on the polarizing influence of the electrified rod, the electrons in the insulating chain are redistributed in such a way that localized electron-rich and electron-poor areas arise. These examples illustrate the induction mechanism.

GILBERT N. LING

As part of the association induction hypothesis, I have proposed that the ability of protoplasm to function coherently depends on a fundamentally similar induction mechanism. The polarizable material is actually the polypeptide chain and its appendages, the side chains. 2. The fundamental action of mechanical mechanics.

(Video) Biology: Cell Structure I Nucleus Medical Media

Acetic acid (CH,,COOH) is a weak acid. This means that its carboxyl group strongly interacts with a proton in aqueous solution, and the proportion of carboxyl groups in the dissociated state is relatively small. Trichloroacetic acid (CC1,C O O H), on the other hand, is a very strong acid. This means that its carboxyl group interacts only weakly with a proton; the proportion of carboxyl groups in the dissociated state is large. Trichloroacetic acid is obtained by replacing the three hydrogen atoms in the methyl group with chlorine atoms. Because chlorine atoms are more electronegative than hydrogen atoms (that is, the chlorine atom is more likely to attract electrons than the hydrogen atom), this substitution results in a reduction in the electron density of the carboxyl group (or i.e. a reduced c-value). Since the interaction energy between the carboxyl group and the proton is mainly electrostatic in nature, decreasing the c value leads to a weakening of the interaction energy and more H+ ions are found in the dissociated state. Thus, the replacement of hydrogen atoms by chlorine atoms produces an inductive change in the dissociation constant of an acidic group in another part of the molecule. This inductive effect is a general property of organic compounds and its consequences are not limited to changing the dissociation constant of acids. For example, Taft showed that a variety of kinetic and equilibrium properties of organic compounds are predictably influenced by the inductive effects of a long list of substituents, including hydrogen and chlorine atoms (Taft, 1960; Taft and Lewis, 1958; Hammett, 1940 ; Ling, 1964a, b). Among these properties is the strength of the hydrogen bonds formed by many of these compounds (Ling, 1964a,b, 1962, Chapter 7). Therefore, the replacement of a hydrogen atom by a chlorine atom in diethyl ether reduces the oxygen electron density of the ether and thus decreases its proton accepting power (Gordy and Stanford, 1941). 3. How far is the Idr/rtiue effect transmitted?

The dissociation constants of tx-amino-m d a-carboxyl groups of amino acids YH" (R-C-COOH)

they vary due to the variable "electronegativity" of the side chain (R). if

A NEW MODEL FOR THE LIVING CELL

Peptides are formed by joining amino acids together, converting most of the a-carboxyl and a-amino groups into proton-accepting C=O and proton-donating NH groups. However, the 1-carboxyl group and the 1-amino group at the ends of the peptide are left as such. The effect produced by replacing a hydrogen atom with a glycyl group (NH, CH, CO) can be transposed. Table I shows that this substitution has an effect on the carboxyl group uTABLE 1

amino group

YO M NaCl

Água N H ,CH,COO H NH,CH,CONHCH,COOH NH,(CH,CONH),CH,COOH NH,(CH,CONH),,CH,COOH NH.,(CH,CONH),CH,COOH NH ,(CH,CONH),CH,COOH

9,70 R.20 8,00

7,75 7,70 7,60

agua

9,60

9:lc)

2:42

8,13 7,91 7,75

8,07 7,83 7,93

3.13

7,70 7,60

3,00 3,05 3,cr5 3,05

NaCl 1 M 2,3,4 3,06 1,26 3,05

1,05 3,05

3.02

3,33 3,39 3,50 ~

Carboxylic oxygen, although separated from the substituent by a peptide amide group (-CONH-), two saturated carbon atoms, a nitrogen atom, and a carboxylic carbon atom. This example shows that protein chains are exceptionally polarizable. Based on this, it is to be expected that changes in the “electronegativity” of a side chain, arising, for example, from the dissociation of a proton, could have a significant impact on its two neighboring C O N H groups:

e Z

-N-C-C-N-CI H

AND

? ,?I-N-C-C-N-C-+

e1 ho

i h

II O

i h

H+

On the other hand, the r value of the hydroxyl group changes when N-C I I1 HO

The groups change their H bonding partners to those with different hydrogen bonding strengths (or polarization).

4. Euers I n t e s p l q;q), mean entropy models of magnetized iron nails and electrified insulators deal with macroscopic objects; here energy itself plays an important role. such phenomena

GILBERT N. LING

since the adsorption and exclusion of ions are microscopic processes. As such, they depend on both entropy and energy. A simple example is the sublimation of ice at sub-zero temperatures. Thus, both a housewife drying clothes in winter and a biochemist freeze-drying an enzyme depend on the large translational and rotational entropy gain of water when the ice evaporates. From an energetic point of view alone, such a step is highly unfavorable. Now consider a segment of a polypeptide chain containing m peptides. For example, it can be wound into a helix or fully extended (the so-called "random coil"). In a helical fashion, all NHCO groups in the backbone form hydrogen bonds with other C O NH groups in the same polypeptide chain. These groups are therefore internally saturated and protected from further interactions. In the extended conformation, the NHCO groups are free; in an aqueous medium, they have no choice but to form hydrogen bonds with surrounding water molecules. Let's assume that each NHCO group effectively interacts with ?z water molecules. If we ignore all other components of the chain (i.e., the side chains), whether this peptide is in helical or extended form depends on the total energy and entropy of the entire system. In the helical state, the energy and entropy terms to be considered are: (1) The energy of a pair of peptide H bonds; (2) the entropy of the helical peptide; (3) the total free water energy involved in each NHCO group; (4) the total entropy of the free water involved in each NHCO group. Of these four elements, (2), (3) and (4) are constant for all helical proteins; (1) On the other hand, it varies with the type of protein molecule. In the expanded state we must consider: (5) The energy of the peptide (water), complex; (6) the entropy of the peptide (water), complex. However, (5), (6) and the value of n are interdependent. Thus, if the energy of a peptide complex (H20) is known, its entropy and 12 can be defined, as the properties of water molecules do not change. In summary, it essentially depends on the relative magnitudes of the energies of the peptide-peptide bonds and the peptide-water bonds whether the polypeptide is in the helical state or in the extended form. Of course, if these bonds had the same energy in all proteins, they would be found in helical or straight form. This is clearly not the case. The usual explanation for the variability in the conformation of different proteins is the interaction between the side chains and the interfering influence of proline and hydroxyproline on a-helix formation. Tertiary side chain interactions include: (1) disulfide (S-S) formation; (2) hydrophobic bonds between nonpolar side chains; (3) ionic bonds between charged groups (salt bonds); (4) hydrogen bonding; (5) electrostatic attraction between oppositely charged side chains; (6) electrostatic repulsion between equally charged side chains.

A NEW MODEL FOR THE LIVING CELL

Of these, all but (6) and sometimes (5) favor the formation of the helical structure. Electrostatic repulsion can be effectively eliminated when the protein is at its isoelectric point (IEP). Thus, if the conventional interpretation is entirely correct, a comparison of the IEP helical stability of different proteins should show that those with a wide variety of functional groups form the strongest helix. Those unable to form a tertiary structure would form the least stable helix. An examination of the properties of a specific poly-L-alanine polymer will show that this is not the case. 5 . Evidences for the direct inductive influence of

SMALL

the side chain in

- OC Boud in a-Hciicccll structure

It is well known that most proteins contain a large number of side chains capable of forming tertiary helix stabilizing structures. However, some of these proteins, such as oxidized ribonuclease, do not form the helical structure in an aqueous medium (Harrington and Schellman, 1956). Many others that form a helical structure lose it in the presence of 8 M urea, 5 M cuanidine HCl, or 0.1 M sodium dodecyl sulfate. expect that a polypeptide that cannot form a tertiary helix stabilizing structure would be entirely in the extended conformation. Doty and Gratzer's results proved the opposite (Doty and Gratzer, 1962). These authors found that a poly-L-alanine polymer made soluble in water by joining two poly-D,L-glutamic acid block polymers at each end is entirely in the form of a helix. The stability of this helix is such that it resists all common denaturants, including 10 M urea, 4 M guanidine HCl, and 0.1 M sodium dodecyl sulfate. However, as Doty and Gratzer point out, this stability cannot be the result of chain interaction side effects, since the methyl side chains are too short to interact with the next neighboring methyl side chains. Since the polypeptide structure of all proteins and polypeptides is the same, the unusual strength of the polylalanine helix can only be the result of an attribute derived from the properties of the CH1 side chain. Therefore, we are forced to find a new mechanism by which the CH group can reinforce the helical structure. The methyl side chain is an electron-donating substituent (compare the pK values of HCOOH, 3.8; CH,COOH, 4.75; and CH,CH,COOH, 4.87). We showed earlier that an inductive effect can be transferred from a side chain to at least the two flanking NHCO groups. From this we must conclude that the proton donating capacity of the NH group and the proton accepting capacity of the CO group immediately adjacent to the side chain are influenced to different degrees by the inductive effect emanating from the side chain and the consequence of this donation of side chain electrons being the helical NH-OC - Strengthening the bond.

GILBERTO

N. Ling

Let's consider this issue in more detail.

The methyl side chain donates electrons to the immediately adjacent C=O and NH groups, increasing the proton accepting power of the CEO group and weakening the proton donating power of the NH group. If these effects were exactly the same, it would be difficult to understand how the helix could be strengthened, since the strength of the CO-HN bond must depend on the proton accepting power of CO and the proton donating power of NH. clusters. The fact that the helix is strengthened has a twofold effect: (1) the proton accepting power of the CO group increases more than the proton donating power of the NH group decreases, and (2) the increase in the proton accepting power . The strength of the CO group increases the free energy of the CO-HN helix bond more than that of the CO-(H,O),,,, bond. You will recall that we showed in Section 111, A.3 how a similar increase in electron density (that is, an increase in the r value) of the parent group of the CO group, the -COO group, also has a differential, whereby the free energy of association of the Na+ ion increases more than that of the K+ ion.]

The first statistical-mechanical treatment of a cooperative transition was performed by Bragg and Williams (Bragg and Williams, 1934) for the order-disorder transition of (3-brass) with increasing temperature. More recently, it has been recognized that protein and DNA denaturation also represents a cooperative phenomenon (Schellman, 1955; Zimrn and Bragg, 1958; Gibbs and DiMargio, 1958). In all these cases, the nearest neighbor interaction energy was considered to be primarily entropic in nature, referring to the increase in entropy than the native helix. the transition is no longer considered between an ordered state and a disordered state, but between two alternative adsorption states that may be ordered or ordered is disordered and that the nearest neighbor interaction has an energy component and an entropy component. model For example, it will be useful to define the / value as a measure of the positive charge on a group of cations (NH: group). This parameter is analogous to the c-value, which was developed to measure the electron density of an anionic oxyacid group. The accepting power of protons from the C=O group and

A NEW MODEL FOR THE LIVING CELL

the proton-donor power of the NH group can also be represented by a c-valued analogue or a c'-valued analogue. Now consider the model shown in Figure 24 of a small segment of a protein chain containing a control (cardinal) site. Backbone amide groups have two choices of partners, either amide groups on an adjacent protein segment (a + a - ) or free hydrogen bonding molecules such as water (bt, b-). In the absence of an adsorbent at the cardinal site, the c-valued analogues of the amide groups are CO as well as the 1-valued analogues.' of the NH amide groups all have a low value of, for example, 1 (see inset). In this state, the electrons are fairly evenly distributed (as in the case of the series of insulators before approaching the electrified rod, Fig. 23B), and the main groups prefer to mix with the fixed groups ;I+ and a- (diagram higher ). So, when a cardinal C adsorbent is introduced, it reacts with the cardinal site. Then an inductive effect raises the c' - v he analogue of the nearest neighboring NH group from 1 to 2. At this value of c, that site no longer prefers a to b and an a + h switch takes place. Since b- is more polarizing than a-, replacing a- with b- has the effect of removing an electron from the neighboring CO group. The analog value of this group increases from 1 to 2, resulting in an a++b+ switch. This process continues until all a+ and a are replaced by 13-t and h-. The result of cardinal adsorption is to create a series of water adsorption sites in an all-or-nothing manner and cause (Ling, 1962) an all-or-nothing dissociation from the neighboring protein and the adoption of a new discrete conformation. . If the numerous hydrogen, ionic, and other binding sites on a protein molecule were independent, each site would have a variety of partner options. Such a protein can exist in a variety of conformations that are not clearly distinguishable from one another. In the present model, there is a site-to-site interaction between the nearest neighbors, so that the occupation of neighboring sites by the same adsorbent is favored. This self-cooperative interaction leads to the existence of discrete molecular states (conformations) of protein molecules (see next section). It is the fundamental self-cooperative nature of protein adsorption that allows modulation and control of many sites by a small number of cardinal adsorbents. These cardinal adsorbents can be hormones, drugs, ATP, etc. Their action and inactivity form the fundamental step in the transmission of energy and information over long distances (Ling, 1962). 7.

Un iModel /More Sper.ific; La Coopemtiz'e ALJsorptioii de K + aiid N a f lo71

Figure 2-5 shows a protein segment containing anionic side chains that exist in two alternate states in an aqueous environment containing K+ and Na+ ions. In one state (B), the carboxyl groups have a relatively low charge density (low

GILBERT N. LING

Value c) and prefer the K+ ion to the Na+ ion. The proximity of the positive charge of the K+ ion to the anionic charge of the carboxylic oxygen makes the entire carboxylic K+ ion complex a weaker electron-donating source than an ionized carboxylic group itself. Based on the polyalanine evidence, we can expect a weakening of the helical structure. Consequently, the protein segment is in elongated form. The water in the vicinity of the chain is in the polarized multilayer state. In the alternative state (A), the anionic side chains are occupied by the Na+ ion, which our previous calculations have shown (in the low c-value ranges considered here) tends to adopt a configuration in which more water molecules are present as a separate cation. the carboxyl group. The result is that the H,O-Na+ ion complex of the carboxyl group acts as a stronger electron donating group (than the K+ ion complex of the carboxyl group), thus stabilizing the helical conformation. In that case, the surrounding water molecules would be in a free state. The transition between the two states A and B is controlled by the cardinal adsorbent C. 8. The equation for cooperative adsorption on proteins

In 1964, Yang and Ling published an equation based on Ising's one-dimensional model method for cooperative adsorption on a protein chain (see Ling, 1964a). In this model, similar sites on a long chain have similar properties and there is nearest neighbor interaction between these sites. Each location has two sorbent options. For the adsorption of K+ ions in the presence of Na+ ions, the equation has the following form (see Ling, 1964a, for the general equation)

As will be recalled, If] represents the concentration of adsorption sites. K& and Ki are the intrinsic adsorption constants of Na+ and K+ ions, respectively. Figure 21. Schematic representation of a cooperative transition induced by a cardinal adsorbent. The upper panel shows the variation of adsorption free energies with changes in the analogue of the r value of the C=O group and in the analogue of the α value. of the NH group. The figures below show the gradual displacement of b+ and b- (NH and C=O groups in the lower polypeptide) by a-t- and a- as a result of interaction with the leftmost cardinal adsorbent. The overall result of the cooperative transition is the dissociation of the two peptides (or the unwinding of a helix).

A NEW MODEL FOR THE LIVING CELL

. B',

B-

a+, aL

c-y c

analog value

GILBERT N. LING

and are related to the intrinsic standard free energies of adsorption A": and A P i 0 by the relation

- A i c z = RT In i Y ; . ~

(22)

AI;"" I< = RT En KE

(23)

The free energy of the nearest neighbor interaction is -y/2. It is equal to the change in free energy each time a new adjacent pair of different adsorbents is created. Thus, in a series of three adjacent sites, if the intermediate site can adsorb its Na+ ion against a K ion (N a +, K + ), all the Na+ ions (N a f, Na +, Na +) will adsorb . , N a + ), the total energy change is the difference in free intrinsic -AP,") p h 2 ( - ~ / 2 ) or -y, due to the two neighboring new energies >:I creating dull pairs of "I+ , K-t. On the other hand, a change from Na+, Nn+, K+ to Na+, K+, K+ only implies AFqo - AF"", as there are no new Na+, K + Si! neighboring pairs appear. An important feature of the cooperative adsorption equation is that when log ( I K + ] n , l / [ N a + ] nd ) is plotted against log ( [K1,JINaJeS) (as in Fig. 28 ) the tangent a a The resulting curve through the point where [ K + ] 1Na+Inll= 1 is described by the following equation (Ling, 1964a, 1965b, 1966):

The slope 71 is related to the free energy of the nearest neighbor interaction by the explicit relationship 11

= acre

(-H)

when I I = 1, -y/2 = 0 and there is no nearest neighbor interaction. In this case, the adsorption is effectively non-cooperative and in fact follows the well-known Langmuir adsorption isotherm (Figure 26). When 12 < 1, -y/2 > 0 and the nearest neighbor interaction is such that different adsorbents are favored at neighboring locations (i.e. K, Na, K, Na, etc.), we refer to the interactions as Hetero -cooperatives (Ling, 1962, pp. 1 0 2). If 12 > 1, -y/2 > 0; this means that the nearest neighbor interaction is such that similar adsorbents are favored at neighboring locations (eg K + , K-I-, K + , K + or Na + , Na + , Na + , Na + ). Similar

CARDINAL SITE7

COOPERATIVE TRANSITION

+ of

with

Cowardly. 25. Diagram of part of a protein molecule undergoing a self-cooperative transformation. Simplify. Water molecules adsorbed on multiple layers are shown as a single layer. The W-shaped symbol represents a cardinal adsorbent.

GILBERT N. LING

The interactions are referred to as self-cooperative. It is an important theme of the association-induction hypothesis that this mode of cooperative adsorption underlies most, if not all, of the known all-or-nothing phenomena in cell physiology (Ling, 1962, 1964b). 10

0,8

XP 0,6

0,2

0. years

COWARDLY. 26. Cooperative adsorption isotherm for a one-dimensional chain. Line graphs of theoretically calculated isotherms. For -y/2 = 2.30 kcal./mol the isotherm is self-cooperative and shows a sinusoidal shape. For -y / 2 = 0.0 kcal./mol (the Langmuir adsorption isotherm), the curve resembles a hyperbola (Ling, 1%6b, with permission from Federatiorr Proceedings),

9. The general equation for S o h t Disfribiitioiz

jti

LiiJing Cell1l.c.

Equations (13) and (14) are specific equations for the distribution of K+ and Na+ ions in case there is no site-to-site interaction (ie, the adsorption follows the Langmuir adsorption isotherm). The following more general equation for the intracellular K+ ion applies to the case where the sites also have nearest neighbor interaction: [K+]iii

= ayR [ K + l r x

L=l

[fl”-{I+ 2

5" - 1 1(5L-1)2+451,exp(yL/RT)]~

(26)

A NEW MODEL FOR THE LIVING CELL

where [ f l L , 5'. and y" refer to the Lth type of sites. The equation for the intracellular Na + ion is

CQL 2

5" - 1

yo(lj'2-

l)2+4E1,exp

(yL/RT)]x (27)

10. Cooperative biological adsorption system; Experimental Evidence

Two types of plots are useful for distinguishing cooperative adsorption isotherms from Langmuir adsorption isotherms. We have already mentioned the use of the log-log graph, that is,

1og l K + l a d versus log [K+ 1ex "a+ 111d lN"+lI'Y Although the model used to derive this equation for cooperative adsorption was based on a chain of similar sites, it is often possible to replicate the adsorption on heterogeneous proteins by Analyze Plot the data on a log graph as if there were only one type of adsorption site. The result appears as a series of straight line segments connected at rather acute angles (Ling, 1966b). For the combination of isotherms shown in Figure 27, the extreme segments of the curve have unity slopes. The two inner segments abruptly change from a slope of less than one to a slope of more than one. This change marks the point at which adsorption transitions from hetero-cooperative to self-cooperative. The line graph is perhaps more familiar to most biologists. We showed earlier that on such a plot, a Langmuir adsorption isotherm has the shape of a hyperbola. A cooperative (automatic) curve, on the other hand, is sigmoid. Such curves are seen in the binding of oxygen to hemoglobin (Wyman, 1964), the effect of many feedback inhibitors on enzymatic reactions (Monod et al., 1965), and the adsorption of K+ ions by living cells (Ling, 1966b). ). Let's take a closer look at some of these examples. For. Binding of oxygen at hemoglobirz. It has long been known that the curve for oxygen binding to hemoglobin is not hyperbolic but sigmoidal and that this indicates the interaction between the four heme groups to which oxygen is adsorbed (Ling, 1964b, 196613; Wynian, 1964). A.V. Hill introduced an empirical equation to describe this sigmoidal adsorption:

log __- = tz log PO2 1 -y

+ n K-Register,

(28)

where y and PO represent the number of oxygen molecules adsorbed by the blood.

GILBERT N. LING

globin molecule or the partial pressure of oxygen and K, is a constant (Hill, 1910). As is well known, n is a kind of measure for the degree of interaction. This equation is analogous to equation (24). Therefore, the Hill coefficient n is related to the free energy of the nearest neighbor interaction according to Eq. (25). The reaction of oxygen and hemoglobin does not take place in a vacuum, but in an aqueous solution. Thus, from equation (28), K is the intrinsic adsorption constant of oxygen divided by the intrinsic adsorption constant of the alternative adsorbent, ie, water.

lo4

10Xi -

10-1

COWARDLY. 27. Complex cooperative adsorption isotherms. The adsorption isotherms for two linear polymers, one self-cooperative (8 = 100) and the other heterocooperative (0 = & I), are shown in curves 111 and 11, respectively contains polymer. Curve I was drawn as a series of straight lines showing the significant parts of the more precise curve that would connect all the circles. Note that the two outermost straight lines have unity slopes; The slopes of the two central lines have imprecise but significant values that reveal the hetero- or self-cooperative nature of the component systems (Ling, 1966b, with permission from the Federation Proceedings).

A NEW MODEL FOR THE LIVING CELL

If we take into account the fact that there are four heme groups per hemoglobin molecule, the complete equation for the adsorption of oxygen to hemoglobin is: PO2

x K, - 1 (29)

In Fig. 28, Lyster's data (see Rossi-Fanelli et al., 1964) for oxygen

p 0 2 (rmmHg)

COWARDLY. 28. Log plot of Lyster data on oxygen consumption by human hemoglobin at pH 7.0 at 19°C. In the review by Rossi-Fanelli et al. (1964). Points are experimental; the line is theoretical according to equation (29) with K = 5.88 x 10-6 M and -y/2 = 0.67 kcal./mol.

Hemoglobin binding was plotted again on a logarithmic scale. The line is theoretically calculated according to the equation. (29) with K equal to 5.88 x 10 Wg M and -y/2 equal to 0.67 kcal./mol. In 1965, Monod, Wyman and Changeux (Monod et al., 1965) presented a model of “allosteric transitions” that bears some resemblance, at least superficially, to our cooperative adsorption model (see also Haber and Koshland, 1967). A brief discussion of your model can help to avoid further confusion. Basically, Monod and coworkers make the following assumptions: (1) A protein molecule capable of allosteric transitions contains symmetrical subunits with which ligands react symmetrically. (2) that

GILBERT N. LING

The protein molecule is capable of existing in at least two separate states that are in equilibrium with each other. (3) The affinity of the protein molecule for the ligand differs in the two states. (4) The binding of any ligand molecule is independent of the binding of any other. Based on this model, Monod et al. are able to predict the types of sigmoid curves observed in allosteric interactions in enzymes (Monod et al., 1965) and the Lyster curve for oxygen binding to hemoglobin. (See also Changeux et al., 1967). The Monod, Wyman and Changeux model differs from the model presented above in the following respects: (1) the existence of a small number of discrete states of the protein molecule is an a priori assumption; The reason for the existence of a small number of discrete states rather than a large variety of continuously different states is not clear. We noted above that it is the nearest neighbor interaction that gives the protein its ability to assume such discrete states. Monod et al. they do not include this nearest neighbor interaction as part of their model. (2) Monod et al. they refer to "cooperative effects" by what they seem to mean interactions that enhance the binding of substrate molecules; such interactions are therefore similar to the self-cooperative interactions described above. However, the mechanism they postulate for these effects is not the type of cooperative interaction discussed above, since, again, nearest neighbor interactions, an integral part of classical cooperative interactions, are not part of their model. ( 3 ) The Hill coefficient 12 in the hemoglobin oxygen binding equation is unrelated to the values reported by Monod et al. parameters used (L and c). to describe their oxygen binding curves. (4) In the Monod-Wyman-Changeux model, the conformational change of the protein and the binding of a single ligand are considered separate events (L and c are independent parameters). It is believed that binding of the ligand does not cause the conformational change. In the model presented above, ligand binding and protein conformational change are interrelated processes, not only because of the role of nearest neighbor interaction, but also because ligands compete for the same sites that maintain protein conformation. ( 5 ) The Monod-Changeux-Wyman model is only applicable to symmetric molecules; our model can be applied to all proteins (for experimental data on cooperative adsorption to denatured proteins, see Ling, 1966b).

B. Detergent Biizdilr'g 012 Bavitze Serzlm Albi~ziiz. There is a fairly large body of experimental data on in vitro adsorption to proteins which does not agree with the Langmuir adsorption isotherm but can be explained on the basis of cooperative adsorption (Ling, 1962). As an example, we show the Pollansch and Briggs data on the adsorption of in Fig. 29

A NEW MODEL FOR THE LIVING CELL

Detergent, dodecyl sulfate, based on bovine serum albumin. Theory predicts that the point of abrupt change to a higher slope, indicating the start of self-cooperative adsorption, should coincide with the start of a conformational change. Indeed it is: at this point the electrophoretic limit is divided by one for two (Pallansch and Briggs, 1954) (for other examples see Ling, 1962).

double electrophoretic limit

XDodS ‘-xDod S

lo-'

Limit electrohoretic signal

10" 10-5

10-4

Dodecylsulfatkon concentration (mol/liter)

COWARDLY. 29. Adsorption of dodecyl sulfate to bovine serum albumin. The ordinate represents, on a logarithmic scale, the molar fraction of sites that bind to the anionic detergent dodecyl sulfate divided by the molar fraction of sites that do not bind to the detergent. The abscissa represents the concentration of dodecyl sulfate also on a logarithmic scale. The total number of binding sites, 102, is the sum of the number of arginine, lysine, and histidine residues per protein molecule. Note that the change from a single to a double electrophoretic threshold (dashed line) occurs at a dodecyl sulfate concentration corresponding to the abrupt change in the hetero-cooperative to self-cooperative detergent adsorption slope. The open, half-filled circles represent two different sets of experiments (Pallansch and Briggs, 1954, with permission from The Youngal of Amerii-an Chernjcal Sol- i ely ).

C. Cooperative adsorption of K + l or n in Livjiig cells. you frog muscles. It takes 3 to 4 days of incubation at room temperature for frog muscles to reach new equilibrium ion concentrations when external ion concentrations are varied. In our laboratory, using tissue culture techniques, we managed to keep the frog muscles under normal in vitro conditions for 7 days or more, more than enough time to reach these new levels. In Fig. 30, we show the results of an experiment in which the external K+ ion concentration varied while the external Na+ ion concentration remained constant. The achieved equilibrium concentrations of internal K+ ions are plotted as a function of the external K+ ion concentration on a linear diagram. The sigmoid curve obtained can be compared to the similar curve obtained for oxygen binding by hemoglobin.

GILBERTO N. L I N G

(Figure 28). The experimental data were fitted to a theoretical cooperative adsorption curve [Eq. (20)] calculated with a value of 665 M-l for K i /K i a and 0.76 kcal./mol for -y/2. This means that K + adsorption takes place

[K + Iex (mmol/liter) FIG. 30. Equilibrium concentration of K+ ions in toad sartorius muscle in solutions with low concentrations of K+ ions but high concentration of Na+ ions. Isolated and sterile Sartorius muscles were shaken at 25°C for 72 hours. in Ringer's solutions containing a fixed concentration (100 mmol/liter) of Na+ ions and varying concentrations of K+ ions. K+ and Na+ ions were analyzed by flame photometry in muscle HCl extracts. Total intracellular ion concentration was obtained from raw analytical data after correction for extracellular space (10%). The adsorbed ion concentration in millimoles per kilogram of fresh tissue was further calculated from the total intracellular concentration by subtracting the interstitial ion concentration (estimated to be 10.4% of the external equilibrium ion concentration; this number represents an average of all values determined up to this point). Each point represents a single determination. Inset shows oxygen consumption by human erythrocytes (dashed line with filled circles) and by myoglobin (solid line) (from Eastman et al., 1933) (Ling, 196613, with permission from Federation Proceedings).

becomes more favorable at 2 x 0.76 = 1.52 kcal/mol if the two flanking sites are occupied by K+ ions rather than Na+ ions. ii. Mammalian smooth bones. Jones showed that the constant uptake of K+ ions in canine arterial smooth muscle also obeys the equation. (20) (confirmed by Gulati, publication pending). The K i /K i a y - y/2 values are 93 M - 1 and 0.61 kcal./mol, respectively (Jones and Karreman, in press). iii. Esberichiu coli. Damadian showed that a sigmoid curve is generated by plotting the constant level of K+ ions accumulated in an E. coli mutant (RD-2) versus the external K+ ion concentration ranging from 0 to 0 external K+ ions becomes: 0, 3mM. At higher concentrations, there appears to be a second set of cooperative adsorption sites (Damadian, 1968).

A NEW MODEL OF LIFE FOR CELI.

Coitsol-Rinde[email protected]de Cooper.crtitie Ad.sosptioFi

For. K-Nci Adsosptioii Coitsol bq' Castliac Glycosides. The theoretical model shown in Figure 2 predicts that, in the same ionic environment, the interaction of the protein with a cardinal adsorbent can change the system from a relative affinity for the K+ ion to a relative affinity for the Na+ ion of the whole or Nothing form. Figure 31 shows that the cardiac glycoside lanoxin at pharmacological concentrations produces a change in the cooperative adsorption isotherm, such that the ratio of intrinsic equilibrium constants (KO/KO) is shifted towards a lower preference of the KNn K+ ion and a higher preference of the Na+ ion by a factor of 20 The self-cooperative character of the curve (slope > 1 in [K+].,/[Na+Isd = I) is preserved. This effect has a deep physiological significance. The sine curve shown in Figure 30 shows that muscle can change more or less all or nothing from adsorbing all K+ ions to adsorbing all Na+ ions. However, under physiological conditions there are no large changes in plasma K+ concentrations.

me and 10-3

10-2

AND-'

FIG.31. Logarithmic representation of the distribution of K+ and Naf ions in the Sartorius muscles of the frog in the absence (left) and in the presence of lanoxin. The experimental procedures used were similar to those described in FIG. Lanoxin concentration was 2.5 pg/ml. X and X refer to the mole fraction of adsorbed Kf and Na+ ions, respectively.

GILBERT N. LING

or Na+ ions. Under these conditions, the changes in the adsorbents must be caused by some other agent, preferably an agent that is effective in very small amounts and alone can affect the adsorbents in many places. In the present experiment, Lanoxin is one such agent. Thus, in an ionic invariant environment, the interaction with Lanoxin changes the location from a state where the K+ ion is the main adsorbent to a state where the Na+ ion is the main adsorbent. In Section 111, C.b. Eergization of biological activators. In the history of biology, one of the most notable developments of the 1930s was the way in which leading scientists in the field of metabolism, A. V. Hill and O. Meyerhof, reacted to the discoveries of the then relatively unknown Lundsgaard. Contrary to the lactic acid theory of muscle contraction proposed by Hill and Meyerhof, Lundsgaard found that muscles can contract without lactic acid production (Lundsgaard, 1930; Henriques and Lundsgaard, 1931). The immediate confirmation of Lundsgaard's discovery in Meyerhof's laboratory prompted Hill to write a paper entitled A Revolution in Muscle Physiology (Hill, 1932), which paved the way for recognition of the important role of ATP in cell function. Further development of the concept of the high-energy phosphate bond led to the assumption that enzymatic hydrolysis of this bond releases energy for doing biological work (Lipmann, 1941). Evidence that the enthalpy (see footnote 3) of the high-energy phosphate bond is not 12 kcal/mol, as previously assumed (for a possible source of error, see Ling, 1962), but only 4.7 kcal ./mol (no more than one common phosphate bond, Podolsky and Kitzinger, 1955; Betzinger and Morales, 1956) made this theory of energizing biological work untenable. According to the association-induction hypothesis, ATP is designed to perform its energizing function not through hydrolytic cleavage, but through adsorption at cardinal sites that create or maintain a particular cooperative state of cellular protein (Ling, 1962 ). The unusually high enthalpy of ATP adsorption on G-actin [-24 kcal/mol (Asakura, 1961)] indicates that energization can be achieved in this way. The effect of ATP on the adsorption of K+ ions would be essentially similar to the demonstrated effect of Lanoxin, but in the opposite direction. One prediction of this model is that the concentration of K+ ions in living cells does not depend on the rate of ATP hydrolysis, but is determined by the ATP concentration in the cell itself. Thus, each time an ATP molecule is adsorbed at a cardinal site, a fixed number of anionic sites (see Figure 25) will cooperatively adsorb the K+ ion. Without ATP as the main adsorbent, some other ionic components will occupy the anionic sites; these can be Na+ ions or cations bound to nearby proteins. This prediction was confirmed for the K+ ion

A NEW MODEL FOR THE LIVING CELL

Distribution in frog muscle treated with iodoacetate and nitrogen, in human erythrocytes and in E. roli (Ling, 1962, ch. 9). Figure 32 shows the concentration of K+ ions in frog muscles as a function of cellular ATP concentration. Once the dependence of K+ ion accumulation on ATP adsorption is understood, 100

[ATPI em (pmol/g)

COWARDLY. 3 2 . Correlation of intracellular concentration of K+ ions and ATP concentration in voluntary muscles of frogs. Muscles were treated with 1 mM iodoacetic acid for different periods of time at room temperature and then cooled to 0°C in the same bathing solution. and left to equilibrate at this lower temperature for 1 hour, after which their K+ ion and ATP content were analyzed (Ling, 1962, by permission of the Blaisdell Press).

ATP's physiological role is becoming clear again. Thus, the normal resting cell hydrolyzes ATP slowly, but the activated cell hydrolyzes ATP very rapidly. (This indicates that ATPase activity itself is under physiological control.) A cyclic event can be visualized as follows: Adsorption of ATP by enzymes

Non-active cardinal digits

ATP-Synthesis

Cooperative adsorption of K+ ions

cooperative desorption of K+ ions

\ ATP-Hydrolysis

Stimulus that activates ATPase

GILBERT N. LING

ACKNOWLEDGMENTS The preparation of this review and the reported new research was supported by National Science Foundation Research Grants GB3921, GB7095, National Institutes of Health Research Grants 2RO1-GM11422-04 and HE-07762-64, and Naval Research Grant Office Telephone 4371 (00)-105327. The author is supported by the Public Health Service Research Career Development Award K3-GM-19032. The author is grateful to Dr. Frank Elliot, Dr. Margaret C. Neville, Margaret M. Ochsenfeld. Grace Bohr and Marie Bowers for their invaluable help; and the John A. Hartford Foundation for providing basic research equipment.

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A NEW MODEL FOR THE LIVING CELL

Doty, P. and Gratzer, W.B. (1962). In "Polyamino Acids, Polypeptides, and Proteins" (M.A. Stahmann, ed.), p. 111. University. by Wisconsin Press, Madison, Wisconsin. Eastman NJ, Geiling EMK, DeLander AM. (1933). Bull. Johns Hopkins Hospital. 53, 246. Eisenman, G. (1961). Membrane Transport Metabolism, Proc. Symp., Prague (A. Kleinzeller and A. Kotyk, eds.), p. 163. Czechoslovak Academy of Sciences, Prague. Eisenman, G., Rudin, D.O. and Casby, J.U. (1957). Science 126, 831. Epstein, E. and Hagen, C. . And (1952). Attachment. physiol. 27, 457. Falk, G. and Gerard, R.W. (1954). j comp. cellular physiol. 43, 393. Fenichel, I.R. and Horowitz, S.B. (1963). Acta Physiol. To stop. 222, 1. Fischer, H. and Moore, G. (1908). I am J Physiol. 20, 330. Fritz, O.G. and Swift, T.J. (1967). Biography. j 7, 675. Gibbs, J.H. and DiMargio, E.A. (1958). j physical chemistry 28, 1247. Gordy, W. and Stanford, S.C. (1941). j Cbem. Physical 9, 204. Grundfest, H., Kao, C.Y. and Altamirano, M. (1945). j Genesis 1:1 Physiol. 38, 245. Haber, J.E. and Koshland, D.E. (1967). perc. National Academy. Science 58, 2087. Hallett, J. (1965). federation process. 24, S-3 Burger, HJ (1889). 2. Supply 26, 414. Hammett, LP (1940). "Physical-Organic Chemistry". McGraw Hill, New York. Harkins, W.D. (1945). Science 102, 292. Harrington, W.F. and Schellman, J.A. (1956). Compt. To tear apart. dare Laboratory. Carlsberg, Ser. quim 30, No. 6, 21. Harris, E.J. (1950). Trans. Ser Faraday. 334, 872. Harris, E.J. and Burn, GS (1949). Electric. Ser Faraday. 45, 508. Heckmann, K. (1953). Natural Sciences 40, 478. Henriques, V. and Lundsgaard, E. (1931). Biochemistry 2. 236, 219. Heppel, L.A. (1940). I'm J Physiol. 128, 449. Hermann, L. (1879). Handbook of Physiology, Vol. 2 (F.C.W. Vogel, ed.), p. 3. L. Hermann, Leipzig. Hill, A.V. (1910). J Physiol. (London) 40, iv-vii. Hill, A.V. (1930). perc. Roy. Bag. (London) B106, 477. Hill, A.V. (1932). physiol. Rev. 12, 56. Hinke, J.A.M. (1959). Nature 184, 1257. Hodgkin, A.L. (1951). Soft . Rev. Cambridge Phil. Sor. 26, 339. Hodgkin, A.L. and Horowicz, P. (1959). J Physiol. (London) 148, 127. Hodgkin, A.L. and Katz, R.D. (1949). j physiol. (London) 108, 37. Hodgkin, A.L., and Keynes, R.D. (1953). j physiol. (Londotr) 119, 513. Holter, H. and Holtzer, H. (1959). ex. Cell Resolution 18, 421. Holwill, M.E.J. and Burge, R.E. (1963). Bow. Biography of Biochemistry. 101, 249. Horowicz, P. and Hodgkin, A. (1957). j Pbysiol. (London) 145, 405. Huxley, H.E. (1957). J. Biophy.r. biorhem. cilene. 3, 631. Jacques, M. (1936). J. Gen. Physiol. 19, 397. Jones, A. and Karreman, G. (1969). BiophyJ. j (in press). Keynes, R.D. and Maisel, G.W. (1945). perc. Roy. Asian (Lodon) B142, 383. Koketsu, K. and Kimura, Y. (1960). j comp. cellular physiol. 55, 219. Krogh, A. (1946). perc. Roy. society (London) B133, 140. Langmuir, I. (1917). Jelly . Chemistry Soc. 39, 1848. Levi, H. and Ussing, H. H. (1948). Acta Physiol. 16 beach, 232. Lewis, M.S. and Saroff, H.A. (1957). Jelly. chemistry ser. 79, 2112.

GILBERT N. LING

Ling , G. N. ( 1951 ). I am J Physiol. 167, 806. Ling, G.N. (1952). In "Phosphorus Metabolism" (W.D. McElroy and B. Glass, eds.), vol. 2, p. 748. Johns Hopkins Press, Baltimore, Maryland. Ling , GN ( 1955 ). J Physics. Medicine. 34, 89. Ling, G.N. (1960). J. Gen. Physiol. 43, supplement, 149. Ling, G.N. (1962). "A physical theory of the state of life." Ginn (Blaisdell), Boston. Ling , G. N. ( 1964a ). Biopolymers, Symp. 1, 91. Ling, G.N. (1964b). Representative of Texas Biol. Medicine. 22, 244. Ling, G.N. (196Sa). ann. n And Arad. Sri. 125, 401. Ling, G.N. (1965b). federal pror. 24, Supplement 15, p-103. Ling, G. N. ( 1965 ~ ) Prospects . biol. Medicine. 9, 87. Ling, G.N. (1966a). ann. N.Y. Acad. Sci-i. 137, 837. Ling, G.N. (1966b). federal pror. 25, 958. Ling, G.N. (1967a). In "Glass Electrodes for Hydrogen and Other Cations" (G. Eisenman, ed.). Decker, New York. Ling , GN ( 1967b ). Naturally. Roundschau 20, 415. Ling, G.N. (1967~) In "Thermobiology" (A. Rose, ed.), chap. 2. Academic Press, New York. Ling, G.N. and Ochsenfeld , M. ( 1965 ). Biography. J. 5, 777. Ling, G. N. and Ochsenfeld, M.M. (1966). J. Gen. Physiol. 49, 819. Ling, G.N., Ochsenfeld, M.M. and Karreman , G. ( 1967 ). J. Gen. Physiol. 50, 1807. Genus. G.N. and Ochsenfeld, M.M. (196a.). process of federation. 27, 702. Ling, G.N. and Ochsenfeld, M.M. (1968b). prof-. Internal. physiol. Cungr. Us'aihittgton DC, 24, 266. Ling, G . N. and Woodbury, J. W. (1949). J.Comp. Cell phone. physiol. 34, 407. Lipmann, F. (1941). Advanced Enzymol. 1, 99. Lundsgaard, E. (1930). Biochemistry 2.227, 51. McBain, J.W. and Peaker, CR (1930). 1. Physics. Chern. 34.1033. McDonald , JS ( 1900 ). pror. Roy. dor (London) 67, 310. McLaren, A.D., Jensen, W.A. and Jacobson , L. ( 1960 ). Annexure. Phy.rio1. 35, 549. Mellon, S.R. and Hoover, E.F. (1950). Geléia sor of chemistry. 72, 2562. Mond, R. and Netter, H. (1930). Pfuuger's Bow. 224, 702. Monod, J., Wyman, J. and Changeux, J. (1965). J Mol biol. 12, 88. Mysels, K.J. and McBain, J.W. (1948). 1. Colloid Sri. 3, 41. Nielsen, J.M., Adamson, A.W. and Cobble, J.W. (1952). Geléia. sor of chemistry. 74, 446. Osterhout, W.JV (1936). barteriol. Rev. Fr. 2, 283. Oberton, E. (1899). quarterly Natural Resource Rh. GeJ. (Zurich) 44.88. Overton , E. ( 1907 ). In "Handbook of Human Physiology" (W. Nagel, ed.), Vol. 2, p. 744. Vieweg, Brunswick. Pallansch , MJ and Briggs , DR . (1954). Geléia Chemical Soc. 76, 1396. Pfeffer, W. (1921). osmotic substances. 2nd edition Engelmann, Leipzig. Podolsky, R.J. and Kitzinger, C. (1955). Federation Pror. 14, 115. Rapatz, G. and Luyet, B.J. (1958). Biodynamir 8, 121. Rossi-Fanelli, A., Antonini, E. and Caputo, A. (1964). Notice. Protein Chemistry 19, 73. Rotunno, CA, Kowalewski, V. and Cereijido, M. (1967). Biorima. Biography. Artha 135, 170. Ruhland, W. and Hoffman, C. (1925). Plant 1, 1. Riser, H.J.P. (1968). Science 159 , 390 .

A NEW LIVING CELL MODEL

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LEONARDWEISS Cell Periphery Department

von

Experimental Psychology, RoJiuell Park Memorial Institute, Bruffalo, New York

Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Lipid bilayers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Other models. . . . . . . . . . . . . . . . . . . . . . . . . ... IV. Cell surface charge. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Measurement of Electrophoretic Cell Mobility. . . . B. Sialic acid residues. . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Ribonuclease Sensitive Groups. . . . . . . . . . . . . . . . . . D. Amino groups. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Other peripheral ionic groups. . . . . . . . . . . . . . . . F. Dynamic aspects of surface charge. . . . . . . . . . . . . . . . g load sharing. . . . . . . . . . . . . . . V. Enzyme activity and cell periphery. . . A. Sublethal autolysis. . . . . . . . . .

63 64 70 78 78 81 82 85

86 87

....

VISA. The peripheries of malignant cells. . . . . . . . . . . . . . . . . . A. Fine structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Calcium fixation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Surface charge. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . references . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

94 95

96 97 99

I. Introduction The structure of the cell periphery is of interest to researchers in many seemingly disparate fields, and a comprehensive discussion of the topic should ideally present an integrated picture. This is currently not possible and each review is inevitably weighed against the reviewer's own interests. My own work deals with interactions between living cells in cancer and morphogenesis and includes biophysical analysis of cell contact and recognition phenomena (Weiss, 1967a). In these studies, it is useful to make a physical distinction between cell contact, cell adhesion, and cell detachment (Weiss 19621, 1967b). This approach requires a distinction between the cell surface, which is an approximately flat, two-dimensional structure that surrounds the cell and is in contact with its surroundings, and the cell periphery, which is a three-dimensional area that surrounds the cell and includes the flat membrane(s) or permeability barrier(s). All these defined regions must be considered dynamically. 1 Some of my own work described here was supported in part by the American Cancer Society Grant No. P-403A.

LEONARD WELSS

No attempt is made to review the transport phenomena or the large body of immunochemical data that are clearly fundamental to presenting an integrated view of the structure of the peripheral region of the cell. In this and other respects, this review is incomplete. In this field, some of the techniques may not be familiar to the general reader. As the techniques themselves influence the interpretation of experimental data, I have occasionally used the work of my colleagues and my own extensively to illustrate interpretation difficulties as I am more familiar with our own techniques. This is not to say that I don't know about or want to ignore the work of others. 11. Lipid bilayers

Gorter and Grendel (1725, 1726) observed that when acetone-extracted erythrocyte lipids were distributed at the air/water interface of a Langmuir channel, the area of the compressed film was twice the calculated surface area of the erythrocytes. Based on this, it was proposed that the erythrocyte membrane consists of a lipid bilayer (Fig. 1). It has since been noted by Winkler and

COWARDLY. 1. The Gorter-Grendel model

Bungenberg de Jong (1741) and Hoffman (1762) that Gorter and Grendel underestimated the surface area of erythrocytes by about 50% and that the ratio of total lipid surface area to cell surface area is 1:1 rather than of 2:1; however, because acetone does not remove all lipids from erythrocytes, the original bilayer estimate is probably correct. Davson (1962) cited more reliable data derived from lipids extracted from rabbits, guinea pigs and human erythrocytes in which the ratios of film areas to calculated cell areas are all about 2:1. Danielli and Davson (1735) also independently proposed a modified lipid bilayer hypothesis. Danielli and Harvey (1934) observed that tensions measured at the periphery of various oil droplets and cells corresponded to maximum estimates for their surface tensions of 1 to 3 dynes per cm. The surface tensions of lipid films were expected to be on the order of 10 to 30 dynes per cm, and experiments have shown that various proteins can significantly lower the surface tension of lipids. The well-known model was

THE PERIPHERY OF THE CELL

Sixty-five

Therefore, advances have been made in the plasma membrane, in which a lipid bilayer is coated with proteins on its inner and outer surfaces. Earlier and later versions of this model are shown in Figures 6 and 7. 2 and 3. It is not my intention here to review in detail the extensive literature on the lipid bilayer model for the cell membrane. It's hard to resist a discussion about hydrocarbons

FEIGE. 2 .

pole groups

The Danielli-Davsorl model [Adapted from Danielli and Davson (1935)l.

of the fascinating experiments performed on black lipid films originating from the work of Mueller and colleagues (Mueller et al., 1962; MueIler and Rudin, 1963) and recently revised by Tien and Diana (1968). However, although these films mimic certain natural membrane functions to a remarkable degree, particularly with regard to the action potentials observed in the nervous system, their role in elucidating the structure of cell membranes in general is unclear. Likewise, the lipid vesicles studied by Bangham and colleagues (Bangham et al., 1965; Bangham and Haydon, 1968) are not discussed. If it is technically possible to incorporate membrane proteins and carbohydrates into these lipid systems or produce them from lipoglycoproteins, membrane models other than the lipid bilayer can be evaluated. Much of the other relevant work on model systems has been reviewed by Kavanau (1965) and myself (Weiss, 1967a), among others. In 1962, Danielli stated: "The problem of the basic structure of the plasma membrane was essentially solved around 1940.

LEONARD WEISS

sufficient evidence that the membrane was a bimolecular lipoid layer with protein layers adsorbed on both surfaces. . . .” Since this model has dominated much thought and work on membrane structure for nearly 30 years, it is pertinent to critically examine the foundation upon which it rests to determine whether it is hypothesis or fact. lipoid molecule

protein molecule

polar pore

COWARDLY. 3. The Danielli-Davson-Harvey model modified by the addition of polar pores (Danielli, 1958). [Reprinted with permission from "Surface Phenomena in Chemistry and Biology" (J.F. Danielli, K.G.A. Pankhurst and A.C. Riddiford, eds.), Pergamon Press Ltd. (195E).l

While other techniques are of considerable historical interest, it appears that current concepts of the validity of the lipid bilayer model rely heavily on observations made using electron microscopy and X-ray diffraction, as well as previous work that required considerable technical and interpretive ingenuity, given the techniques current. somewhat ambiguous experience and will not be repeated here; for more details, the reader is referred to Kavanau's (1965) monograph. If you look at the peripheral regions of cells under an electron microscope, you can see the familiar three-layer structure. Robertson (1960) reviewed the many observations and variations on the technique that revealed two dense lines of 20-A electrons. wide separated by 35 A., and this led to the concept of a simple "75-A. unit." However, this concept is not tenable, given the different latitudes reported by Zetterqvist (1956), Freeman (1959),

THE PERIPHERY OF THE CELL

Among others Karrer (1960), Smith (1961), Sjostrand (1963b), Yamoto (1963), Cunningham and Crane (1966) and Parsons (1967). Sjostrand's (1963b) observations are particularly valid, as all of his measurements of the width of trilaminar structures on adjacent membranes were made within the same small field. In mouse kidneys and pancreas, mitochondrial membranes and cytomembranes1 measure 50 Å in osmium-fixed material and 60 Å in permanganate-fixed samples. In the same material, smooth cytomembranes measure 60 Å in osmium-fixed preparations and 70-80 Å in permanganate-fixed preparations, while plasma membranes and membranes surrounding zymogen granules measure 90-100 Å. In addition to the usual artifacts associated with electron microscopy (Weiss, 1962b; Elbers, 1964; et al.), the interpretation of trilaminar structure in terms of lipid orientation presents many difficulties. On the one hand, the arrangement of lipid molecules is partially determined by the amount of water present. Bangham (1963) reviewed much of the literature on model systems that indicate the formation of different phases as a function of lipid concentration. When water is removed from such systems, lipids tend to form bilayers because they have the lowest free energy configuration (Haydon and Taylor, 1963) and are therefore the most stable structures. Therefore, regardless of the initial arrangement of lipids in the model systems, a bilayer coverslip would tend to result as a preliminary artifact after dehydration. Sjostrand (1967) felt that knowing such model systems, it is not possible to draw any conclusions about the phase transitions in membrane lipids, where they represent only about 30% of the dry weight. However, work on model systems indicates that lipids can exist in a more dynamic state than the bilayer concept implies (Weiss, 1962b; Lucy and Glauert, 1964), and polymorphism in lipids in the aqueous phase has been extensively discussed by Luzzati and his colleagues (Luzzati et al., 1957, 1958, 1960, 1962). Chapman et al.]. (1967) and Clifford et Studies on amoebae, they examined the reactions of various dyes with lipids, which parts of the lipid molecules are labeled, whether lipid or a marker is present in the sample when examined under an electron microscope, and whether or not

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the position of the lipid or its marker is not as oriented as in the untreated cell. Their experimental results show that Acunthumoebu cells fixed in glutaraldehyde by standard preparation methods cannot contain any lipids at the time of microscopy, and cells fixed in osmium tetroxide or potassium permanganate lose most of their neutral lipids and a significant proportion of their phospholipids. Casley-Smith (1967) agrees that there is no general binder for lipids. Despite this lipid loss, regular trilaminar structures can be seen at the cell periphery. These results are reminiscent of the work of Green and Fleischer (1963), in which no electron microscopic changes were observed in the trilamellar structure of mitochondrial membranes after acetone extraction of about 85% of their phospholipids. The work of Fleischer et al. (1967) on the fine structure of lipid-poor mitochondria is also difficult to reconcile with the bilayer model. These authors observed the persistence of trilaminar structures after removal of 95% of the lipids from the mitochondrial membranes, whereas, if the bilayer model was correct, they would expect the collapse or separation of the two electron-dense bands of the membrane unit. If, as Sjostrand (1963a) suggests, the crosslinks between the outer layers of proteins hold the trilamellar structures together, then one would expect these crosslinks to be visible after lipid extraction. Fleischer et al. I didn't watch her. Although these observations argue against the applicability of the bilayer model to mitochondrial membranes, the authors note that erythrocyte ghosts, for example, collapse when lipids are removed using similar techniques. These results seem to argue against a common "unit" membrane, rather than providing a clear refutation of the bilayer model. Wigglesworth (1947) and Baker (1958) proposed that OsO acts by linking the double bonds of fatty acids to form stable diesters. This proposal is supported by the work of Stoeckenius and Mahr (1965) who failed to demonstrate the direct reaction of OsO with polar groups of phospholipids other than phosphatidylserine; and by Korns (1967) chromatographic and spectroscopic studies on unsaturated lipids, which also show diester bonds with unsaturated fatty acid chains through OsO. However, Stoeckenius and Mahr also showed using infrared spectroscopy that side reactions with hydrophilic groups actually occur when phospholipids react with OsO; and Stoeckenius (1962) had already interpreted electron micrographs of lipid-water systems "fixed" with OsO to indicate the deposition of osmium on the polar heads of phospholipids after decomposition of the osmium ester. Korn's data show the formation of only one esterified osmium molecule for every two fatty acid molecules, indicating that one cannot necessarily assume that osmium reacts or marks polar groups of phospholipids during sample fixation. Furthermore, in cell membranes, marked

THE PERIPHERY OF THE CELL

polar groups, unlike phospholipids, can be related to proteins and carbohydrates. The general impression is that electron microscopy of stained lipid preparations can be used to deduce their molecular arrangement in defined model systems, but this is not the case for cell membranes. X-ray diffraction studies of the myelin sheath, pioneered by Schmitt, Bear, and Clark (1935), are often considered the strongest direct support for the lipid bilayer membrane model. Geren (1954), Maturana (1960) and Peters (1960) showed that the myelin sheath is formed by extensive turnover of Schwann cells; however, whether the myelin sheath can be considered a viable model for other membrane structures seems doubtful, given its highly specialized functions and electrical properties. Finean (1962) performed extensive studies correlating the electron microscopic appearance of myelin sheath material with X-ray diffraction patterns. This work was widely accepted as unequivocal evidence for the existence of a lipid bilayer structure in the myelin sheath. Some authors have used the results of work with myelin as strong supporting evidence in favor of similar structures at the periphery of other cells. The work by Finean and colleagues on X-ray diffraction of myelin sheath material has been interpreted in terms of a membrane approximately 80-90 Å thick, where the distance between the polar phosphate heads and the phospholipid content is 50 Å. (Finean, 1962) as shown in Fig. 4. Finean and Burge (1963) studied the X-ray diffraction patterns of the myelin sheath in different degrees of swelling. From their measurements of the intensity distributions under different swelling conditions, they tried to assign phases to the different peaks in the normal X-ray pattern.

nyelin

171A

myelin

COWARDLY. 4. A proposed structure for the myelin sheath (Finean and Robertson, 1958). L, lipid; Pr, protein; P, phosphorus.

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The distribution is subject to criticism because it assumes that the only parameter that changes as myelin increases is distance. As myelin inflammation is not well understood and is not uniform, it seems unwise to assume that it is not accompanied by molecular rearrangements. That the interpretation of intensity distributions of swollen materials can be ambiguous is evident from Perutz's (1954) early discussions of his X-ray diffraction data on hemoglobin, in which phase data obtained from swollen hemoglobin crystals are compared with those obtained using were isomorphic substitution technique obtained. Serious difficulties arise when trying to extrapolate data from electron microscopy and X-ray diffraction of myelin fixed in osmium permanganate to fresh unfixed myelin. First, these two reagents alter the properties of the membrane, as shown by Schmidt (1936, 1938) using the polarizing microscope, although the exact nature of the alteration cannot be interpreted, since in all polarization measurements, the birefringence of the form cannot be satisfactorily separated from intrinsic birefringence. . Shah's (1968) experiments with lipid monolayers also indicate a change in osmium binding. Stockenius et al. (1960) showed that when phospholipid lamellar phase systems react with OsO, the repeat pattern indicates that their lamellar structure is preserved. However, under these conditions, there is a loss of 4.5 A. Banda identifying the hydrocarbon chains of the fatty acid component of phospholipids (Luzzati and Reiss-Husson, 1962). Therefore, it has been suggested that the lamellae are held together after binding by relatively few crosslinks between the fatty acids on opposite sides of the bilayers, but that the packing of the remaining chains must be "heavily perturbed". Parsons and Akers (1968) studied the effects of different concentrations and reaction times of OsO on the myelin sheath. Their electron microscopy and X-ray diffraction data show that there are obvious rearrangements within the envelope under conditions routinely used to prepare electron microscopy preparations. The evidence cited suggests that X-ray diffraction data on the myelin sheath may not be unambiguously interpreted as previously thought. Various chemical fixation methods used to prepare samples for electron microscopic observation can seriously alter the original molecular arrangement, and interpretation of fine structural details must be done with great care.

111. Other Models Although the lipid bilayer model may not have been conclusively disproved, the fact that it cannot in any way be considered proven allows other concepts to be examined more openly than before. A variety of models have been proposed, ranging from complete lipid structures (Osterhout, 1940) to lipid/protein mosaics (Nathanson, 1904).

THE PERIPHERY OF THE CELL

layered structures proposed by Danielli and Davson. Among the most conceptually interesting of these models is the one postulated by Parpart and Ballentine (1952) based on indirect evidence and shown in Figures 5 and 6. This membrane model is of particular interest because it shifted the focus from lipid to protein structures by concentrating on se in it suggested that lipids may exist primarily in a protein scaffold with their polar heads oriented in aqueous spaces.

r---------3

50 a.

COWARDLY. 5 . Model by Parpart and Ballentine (1952) in cross section showing the polar heads of phospholipids (small circles) interacting with membrane proteins (large circles) and aligned with aqueous spaces to form pores.

50A

COWARDLY. 6 Model by Parpart and Ballentine (1952) in tangential section. Protein is shaded; stained water; non-aqueous phase per clear area; phospholipids by rounded head rectangles; and cholesterol by rectangles.

Among other things, Bakerman and Wasemiller (1967) performed detailed analyzes of human erythrocyte membranes. Their work shows that the membrane material is a lipoglycoprotein containing 55% protein, 35% lipids and 10% carbohydrates. The average molecular weight of the complex was approximately 44,400 and 22,200 for the protein. These authors see their evidence

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as an indication of repeating units of the structural membrane of the erythrocyte. Although the averaged parameters may represent a simplified view of the cellular peripheral structure, treating the membrane as an integrated complex that encompasses all its main components seems to be the most promising approach at the moment. An indication of the diversity of possible membrane structures of lipoproteins compared to phospholipids comes from reviews by WH Cook and Martin (1962) and Oncley (1964). Although lipoproteins are known to contain phospholipids, neutral lipids, and proteins in varying proportions, little is known about their structure or binding except that they are joined together in non-stoichiometric proportions by forces weaker than covalent bonds. This in turn indicates that the determined composition of the complexes is very sensitive to the techniques used for their isolation and cautions against hasty acceptance of analytical data when drastic methods have been used to isolate them from membranes. Cook and Martin classify lipoproteins into those with less than 33% protein content (LPL) and those with more than 33% protein content (HPL). In the LPL class, the neutral lipid-phospholipid ratio ranges from 1:1 to 10:1, but this ratio remains 1:1 in the HPL class. LPL beads tend to have proteins and phospholipids at their interface with an aqueous environment and resemble micelles. The HPL class maintains a structural integrity that approaches that of protein molecules. Conversion from LPL to HPL classes is problematic and would imply more profound changes than simple lipid gain or loss. The studies by Lenard and Singer (1968) on erythrocyte membranes treated with phospholipase C indicate the relevance of the reviews by Cook, Martin and Oncley to cell membranes. intact as observed by phase contrast microscopy, and the average conformation of proteins in them, as determined by ultraviolet circular dichroism measurements, remains unchanged. These results are interpreted by the authors as indicating that the phosphoester bonds of membrane phospholipids are easily accessible to the enzyme and that electrostatic interactions between these phosphate groups and membrane proteins play a minor role in maintaining the integrity of the membrane of these phospholipids and the membrane. membrane. conformation. These results are considered more consistent with a scheme proposed by Lenard and Singer (1966) (Fig. 7) based on their conformation studies of membrane proteins using optical spin scattering and circular dichroism techniques, the Danielli scheme model de Davson, Lenard, and Singer (1966) suggested that the polar and ionic heads of lipid molecules, along with any ionic side chains of the structural protein, lie on the actual surface of the cell in contact with the environment. The non-polar residues of the protein together with the hydrophobic "tails" of the phospholipids and relatively non-polar lipids, e.g.

THE PERIPHERY OF THE CELL

cholesterol, reside within the membrane. They also postulate that the helical parts of the membrane protein reside within the membrane and that the interactions of the enumerated types determine the general conformation of the structural proteins. They speculate that subunits could be formed with these general arrangements that could aggregate on an intact membrane in a similar way to the pro-

e 86

Hellcal coil parts

/vvv\ Random call

portions

LIPID

COWARDLY. 7. Lenard and Singer's (1966) model (see Fig. 2). The proteins on the outer surfaces of the membrane are composed of random helical and spiral sections. Polar lipids are oriented in a bimolecular sheet with their polar heads (circles) facing outward. The shaded areas are assumed to be occupied by relatively non-polar components (lipids or hydrophobic amino acid residues).

raised by Green and Perdue (1966). When evaluating this model, care must be taken to correlate the accessibility of the phospholipid-phosphoester bonds with their position relative to the cell surface, as Seaman and Cook (1965) explained the electrical charge on the surface of erythrocytes as a carboxyl. of sialic acids and glutamyl residues, and there is no direct evidence of the existence of positively charged groups associated with the polar heads of phospholipids in this region. Benson (1966) (Fig. 8) presented a model similar to that proposed by Lenard and Singer for data derived from plant chloroplast layers, which consist of sets of subunits called genomes. Within the quantosomes are four amphophilic lipids, each containing a limited and specific group of fatty acids. Benson proposes that the hydrophobic hydrocarbon chain of these fatty acids may associate with specific hydrophobic amino acid residues of the membrane protein. In this model, a protein scaffold occupies the entire thickness of the membrane; the polar groups of proteins and lipids are on its outer sides, while the central region is hydrophobic. The molecular arrangement would explain the trilaminar structure seen in electron micrographs of stained membranes as the spots would grow together.

LEONARD WEISS

lect in the polar regions, leaving the central region transparent to electrons. Benson makes an interesting speculation that metabolically induced changes in the conformation of a flexible lipoprotein ion exchange membrane may play a role in transport phenomena.

COWARDLY. 8. Model of Benson (1966) showing a protein structure containing phospholipids.

Studies performed on mitochondrial membranes may be relevant to the structure of the cell periphery, although extrapolation from one membrane system to another is obviously inadvisable. Mitochondrial data are extremely useful because, as Parsons (1967) pointed out, at least 60-70% of proteins have been identified in mitochondrial membranes, as opposed to other membranes. Green and others. (1961) and Richardson et al. (1963) showed that 40-50% of mitochondrial protein is present in an insoluble form which they termed "structural" protein. Of great interest is that this structural protein has a molecular weight of 22,500 (Criddle et al., 1962, 1966), which corresponds approximately to the protein with molecular weight of 22,200 isolated from the erythrocyte membrane by Bakerman and Wasemiller (1967). . Mitochondrial protein readily polymerizes, which may explain why trilamellar structures persist in mitochondria after 95% of their lipids have been removed (Fleischer et al., 1967). Mitochondrial protein also forms strong complexes with phospholipids. Parsons (1967) cites the unpublished work of Racker and Stoeckenius showing that the lipid-free protein has an amorphous electron microscopic appearance similar to the structural protein, but with the addition of a small amount of phospholipid to the protein.

THE PERIPHERY OF THE CELL

The system leads to the formation of sheets and vesicles that resemble fragments of the inner mitochondrial membrane. Of great importance is the observation that membrane-like structures apparently can only be formed by structural proteins. McConnell et al. (1966) described how membranes can be formed by so-called mitochondrial cytochrome oxidase repeat units. The "repeating units" are "the last lipoprotein units of the membranes". . (Green et al., 1967) and can be prepared by treating membrane structures with bile salts. Membrane-like structures are formed as a result of the removal of bile salts. Under controlled conditions, the ability of the repeating units to form membranes is lost with the removal of phospholipids and can be recovered with the addition of lipids to the system. Green and others. (1967) presented evidence from electron microscopy for the formation of membrane repeating units from mitochondrial membranes, the chloroplast membrane of spinach, the outer segments of bovine photoreceptors, a microsomal membrane of bovine liver, and the plasma membrane of bovine erythrocytes. In all of them, lipid depletion reversibly inhibits membrane formation. It is believed that, even in the case of mitochondrial membranes, the repetitive units are the so-called building blocks and can be visualized as approximately cubic and parallelepiped protein structures (Fernández-Morin et al., 1964) measuring 114 x 50 x 114 A ( Figure 9). Green and his colleagues postulate that, when lipid is present, it is confined to two of the parallelepiped faces and that the repeating units can only interact on the remaining four non-contacting faces.

Membrane repeater units (base parts)

Less lipids (bulk phase)

More lipids (membrane)

COWARDLY. 9. The Green et al. (1967) showing that nested repeat units assemble in a membrane with the polar (dark) heads of lipids aligned to be present on its inner and outer surfaces.

LEONARD WELSS

They contain lipids, with the consequent formation of membranes. In the absence of lipids, the repeating units can interact on all sides, resulting in an amorphous aggregate. According to this ingenious hypothesis, a protein must meet four conditions to form membranes. It must be able to polymerize into three-dimensional aggregates; must hydrophobically associate with negatively charged phospholipids; must associate asymmetrically with phospholipids to cover only 2/6 faces (see siipra); and, finally, the phospholipid-coated protein repeat unit must be able to hydrophobically bond to other units to form a curved layer. Green and others. point out that they did not find any proteins other than the repeating units themselves that can give rise to “authentic” membrane structures when interacting with phospholipids. These authors criticize Kagawa and Racker's (1966) report that structural proteins can form membrane-like vesicles under the influence of phospholipids because their published electron micrographs do not allow distinguishing between domains of phospholipid micelles that are present and "true" vesicles for distinguish. Undoubtedly, this important question will be resolved through experimentation. In general, proteins that connect to phospholipids via hydrophobic bonds can be expected to contain a high proportion of amino acid residues with lipophilic side chains. Interestingly, these conditions are met by mitochondrial structural proteins (Criddle et al., 1362), myelin protein (Hulcher, 1963), and protein isolated from erythrocyte membranes (Bakerman and Waserniller, 1967). All recently proposed membrane models tend to favor membrane proteins that are in globular shape, in contrast to the Danielli-Davson model, which depicts the protein in the 0 configuration extended alongside the polar heads of phospholipids. It is therefore of interest that examination of erythrocyte ghosts by Maddy and Malcolm (1965) by optical rotation scattering and infrared spectroscopy does not show evidence of protein in the (3) configuration. Studies of the inner mitochondrial membrane by several researchers indicate that it is only 5 5 Å thick. This value is too low to be explained by a lipid bilayer model. Parsons (1967) suggested that phospholipids could be packaged in these membranes in the form of "flat micelles" with a minimum thickness of 9 Å, with their polar heads positioned in the center and their nonpolar tails forming a network of variable dimensions in the membranes that could fit the proteins (Fig. 10). It remains to be seen whether this concept will be compatible with others providing details of the protein-phospholipid combination. Additional weight for the suggestion that Jome membranes can be constructed from repeating units comes from the electron microscopy studies reviewed by Sjostrand (1967). In sections of permanganate-fixed cells from proximal tubules of the mouse kidney, Sjostrand observed mitochondrial membranes and

THE PERIPHERY OF THE CELL

Smooth-surfaced membranes adjacent to the plasma membrane. Although the plasma membrane appeared as a trilaminar structure, the other two membranes showed a well-defined spherical substructure in which blood cells were approximately 50 Å in diameter in mitochondria and 60-70 Å in smooth surface membranes. The lyophilized material also showed a similar substructure.

COWARDLY. 10. Model by Parsons (1967) for the inner membranes of mitochondria1. Speculative diagram for "flat micelle" membrane phospholipid packaging type for devices containing six phospholipid molecules per micelle (A,B) and eight molecules per micelle (C,D). A and C are densely packed arrays, B and D are more open arrays. These arrangements could occur in membranes with a low phospholipid content and would have the advantage of leaving enough space for cytochromes and enzymes in globular form. The models are only approximately to scale and do not show bends in the hydrocarbon chain due to unsaturation. Actual models indicate that the polar base portion of the phospholipid molecule readily adopts a nearly central position, as indicated in the diagram.

Sjostrand emphasizes that he has not visualized the globular substructure in either the plasma membrane or the myelin sheath, noting that this in itself suggests that there are at least two types of membranes beyond this detailed substructure at the molecular level. It is of some interest that the myelin sheath does not represent the globular substructure, as Folch-Pi (1967) concluded that in these structures the phospholipids are linked to the protein by ionic and electrostatic bonds, which, contrary to other opinions expressed stand structures . Membranes It should be mentioned here that the electron by Rendi and Vatter (1967)

LEONARD WEISS

Microscopic studies of mitochondrial membranes1 also led them to postulate a spherical substructure, but they visualize their results in terms of discrete 'granules' of phospholipids and proteins with a diameter of about 20 Å (Fig. 11). Although they emphasize that they cannot interpret their observations in terms of molecular arrangements, they point out that they are two different grain layers.

COWARDLY. eleven . Model by Rendi and Vatter (1967). A schematic representation of the unitary membrane image interpreted by Robertson (left). Observation of the fine structure of the cut and negatively stained membranes shows that the membrane can be interpreted as a granular mosaic. The granules (right) are labeled to indicate phospholipids (PL) and "structural" protein (SP). (The arrangement of the two types of units does not indicate their organization, but it does explain the proportions of the two components in the membrane.)

separated by 30 Å would give the same image of a trilaminar structure, supporting the Danielli-Davson model. The difficulties in interpreting electron micrographs of membranous regions, extensively reviewed and discussed by Elbers (1964), leave me with the strong impression that the problems of their detailed molecular assembly cannot be solved by existing techniques of electron microscopy, although, as Sjostrand (1967) has stated: "The development of any concept related to the structure and function of living systems depends on research into available techniques, however rudimentary they may be."

IV. Cell Surface Charge A. MEASUREMENT OF CELL ELECTROPHORIC MOBILITY All vertebrate cells studied to date carry a net negative charge on their surfaces. When such cells are suspended in an electrolyte solution through which a direct current flows, they migrate to the anode. In an electrophoresis device, the velocity of individual cells residing in the "stationary layer" relative to the walls of the observation chamber in which they are migrating can be measured directly. The electrophoretic mobilities of the cells are then expressed as the observed velocity in microns per second per volt per centimeter of potential gradient (ps - 1 volt - 1 cm). the different

THE PERIPHERY OF THE CELL

the techniques used are discussed in the symposium report edited by Ambrose (1965) and are not described here. For the biologist, the main question is: what do electrophoretic mobility measurements mean and what information about the cell periphery can be derived from them? Some aspects are covered here, but for a complete discussion of the underlying theory, the reader is referred to recent detailed reviews by Overbeek (1950), Booth (1953), James (1957), Brinton and Lauffer (1959), Lyklema and Overbeek (1963). ), Haydon (1964) and Wiersema, Loeb and Overbeek (1966). When a cell moves through an electrolyte solution in an electric field, some of its surroundings move with it. The interface between the environment that moves with the cell and most of the environment is the so-called "hydrodynamic slip plane", and electrophoretic mobility measurements reflect the potential in this plane (the zeta potential) 5 which can be accounted for as surface electrokinetics of the cell. A charged surface preferentially attracts oppositely charged ions, creating a diffuse electrical double layer. The effective thickness of this layer can be defined in terms of the Debye-Huckel parameter 1/K, which is the distance from the true plane of surface charges at which the potential drops from I#~ to l/e x I$~. The value of 1/K depends on ionic strength and valence as indicated by

I/K = 3.05.1-1/2 Donde I

= 1/2 Br 22 (Lewis and Randall, 1921), c = ion concentration, y i i i

= value. In "physiological" saline solutions it is probably 1/K

8-10 A. (Heard and Seaman, 1960). A cell is not a spherical particle, and the question of what value should be assigned to the radius of curvature in electrokinetic studies of cells and what effect cracks and filaments have on the interpretation of mobility measurements has been raised repeatedly. If the fluid flows freely through the cracks and around the filaments, the dimensions of the cracks or cavities and the radii of the filaments must be taken into account from an electrokinetic point of view. However, as discussed by Haydon (1964), among others, in the absence of a highly filamentous surface and considering the periphery of the cell as a porous macromolecular assembly, the fluid movement can be considered flat and not superficial. defined, and the radius of curvature considered is that of the entire cell. These considerations pose extraordinarily difficult hydrodynamic problems to consider for any type of cell surface geometry and seem rather intractable. for example

LEONARD WEISS

Where u is the total radius of a particle when Ku > 300, which is said to be the case for cells in saline solution, then the electrophoretic mobility p can be related to the zeta potential 5 by the Smoluchowski equation

where E and TI are the dielectric constant and viscosity in the area of the hydrodynamic slip plane. It should be noted that the use of global phase values for E y '(1) can certainly be a source of numerical errors (Henniker, 1949). Haydon (1964) states that at Ka > 300, 5 can be found for any shaped particle using the above equation if the charge distribution on the electric double layer is not affected by the field applied in electrophoresis and if the cell surface conductivity is not é grande Gittens and James (1963) drew attention to the influence of the electrical conductivity of the surfaces of bacteria on their electrophoretic mobilities. The higher the conductivity, the lower the observed mobility. These investigators show that although in NaCl solutions below 0.02 M, erythrocyte mobilities are significantly greater than when changes in conductivity are ignored, no significant changes attributable to conductivity are found in the solutions. determined. with ionic strength greater than 0.05 M NaCl. Therefore, it seems highly unlikely that the surface conductance is high enough to significantly affect the interpretation of mobility data performed in physiological saline solutions (10.145 M NaCl). Electrophoretic mobility can also be related to the surface charge density (T per

to =-

The use of these equations in the present context allows for the biologically unjustifiable assumption that cell peripheries are impenetrable to counterions. Haydon (1961) examined this problem and came to the conclusion

where a is the fraction of the total space within the "surface" that cannot be occupied by counterions; k is the Boltzmann constant; T is the absolute temperature; and YZ is the number of ions per unit volume of the main phase. Thus, neglecting Haydon's "a" could lead to an underestimation of the charge density by a factor of 2.

THE PERIPHERY OF THE CELL

In the above equation it can be seen that the symbol v,, appears for the surface potential. Consideration of the diffuse bilayer shows that the cell electrophoresis technique provides measurements of the zeta potential, not the surface potential, and that 5 < *q~". Haydon's (1960) 5 measurements for oil droplets in surface aqueous solutions - The ions actives are the same as the phyllo measurements obtained by surface potential measurements of the corresponding flat plates, provided that the surface potential is less than 50 mV Since it is assumed that the surface potentials of the cells studied so far are lower at -50 mV, Haydon equals +,) and 5 in the field of cell electrokinetics. This can lead to significant errors. It appears that the technique of measuring cell electrophoretic mobility can be used to advantage when comparing the mobilities of similar measured cells under similar environmental conditions. Although it is an estimate of cell surface potential, surface charge density and zeta potential are sufficient measures of motility that can be obtained and these estimates can be used for comparison purposes, one cannot rely too heavily on numerical estimates themselves . Some indications of the usefulness of electrophoretic techniques in investigating the chemical nature of the electrokinetic surface of the cell will now be given.

B. SIALIC ACID MOVEMENTS Burnet et d. (1746) showed that Vibvio chokvue and Clostridjum zuelchii filtrates destroyed influenza virus receptor sites on the surface of human erythrocytes. Hanig (1748) observed that when erythrocytes adsorb the PR8 virus, their electrophoretic mobilities are reduced. Therefore, some of the negative charges on human erythrocytes were somehow related to virus adsorption. Ada and Stone (1750) showed that the receptor-destroying enzyme V. chdeme reduced the net negativity of the erythrocyte surface by more than 80% and suggested that the enzymatically cleaved fraction of the erythrocyte surface contained acidic groups. In 1958, Klenk suggested that acylated neuraminic acids could contribute to the negative charge of erythrocytes. The final steps in proving this conjecture followed Ada and French's (1759) purification of a receptor-destroying enzyme, which turned out to be neuraminidase, and Gottschalk's (1957) characterization of its specificity, i.e., hydrolytic cleavage of the glycosidic bond. that binds to keto. N-acetylneuraminic acid group to a sugar or sugar derivative. In 1960, Heard and Seaman demonstrated an 80% decrease in the electrophoretic mobility of human erythrocytes with purified neuraminidase and showed by analysis that such incubation resulted in the release of free sialic acid from the cells into the medium. It was later shown (Weiss, Igblc, 1963b) that after incubation with

LEONARD WEISS

Neuraminidase-pure mouse fibroblasts grown in glass detach more easily than their controls. This was taken as an indication that sialic acid residues were also present on the tissue cell surface. The most direct evidence for the presence of sialic acids on the cell surface comes from experiments in which there is a significant reduction in electrophoretic cell mobility, indicating the loss of surface anionic groups after incubation of cells with neuraminidase. In studies by Wallach and Eylar (1761) on normal and tumor cells, G.M.W. Cook et al. (1962, 1963) and Miller et al. (1963), among many others, only the N-acetyl and N-glycolyl derivatives of neuraminic acid were observed. An additional cautionary note against considering any cell membrane as a good model for any other comes from observations that the electrophoretic mobilities of a variety of cells are not altered by neuraminidase treatment (Naaman et al., 1965; Chaudhuri and Lieberman, 1965 ; Gelding and Pérez-Esandi, 1964). Of the intracellular membranes, those of the nuclei of liver cells (Marcus et al., 1965) and Ehrlich's ascites cells (Mayhew and Nordling, 1966) were reduced in their motility by neuraminidase, while the charge properties of the membranes of the endoplasmic reticulum of the Ehrlich's ascites cells remained unaffected by such enzymatic treatment (Wallach and Kamat, 1966). Mayhew and Nordling (1966) also made the interesting observation that although the electrophoretic mobilities of murine Ehrlich ascites, sarcoma 37 ascites, and liver cells and their homologous isolated nuclei were similar (indicating surface charge densities), in the periphery of cells and the like in their cells were their own nuclei). membranes), the reduction in mobility produced by neuraminidase differed between peripheries and nuclei, indicating that the similarities in mobility were attributable to different species of anions. C. RIBONUCLEASE-SENSITIVE GROUPS Lansing and Rosenthal (1952) proposed that RNA was present in the periphery of Arburia eggs and Elodeu cells; de Kloet (1961) suggested that it was present on the periphery of protoplasts Sacchaomyces cnrlrbeygensir and Chaudhuri and Lieberman (1965) described RNA on the surface of the nuclear membrane of liver cells. Mayhew and I systematically attempted to demonstrate the presence of RNA at the periphery of mammalian cells (Weiss and Mayhew, 1966, 1967; Mayhew and Weiss, 1968). Earlier work had shown that cells detached from the glass left fragments of their peripheral areas on the glass surface (Weiss, 1961a,b; Weiss and Coombs, 1763; Weiss and Lachmann, 1964). When cells grown in vitro on tritium uridine-labeled glass slides were carefully removed from the glass, they left radioautographically detectable "prints" on which the contours of the cells could be superimposed. These "footprints" have been removed

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of the vessel by treatment with RNase. Separation of similar cells from the glass was facilitated by treatment with active RNase but was unaffected by incubation with inactivated enzyme. These results suggest that RNA may be present as a structural component of the periphery of the studied cells, although other interpretations of the experimental data are possible. The electrophoretic mobilities of some cells were significantly reduced after incubation with RNase, but were not reduced in cells incubated with RNase inactivated by the method of Barnard and Stein (1958). Interpretation of these electrophoretic data is instructive as it illustrates some of the more general issues associated with this experimental approach. On the one hand, the reduction in electrophoretic cell mobility after RNase incubation may be the result of loss of enzyme-associated and susceptible negatively charged surface groups; Bovine pancreatic RNase acts on the phosphodiester bond between the 5' positions of ribose residues in RNA (Brown and Todd, 1955) and is highly specific for linkages of pyrimidine nucleosides (Volkin and Cohn, 1953). Therefore, if it can be shown that the reduction in net negativity of RNase-treated cell surfaces is the result of its enzymatic activity, this finding can be interpreted as indicating the presence of RNA in the cell periphery. On the other hand, it is known that RNase is a basic protein and its nonspecific adsorption on the cell surface would also cause a reduction in electrophoretic mobility due to the drop in negativity of the surface on which it is adsorbed. A very strong indication that the effects observed by Mayhew and I were not the result of non-specific enzyme adsorption comes from a detailed consideration of the inactivated enzyme, which had no detectable effect on the cell surface. The inactivated enzyme we used was prepared by carboxymethylation of native RNase with bromoacetic acid, following the procedure of Barnard and Stein (1958), removing the positively charged group at residue histidine 119 (Crestfield et al., 1963). Therefore, of the 19 positively charged RNase clusters, only one is removed by the inactivation process. This small change in net charge is evident from the fact that both inactive and active enzymes have isoelectric points near pH 9.6 and there is little difference between them on ion exchange columns (Glick et al., 1967). From an electrostatic point of view, it is unlikely that the differences between the active and inactive forms of the enzyme are due to their different adsorption capacities. Karthas (1968) X-ray diffraction studies of active RNase and enzyme inactivation by the Barnard and Stein technique show only very small conformational changes, as indicated by the pattern of electron density in 4-A. Resolution. The changes found in the inactive molecule are centered around the active site where it reacts with RNA phosphate. If the changes in electrophoretic cell mobility that we observed were the result of preferential adsorption of the active RNase

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in inactivated enzymes resulting from conformational changes, it can be argued that only the phosphate groups on the cell surface would show this level of discrimination between the two adsorbents and that they are more likely to be associated with phospholipids or RNA. Adding weight to the suggestion that non-specific RNase adsorption does not explain the reduction in net negativity on cell surfaces comes from the observation that the electrophoretic mobilities of several different cell types, including erythrocytes of three species, are demonstrably unaffected. by incubation with active RNase, although all contain phospholipids in their peripheral regions. Peripheral RNA postulated by Mayhew and I is not an adsorbed contaminant, since attempts to remove it by washing cells up to 12 times have been unsuccessful, and deliberate attempts to contaminate cells before and after RNase treatment by incubation in lysed cell suspensions did not work. show no RNA adsorption reflected in electrophoretic mobility measurements. Peripheral RNA cannot be attributed to the presence of PPLO-like organisms on the cell surface, since careful reexamination of cells obtained from suspension cultures and mouse ascites tumors by culture techniques consistently gave negative results for mycoplasma during periods of study. . In the case of Ehrlich ascites tumors, many electron micrographs failed to show the presence of virus (RNA) on the cell surface, which peripheral RNA could have explained. Recent work by Mayhew and I has shown that the net surface negativity of a variety of mammalian cells can also be reduced by incubation with ribonuclease TI, which, unlike ribonuclease A, has a net negative charge. Therefore, the effects of RT ribonuclease are not attributable to its adsorption, but are consistent with other data indicating the presence of RNA- and ribonuclease-sensitive anionic groups in the periphery of some cells. Ion binding studies (Weiss and Mayhew, 1967) have shown that calcium binding to the cell surface is reduced after incubation of cells with RNase. Our results show that calcium binds more strongly to RNase-sensitive groups than to neuraminidase-sensitive groups, and that it binds more strongly to yet unidentified acidic groups. To date, RNase-sensitive acid groups have been detected by cell electrophoresis on the surfaces of murine ascitic tumors (Ehrlich L1210 and Sarcoma 37), permanent cell lines derived from human osteogenic sarcoma, and murine mast cells, lymphocytes, and possible polymorphonuclear blood leukocytes. freshly isolated mouse thymocytes and liver cells. Additional supporting evidence for the presence of RNA within the cell periphery comes from other analytical approaches. Warren et al. (1967) has

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showed that 1-2% of the total RNA of L cells is associated with their isolated peripheral membranes; Lansing (1966) found constant small amounts of RNA associated with "clean" preparations isolated by electron microscopy from peripheral membranes of liver cells; and Burka et al. (1967) reported RNA in reticulocyte membranes. The disadvantage of all these latter techniques is that, in addition to the problem of contamination of isolated membrane samples by intracellular contents, conventional analyzes do not indicate where the RNA is located in the peripheral zone of the cell. Using electrokinetic techniques, Mayhew and I were unable to detect surface RNA in human, mouse, and chicken erythrocytes, human monocytes and platelets, mouse peritoneal macrophages, or in cultures of cells derived from two Burkitt tumors. We are currently trying to study as many different cell types as possible. An obvious and important issue regarding surface RNA concerns its type and function. While it is possible to speculate about some of the possible consequences of surface RNA (Weiss, 1968 ~ ), it must be emphasized that the validity of such speculations depends very much on its characterization, which has not yet been achieved. The crawling movements of cells over or across cellular or non-cellular substrates can only be achieved when actively moving cells make and break continuous contacts with their substrates. For theoretical reasons, it has been postulated (Weiss, 1962a) that an inevitable part of active cell movement consists of small pieces of peripheral material being torn off by the moving cell and left behind on the surface over which it is moving; Portions of the substrate on which a cell is crawling may be broken off and pulled to the surface of the crawling cell, or both processes may occur, resulting in a mutual exchange of peripheral material. If the postulated surface RNA is "informational", then its transfer from one cell to another could potentially be seen as information transfer.

D. AMINO GROUPS Bangham et al. (1958, 1962; Bangham and Pethica, 1960) were unable to detect significant changes in the electrophoretic mobilities of a variety of cells in the pH range 7-9. Since the pKa of amino groups is in the pH range 7-10, increases in net cell surface negativity are expected to become apparent as ambient pH approaches these values. Although small increases in mobility have been observed at values around pH 10, the generality of this type of experiment is questionable, as at such high pH values other surface changes besides ionization of the clusters are likely (Pulvertaft and Weiss, 1963). This opinion is supported in the case of human erythrocytes by observations that their motility was not affected by treatment with formaldehyde, acetaldehyde (Heard and Seaman,

LEONARD WEISS

1961), p-toluenesulfonyl chloride (Seaman and Heard, 1960), or dinitrofluorobenzene 2:4 (Seaman and Cook, 1965), as all these reagents are expected to react with accessible amino groups, resulting in loss of positivity. Recently, Weiss, Bello, and Cudney (1968) analyzed the electrophoretic mobilities of human and mouse erythrocytes and ascites and tumor cells cultured after treatment with freshly generated formaldehyde, 2,4,6-trinitrobenzenesulfonic acid, 2-chloro-3,5 - dinitropyridine, or 2-chloro-3,5-dinitrobenzoic acid. None of the reagents lysed red blood cells, and all three aromatic reagents were used at concentrations that were non-lethal to nucleated cells. Of the possible basic groups present in the cell periphery, Gasic et al. (1968) listed the side chain amino groups of lysine and hydroxylysine, the terminal protein amino groups of arginine, and the names of phospholipids and glycolipids. The aromatic compounds we use do not react with the guanidine groups of arginine, but formaldehyde does (Fraenkel-Conrat and Olcott, 1948). None of the reagents react with the positively charged quaternary ammonium ion of lecithin, and their reactions with other peripheral phospholipids are problematic due to the possibility of phosphate-amine interactions. In none of the four cell types examined was electrophoretic mobility consistently increased by the reagents, despite evidence of a response. This lack of loss of surface positivity was not due to detection by groups sensitive to sialic acid or trypsin, since treatment with the different reagents did not cause significant changes in mobility after incubation of cells with neuraminidase or trypsin, different from those treated differentiated cells. with the enzyme alone. From this, it was concluded that the positively charged groups associated with proteins were not present in detectable amounts on the electrokinetic surfaces of cells. It must be emphasized that these electrokinetic studies do not indicate that positively charged amino groups associated with proteins are not present in the peripheral zones of the studied cells, but rather that they are not detectable on their electrokinetic surfaces. Gasik et al. (1968) suggested in electron micrographs that positively charged clusters reacting with electron-dense and negatively charged colloidal particles are within the deep peripheral zone for surface sialic acid residues, and our own results are consistent with these conclusions.

E. OTHER PERIPHERAL IONOCENE GROUPS Considering some of the recent models for surface membranes, negatively charged lipid phosphate groups are expected to be present on the electrokinetic surfaces of cells. Bangham and Pethica (1960) who studied the concentrations of

THE PERIPHERY OF THE CELL

various cations needed to induce charge reversal. From this work, they concluded that phosphate groups were present on the electrokinetic surfaces of several different cells. It now appears that this technique is not as specific as previously thought, and that simple charge inversion spectra do not allow unambiguous identification of ionic surface groups. In the case of human erythrocytes, where the surface charge was previously thought to be largely due to ionized phosphate groups, Seaman and Cook (1965) showed that the dominant ionogenic group on the electrokinetic surface is the carboxyl noacetylneuraminic acid, with a lesser contribution. of the a-carboxyl of glutamic acid. These investigators also showed that aldehyde-fixed erythrocytes were isoelectric between pH 6 and 8 when treated with diazomethane, which esterified the acidic groups. If this drastic treatment can be assumed to leave the erythrocyte surface in a state consistent with that of normal cells, then this observation would argue against the asymmetric and nonspecific distribution of environmental ions near the cell surface, which contribute significantly to cell growth. . electrophoretic mobility. It should be noted that after treatment of a cell line with neuraminidase and RNase, the cells do not become isoelectric (Weiss and Mayhew, 1967), indicating that other groups are present on their electrokinetic surfaces. The nature of these groups remains unclear; they may well be phospholipid phosphates. Positive identification of these ionogenic species using electrophoretic techniques such as those described above will depend on the availability of highly purified enzymes that will separate them from the cell surface.

F. DYNAMIC SURFACE LOAD ASPECTS

Until now, the periphery of the cell has been discussed rather statically. Like any other organelle, this region of the cell must be in a dynamic state with respect to anabolic and catabolic processes, and any part must be considered in terms of half-life. A detailed study of the biochemical synthesis involved in maintaining a steady state of membranes is beyond the scope of this review; Indeed, investigations into this aspect of membrane physiology in metazoan cells are just beginning and are complicated by the inherent difficulties in obtaining pure membrane fractions for analysis. Changes in the cell periphery associated with viral cytodifferentiation, induction, modulation and infection have been extensively reviewed elsewhere (Weiss, 1967a) and will not be discussed here either. 1962 Eisenberg et al. tried to relate the electrophoretic mobilities of rat liver cells to their growth rate. Liver cells were isolated from their original organs after partial hepatectomy and during postnatal growth. Mobilities in the regenerative phase after hepatectomy and in the

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early neonatal period were significantly higher than in normal adult cells. These results generally agree with those of Ben-Or et al. (1960), Heard, Seaman and Simm-Reuss (1961) and Ruhenstroth-Bauer and Fuhrmann (1961) who observed that some cells derived from embryonic or regenerating tissues exhibit significantly greater mobility than their "normal" or adult counterparts. From their own studies in cultured cells, SimonReuss et al. (t964) concluded that it is impossible to generalize the effects of aging and regenerative processes on electrophoretic mobility. Later, more sophisticated experiments, to be described shortly, suggest that it may be possible to make some general statements about the effects of metabolism and other intracellular events on electrophoretic mobility, and that these predictions lend themselves to experimental testing. When examining cells isolated from solid tissues, there is always a risk that dissociation processes alter the cell periphery. This is particularly true for cells isolated from tissues by trypsinization, as trypsin can remain on electrokinetic cell surfaces for several hours and reduce net negativity due to its own net positive charge (Barnard et al., 1969). Mechanical isolation of cells from their original tissue can lead to changes in their surfaces, since the plane of separation does not coincide with the plane of the cell surface (Weiss, 1967b). If cells are separated from their original tissues without irreversible damage and cultured for comparatively short periods of time to allow for their recovery, one might ask whether an isolated cell could rebuild its peripheral regions in the same way as in the solid state. Tissue, as it must adapt to the demands of its existence as a single-celled organism, rather than being surrounded by other cells with which it can communicate (Loewenstein, 1967) and a varying amount of connective tissue. It also seems reasonable to argue that some cells adapt faster and/or better to our various culture media of choice than others, and that hypothetical recovery times for isolated cells in such media vary accordingly. Mayhew and O'Grady (1965) and Mayhew (1966) carried out a series of studies on cells in suspension culture in which parasynchrony was induced and conclusively showed that in the cell line studied the electrophoretic mobility during the mitotic phase is significantly greater. peak phase than at any other point in the mitotic cycle. Regardless of their phase in the mitotic cycle, treatment of these specific cells with neuraminidase reduced their electrophoretic mobilities to a common value, suggesting that the observed increases in liquid surface negativity observed in the mitotic phase correspond to an increase in the density of ionized carboxyl groups. to sialic acid units on the electrokinetic surface of the cell. Analytical data from Kraemer (1967) suggest that there is a constant amount of sialic acid per unit of cell volume independent of the mitotic phase. Mayhews

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The interpretations are fully in agreement with Kraemer's data, since Mayhew's electrokinetic data only measure charged debris located no more than 10 Å from the hydrodynamic slip plane, while Kraemer's measurements refer to sialic acid emanated from cells released when incubated with neuraminidase. The sialic acid fractions detected by Kraemer may be located somewhere deep in the three-dimensional peripheral zone of the cell, consistent with Seaman's concept that this region can be seen as a polyanionic sponge; and Mayhew's observation could be interpreted as suggesting that the sialic acid-dependent increase in surface charge density in the peak mitotic phase is the result of a structural rearrangement of sialic acids within the peripheral zone versus the mitotic zone. O. On the other hand, Kraemer hypothesizes that when cells are incubated with neuraminidase, this enzyme only reacts with their surface regions and does not enter the cells as suggested by Wallach and Eylar (1961), nor does it react with intracellular membranes. after entry. Nordling and Mayhew (1966) convincingly showed that neuraminidase enters cells and, upon entry, reduces the surface charge density of their nuclei; this raises the possibility that part of the sialic acid released when cells are incubated with neuraminidase originates from intracellular structures. However, it seems likely that one of the main reasons for the discrepancy between "chemical" and "electrokinetic" estimates of cell surface sialic acid lies in deficiencies in the relationship between zeta potential and surface charge density. An attempt was made to correlate electrophoretic mobility with cellular metabolic activity in a series of tumor cells growing in suspension culture by examining the effects of ambient temperature:tl on both parameters. A good correlation between mobility and oxygen consumption was observed in the temperature range from 2 °C to 600 °C, and it was speculated that there could be a causal relationship between energy-dependent conformational changes at the periphery of the cell and charge density. on its electrokinetic surface (Weiss, 1966). Intensive studies performed over 2 years (Weiss and Ratcliffe, 1968) on two types of tumor cells maintained in suspension culture and on a murine ascitic tumor confirmed that the same cells show a real and rapid increase in electrophoretic motility in showing increases in room temperature . increased from 10 to 37°C, but that the changes, although statistically significant, are small. Temperature-dependent motility changes, when they occur, are associated with increases in mean cell volume and can also be induced by exposure to hypotonic media in susceptible cells; Based on this and other evidence, it has been suggested that motility changes are the result of unspecified cell surface interactions with serum components along with expansive cell surface movements. The influence of temperature is not observed in all cells; thus, on the one hand, Merishi and Seaman (1966) failed to discover it, but on the other hand, Nordling (1967). Weiss and Ratcliffe treated the cells with a variety of anti-

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metabolites and found that severe suppression of oxygen utilization, anaerobic glycolysis and uncoupling of oxidative phosphorylation can occur in the cells studied without appreciable changes in electrophoretic mobility. There seems, therefore, to be no direct causal relationship between short-term metabolic changes of the type mentioned and cell electrophoretic mobility. Long-term studies by Mayhew and Weiss (1968) on a cell line maintained in suspension culture showed that when their growth rates are increased over periods of several days by increasing the amount of serum in the culture medium, a reversible increase occurs. in electrophoretic mobility. Studies on the effects of neuraminidase and ribonuclease on the motility of these cells strongly suggest that the surface density of RNase-sensitive clusters increases with growth rate, whereas the surface density of sialic acid residues remains relatively constant, regardless of growth rate. . When cells die or starve to death, the RNase-sensitive clumps on the cell surface are undetectable; however, RNase-sensitive pools reappear within a generation after previously starved cells receive fresh medium. Of the 13 cell types described by Mayhew and myself, only actively growing cells showed a significant reduction in electrophoretic mobility (20% or more) after incubation with RNase. The growth rate cannot always be correlated with the possible presence of RNA on the cell surface, since RNase treatment alone decreases the electrophoretic mobilities of L1210 cells by 4-9%, even when these cells grow very rapidly in vitro.

G. CHARGE DISTRIBUTION Consideration of electrophoretic mobility measurements shows that they provide, at best, an approximate index of surface charge density. Measurements do not show the arrangement of ionogenic groups on the cell surface. Experiments on the activities of penicillinase on the surfaces of B U C ~ ~ ~ Z L J . siibtilis proposed that charged group densities were greater in some surface regions than in others (Weiss, 1963a). Subsequent work on the deformability of mammalian cells showed that cells deform more easily after incubation with neuraminidase, but no change was found after treatment with ribonuclease (Weiss, 1965a, 1968a). One explanation offered for these experimental data was that the expansion of the peripheral zone of the cell in a suction micropipette was achieved by an "unfolding" process and that the partial resistance to "unfolding" was attributed to electrostatic repulsion between the ionized sialic carboxylic acids. that were present above average density over the folds. Studies of the effects of temperature and hypotonic media on electrophoretic mobility have also led to the suggestion that some of the charged groups on the electrokinetic cell surface exhibit a zonal distribution in

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Regions with above-average surface charge density (Weiss, 1966; Weiss and Ratcliffe, 1968). It has been suggested that the phenomena of contact between cells may be regulated in part by electrostatic repulsion between their surfaces (for a review, see Weiss, 19672). In many contact time-lapse cinemicrographic studies between a variety of animal cells in culture, the cells are observed to explore each other's surfaces in a way that strongly suggests some kind of spatial specificity. In a sense, spatial specificity implies structural heterogeneity. Electron microscopic evidence for such surface specialization comes from the work of Farquhar and Palade (1963), Fawcett (1965), Roth and Porter (1964), and Bowers (1954), among others. It is known from the work of Chambers and Fell (1931) and Ambrose (1961) that when cells come into contact with glass substrates, they do so across small areas of the glass/whole cell interface. The correlation of experimental observations on the contact interactions of different cells with glass surfaces and the calculated interaction forces between cells and glass strongly suggest that close contact at distances that allow the formation of bonds cannot be achieved by cells with ionic moieties. evenly distributed over their surfaces. (White, 1968b). The various evidence in favor of heterogeneity in surface charge distribution presented here is indirect; however, knowledge of the possibility of their existence may have some relevance in the biophysical analysis of surface-dependent cell contact phenomena.

V. Enzyme Activity and Cell Periphery A. SUBLETAUTOLYSIS Another aspect of cell periphery alteration is attributable to enzymes. Fell and Mellanby (1952) showed that, in the presence of excess vitamin A, cultures of cartilaginous skeletal rudiments from chicken embryonic limbs show a loss of metachromasia when stained with toluidine blue, due to loss of intercellular matrix. Thomas et al. (1960) showed a histological picture similar to that of hypervitaminosis A with papain. Other studies by Lucy et al. (1961) suggested that normal chondrocytes contain enzymes capable of degrading the cartilage matrix, producing an effect similar to vitamin A. Dingle (1961) showed that the enzymes involved were indeed lysosomal hydrolases as defined by Duve (1959). and colleagues. The observation that pretreatment of mouse dermal fibroblasts in glass cultures with excess vitamin A facilitated their release from this substrate also suggested that the released lysosomal enzymes may attack and weaken the peripheral zones of the cells (Weiss, 1962a ,B). Following the work of Bitensky and colleagues (Bitensky, 1963) on the cytotoxic activity of antibodies, Weiss and Dingle (1964) examined the

LEONARD WEISS

Effects of exposing rat liver lysosomes in suspension, rat liver slices and rat "fibroblast" cultures to an antiserum produced by injecting partially purified rat liver lysosome preparations into rabbits. Loss of acid (lysosomal) phosphatase after antiserum challenge was detected in both liver slices and cell cultures, but interestingly not in the lysosome suspension. This finding that antisera had no direct effect on isolated lysosomes, as reported later by Dumonde et al. (1765) concluded that the lysosomal activation within cells caused by the antiserum was not the result of a direct effect on the lysosomes themselves. This conclusion supported Bitensky's (1963) suggestion that antiserum affects lysosomes indirectly by increasing cell membrane permeability. Dingle et al. (1967) postulated that adsorption of antiserum with sufficient complement to the plasma membrane produces local changes that facilitate membrane fusion with primary lysosomes, and Dingle (1968) discussed such fusion in terms of emulsion stability. More direct evidence that antisera could act on cells to cause the release of enzymes capable of degrading the intercellular matrix came from citrus studies on the effects of antisera on mouse fetal bones (Fell and Weiss, 1965) and on fetal bone rudiments, embryonic limbs of chickens (Fell and Weiss, 1964). These degradative changes were inhibited by hydrocortisone. Weiss (1965b) found that detachment of cells growing on glass substrates could be facilitated by exposure to low concentrations of antisera and that this facilitation was inhibited by micrograms of hydrocortisone, which also reduced the loss of intracellular acid phosphatase. Based on this and other evidence, it has been suggested that the cell periphery may undergo continual modification by sublethal autolysis and that this may be under endocrine and physiological control. This concept of "chronic purulent lesions" at the cellular level has been discussed in terms of cellular interactions (Weiss, 196713). The reviews by Weissmann (1965) and Straus (1967) et al show that many agents can induce lysosomal activation in a wide variety of pathological and physiological circumstances. This may indicate that sublethal autolysis of the cell periphery occurs frequently and may dictate the need for a high peripheral turnover rate in many cells.

B. SUPERFICIAL PH In addition to the sublethal autolysis discussed above, the enzymatic degradation of intercellular materials by enzymes is believed to play an important role in the infiltrative and metastatic processes in malignancy (Sylvin and Malmgren, 1957). As enzymatic activity can be controlled by environmental pH, the issue of hydrogen ion concentration near the periphery of the cell is of concern. In the case of aqueous solutions of sulfonated acid dyes, they are stirred with benzene

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form an emulsion there is a color change indicating a lower pH at the benzene/water interface than in the main aqueous phase (Deutsch, 1927, 1928). Studies on the relationship between the tensions at the fatty acid/water and oil/water interfaces and the pH of the main phase also indicate the existence of differences between the main phase and the pH of the interface (Reinders, 1910; Jahrisch, 1922). ; Hartridge and Peters, 1922; Peter, 1931; Daniela, 1937). The studies showed that although the curves related to pH and fatty acid dissociation were similar in shape to those related to pH and interfacial tension, there was a change of approximately 2 pH units between the two curves, suggesting that the concentration of ions of hydrogen near the fatty acid water/water interface was 100 times greater than that in the main phase. A more mathematical approach to the question of surface pH was presented by Hartley and Roe (1940), who postulated that the concentration of hydrogen ions near a negatively charged surface is the product of the total phase concentration and the factor exp( - -eS/ kT is ) where e = electronic charge; = zeta potential; R r Boltzmann's constant and T = absolute temperature. The effective dissociation constant at the surface, K, is given by

K , = K,, exp (-ec/KT) = K , exp (-F
= pH,,,,,,

+ S/60

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may deviate from those calculated from average electrokinetic data due to the presence of above and below average charge density zones (Weiss, 1963a). Further evidence showing that surgeons in one! The substrate can affect its enzymatic degradation from experiments carried out or digestion of thin films of lecithin in a Langmuir channel by lecithinase (Bangham and Dawson, 1958; Dnwson and Bangham, 1959). These experiments do not show whether the charging effect on lecithin films is mediated directly by ApH or by the electrostatic orientation of lecithinase relative to lecithin. It is now generally accepted that many cells are surrounded by carbohydrates and that the use of the term "glycocalyx" for this region is appropriate (Bennett, 1963). This material may have antigenic properties, as discussed by Watkins (1967), and may be detected as "fluff" by electron microscopists, as discussed by Revel and Ito (1967), among others. It is often forgotten that in tissues the dividing line between the cell surface and the intercellular matrix is purely arbitrary and that it may be considered beneficial for a cell to overcome an ill-defined distance in its connective tissue domain. The volume of these domains can be large due to the water associated with them; for example, the specific hydrodynamic volume of hyaluronate is on the order of 200-500 ml/g. (Rogers, 1961) due to the "tangled sheep" configuration of the hyaluronate complex, which occupies a very large solvent domain. Rogers considered the role of hyaluronate in terms of its effect on water retention, diffusion rates and inhibition of enzyme activity due to its macroanionic properties. Weiss (1962b, L967a) extensively considered these regulatory functions of enzymes in relation to mammalian cells, where it is postulated that hyaluronates and similar polyanions might inhibit more enzymes than the "physiological" pH optimum on the one hand and on the other hand might be expected. due to the effects of ApH that optimizes the activity of enzymes that work better at a pH lower than physiological. By limiting diffusion out of the cell, hyaluronate would also tend to localize exoenzyme activity to the immediate pericellular region. Furthermore, as Rogers suggested, removal of the macroanionic matrix via a hyaluronate-hyaluronidase interaction in this region could also control pericellular enzymatic activity. Changes in electrical charge density on cell surfaces have been previously described and associated with cell growth rate and mitotic cycle. Therefore, all of these activities may also play a role in controlling enzyme activity at the periphery of the cell and in integrating this activity with other aspects of cell function.

VISA. The Periphery of Malignant Cells Many claims have been made in the past describing structures and properties unique to the periphery of malignant cells. Majority

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Some of these postulated differences between the peripheral regions of normal and malignant cells turned out to be unfounded or just differences of degree. The sheer volume of literature on the subject precludes an exhaustive review, and only some of the conclusions reached from a detailed review of the literature can be presented here (eg, Weiss, 1967a). It should be noted that approximately 273 human neoplasms are cataloged in the Illustrated Nomenclature of Tumors of the International Union Against Cancer (UIC.C.). This large number allows for great individuality in the nature of the periphery of cells representative of a particular neoplasm and cautions against uncritically extrapolating data not only from one human tumor type to another, but also data obtained from animal tumors. to the human situation. Those unfamiliar with histopathology often overlook the fact that tumors are not homogeneous structures containing only viable cells. In any malignant tumor, there is often apparent cytological and histological variation between apparently viable cells, in addition to multiple necrotic foci. This requires a careful definition of the neoplastic material under study, which is often absent in works of a biophysical nature. When studies are performed on mechanically isolated tumor cells, there is a distinct possibility that the plane of separation between cells is spatially distinct from their adhesion interface (Weiss, 1967b). While this is not expected to affect raw analytical data, it could profoundly affect actual surface parameters such as electrophoretic mobility. FOR.

INTESTINE

STRUCTURE

As summarized by Mercer (1963), many of the published electron microscopy data represent changes typical of cell death or degeneration, as opposed to changes unique to malignancy per se. Mercer believed that no unique features of the periphery of malignant cells had been observed in fine electron micrographs. As far as I know, this conclusion is still valid. In electron micrographs of rapidly proliferating neoplasms, considerable variation in the substructure of the junctions is seen, with loose connections and simplifications of peripheral structures being prominent features. However, Lane and Becker (1966) observed similar changes in the regeneration of rat livers after partial hepatectomy. Some of the changes observed in neoplasms may also be the result of sublethal autolysis, as Overton (1962) described the disappearance of desmosomes in enzymatically dissociated chicken embryonic cells. Coman and Anderson (1955) made a first attempt to study the surface contours of malignant cells using a replication technique. By comparing the cell surfaces of rabbit VX2 squamous cell carcinoma with normal squamous cells from depilated skin, Coman and Anderson found that this was the case.

LEONARD WELSS

ma1 cells showed surfaces uniformly covered with plaques about 30-60 Å in diameter. Malignant cell surfaces were much more heterogeneous and covered by plaques of 30 to 300 Å. These observations were essentially confirmed by Berwick (1959), who also reported that the cell surface contours of Shope's papillomas, which can be seen as interfaces between normal epithelial cells and those of the VX2 tumor, were homogeneous particles. However, comparisons of the surface morphology of lymphocytes from normal and leukemic mice by Nowell and Berwick (1958) did not reveal consistent differences, as both cell types were covered with irregularly distributed plaques approximately 100-300 Å in diameter. Likewise, Easty and Mercer (1960) and Catalano et al. (1960) did not find significant differences between normal cells from the hamster renal cortex and tumor cells from the same region. (1960) observed no difference in surface substructure that could be correlated with the very different j u i i i w behavior of two MCIM mouse sarcoma sublines. It will be interesting to see whether differences in surface structure between malignant cells and their normal counterparts can be revealed by the newer freeze-fracturing techniques described by Bullivant and Ames (1966), Branton (1966), and Weinstein and Bullivant (1967). among others, although current results suggest that differences attributable to malignancy per se are unlikely. With regard to possible connection differences between normal and malignant cells, it is interesting that Loewenstein and Kanno (1966) demonstrated low electrical connection resistance between microelectrodes placed on adjacent normal liver cells, but found no evidence of low resistance connections between microelectrodes. . In addition to the obvious errors in extrapolation to other tumors, these results should be treated with great caution, as the "negative" finding of high binding strength could be a result of the inherent difficulties of this technique.

SOCCER

BINDING

Beebe (1904) and Clowes and Frisbie (1905) found that the total calcium content of a limited number of malignant tumors was somewhat less than that of normal tissues. Carruthers and Suntzeff (1944) observed that, after treatment with the carcinogen methylcholanthrene, the calcium concentration in the epidermis of mice decreased during the benign hyperplasia phase. When the lesions became frankly malignant, a second drop in calcium levels occurred. Consistent with these results, Brunschwig et al. (1946a) found that human gastric carcinoma contained less calcium than the adjacent unaffected mucosa, but that in a benign papilloma located between two discrete carcinomas, the calcium level was the same as in malignant lesions. Brunschwig et al. (1946b) also found that several human colon carcinomas contained less calcium than the two adjacent ones.

THE PERIPHERY OF THE CELL

normal mucosa and benign papillomas. The most recent studies by Kalant et al. (1964) in DAB-induced hepatomas did not show a positive correlation between malignancy and low tissue calcium. Electrokinetic studies by Bangham and Pethica (1960) showed that the ambient concentration of calcium required to induce surface charge reversal in mmse cells is approximately the same for Ehrlich ascites tumors and for those derived from normal livers, and that lymphocytes and erythrocytes require even higher concentrations, indicating even lower calcium binding capacities than tumor cells. Therefore, the general assertion that tumor cells generally contain less calcium or bind this ion less tightly than normal cells is unacceptable on the basis of existing evidence. It would be very reassuring if more tissue calcium levels could be determined in intact tumors of a wide variety with modern analytical techniques. In 1900, Herbst demonstrated that the blastomeres of Echinus mirrotiiberri/latl/s could be disassembled into individual cells by treating them with calcium-free seawater. Since then, many researchers have shown that removing calcium facilitates the separation of cells from each other and from a variety of substrates. Coman (1953) suggested that the reduced "adhesion" he observed between malignant cells may be related to their reduced calcium content, and Zeidman (1947) showed that removal of calcium facilitated cell detachment. Coman therefore postulated a sequential relationship between low calcium content of tumors, facilitation of malignant cell separation, and metastasis. Calcium's role in cell maintenance has been viewed as a "bridge" connecting anionic sites in neighboring cells (Steinberg, 1958) in terms of its effects on the electrical double layers that surround cells (Weiss, 1960). desolvation (Schmitt, 1941) and in the stabilization of mucostances coacervates (Rinaldini, 19j S). All of these interactions are more complex than commonly believed, as discussed by Katchalsky (1964). Recent work on murine sarcoma-37 cells (Weiss, 1967) and cultured cells (Weiss, 1967d) suggests that calcium binds to their periphery, increasing their mechanical resistance and thus making their separation more difficult, but this has not been demonstrated. that affect their mutual relations. membership . . Thus, it is considered that calcium will bind "tangentially" within the periphery of individual cells, as opposed to "radially" where it may bind a cell to another cell or to an intercellular substance. Therefore, the current status of calcium in the periphery of tumor cells, its mode of binding and its role in periphery-dependent contact phenomena are unclear.

C. SURFACE LOAD Ambrose (1967) reviewed some of the various tests comparing the surface load of "normal" cells with their malignant counterparts. cells from

LEONARD WEISS

Stilbestrol-induced hamster kidney tumors and buttery yellow rat hepatoma had higher electrophoretic mobilities than their normal analogues. When cultured hamster fibroblasts were transformed by polymavirus, they fell into two types of colonies. One type had electrophoretic mobilities indistinguishable from the original fibroblasts, while the other cell type showed an increase in neuraminidase-sensitive motility. This latter observation was consistent with that of Defendi and Gasic (196.5) who found that polyoma-induced conversion of W.IS hamster embryonic cells was associated with the acquisition of a dense pericellular region sensitive to acid mucopolysaccharide neuraminidase, detectable by cytochemistry. Techniques Other cell surface changes after viral infection that may correlate with those mentioned above are related to erythrocyte adsorption (Marcus, 1962) and the development of specific viral antigens (Vogt and Rubin, 1962; Haughton, 1965; Pasternak, 1965 ; Tevethia et al., 1965). The question of whether or not increased cellular electrophoretic mobility correlates with malignancy, as Ambrose so often suggests, is a complex one. Fuhrmann (1965) does not consider the increase in electrophoretic motility as a nonspecific expression of cell proliferation, as there is evidence that the motility of both proliferating liver cells and cells of an ascitic hepatoma is high, but only the mobilities of malignant cells are high. reduced by incubation with neuraminidase. Thus, this suggests that malignancy is somehow associated with sialic acid-mediated increases in cell surface charge density. However, unlike Fuhrmann, Chaudhuri and Lieberman (1965) showed that the increased cell motility of regenerating rat livers actually results from increased amounts of surface sialic acids. The work cited above also requires that the cells themselves exhibit similar growth rates and mitotic indices before valid comparisons can be made between the mobilities of normal and malignant cells. If these conditions are not met, it is impossible to attribute increased mobility to malignancy per se. This last suggestion is supported by the work of Vassar (1963) and Vassar et al. strongly supported. (1967), in which a consistent difference was not demonstrated between the mobilities of cells mechanically isolated from gastrointestinal carcinomas and the adjacent normal epithelium, also of rapid proliferation. Purdom et al. (1958) made a remarkable attempt to relate the degree of malignancy, which is generally understood as the time required to kill the host, with the electrophoretic mobility of tumor cells. In the panel of mouse tumors examined, cells with increased malignancy showed a tendency to exhibit increased electrophoretic mobilities. The growth pattern of a tumor in a host depends in part on the host's response. If increased motility reflects the presence of increased amounts of sialic acid moieties on the cell surface, one wonders whether they might somehow mask antigenic or other sites in this region and thus prevent motility.

THE PERIPHERY OF THE CELL

Destruction of tumor cells by host defense mechanisms. Sanford (1967) observed that neuraminidase-treated ascites tumor cells from TA3 mice caused significantly fewer seizures in mice than untreated controls. She interpreted these findings as indicating loss of graft specificity in untreated neoplastic cells due to masking of surface isoantigens by sialomucins that were eliminated by neuraminidase treatment. However, when Hauschka and Weiss (1968) tested this interpretation in our own laboratory with similar cells and mice, it could not be confirmed, as neither untreated controls nor neuraminidase-treated cells adsorbed H-2-specific isoantibodies. Face. When the same cells were injected into six different strains of mice, their survival times ranged from 7.2 to 12.6 days. Despite these highly significant differences, it was not possible to find a correlation between electrophoretic mobilities of cells obtained before or after incubation with neuraminidase and survival time. Therefore, it appears that in this experimental system the overall host response is not governed by a first-order relationship with tumor cell surface charge density. The faster tumor cells grow in their respective hosts, the faster they can kill them. From the discussion of cell proliferation, one would expect that a faster growth rate would cause higher mobilities, rather than higher mobilities causing higher growth rates. However, our own studies of the TA3 tumor in different hosts, where there were large differences in growth rates, did not show any significant correlative changes in electrophoretic mobility. Currently, it seems premature to correlate malignancy or degree of malignancy with electrophoretic cell motility alone. This is not surprising, given the complexity of the malignant process and its dependence on host-malignant cell interactions and the isolated properties of the malignant cells themselves.

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I04

LEONARD WEISS

Smith. F.O., Bear, R.S. and Clark, G.L. (1935). Radiology 25, 131. Seaman, G.V.F., and Cook, G.M.W. (1965). In "Cell Electrophoresis" (E.J. Ambrose, ed.), p. 48. Churchill, London. Seaman, G.V.F., & Heard, D.H. (1960). J. Gen. Physiol. 44, 251. Shah. D.0. (1968). Biography. J. Summary A-189. Simon-Reuss, I., Cook, G.M.W., Seaman, G.V.F., and Heard, D.H. (1964). cancer res 24, 2038. Sjostrand, F.S. (1963a). Nature 199, 1262. Sjostrand, F.S. (1963b). J. Ultrastruct. Resolution 9, 561. Sjostrand, F.S. (1967). Protoplasm 63, 248. Smith, D.S. (1961). J. Biophys. Cytol Biochemistry. 10, 123. Steinbeg, M.S. (1958). I'm a naturalist 92, 65. Stoeckenius, W. (1962). J. Cell Bidding. 12, 2 2 1 . Stoeckenius, W. and Mahr, S.C. (1965). Laboratory. sec 14, 1196. Stoeckenius, W., Schulman, J.H. and Prince, L.M. (1960). colloid-2. 169, 170. Strauss, W. (1967). In "Enzyme Cytology" (D. 8. Roodyn, ed.), p. 219. Academic Press, New York. Sylvh, B. and Malmgren, H. (1957). With Izadiol. Scanning. Su/~pl.154. Tevethia, S.S., Katz, M. and Rapp, F. (1965). stop. pain ex. biol. No. 119, 896. Thomas, L., McCluskey, R.T., Potter, J.L. and Weissmann, G. (1960). J. Expl. yo 111, 705. Tieo, H.T. and Diana, A.L. (1968). Chemistry Phy.1. Lipids 2, 55. Vassar, P.S. (1963). Laboratory. Invest. 12, 1072. Vassar. P.S., Seaman, G.V.F. and Brooks, D.E. (1967). pro Performance. res of cancer Conj. 7, 268. Vogt, P.K. and Rubin, H. (1962). Cold Spring Harbor Symposium. Soft quantum . 27, 395. Volkin, E. and Cohn, W. 1:. (1953). J. Bid Chemie 205, 767. Wallach, D.F.H. and Eylar, E.H. (1961). Biorbim. Biography. Arya 62, 594. Wallach, D.F.H. and Kamat, V.B. (1966). jCidl Biol. 30, 660. Wallach, D.F.H., and Perez-Esandi, M.V. (1964). Biochemistry Biography. Accra 52, 594. Warren, L., Glick, MC. and Nass, M.M.K. (1967). In "The Specificity of Cell Surfaces" (B.D. Davis and L. Warren, eds.), p. 109. Prentice Hall, Englewood Cliffs, New Jersey. Watkins, W.M. (1967). In "The Specificity of Cell Surfaces" (B.D. Davis and L. Warren, eds.), p. 257. Prentice Hall, Englewood Cliffs, New Jersey. Weinstein, RS. and Bullivant, S. (1967). Blood 29. 780. White. L (1960). ex. crll res. 21, 71. Weiss, L. (1961a). ex. Cell Re.r, Suppl. 8, 141. Weiss, L. (196TB). ex. Cell resolution 25,504. 191, 1108. Weiss, L. (1961~) Natur Weiss, L. (1962a). J. The O w Delicate . 2, 236. Weiss, L. (1962b). Sor. of Biochemistry. Symptom (Cambridge, Engl.) 22, 32. Weiss, L. (1963a). J. Gen. Microbiol. 32, 331. Weiss, L. (1963b). ex. Cell 30, 509. Weiss, L. (1965-1). J, Cell Biol. 26, 735. Weiss, L. (1965b). ex. ReJ- cell. 3'1, 540. Weiss, L. (1966). J.Null. Cancer Institute 36, 837. Weiss, L. (1967a). "The Cell Periphery, Metastases, and Other Contact Phenomena." North Holland Publ., Amsterdam.

THE PERIPHERY OF THE CELL

Weiss, L. (1967b). Yroc. Desempenho. restauração res. Conf. 7, 292. Weiss, L. (1967~) J. . Cell B i d 33, 341. Weiss, L. (1967d). biol celular j. 35, 347. Weiss, L. (1968a). j Theorie Biol. 18, 9. Weiss, L. (1968b). Expile Cell Resolução 53, 603. Weiss, L. (1968~) IP”. eu v s/. S.ynip. Mo>io,qr. 8, 7.1. Sábio, L. e Coombs. R. A. (1963). ex. Cell Resolution 30, 331. Weiss, L. e Dingle, J. & T. (1964). Sou Chemical Research 23, 57. Weiss, L. e Lachmann. PJ (1964). Expression Cell Resolution 36, 86. Weiss, L. e Mayhew, E. (1966). célula j. Phy.rio1. 68, 3/15, Weiss, L. e Mayhew, E. (1967). célula j. fisicamente. 69, 281. Weiss, L. e Ratcliffe, T.M. (1968). J. Natl. Canrer Itist. 41, 957. Weiss, L., Bello, J. e Cudney, T.L. (1968). Interno. J. Câncer 3, 795. Weissmann, G. (1965). Novo Etigl. J.Med. 273, 1084, 1143. Wiersema, P.H.. Loeb, A. L. e Overbeek, J.T.G. (1966). J. Colloid Interface Sci. 22, 78. Wigglesworth, V. €3. (1947). YYOC. Roy. Sr. (Lnadoiz) B147, 185. Winkler, K.C., e Bungenberg de Jong, H.G. (19.itl). Arco. Precisar. fisiol. 25, 431, 467. Yamoto, T. (1963). j célula B i d 17, 413. Zeidman. Eu (1947). Cizncer Res. 7. 386. Zetterqvist, H. (1956). "A organização litrastructural das células absortivas colunares do intestino do rato." Godvil, Estocolmo.

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Mitochondrial DNA: physicochemical properties, replication and genetic function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Basic composition of mitochondrial DNA. . . 111. Frequencies of nearest neighbors of mitochondria IV. Differences in base composition and base sequence of complementary strands of mitochondrial DNA1. . . . . . . . V. Size and structure of mitochondrial DNA from animal tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Introduction .................... B. Characterization of mitochondria [ D N A closed duplex and its derivatives . . . . . . . . . . . . . . . . . . . . C. The number of superhelical turns in mitochondrial DNA.........._.._..l......._...,......... . D. Size and roundness of mitochondrial DNA from animal tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Mitochondrial DNA oligomers. . . . . . . . . . . . . . . . . F. The behavior of mitochondrial DNA in alkali. . . . . . g Composition of mitochondrial DNA from animal tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VISA. Size and structure of mitochondrial DNA in plants and protozoa. . . . . . . . . . . . . . , . , . . . . . . . . . . . . . . VIII. The amount of mitochondrial DNA per mitochondrion and per cell. , . . . . . . . . . . ................................VIII. Mitochondrial DNA replication. . . . . . . . . A. Timing of mitochondrial DNA synthesis in the cell replication cycle. . . . . . . . . . . . . B. Mitochondrial DNA turnover. C. The mechanism of intact mitochondrial cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Incorporation of deoxyribonucleotides into the DNA of isolated mitochondria. . . . , . . . . . . . . . . . . . . . . . . . . . . . IX. Effects of anaerobic agents, glucose repressors and mutagens on yeast mitochondrial DNA. . . . . . . . . . . . . . . . FOR . anaerobiosis. . . . . , . . . . . . . . . . . . . . . . . . . B. Glucose Suppression ....................... C. Mutagens. . . . . . . . . . . . . . . . . .. . X. Mitochondrial DNA recombination. . . . . . . . . . . . . . . . . XI. Renaturation Studies Using Mitochondrial DNA XII. Evolution of mitochondrial DNA and the relationship between mitochondrial and nuclear DNA. . . . . . . . . . . . . XIII. Genetic function of mitochondrial DNA. . . . . . . . . . . . . . . The introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . B. DNA-RNA hybridization experiments. . . . . . . . . . . . . . 107

108 109 117 117

118 118 121 128

130 133 136 137

139 143 145 145 146 149 152

154 154 155

156 163 165 167 168 168

169

108

S. BORST AND AM

XIV.

KRON

C. The product of mitochondrial protein synthesis. . . . . . D. Identification of mitochondrial proteins encoded by nuclear DNA or synthesized outside the mitochondria. E. Mitochondrial enzymes found in small cytophysical mutants of yeast. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Correlations of changes in mitochondrial proteins with changes in mitochondrial DNA. . . . . . . . . . . . . . . . . . . G. Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . Attachment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . references . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

172 171 174 176 178 179 181

I. Introduction Research over the last two years has provided increasing evidence for the concept that the biosynthesis of functional mitochondria requires the cooperation of two genetic systems: the nuclear system, involving nuclear mRNA, which is likely to be translated on extremitochondrial ribosomes, and a system mitochondrial located in the space of the mitochondrial matrix. The genetic continuity and expression of the mitochondrial system seems to be guaranteed by the presence within the inner mitochondrial membrane of the enzymes necessary for DNA and RNA synthesis and the complete machinery necessary for protein synthesis. Because several important parts of this machinery differ from their extramitochondrial counterparts (e.g., ribosomes), the mitochondrial system for macromolecule synthesis is partly unique and not simply a copy of the extramitochondrial system that resides in the space of the mitochondrial matrix for the benefit of stalled translation. is the information present in the mitochondrial DNA. The fact that two genetic systems are involved in mitochondrial biosynthesis raises obvious questions. What good is such a complicated configuration for a cell that apparently manages to solve other complex cytological problems, such as peroxisome or lysosome biosynthesis, without using a second genetic system? What is the evolutionary advantage of having DNA in the cytoplasm? Why is it necessary to equip this DNA with a unique protein synthesis system rather than the extrachondrial system? How is coordination between nuclear and mitochondrial DNA contributions achieved in the cell? The answers to these questions lie in mitochondrial DNA and, in our opinion, a complete understanding of the genetic function and evolution of mitochondrial DNA and the control of its replication and transcription will be an important step forward in understanding mitochondrial biogenesis and the possible genetic functions of DNA. mitochondrial DNA in light of our current knowledge of the physicochemical properties of mitochondrial DNA from a variety of sources. Various aspects of mitochondrial biogenesis have been extensively reviewed in

DNA mitocondrial

109

the last few years. Jinks (1964), Wilkie (1964), Gibor and Granick (1964), Granick and Gibor (1967), and Roodyn and Wilkie (1968) discussed the genetic aspects and general biological implications of cytoplasmic inheritance. The general problem of mitochondrial biosynthesis has recently been summarized by Luck (1965, 1966), Kroon (1966a), Tuppy and Wintersberger (1966), Granick and Gibor (1967), and Roodyn and Wilkie (1968) and discussed extensively in the round-table of discussion on biochemical aspects of mitochondrial biogenesis, held in Polignano in 1967 (Slater et al., 1968). As earlier work on mitochondrial DNA was also published by Swift (1965), M.M.K. Nass et al. (1965), M.M.K. Nass (1967) and Borst et al. (1967a) we limit this review to a critical and detailed discussion of recent work on the physicochemical properties, replication, and genetic function of mitochondrial DNA. 11. Basic composition of mitochondrial DNA

The basic composition of mitochondrial DNA from a variety of organisms has been studied by direct analysis, measuring the density in CsCl and determining the T, [midpoint of the DNA melting curve (see Marmur and Doty, 1962)] under standard conditions. A summary of the published results is presented in Tables 1-111. To make the results obtained in different laboratories comparable and to limit the scope of Tables 1 and 11, we recalculated all buoyancy densities on a common basis and grouped the results of different authors as explained in the footnotes of the tables. Several useful generalizations follow from these data: (1) Mitochondrial DNA is double-stranded. For most of the cases shown in Tables 1-111, this is derived from melting behavior or the characteristic density increase in CsCl after denaturation. (2) Mitochondrial DNA is homogeneous in its basic composition. This is due to the narrow unimodal bands and sharp melting curves observed in CsCl. The only exception to this generalization reported so far is the Neztrospora mitochondrial DNA (Table 11). Mitochondria from Neiiro.rporci rrussa contain equal amounts of DNA components with equilibrium densities of 1.698 and 1.702 g/cm3, while mitochondria from Neziro.rpom sitophilu contain a third major component with bands of 1.692 g/cm3. The cause of this heterogeneity is not known (3), although the fluctuating density of all investigated mitochondrial DNA ranges from 1.683 g/cm. (Tetrabymenu) and 1.716 g/cm3 (peanut), the mitochondrial DNA density of related organisms is similar in all cases. (4) There is no apparent relationship between mitochondrial and nuclear DNA densities present in the same cell. They can be almost the same (eg in rodents) or the mitochondrial DNA density can be up to 21 mg/cm3 less.

TABLE I DENSITY IN CsCI. T-. AND BASIC COMPOSITION OF MITOCHONDRIAL AND NUCLEAR DNA

VON

ANIMAL QUESTIONS

T,, em SSC Dichte in CsCl (gm./cm.3)a

mitochondrial nucleus

Manirnals DNA Male (Leukemic Leukocytes) Male (Liver Cells) Male

BOI

Sheep Mouse Mouse (L v e r) Camundongo (cells L) Giiinri Pork Coelho Birrls Frango Duck Pombo

Amphibian frog (R a m pi pi e m) Toad (Xenoprrs laeuis) Carp Echinoderm Sea urchin

1.705 1.688

1,702 1,703 1,701 1,701 1,698 1,702 1,703

ADN 1.695 1.699 I .700 1.704 1.703 1.703

("C.) b Mythos-

Nuclcar Chonsatellite Trial ADN Nuclear ADN ADN

bunny composition

direct analysis

(5)GK)c

Density4

Von

TMC

METRO

norte

METRO

norte

Referencescsb'

43 44

3 4,5~5

87 85,6

5 44 14 44

1.701

1.703

81,7

1. OTHERS 1,701

1,708 1,711 1,707

1.701 1.700 1.700

1.702

1.701

1.702 1.702

1.703

1-69?

1.704

1.694

7 8,5,6,9,10 7,6,11.5.6,12

TO PERCEIVE

-I1

14-5

90,0

42 41

16,7.17,18

43 45

43 43

19,18,20,21

87.3 years

86,8

52 48

19,18,10,21

All densities were recalculated using Sueoka's formula (1961) and a reference density of E. coli DNA 1 1,710 Gin./cm.3. Tm = midpoint of the melting curve (see Marmur and Doty, 1962); SSC = 0.15 M NaCl and 0.015 M sodium citrate (pH 7.0); the T given for the nuclear DAC is the T of the main component of the nuclear DNA. % GC = molar percentage of guanine-cytosine. 0.098 ( G C ) 1.660 gm./cm.-3, where p = buoyancy density in gm.:cm.-:'b. calculated as described Calculated using the formula p in footnote a, y (GC) = mole fraction of guanine plus cytosine. This formula was empirically derived!). by Schjldkraut et al. (1962) for DNAs containing only the four standard bases. Calculated using the formula T m = 69.3”C. 0.41 (GC). empirically derived from Marmur and Doty (1962). f The values presented in the table are declared or recalculated. the first specified reference. It is believed that the data presented in the other cited references do not differ significantly from the values presented here. K q 10 re!er.enr.c.r; (1) Clayton and Vinograd (1967); (2) Koch and Stokstad (1967); (3) corneal allergy. (1967); (4) Crown (1966b); (5) Corneo was one]. (1966); (6) Sinciair (1966); (7) crown and? Alabama. (1966); (8) Schneider and Kuff (1965): (9) Suyama and Bonner (1966): (10) S. Nass (1967); (11) Borst and Ruttenberg (196 ha); (12) Sinclair and Stevens (1966); ( 1 3 ) Hello. hf. K. Nass (1968); ( 1 4 ) Borst and Ruttenberg (1961%): ( 1 5 ) Borst el a!. (1967a); (16) Borst et al. (1967b); (17) Rabinowitz et al. (1965): (18) Dawid and Wolströnholme (1968a); (19) Dawid (1965), (20) Daaid (1966); (21) Dawid and Wolstenholme (1967); ( 2 2 ) Van Bruggen ef a / . (1968): (23) Piko et al! . (1967). 0 satellite detected in entire Dh'A cell; unknown intracellular location. For

DENSITY IN CSCI.

T,,

AND BASIC COMPOSITION 01'-~

TABLE 11 MITOSTIC AND NUCLEAR DNA

T and SSC ("C.) Dtnsity

111

CsCl ( g m . / cm . ~ ) ) "

DNA mitocondrial

DNA nuclear

nuclear satellite DNA

Mitochnn-

FKOM ~ ~ K l C I ! l LiI.AR .I ORGAhtSMS

Base Composition (% GC)c Direct

From to was dense

(Video) Introduction to Cells: The Grand Cell Tour

To analyze

teste de DNA nuclear M h

METRO

norte

Von

norte

1.6g+

0.7 years 3

1.702

1.692+ 1.698+

.713?

1702

1.686

0,700

1.706"

-633"

1.686 1.685

0,692

1.686

.mz

,685

Ich1

26 25 26

0 - p See footnotes LO Table I. 1 Values shown in the table are the data or derived from the hrsr references provided. It is believed that the data presented in the other cited references do not differ significantly from the values presented here. Keys to rejection: (1) fdelman et al. (1966); (2) Edelman et al. (1965); (3) Ray and Hanawalt (1965); (4) Tewari ei d. (1966); (5) Corneo et al. (1966); (6) Moustacchi and Williamson (1966): (7) Mounolou?l al. (1966); (8) Carnevali et al. (1966); (9) Borstet al. (1067a); (10) Hollenberg, Ruttenberg and Borst, unpublished observations; (11) Rich and Fortune (1966); (1 2) b.vnns (1966), (1 3) Suyama and Preer (1965); (14) Suyaina (1966): (15) Sinclair (1966). The satellite's nuclear location has not been proven. The density difference observed between this satellite and the nuclear DNA varies in different congenital between d mg./crn.3 (Cnrneo et mi., 1966) and II rng.icm.3 (Carnevali et al., 1966). h The values given by the authors of rhr are 1702 and 1689 grn./cm.3. However, in a later article, Suyama (1966) mentions this due to a calibration error. Trahywend DNA densities reported by Suyama and Preer (196j) were very low at 4 mg/cm 3 . We assume the same ilolrls for the deutty vf Purume~~jrrmDNA reported by Suvama and Preer in the same paper.

norte

114

BOOK PAGE . RUBS YES. M. CROWN

TABLE 111

CSCl DENSITY OF MITOCHONDRIAL AND NUCLEAR DNA

VON

Higher Plants

Density in CsCl (gm./cm.3) Nuclear DNA Calendula (Tagetes patula) Tobacco (Nicotiuna tubacrum) Sweet potato (Ipomoea b a t a t a ) Mung bean (Phaseolus aureus) Turnip (Bra.r.rica rapu) Onion (Allium cepa) Spinach ( Spinarea olerureu) Spinach (Spinucea olerarea) Beetroot (Bela vulguri.r) Chard (Beta vulgaris var. cicla) Lettuce ( L t u l - a sativu ) Fava bean ( Viciu fuhu ) Pea ( Lathyrus 0doi.alu.i ) Peanuts ( Ararhis hypoxaeus)

Cloroplasteno-DNA

1.692 1.696 I .692 1.691 1.692 1.688 1.695 1.692 1.605

1.702 1.706 -_

1.705? 1.6515 1.705

1,690 1,692 1,692 1,692 1,705

1.700 1.695 1 . 6 ~ 1.605

_. _. _.

Mitochondrial DNA 1,707? 1.71 n? 1,706 1,706 1,706

Analysis method references"

I.719? 1.705 1.719?

A A 3€ B B B A C A

3 3 3 3 4,5 6 4

1.705? 1.705 1.70) 1.705 1.716

D C C C E

7, s6 e 6 9

1.706 (1.718)

1 1.2

a The nuclear DNA of higher plants can contain up to 6% methyl-C. The replacement of methyl-C by C reduces DNA density (Kirk, 1967). 1, (A) Reference E. c-oli D NA = 1,710 gm./cm.3; Density calculated using the Sueoka formula (Sueoka, 1961). (B) Like (A). In a later article, Suyama (1966) mentions that, due to a calibration error, the previously reported density for Tetrahytnena D NA was wrong at 4 mg/cm3. As this Tetrahymena DNA was also used as a second reference DNA in plant DNA studies, it is likely that the Suyama and Bonner densities given in this table should also be increased by 4 mg/cnl.3. (C) Methods not provided. ( D ) As ( A ) cabin density calculated according to Vinograd and Hearst (1962). [In contrast to Sinclair (1966) in the same laboratory.] (E). Reference Pseudomonas aerugnosa D N A N15; Density calculated using the Sueoka formula (Sueoku, 1961). C Key/o References: (1) Green and Gordon (1967); (2) Green and Gordon (1966); (3) Suyama and Bonner (1966); (4) Chu et al. (1963); (5) E. Englert, quoted in Green and Gordon (1967); (6) Wells and Birnstiel (1967); (7) Fast (1965); (8) Kislev et al. (1965); ( 9 ) Breidenbarh-t-ul. (1967).

(N.dophiku) or up to 16 mg./cm.3 greater (Englend gmciilis) than nuclear DNA. (5) Direct analysis of the base composition of mitochondrial DNA from yeast, rat, mouse and chicken tissues showed approximate molar equivalence of A to T and G to C. No unusual bases were detected at the 1% sensitivity level. however, the presence of minor amounts of methylated bases has not been investigated. Absence of significant amounts of unusual bases in the mitochondrial DNA of sheep, frogs, toads, sea urchins and tetruhymenu

DNA mitocondrial

115

this is suggested by the base composition agreement calculated from the density of these DNAs in CsCl and their T in 0.15 M sodium chloride, 0.015 M sodium citrate (Tables I and 11). It is unclear why the Tm of yeast mitochondrial DNA is much lower than expected from the base composition of this DNA (Table 11). Early work on yeast mitochondrial L-lactate dehydrogenase (cytochrome b) suggested that this enzyme contained specific low molecular weight DNA. Recently, however, Burgoyne and Symons (1966) have convincingly shown that the association of DNA with this enzyme is the result of non-specific binding of small fragments of DNA to the enzyme produced in autolysing yeast. Whether the high proportion of 5-methylcytosine in the nuclear DNA of some plants is also present in the mitochondrial DNA of these plants is unclear. Although the fluctuating densities determined in different laboratories for mitochondrial DNA matched reasonably well in most cases, discrepancies were observed in four cases. Kalf and GrPce (1966) reported for the first time that the density of mitochondrial DNA from sheep hearts was identical to that of nuclear satellite DNA from sheep tissue (1.714 g/cm3). Krone et al. (1966) subsequently showed that the purified, closed, circular double fraction of sheep heart mitochondrial DNA formed bands of 1.703 g/cm3 without any material at 1.714 g/cm3, while the 1.714 DNA was present only as a trace contaminant in crude preparations of sheep heart mitochondria was DNA. Based on these results, it is likely that the value of 1.714 for sheep mitochondrial DNA is the result of experimental error and therefore we omit it from Table I. We also omit the value reported in a brief note by Parsons and Dickson (1965) . for mitochondrial DNA from Tetrdymetza pyifofarmis, Syngen 6 (1.671 gm./cm.:i versus 1.685 gm./cm.3 found by Suyama). It seems likely that in this case a carbohydrate spike (see Counts and Flamm, 1966) was mistaken for mitochondrial DNA in the density gradient. The situation is less clear for mitochondrial DNA from human and plant tissues. Clayton and Vinograd (1967) reported a density of 1.705 g/cm3 for mitochondrial DNA 1 of leukocytes from three different patients with leukemia. DNA was isolated on the basis of its restricted uptake of ethidium bromide in a preparative CsCl gradient; therefore, it consisted exclusively of closed circular duplex DNA and, as only circles of 5p or multiples of 5 were present in electron micrographs of this DNA fraction, the conclusion that it is of leukocyte mitochondrial origin seems well founded. Likewise the density of 1,705 gm./cm.?. this DNA agrees well with the density found for mitochondrial DNA1 from other mammals. In contrast to these results, Koch and Stokstad (1967) isolated DNA with a density of 1.688 g/cm3 from the mitochondrial fraction.

116

PAG BORSTY D A. M Krone

of cultured human Chang liver cells. A smaller satellite component of this density, accounting for less than 1% of total cellular DNA and of unknown subcellular location, had previously been reported by Corneo et al. detected in human bone marrow cells. (1967). This makes it highly unlikely that the DNA could have come from contaminating bacteria or viruses. Furthermore, recent work by Koch et al (private communication) showed that the isolated DNA fraction contained circular DNA with an outline length of approximately 5.3 p. Most of this DNA formed bands at the position of the closed circular duplex DNA on CsCl gradients containing ethidium bromide. It is difficult to reconcile these results with those of Clayton and Vinograd. If the banding of DNA at 1.688 g m . /~m. ~ is indeed the mitochondrial DNA of Chang's liver cells, it must have undergone a radical change in the base composition of the liver cells from which these cells originated, assuming that the normal equilibrium density of human mitochondrial DNA is 1.705 g m. /~m. ~A change in density of 17 mg/cm3 may result from the presence of a fixed amount of covalently bound protein or carbohydrate, replacement of all C by methylLC, or a drastic decrease in GC content. Alterations in GC content have been observed in small yeast cytoplasmic mutants (see Section IX), but in these cases functional mitochondria are not formed, whereas mitochondrial energy production responses have been shown to be normal in all tumors examined (see reviews by Borst, 1961; Wener, 1967). It will be of interest to examine the basic composition of mitochondrial DNA from normal human tissues. There is considerable controversy regarding the CsCl density of mitochondrial DNA from higher plants, as shown in Table III, which contains chloroplast DNA data. In this case, due to lack of data, no attempt has been made to recalculate the densities on a common basis, but where possible the method used to determine the buoyancy density has been provided. The main difficulty is to distinguish between chloroplast and nuclear DNA. According to Wells and Birnstiel, the chloroplast DNA of spinach, lettuce, broad beans and vetches has a buoyant density of only 3 nig. / ~ m .greater ~ than nuclear DNA density, while mitochondrial DNA 1A bands at 1.705 g/cm 3 indicate that the 1A bands reported by Chun et al. and Shipp et al. (1965) was indeed mitochondrial. They support this proposal with renaturation studies of their chloroplast DNA, showing that it renaturates much faster than nuclear DNA but slower than mitochondrial DNA from animal tissues. The only other study of extranuclear plant DNA that characterized the DNA by renaturation analysis was done by Tewari and Wildman (1766). In this case, no quantitative renaturation studies were performed, so it is not possible to decide from their results whether they examined mitochondrial or chloroplast DNA. Furthermore, it is noteworthy that they found a difference in density between nuclear DNA and chloroplast in tobacco of only 5 µg. /~m. ~this is closer to 3 mg./cm.3

DNA mitocondrial

117

found by Wells and Birnstiel for other plants greater than differences of 10 and 13 mg/cm." found by Green and Gordon and Shipp the ul, respectively. Wells suggested that the "nuclear DNA contamination observed by others in their purified fraction of chloroplast" actually was mostly chloroplast DNA, while the enriched satellite bands were the result of enrichment of mitochondria in the chloroplast fraction. clean separation of subcellular components using marker enzymes to assess the level of cross-contamination and quantitative renaturation studies to characterize isolated DNA. Recently, Whitfeld and Spencer (1968) also showed that chloroplast and nuclear DNA densities are identical in tobacco ( 1.697 g/cm3) and very similar in spinach (1.696 and 1.694 g/cm3), which confirms the conclusions of Wells and Birnstiel. 111. Nearest neighbor frequencies of mitochondrial DNA

The nearest neighbor frequencies ending in G were found by Cummins et al. (1967) for mitochondrial and nuclear DNA from the slimy fungus Physurmz polycephalrLm using RNA copies of these DNAs made in v h or with RNA polymerase. While the nuclear DNA contained the low CpG content characteristic of the nuclear DNA of all eukaryotic organisms (Swartz et al., 1962), the mitochondrial CpG content was not significantly different from chance. As bacterial DNA is also characterized by a CpG content equal to or greater than expected for a random sequence of bases (Swartz et al., 1962), the authors conclude that their observations add to the list of similarities between mitochondria and bacteria. However, it remains to be demonstrated that RNA polymerase makes complete copies of both strands of mitochondrial DNA. It will be interesting to determine the nearest neighbor frequencies of other mitochondrial DNA to see if a difference between nuclear and mitochondrial DNA is also found in animals where the difference in base composition between the two is much less than 15% GC has been found. In P. polycephalus.

IV. Differences in base composition and base sequence of complementary strands of mitochondrial DNA DNA from different bacteriophages forms two bands in neutral CsCl after denaturation. Marmur and Cordes (1963) showed that this is due to an uneven distribution of purines and pyrimidines in the complementary chains, the heavy chain being relatively rich in pyrimidines. Band cleavage in neutral CsCl was not observed for any mitochondrial DNA after denaturation. As-

118

S. BORST Y A. M. KRONE

However, Dawid and Wolstenholme (1967) reported that frog mitochondrial DNA separates into two bands on CsCl-alkaline equilibrium gradients differing in density by 13 mg/cm3. Smit and Borst (unpublished observations) found a much greater difference (31-33 mg/cm3) for rat liver mitochondrial DNA in alkaline CsC1. It seems very likely that the two bands represent complementary strands of mitochondrial DNA. This point will be further explored in our laboratory, as the preliminary separation of complementary strands of mitochondrial DNA will be of interest both for experiments on mitochondrial transcription and for analyzing the evolution of mitochondrial DNA1. Recently, Ruttenberg and Borst (1968) tried to separate the complementary strands of chicken liver mitochondrial DNA in CsCl by complexing them with polyribonucleotides according to the technique developed by Szybalski (Kubinski et al., 1966; Hradecna and Szybalski, 1967) . In the presence of poly-U, the buoyant density of CsCl-denatured mitochondrial DNA increased from 1.723 to 1.751 g/cm:'; in the presence of poly-IG with different molecular weights, the density increase varied between 8 and 13 mg/cm³. Strand separation was not achieved with any of the ribopolynucleotides.

V. Size and structure of mitochondrial DNA1 from animal tissues A. INTRODUCTION In early 1966, Borst and Ruttenberg (1966a) and Van Bruggen et al. (1966) reported that mitochondrial DNA from chicken liver, mouse liver, and beef heart consists of a homogeneous population of circular molecules with a mean contour length of 5.45 p, which corresponds to a molecular weight of 10- 11 x 106 daltons (sodium salt). . . Circular DNA was independently converted to mouse liver mitochondrial DNA by Sinclair and Stevens (1966) and to L cell mitochondrial DNA by M.M.U. Wet (1966). Van Bruggen and others observed two main types of circular molecules. (1966) on electron micrographs of purified chicken liver mitochondrial DNA: open (or semi-open) circles as shown in Fig. 1A and severely oblique circles as shown in Fig. IB, the first pelletizing with a J~, ,= , ~ 27 S (component I), the latter with a .r2,,,,,, = 39 S (component I) , as shown in Fig. 2. The authors suggested that the twisted circles represent the shape of a closed circular duplex of mitochondrial DNA, in Fig. 1. A. Electron micrograph of an open circle of chicken liver mitochondrial DNA elongated according to Kleinschmidt's protein monolayer technique (see Van Bruggen et al., 1968). B. Electron micrograph of a circle of twisted chicken liver mitochondrial DNA (see Van Bruggen et al., 1968). The marks indicate 0.2p.

DNA mitocondrial

119

120

BOOK PAGE . BORST Y A.M. KROON

both chains are covalently continuous, while the open circles represent molecules with one or more nicks, in analogy to the situation previously observed in DNA polyomas (Vinograd et al., 1965). Over the past two years, the findings of Van Bruggen et al. (1966) were confirmed and extended in several laboratories, mainly by Sinclair and coTop

Meniscus

Sob

COWARDLY. 2. Sedimentation of mitochondrial DNA bands1 by neutral CsCl in the analytical ultracentrifuge. Above: Densitometer tracing of a UV absorption photograph of chicken liver DNA 3 2 min after reaching maximum speed (from Borst et al., 1967) Below: . Densitometer tracing of a UV absorption photograph of rat liver DNA 28 min after reaching maximum speed (unpublished experiment by E. M. Smit).

workers (Sinclair and Stevens, 1966; Sinclair, 1966; Sinclair et al., 1967a.b; Swift et al., 1968b); Borst et al. (Kroon et al., 1966; Borst et al., 1(67a,b,c; Borst et al., 1968; Ruttenberg et al., 1968; Van Bruggen et al., 1968), Dawid et al. (Wolstenholme and Dawid, 1967; Dawid and Wolstenholme, 1967, 1968a,b) and Vinograd et al. (Radloff et al., 1967; Hudson and Vinograd, 1967; Clayton and Vinograd, 1967; Piko et al., 1967). The current state of the research field can be summarized in three points: (1) The DNA of the mitochondria1 of all examined animals consists of ring-shaped molecules of the same size. (2) Most of this DNA is present in situ as closed circular duplex DNA. After extraction, this DNA is obtained in a compact, twisted form containing right-handed superhelical turns. In the case of chicken and rat liver mitochondrial DNA, the number of turns per unit length is approximately the same as in polyoma DNA and replicative form DNA of phage XI74.

DNA mitocondrial

I21

In addition, two other classes of molecules are found in DNA isolated from mitochondrial preparations, open circles and mitochondrial DNA multimers. All available evidence indicates that most open loops arise from closed loops during mitochondrial isolation and DNA purification. The proportion of multimers is low in all normal cells tested, but in tumor cells up to 50% of the total mitochondrial DNA may be present as multimers. (3) The DNA size of a variety of animals is remarkably constant, ranging from 5 to 5.5 p. Evidence for these points is discussed below.

B. CHARACTERIZATION OF CLOSED CIRCULAR MITOCHONDRIAL DUPLEX DNA AND ITS DERIVATIVES The characteristic properties common to all circular duplex DNAs are best illustrated by a numerical example. Imagine a closed circular duplex containing 110 base pairs. In the A configuration, with about 11 base pairs around the Watson-Crick helix, the molecule contains 10 complete turns of one strand around the other. A configuration change to the B configuration with 10 base pairs per turn of the helix requires the formation of an additional complete turn of one strand around the other. This can only be achieved in a closed circular duplex by introducing a right-hand superhelical twist to the molecule as a whole. By definition, a superhelical turn results in the twisting of a Watson-Crick helix turn, and when such a molecule adsorbs to a protein monolayer, it contains, in principle (see below), a type-eight compound. Premature DNA denaturation results in an increase in the number of base pairs around the Watson-Crick helix and thus unwrapping the superhelix turns to the right until the circle is fully open. As denaturation progresses, super helical curves to the left are introduced. The molecule with superhelical turns has a higher free energy than an identical molecule without superhelical turns. As a result, any process leading to unwinding of superhelical turns occurs more easily in the twisted circular duplex than in a linear DNA molecule of the same base sequence; the reverse is true for any process that leads to the introduction of superhelical twists. A single wire break on one of the threads of a closed duplex loop is sufficient to release any super-helical twist that may be present, as a “twist” is created in which the two ends freely rotate towards each other and towards each other. thread , it is possible. This brief introduction to the properties of closed circular DNA duplexes can serve as a basis for the experimental results discussed in this section. A detailed discussion of this problem can be found in the articles by Vinograd and Lebowitz (1966), Vinograd et al. (196S), Bauer and Vinograd (1968), Wang et al. (1967) and Wang (1969). All closed circular duplex DNAs synthesized in the intact cell contain superhelical right turns when tested in vitro. This results in a series of

122

S. BORST AND AM. Crown

very distinct properties listed in Table IV. Several of these properties have been investigated for mitochondrial DNA from chicken liver, rat liver, frog eggs, horseflies and sea urchins, and malignant cells of human origin. The results obtained are summarized in Table IV and discussed briefly. (1) "Twisted" circular molecules were observed in electron micrographs of mitochondrial DNA from all animal tissues listed in Table VI. Unfortunately, the comparatively large circles of mitochondrial DNA also show varying degrees of entanglement during open circle expansion. Therefore, the presence of closed duplex molecules in mitochondrial DNA can only be determined by electron microscopy if rigorous criteria are developed to distinguish between tightly twisted JBZJZI molecules and open entangled molecules, and if these criteria are verified in samples of open and open circular molecules. clean closed circles. D N A duplex isolated by preparative band sedimentation. So far, this has only been reported by Borst et al. ( 1 9 6 7 ~ ) and ~ showed that, with their classification of molecules in electron micrographs, only 4% of the molecules in an open circular sample of duplex DNA were classified as twisted, whereas more than 80% of the molecules in a closed sample purified sample. D N Circular Duplex A sample was found crooked. Recently, Ruttenberg et al. (1968) showed that the distinction between closed and open circular DNA by electron microscopy can be greatly simplified by spreading DNA samples in a low concentration of salt containing a high concentration of ethidium bromide. Under these conditions, the configuration of the closed molecules is so characteristic that they cannot be confused with open and interconnected circles (see below). (2) As shown in Fig. 2, chicken and rat liver mitochondrial DNA consists of different proportions of two homogeneous components sedimented at 39 S (Component I) and 27 S (Component II). Similar components have been identified in mitochondrial DNA from duck liver (Kroon et al., 1966), sheep heart (Kroon et al., 1966), frog eggs (Dawid and Wolstenholme, 1967), sea urchin eggs (Piko et al., 1967) and human leukemic leukocytes (Clayton and Vinograd, 1967). Pancreatic deoxyribonuclease treatment of mitochondrial DNA from chicken liver (Borst et al., 1967b), rat liver (Smit and Borst, unpublished observations), or amphibian eggs (Dawid and Wolstenholme, 1967) leads to conversion of component I in component II. in the latter case, the conversion was shown to follow one-stroke kinetics, consistent with the concept that a single chain break is sufficient to convert component I to component II. (3-6) Limited denaturation of a closed circular D N A duplex results in the correct unfolding of the superhelical turns and a concomitant drop in the sedimentation coefficient to that of the open circle. As the degree of denaturation increases, the sedimentation coefficient increases again, probably because further unfolding of the Watson-Crick helix introduces a left-handed superhelix.

TABLE IV GENERAL PROPERTIES OF CLOSED CIRCULAR DUPLEX OBSERVED FOR INTACT MITOCHONDRIAL DNA

VON

DIFFERENT SOURCES

DNA mitocondrial-Quelle1

(1) Twisted circular molecules present in the electron

micrographs ( 2 ) 3 9 4 DNA converted to 27-S DNA by one or more single-stranded cleavages ( 3 ) immersion in rhc sed rate melting curve ( 4 ) increased T,a ( 5 ) increased pH for alkaline transition ( 6 ) High sedimentation coefficient in yarn separation solvents (7) High buoyancy density in alkaline CsCl (8) Titration with ethidium bromide results in a characteristic sedimentation rate curve (9) Reduced ability to bind intercalating dyes, like ethidium bromide

chicken liver

rat liver

+ + +

frog or frog egg

sea urchin eggs

HeLa cells

human leukemic leukocytes

+ +

-T

= of

0 use

-k

+ +

Relating to linear DNA of the same base composition and molecular weight.

CH)

124

S. BORSTAND A. M. CROON

it becomes the molecule as a whole. This sequence of events has been shown for component I of mitochondrial DNA by examining its sedimentation coefficient as a function of pH or heating temperature in the presence of formaldehyde. As shown in Fig. 3, the mitotic sedimentation coefficient

COWARDLY. 3. Sedimentation coefficients of mitochondrial DNA from frog eggs (X. laevjs) as a function of pH. The graph shows the uncorrected sedimentation coefficients determined by the analytical band of sedimentation in CsCl with a density of 1.33. Open Circles, Component I; filled circles, component I1 (from Dawid and Wolstenholme, 1967).

Component I of chondria DNA decreases between pH 11.5 and 12; above pH 12 it increases to a value of 87 S for alkaline supercoiling at pH 13. Similar forms of alkaline supercoil from chicken liver and rat mitochondrial DNA were observed in our laboratory by Smit. The effect of heating chicken liver mitochondrial DNA in the presence of formaldehyde on the sedimentation coefficient of I and I1 is shown in FIG. About 50°C. the sedimentation coefficient of I decreases, indicating the evolution of right-handed superhelical turns. At higher heating temperatures, the sedimentation coefficient increases to a maximum of 8 3 s. As shown in Fig. 4, component I1 is heated to 60°C. or higher in the presence of formaldehyde I leads to the appearance of two components with sedimentation coefficients of 32 and 28 S, tentatively identified as single-stranded ring and broken single-stranded ring (Borst et al., 1967). In principle, they are also expected from the sedimentation of the I1 component in alkali, but only one band was found in the mitochondrial DNA of frog eggs (Dawid and Wolstenholme, 1967). Figures 3 and 4 illustrate two other characteristics shared by mitochondrial closed circular DNA with other duplex closed circular DNAs: pH

DNA mitocondrial

125

The pH required for complete denaturation is more than 0.5 pH units higher than for the "clipped" open circle (Fig. 3). The experiment shown in Fig. 4 shows that the T of component I is much larger than the T of component 11.

COWARDLY. 4. Sedimentation coefficients of mitochondrial DNA from chicken liver heated in the presence of formaldehyde at different temperatures for minutes. Solid line: component I; dashed line: component II (modified from Borst et al., 1967~). 10

(7) A high density of alkaline CsCl at equilibrium was observed for polyoma DNA and the replicative form of the 0 x 1 7 4 phage (see Vinograd and Lebowitz, 1966). Attempts to duplicate this result with mitochondrial DNA have failed because all mitochondrial DNA samples isolated to date were too unstable in alkali to survive a 20 h CsCl run at pH 13 without strand breakage. (8-9) The intercalation of ethidium bromide between base pairs of duplex DNA leads to helix winding, the degree of winding being a function of the amount of ethidium bromide intercalated (Radloff et al., 1967); Crawford and Waring, 1967; Bauer and Vinograd, 1968). Therefore, the sedimentation coefficient of component I decreases in the presence of low concentrations of ethidium bromide. At higher concentrations it increases again because the increasing degree of unwinding probably leads to the insertion of left superhelical turns. That this is the case for the mitochondrial component I of chicken liver is the case

126

S. BORST Y D A. M. CROWN

demonstrated by the experiments illustrated in FIG. Similar results were obtained with rat liver mitochondrial DNA. At very high concentrations of ethidium bromide, less ethidium bromide can be intercalated into component I than into component II or linear DNA (Radloff et al., 1967; Bauer and Vinograd, 1968). Since the buoyant density of the ethidium bromide-DNA complex is in CsCl

301

.MI

EU \

DNA mitocondrial1

EU-

005

0 10

0 15

0 20

j025

Ethidium Br present per nucleotide (Molehnole)

COWARDLY. 5 . Sedimentation coefficients of chicken liver mitochondrial DNA and polyoma DNA as a function of ethidium bromide concentration. Open Circles, Component I; triangles, component 11; closed circles, only one component detectable (from Ruttenberg et al., 1968; data for polyoma DNA was obtained from Crawford and Waring, 1967).

lower than that of DNA alone, the buoyancy density of component I of SV 40 DNA in CsCl in the presence of saturated concentrations of ethidium bromide is nearly 50 mg/cm³ greater than that of component I1 (Bauer and Vinograd, 1968). A similar difference in density under these conditions was observed for mitochondrial DNA from HeLa cells (Radloff et al., 1967; Hudson and Vinograd, 1967), human leukocytes (Clayton and Vinograd, 1967), and chicken and rat liver (Borst and Vinograd , 1967). Smit, unpublished results). In view of these results, there is no doubt that components I and I1 represent the double closed and open circular forms of mitochondrial DNA. The fact that slightly denaturing component I lowers its sedimentation coefficient to that of component II can only be rationalized by assuming that component I contains

DNA mitocondrial

127

Right-handed superhelical twists in solution. This conclusion is supported by the observation that in component I chicken liver all turns analyzable in stereo electron micrographs are right-handed (Van Bruggen, unpublished observations, 1967). Super helical curves result in a compact structure that settles 40% faster than open circle of the same molecular weight. Conversion of a circular duplex molecule into a linear duplex of the same molecular weight leads to a 10-15% decrease in the sedimentation coefficient (see Vinograd and Lebowitz, 1966). Treatment of mitochondrial DNA from frog eggs with deoxyribonuclease II, the enzyme that cleaves both strands of duplex DNA at the same point, resulted in the formation of the expected component with an sz0 of 24 S (Dawid and Wolstenholme, 1967). A similar component was found by Borst et al. detected in mitochondrial DNA from "aged" chicken liver. (1 9 6 7 ~). It seems likely that this component represents the linear form of mitochondrial DNA. The relationships between the different forms of chicken liver mitochondrial DNA discussed in this section are shown schematically in Figure 6, adjusted for denatured DNA.

native DNA

1.39

27S

24 S

COWARDLY. 6 Schematic representation of the different forms of modified mitochondrial DNA from a similar diagram for polyoma DNA by Vinograd et al. (1965). Denatured forms are those observed after complete denaturation by heating in the presence of formaldehyde.

Similar scheme for DNA polyomas from Vinograd et al. (1965). Reported sedimentation coefficients for mitochondrial DNA from chicken and rat liver, amphibian eggs, and human leukemic leukocytes are shown in Table V. The agreement is excellent, supporting the idea that there are no differences in structure or molecular weight between these three mitochondrial DNAs. . the relative

128

P. BORSTY D A. M Krone

The sedimentation coefficients of the different forms of mitochondrial DNA and circular viral DNA are very similar (Borst et al., 1967 ~ Dawid; and Wolstenholme, 1967; Clayton and Vinograd, 1967), with the possible exception of Sl neu + ral/ SII ratio ,lr,,+ml, which is 1.4 for mitochondrial DNA and approximately 1.3 for most circular viral DNAs. From our point of view, it is not possible to assess whether this difference is significant or not based on the available data. TABLE V SEDIMENTATION COEFFICIENTS OF DIFFERENT FORMS OF MITOCHONDRIAL DNA Chicken liver DNA source*

rat liver

amphibian eggs

human leukocytes"

I Neutral (No salt)

11 Neutral (N for Salt) 111 Neutral (N for Salt)

39 27

37 27

39 27 24

Components

I alkaline (Cs salt) I formaldehyde (N salt) I1 alkaline (N salt) 11 formaldehyde (N salt) h G

32.28

From Borst al. ( 1 9 6 7 ~ ) . Srnit and Borst (unpublished results). By David and Wolstenholme (1967). From Clayton and Vinograd (1967).

Using Studier's (1965) formula relating rzo,d to the molecular weight of linear duplex DNA, a molecular weight of 10.9 x 100 daltons can be calculated for the sodium salt of the 24-S form of mitochondrial DNA. The mean length of the chicken liver outline and mitochondrial DNA of Xenoptds 1uevi.r is 5.35 and 5.40 p, respectively. Using a value of 1.96 x 10 6 daltons per micron of sodium DNA (Thomas, 1966), these boundary lengths correspond to molecular weights of 10.5 x 10 6 and 10.6 x 10 6 daltons. Therefore, the molecular weights calculated from the boundary lengths and the sedimentation analysis are in excellent agreement.

C. THE NUMBER OF SUPERHELIC TURNS IN THE MITOCHONDRIA. DNA

The number of superhelical turns in the closed circular duplex form of mitochondrial DNA was examined both by electron microscopy and a dye intercalation technique. Van Bruggen et al., 1968 (see also Borst et al., 1968) counted the number of crossovers in electron micrographs of the closed circular duplex form of chicken liver mitochondrial DNA. For purified component I, the DNA extends at 20°C. in 0.1 M ammonium acetate they obtained a value of 33 - + 7 (DP) crosslinks per molecule; by the component I got rid of the myth-

DNA mitocondrial

129

In chondria lysed by osmotic shock at 2°C, the number of crossovers was 35 t 6 , which corresponds to an average of 6.4 crossovers per micron of DNA. For the purified component I of the replicative form of @X174 analyzed in 0.1 µM ammonium acetate, they counted 13 2 2 junctions or 7.6 junctions per micron of DNA. This suggests that the number of superhelical turns per unit length of mitochondrial DNA and OX DNA is approximately the same. These results were extended by analyzing the sedimentation behavior of chicken liver mitochondrial DNA as a function of ethidium bromide concentration. As shown in Fig. 5, the unwinding of the DNA helix induced by ethidium bromide intercalation initially leads to a loss of supercoiling and a concomitant decrease in the sedimentation coefficient from component 1 to component 11. At higher concentrations of ethidium bromide, the o Sedimentation coefficient of component I increases again, probably because increased helix unwinding leads to the insertion of left superhelical turns. It is clear from figure 5 that the titration curves of chicken liver mitochondrial DNA and polyoma DNA, which were determined under identical conditions, do not differ significantly. Since the binding constant of ethidium bromide to DNA is not affected by base composition or DNA length (Waring, 1965), this result shows that complete unwinding of the right-handed superhelical turns of mitochondrial DNA and polyoma requires the intercalation of the same amount of ethidium bromide per nucleotide. Therefore, the number of superhelical turns per unit length of D N A must be the same. It is possible to calculate the number of superhelical turns per DNA molecule from the results presented in Figure 5 using certain reasonable assumptions (Crawford and Waring, 1967). For chicken liver mitochondrial DNA, a value of 40 turns per molecule or 7.5 turns per micrometer of DNA has been found at 20°C (Ruttenberg et al., 1968). This agrees perfectly with the estimate of 6.4 turns per micron made by counting crosses in electron micrographs. Using the dye intercalation technique, Smit and Ruttenberg (unpublished observations) recently found that mouse liver mitochondrial DNA1 contains approximately the same number of superhelix turns per unit length as chicken liver DNA. Two explanations have been proposed to explain the right-handed superhelical twists found in all closed circular DNA duplexes synthesized in the intact cell. (1) When DNA is synthesized, strand-end closure of the newly synthesized strand occurs before complete folding of the two strands of DNA into the Watson-Crick structure is complete. This explanation predicts that there are superhelical twists when? saw that the number of superhelical turns per molecule is constant and that the number of turns per unit length of DNA is therefore greater for small circles than for large circles (Vinograd et al., 1965).

130

S. BORST Y D A. M. CROWN

Our finding that two DNAs that differ in molecular weight by a factor of 3 have the same number of superhelix turns per unit length of DNA strongly suggests that this explanation for the origin of superhelix turns in closed circular duplex DNA is incorrect. (2) The average twist per base pair of the DNA helix is less in the intact cell than in the solvents used to physically characterize the extracted DNA. The intracellular condition that leads to the "coiled up" state of DNA in the intact cell is not known. However, a recent demonstration that the pitch of the DNA helix depends on the temperature and ionic strength of the solvent (Wang et al., 1967; Wang, 1969; Bode and McHattie, 1968) suggests that ionic conditions may be responsible. . . This explanation implies that the superhelical turns found in vitro do not exist in vivo and that the actual number of turns found in vitro depends on the chosen analysis conditions. If this is true, it is remarkable that the intracellular conditions under which DNA is synthesized are so similar in vertebrate mitochondria, mammalian nuclei (polyoma), and Escherirhia coli (0 x 1 7 4 replicative form) that the pitch of the helix is the same in all three cases.

SIZE D

MITOCHONDRIAL DNA CIRCUIT OF ANIMAL TISSUES

VON

The size and roundness of mitochondrial DNA from a variety of animals were examined by electron microscopy and the results obtained are presented in Table VI. Circular DNA has been found in all animal mitochondria analyzed to date, and no difference in contour length has been observed for mitochondrial DNA from mouse liver, brain, kidney, and pancreas (Sinclair et al., 1967b). Most notable, however, is the uniformity in the size of mitochondrial DNA from branches as diverse on the evolutionary tree as mammals, sea urchins, and insects. Even the small size differences reported in Table VI may not be significant: different versions of Kleinschmidt's protein monolayer technique are used in different laboratories; The analyzed DNA samples usually contained large amounts of contaminating material; and results obtained for the same DNA in different laboratories (and even at different times in the same laboratory, see Borst et al., 1968) can produce different contour lengths, as shown in Table VI. To determine if there are actual differences in contour length, highly purified DNA samples must be used and the presence of two size classes in composite preparations from the two animals must be demonstrated. Although such experiments show that the mitochondrial DNA contour lengths are not exactly the same in human tumor cells, chicken liver, carp liver, sea urchin eggs, and housefly flight muscle, the fact that they are so similar requires explanation. . Three possibilities can be considered:

131

DNA mitocondrial

CONTOUR

TABLE V AND CIRCULATION LENGTH OF ANIMAL MITOCHONDRIA ~

Species Chordata Mammalia male

Ox Sheep Rat

Technikb

K K K-F K K-F K

guinea pig baby bird

Echinodermata Echinoidea Sea urchins (L. pirtccs) Arthtopoda Insecta Fly (M.domestira)

4,81 5,3" 5,1

5.6

5,35 5,55 5,1 5,26

K K-F K-F

references

Radloff and a/. (1967) Croon et al.!. (1966) Sinclair et al. (1967b) Croon et al. (1966) Sinslairet al. (1967b) Van Bruggen et al. (1968) Van Brüggen et al. (1968) The Croonet. ( 1966 ) Sinclair and Stevens ( 1966 ) M. M. K. Nass ( 1966 ) M. M. K. Nass ( 1966 ) Sinclair et al. (1967b)

K-F K-F K

5.1

Brush yes. ( 1 9 6 7 ~ ) Van Bruggen auf a/. (1968) A/de Sinclair. (1967b) Sinslairet al. (1967b) Krone der nf. (1966)

K-F K-F

5,56 5,40

Wolstenholme and Dawid (1967) Wolstenholme and Dawid (1967)

5,4 5,4

Van Bruggen e Outros (1968) Van Bruggen e Outros (1968)

4,45

Piko and Others (1967)

5.2

Van Bruggen e

0s Duck Amphibious Toad ( R. pipiens ) Toad ( X. laevir ) Carp Osteichthyes

(with)

5,4 5,1 4,9 5,4 5,1c 4,96 4,74 5,24

0s Maus

medium scope

A/.

(1968)

De Van Bruggen et al., 1968. K, standard protein monolayer technique, purified DNA; K-F, like K, but with 0.5% formaldehyde present (Freifelder and Kleinschmidt, 1965); OS, mitochondria lysed by osmotic shock as described by Van Bruggen et al. (1968). c Values based on less than 10 measurements. 0

132

P. RORST Y D A . M.K.R.O.N

(I) Mitochondrial DNA 1 is highly resistant to any form of mutagenesis that results in changes in DNA length. Constant length is therefore not the result of a rigid selection system, but the result of a lack of change. This explanation is not very attractive, as mitochondrial DNA is not completely immune to mutagenic events. Table I shows that the basic composition of mitochondrial DNA is far from constant, while the results discussed in Section V, E show that mitochondria contain the equipment to form and clean up mitochondrial DNA multimers. (2) The constant size of the mitochondrial genome reflects the constancy of basic mitochondrial structure and functions. According to this hypothesis, mitochondrial DNA specifies a series of proteins involved in oxidative phosphorylation. Because these proteins can look similar in all animal mitochondria, a similar stretch of DNA is needed to specify them. The difficulty with this hypothesis is that at least one component of the respiratory chain, cytochrome c, is specified by nuclear DNA (see Section X11, D). It is likely that the structural genes for some of the Krebs cycle enzymes (eg, aconite hydratase), which are required to transport reducing equivalents into the respiratory chain, are also located in the nuclear DNA. Therefore, it is clear that there is no apparent reason in this hypothesis why animal mitochondrial DNA cannot be greater than 5p and take control of some of the nuclear genes that specify mitochondrial enzymes. (3) If we assume that mitochondria are descended from bacterial symbionts, a possibility favored by most researchers in this field (cf. Lehninger, 1964; M.M.K. Nass, 1967; Granick and Gibor, 1967; Roodyn and Wilkie, 1968). It can be postulated that, in the course of evolution, most bacterial genes disappeared and their role was taken over by nuclear genes. This genomic reduction process could have stalled at the 5-year stage for a number of reasons. To have an evolutionary advantage, mitochondrial DNA must meet two requirements: first, it must specify certain gene products that are essential for mitochondrial biosynthesis, and the fact that the relevant genes are present in many separate copies in the cytoplasm must have evolutionary significance. . advantage of role playing. Second, cytoplasmic and nuclear gene expression must be tightly coordinated, and therefore a specific number of mitochondrial genes must play a regulatory role. It is possible that the minimum number of D N A that satisfy both requirements is 5p. An alternative possibility is that mitochondrial DNA specifies a set of proteins that function as a unit and that cannot be encoded by less than 5 p of DNA. Obvious candidates for such a unit are mitochondrial ribosomes1 and proteins tightly bound to the inner mitochondrial membrane. If the synthesis of such a unit is regulated by a large operon, it is conceivable (but not necessary) that the chances of obtaining an appropriate and coordinated replacement for the individual genes that specify this unit through mutagenic events in the nucleus are practically nil. . FOR

DNA mitocondrial

133

The third possibility is that mitochondrial DNA encodes a set of gene products that are required within or within the mitochondrial membrane and that cannot be transferred across mitochondrial membranes. Again, mitochondrial ribosomes or inner membrane proteins could be such gene products. It is clear that, according to the latter hypothesis, the 5-year-old DNA of animal mitochondria represents a minimum, an evolutionary bottleneck on the way to a complete loss of mitochondrial DNA and the transfer of its informational content to the nucleus. Identifying the genetic function of mitochondrial DNA will show which of these explanations for the constant size of animal mitochondrial DNA is the correct one. From the considerations in this section, it is clear that there is no reason to expect that all mitochondrial DNA is circular in nature and 5p in length. The evolutionary advantage of circular DNA does not appear to be critical, as many viruses survive on linear DNA, and mitochondrial DNA from as-yet-unstudied organisms can do the same. Similar considerations apply to the size of mitochondrial DNA. Even though the general evolutionary trend is towards smaller mitochondrial DNA, occasionally higher organisms at evolutionary dead ends may grow larger mitochondrial DNA, whereas the mitochondrial DNA1 of primitive organisms may have retained more genes than the mitochondrial DNA of their putative ancestor bacterian. In summary, it is not possible to extrapolate from the data presented in Table VI until more is known about the function of mitochondrial DNA.

E. MITOCHONDRIAL DNA-OLIGOMER

Vinograd and co-workers (Radloff et al., 1967; Hudson and Vinograd, 1967; Clayton and Vinograd, 1967) discovered two types of mitochondrial DNA oligomers in the DNA of malignant cells: circular oligomers, that is, rings with an outline length of a multiple of 5p, and linked oligomers, i. H. Circular DNA molecules composed of independent double-stranded circles that are topologically intertwined or linked like links in a chain. Most oligomers found were dimers, but smaller amounts of higher oligomers chained together to form a septum were also observed. The ratio of total mitochondrial DNA and the ratio of strand to circular oligomers varied depending on the DNA source. Mitochondrial DNA from HeLa cells contained approximately 10% concatenated dimers (by weight) and no circular dimers. Leukocyte mitochondrial DNA from three different cases of human leukemia contained the following oligomer weight fractions: Case I, 39% circular dimers, 5% concatenated dimers, and 4.5% larger oligomers; case 2, 7% circular dimers, 10% chain dimers and 6% larger oligomers; Case 3, 3.5% circular dimers, 5 7 0

134

PAGE BORST AND A.

M. KRON

chained dimers and oligomers 0.7% larger. These percentages are based on DNA analysis on the closed circular double band collected from preparative CsCl gradients containing ethidium bromide. As reported by Vinograd et al. performed, these values are likely to be minimum values. The large fraction of oligomers obtained from malignant cells allowed us to analyze in detail the nature of oligomers. Circular dimers were found to have the same buoyant density as monomers, while their length and settling properties in neutral and alkali salt are consistent with a closed circular duplex structure with exactly twice the cut-off length of the monomer. Vinograd et al hypothesize that the circular dimer contains the base sequence of two identical monomers joined in sequence. Mild shear degradation of the circular dimers, followed by denaturation and renaturation at low concentrations, should therefore lead to the formation of 5-11 rings as the main circular product. This experiment has not yet been reported. The nature of the bound molecules has been analyzed in two ways (Hudson and Vinograd, 1967; Clayton and Vinograd, 1967). The concatenated dimers were added to a CsCl gradient containing a high concentration of ethidium bromide in a band with a density exactly between the closed circular duplex and open duplex DNA bands. This proves that these dimers consist of a closed circle (which absorbs less ethidium bromide) and an open circle (which absorbs more ethidium bromide) of approximately equal size and are linked by stable bonds in 6 M CsCl. The same boundary length of molecules bonded in catenanes was confirmed by length measurements in electron micrographs, and the appearance of the binding site was consistent with the assumption that the molecules are linked by a topological bond (similar to the double strand of monomers). . . in chain-separating solvents) and not by chemical bonding. To rule out the presence of chemical bonds, one must show that the bound molecules are completely dissociated by introducing a double-stranded break in one of the circles. This experiment has not yet been carried out. Mitochondrial DNA oligomers are also present in non-malignant cells, although in much lower concentrations. Oligomers (3%) were found in closed circular duplex DNA of normal human leukocyte mitochondria, while an unspecified fraction of sea urchin mitochondrial DNA consisted of concatenated dimers and a small fraction of higher concatenated oligomers (Clayton and Vinograd, 1967). We also observed a very faint middle band in several preparations of chicken and rat liver mitochondrial DNA centrifuged to equilibrate on a CsCl gradient containing ethidium bromide (Borst and Van Bruggen, unpublished observations, 1967). finds chained monomers and dimers with the same frequency, we calculate that the proportion of mitochondrial DNA present as chained dimers is about 1% in chicken liver and even less in rat liver. analysis

MITOCON1)ADN RIAL

135

the fraction corresponding to the middle band in chicken liver preparations was shown to contain DNA in an appreciable fraction of dimers consisting of a 5p open circle and a twisted molecule. Furthermore, the fractions corresponding to the closed band contained about 2% chain dimers and less than 0.5% open dimers. At these very low levels of multimers, it is often difficult to distinguish circular dimers and chain dimers from randomly overlapping molecules, and therefore these percentages are only approximate. Circular DNA oligomers have recently been observed for the replicative form of the 0x1 7 4 phage (Van Bruggen, Jansz and Pouwels, unpublished observations, 1967; Rush et al., 1967; Rush and Warner, 1967) and for a bacterium. plasmid (Roth and Helinski, 1967). They can be formed during DNA replication or during monomer recombination; the last mechanism is proposed by Vinograd et al. As shown in Fig. 7, a symmetrical

COWARDLY. 7. The recombination model for the formation of mitochondrial multimers1. Circular molecules first pair (a) and then "break" once (b) or twice (c), as shown. Once broken, the assembly results in an open dimer (d) that can be cleaved again (e) and recombined. Half of the products of the second recombination are catenas (f), while the other half are separate circles. When interrupted twice (c), half of the recombinations result in separate circuits (i) while the other half are catenas (h) (from Hudson and Vinograd, 1967).

one crosslink results in a circle of double length, while a second crosslink can result in a crosslinked dimer or two unconnected monomers. Multiple crossover events can lead to multimers of different lengths and different degrees of concatenation. This symmetric recombination model organizes the different types of mitochondrial DNA species into a sequence that ranges from monomers to circular dimers to concatenated dimers and higher oligomers. Vinograd et al consider the mitochondrial DNA population as an equilibrium population and the different possible distributions of mitochondrial species as different positions in a manifold equilibrium. The factors that affect the position of this equilibrium are unknown and there is no explanation for the large differences in equilibrium positions in different tissue samples. If the first recombination event is relatively unlikely compared to subsequent recombination events between the original molecules linked as circular dimer or chained dimer, it would be expected that the multimers would eliminate each other and the number of multimers could be kept small. The finding of 39% circular dimers versus 5% linked dimers in a case of

136

PAG BORSTY D A. M Krone

Leukemia does not agree with this hypothesis. Furthermore, one would expect that further recombination events involving the two circles of a tethered dimer would result in tethered dimers in which the monomers intertwine in the interlocking position. This was not observed. Therefore, the possibility that normal mitochondria have additional mechanisms to remove multimers must be left open. It is not yet clear whether the high proportion of oligomers in the mitochondrial DNA of malignant cells is due to the rapid proliferation of mitochondria or to an imbalance in the recombination system of dedifferentiated cells. Since any simple symmetric recombination mechanism leads to the formation of circular dimers from circular monomers, Wilkie's discovery that mitochondrial DNA recombination occurs in yeast (Section X) supports Vinograd's proposal that DNA oligomers mitochondrial arise by recombination. However, if circular DNA replication involves a double-strand break, as predicted in various models of DNA replication (see Lark, 1966; Yoshikawa, L967), oligomers can also arise during DNA replication and there is no experimental evidence direct to support this. Moment that speaks against this possibility. F. THE BEHAVIOR OF MITOCHONDRIAL DNA

ALKALI

Borstet al. (1967) reported that chicken and rat liver mitochondrial DNA is rapidly degraded in alkali to a collection of heterogeneous fragments. Recent experiments carried out by Smit in our laboratory have shown that this is a consequence of the isolation process. If DNA is extracted and purified at 0-4°C instead of room temperature, most of it is stable in an alkaline medium. Although the cause of alkali lability is unknown, it seems likely that radicals present in low concentrations during DNA preparation can occasionally lead to the release of bases at room temperature without breaking phosphodiester bonds (cf. Bode, 1967; Rhaese and Freese, 1968). After exposure to alkali, the strand breaks where the bases are (Tamm et al., 1953) an alkaline supercoil with a half-life measured in hours. The phenomenon observed in the mitochondrial DNA of frog eggs (Dawid and Wolstenholme, 1967), sea urchin eggs (Vinograd, private communication, 1967) and chicken and mouse livers (Smit, unpublished observations) led to unsuccessful attempts to determine the equilibrium density of component I in alkaline CsCl. It seems likely that the slow degradation to alkali results from the same factors underlying the rapid degradation to alkali. We expect that more stringent DNA purification will bring alkaline stability to the same level as polyoma DNA (see Weil and Vinograd, 1963). Recently, Pouwels and Jansz and their collaborators (Pouwels et al., 1966, 1968;

137

DNA mitocondrial

Jansz et al., 1968) showed that the denatured alkaline 0 × 1 7 4 DNA component I in the replicative form normally does not renature after neutralization, even under conditions where the denatured component I1 completely renatures. However, after cutting the denatured I component, it immediately renatures to the native component 11. The authors propose that the inability of the native denatured I component to renature under these conditions is the result of two effects. (1) When all component I hydrogen bonds are broken, the complementary strands are shifted out of register; after neutralization, non-specific hydrogen bonds between the strands are immediately formed, blocking the movement of the two strands relative to each other in the direction of the helix axis. (2) In denatured DNA, the movement of the two separate strands is severely restricted because the original Watson-Crick turns are still preserved in the denatured molecule, probably partly as dextro-twists and partly as levo-twists. provided super-helical twists. These two effects combined prevent the formation of nuclei, which is necessary for successful renaturation. A single tape break is enough to lift the topological constraint, and renaturation follows immediately. This interpretation of the results obtained with OX-DNA implies that alkaline denaturation is pseudo-irreversible for all closed circular duplex DNAs. In this context, the results of Dawid and Wolstenholme (1967) are unexpected. They denatured mitochondrial DNA from frog eggs with 0.1 N NaOH at 0 °C. and found that after neutralization, a fraction of the DNA equal to the amount of the component present still had the equilibrium density of native DNA in CsCl. Smit (unpublished observations) recently re-examined this point in our laboratory. After denaturation of rat liver mitochondrial DNA with alkali and subsequent neutralization, the entire alkali-stable component I was converted into a new component labeled with a J. ) ~ , , 70-75 S, the expected sedimentation coefficient for the neutral denatured form of component I based on the results of Pouwels et al. (1968) with DNA gX. N or A DNA sedimented in the position of the native I component. Therefore, it seems likely that the "reversible denaturation" observed for frog egg mitochondrial DNA must have been the result of incomplete denaturation. In summary, it now appears that the behavior of mitochondrial DNA in alkali may not differ significantly from that of other circular DNAs. G. MITOCHONDRIAL DNA COMPOSITION

VON

ANIMAL QUESTIONS

Figure 2 shows the size distribution in analytical experiments of band sedimentation of DNA extracted from chicken and rat liver mitochondria. Typically, there are three components. (1) Closed circular duplex form of mitochondrial DNA settling with a sedimentation coefficient of 39S.

138

S. BORST Y D A . M. KRON

(2) The double open circular form of mitochondrial DNA in 27S.

(3) Decantation of low molecular weight heterogeneous material with sedimentation coefficients less than 27S. In addition, two minor components are present that do not appear in band sedimentation studies. (4) Mitochondrial DNA oligomers represent only about 1-30% of all high molecular weight DNA from normal tissues studied to date, but in tumor cells up to 507% of all DNA may be present as oligomers (4) . see VJ section). (5) Since continuous mitochondrial DNA synthesis probably occurs in animal cells (see Section VIII), replicating molecules are expected to reside in the extracted DNA. Among the thousands of mitochondrial DNA molecules from various animal sources that have been studied in different laboratories, no convincing examples of branched molecules have been discovered. Possible reasons for this failure are given by Borst et al. ( 1 9 6 7 ~ ) . Furthermore, intermediate stages are said to be present in the formation of oligomers, but these were also not detected.

It is likely that most of the heterogeneous material at the top of the gradient in the upper lane of Figure 2 represents nuclear DNA debris or RNA degradation products that were not removed by the isolation procedure. The amount of this material varies from preparation to preparation, while in mitochondrial DNA from amphibian eggs, where nuclear contamination is not a problem, low molecular weight DNA was practically not observed in density gradients (Dawid et al. Wolstenholme, 1967 ) and 99% of all molecules in electron micrographs were circular (Wolstenholme and Dawid, 1967). The relative proportions of components I and II can vary significantly in different preparations of chicken liver (Borst et al., 1967c), rat liver (Smit and Borst, unpublished observations), frog and toad eggs (Dawid and Wolstenholme, 1967), sheep heart and duck liver (Kroon et al., 1966). Several by Van Bruggen et al. (1968) indicate that most of the component I1 found originates from component I, either during DNA purification or during mitochondrial isolation by an activation of mitochondrial endonuclease1, described by Curtis and Smellie (1966) and Curtis et al. . (1966). We conclude that in the most extensively studied mitochondria, i. H. Mitochondria from chicken liver, rat liver and amphibian egg, closed circular duplex DNA with a cutoff length of 5–5.5 p represents the major component present in mitochondria in siti. Minor components include mitochondrial DNA oligomers, replication intermediates, and oligomer formation intermediates. So far, only the first few have been discovered. Although these conclusions

DNA mitocondrial

139

ments may apply to animal mitochondria in general, there are no a priori reasons to rule out that exceptions may occur. It must also be emphasized that the presence of minor components other than those mentioned above cannot be rigorously ruled out, even in extensively analyzed mitochondrial DNA. Mitochondrial DNA recovery during extraction and purification normally does not exceed 50%. The loss of a minor component that is specifically removed by the isolation methods used and that does not appear in electron micrographs of mitochondrial preparations lysed by osmotic shock (see Van Bruggen et al., 1968) would therefore go unnoticed. However, there is no positive evidence that this occurs.

VISA. Size and Roundness of Mitochondrial DNA1 from Plants and Unicellular Organisms There is no conclusive evidence for the roundness and size of mitochondrial DNA from any of the organisms represented in these categories. Suyama (1966) reported that Tetrdhymelza deposits mitochondrial DNA with a sedimentation coefficient of 40S. Since the same sedimentation constant is obtained when precautions are taken to avoid shear degradation, Suyama concluded that this 40-S component is non-degraded mitochondrial DNA. . While it is tempting to infer from Suyama's results that the mitochondrial DNA of Tetvihymenais is also a closed circular 5p duplex, Sinclair's (1966) attempts to gain support for this conclusion were unsuccessful. In electron micrographs of mitochondrial DNA from Tetrihyvzenu, he only occasionally observed circles with contour lengths from 1 to 13 p.m, with an accumulation of molecules of sizes 3.0, 4.3, 5.8, 8.4, and 13 p.m. However, these experiments are inconclusive because only about one-third of the DNA in Sinclair's mitochondrial DNA samples is renatured under conditions where mitochondrial DNA from animal tissue or yeast is fully renatured. Thus, the possibility remains that the circles observed by Sinclair originated from contaminating nuclei, while the true 5p mitochondrial circles were absent because they had been degraded. Of course, it is also possible that tetrahyncium mitochondrial DNA consists of linear molecules that sediment at 40S. E. gvucih mitochondrial DNA has been discussed at length by Ray and Hanawalt (1964, 1965). DNA was prepared by preparative CsCl gradient centrifugation of whole cell DNA and a molecular weight of 3-4 x 10° was calculated from its sedimentation rate on sucrose gradients, assuming the DNA was linear. The possibility that the DNA was degraded during purification was ruled out by inconclusive control experiments. However, for DNA extraction, euglen cells were first extracted twice with 937;

140

P. RORST Y D A . M. KRON

ethanol, before lysis by sodium dodecyl sulfate. It cannot be excluded that ethanol treatment activates mitochondrial nucleases that selectively degrade mitochondrial DNA without degrading nuclear and chloroplast DNA. Sinclair (1966) occasionally observed circles with a contour length of 3.5 p in the total cellular DNA of E. gracilis. This is twice the maximum length calculated from the sedimentation coefficient for Euglerzu mitochondrial DNA found by Ray and Hanawalt. Furthermore, there is no evidence that the circles observed by Sinclair originate in the mitochondria. Extensive efforts were expended in several laboratories to determine the size and structure of intact yeast mitochondrial DNA. The main results obtained with mitochondrial DNA isolated from yeast are summarized in Table VII. The results of the first four groups are essentially the same. Linear DNA was found only in mitochondrial DNA preparations uncontaminated with nuclear DNA. Sinclaire and others. (1967a) concluded that these linear molecules could not have arisen from breaking the circles during isolation and extraction and suggested that the 5-6 µl linear molecules could represent intact yeast mitochondrial DNA. However, the marked heterogeneity of the obtained sticks makes this interpretation unattractive, and both Slonimski (in the discussion of Swift et al., 1968b) and Borst et al. (Borst et al., 1968; Van Bruggen et al., Ul., 1968) concluded that linear molecules represented much larger (and possibly circular) DNA degradation products. This is not unreasonable, since yeast mitochondria contain approximately 10 times more DNA per milligram of mitochondrial protein than mammalian liver mitochondria (Schatz et al., 1964b). If we assume that the number of mitochondria per milligram of protein and the number of DNA molecules per mitochondrion are the same for purified yeast and liver mitochondria, yeast mitochondrial DNA can be as long as 50 p. The accuracy of this assumption is supported by the following calculation. A diploid yeast cell contains about 5 x 10-14 g. DNA (Ogur et al., 1952); up to 20% of these may be mitochondria (Moustacchi and Williamson, 1966) and the number of mitochondria per yeast cell is about 50 (Avers et al., 1965). Therefore, one yeast mitochondrion contains about 2 x 10-le g. or 120 x 108 daltons of DNA. To further investigate the length of undegraded yeast DNA, we subjected yeast mitochondria to the osmotic shock method of MMK Nass (1966), in which DNA shear degradation is minimized. Most of the DNA was released in flower-like structures containing at least 50p of DNA. However, the DNA in these flowers was not continuous and the longest observed DNA fragment was 18 IL (Borst et al., 1968; Van Bruggen et al., 1968). Since mitochondrial endonuclease may have introduced breaks during mitochondrial purification, Hollenberg recently investigated the possible presence of closed circular duplex mitochondrial DNA in protoplast lysates in our laboratory.

SIZE AND STRUCTURE OF

TABLE VII MITOCHONDRIAL DNA

YEAST ISOLATE ~

Refrentr

Average

.( -0.W

Trwari su al. (1966)

3-3

Sinclair

Alabama. (1967a)

Borst el a/. (1968) Slonimski (see Swift t em.. 196Sb) Avers (1967) a

about 25

< 5 of 5 of 8 -

maximum

5,5 9a

> 20 11

Calculated Mole Weight (Dalton)

x 1 x >2 x 2

107? de7

107

>4x107

Variable

Longer niolcrules (up to 18 p) were observed in mtDNA cleaved by osmotic shock (see text).

Circular

DNA Hektotrogenität I

++ ++ +++

Gift -

me me

8 5

;

142

S. BORST AND AM. Crown

prior purification of the mitochondria. Protoplasts were lysed with dodecylsarcosinate and after addition of solid CsCl and excess ethidium bromide, the lysed DNA was centrifuged to equilibrium. Under these conditions, all mitochondrial DNA joined together in the position expected for linear DNA with a buoyant density of 1.684 g/cni.s in the absence of ethidium bromide. No closed circular duplex DNA band was detected. Although these experiments seem to indicate that yeast mitochondrial DNA is non-circular, this conclusion is still unwarranted for two reasons. (1) It remains to be shown that the procedure for isolating DNA from protoplast lysates excludes strand breaks. (2) Fast-growing yeast generation time is 1 to 2 hours. If yeast mitochondrial DNA consists of 50 p circles replicating at a rate of 1 p per minute, similar to the nuclear DNA of HeLa cells (Cairns, 1966), Viciu fuba (Taylor, 1968) or Chinese hamsters (Huberman and Riggs, 1968), most of the mitochondrial DNA may be replicating DNA in a log-phase culture. Since circular DNA replication requires at least one cut to introduce a vortex (Cairns, 1963), all replicating DNA in ethidium bromide-CsCl gradients is found in the band containing open circles and linear DNA. We are currently investigating mitochondrial DNA from stationary yeast cultures. Because the results obtained in four different laboratories with yeast are so consistent, Avers' (1967) reported that yeast mitochondrial DNA consists of circular DNA molecules whose boundary length varies between 0.5 and 10 p was surprising. In purified yeast mitochondrial DNA, most molecules measured were circular and almost all circles were smaller than 3p. As it is impossible to convert circles smaller than 3p into linear molecules larger than 5p by the method used, the Avers results are incompatible with the other results presented in Table VII. Therefore, we can point out that none of the circular molecules chosen by Avers (1967) to illustrate his work would have been classified as circular in this laboratory. However, even if the circularity of these molecules could be demonstrated by more convincing electron micrographs, the possibility remains that they are identical to the heterogeneous circular DNA previously reported by Sinclair et al. (1967a). Sinclaire and others. (1967a) showed that these circles have the CsCl density of yeast nuclear DNA and are believed to originate from nuclei that contaminate the mitochondrial preparation. Given these considerations, it is clear that the question of the size and structure of yeast mitochondrial DNA remains an open one. Luck and Reich (1964) observed heterogeneous linear DNA up to 7 p in length in electron micrographs of mitochondrial DNA. The interpretation of these results encounters the same difficulties as the other cases discussed in this section. Only a limited number of experiments were conducted to determine the size

DNA mitocondrial

143

and structure of mitochondrial DNA from higher plants. Swift and others. observed heterogeneous linear DNA up to 7 p in length. (1768b) in electron micrographs of mitochondrial DNA from the hypocotyl of common bean (Phuseolus udgaris). More recently, Van Bruggen, Borst, and Talen (unpublished results) analyzed DNA from the spadix mitochondria of the voodoo lily (Saaromutum vemJnm) lysed by osmotic shock (cf. Van Bruggen et al., 1768). Only linear strands of DNA were released, ranging in length from <1 LL to 35 p. Most molecules were larger than 10p and the frequency distribution did not show preferred lengths. Taken together, the results discussed in this section suggest that DNA from Plmts and lower organisms may be larger than 5p and linear.

VIII The amount of mitochondrial DNA1 per mitochondrion and per cell Considerable effort has been expended in different laboratories to determine the amount of mitochondrial DNA per mitochondrion in mitochondria from different sources. Two methods were used. (1) Determination of acid-insoluble deoxyribose in highly purified mitochondrial fractions, usually using the diphenylamine reaction (Nass, S. et al., 1965). The assumptions made in this method are that all material reacting in the diphenylamine test is mitochondrial DNA and that all mitochondrial DNA present is measured. It remains to prove that both assumptions are correct. No satisfactory test has yet been developed to detect the absence of nuclear fragments in mitochondrial preparations. In this laboratory, the most used method, light microscopy of Feulgen-stained smears, proved to be insufficiently sensitive (Kroon, unpublished results). Furthermore, mitochondrial lipids interfere with the diphenylamine reaction, and even after repeated extraction of acid-insoluble material with lipid solvents, the spectrum of mitochondrial material1 that reacts with diphenylamine is generally not identical to the spectrum of the reaction product of diphenylamine in DNA. pure . Therefore, correction of these greenish contaminants is necessary, adding another factor of uncertainty to the determination of mitochondrial DNA by this method. (2) To distinguish mitochondrial DNA present in mitochondrial fractions from contaminating nuclear, bacterial, or chloroplast DNA, the DNA was extracted and characterized by its equilibrium density in CsCl (Suyama and Bonner, 1966; Borst et al., 1967b; Clayton and Vinograd, 1967), restoration behavior (Kroon et al., 1966) or sedimentation rate properties (Borst et al., 1767b). This can only be done after the DNA has been purified and to control DNA loss during purification, it is necessary to add a known amount of marker DNA to the mitochondrial lysate due to this loss of mitochondrial DNA

144

S. BORST Y A. M. KRONE

The DNA and the DNA tag are the same during purification. As mitochondrial DNA has unique physicochemical properties and can bind to mitochondrial membranes, it is uncertain whether marker DNA losses in these experiments are concomitant with mitochondrial DNA losses. The amount of mitochondrial DNA per gram of mitochondrial protein determined by these methods for mitochondria from different sources in different laboratories is shown in Table VIII. In general, most authors find values in TABLE VIII QUANTITY OF DNA FOUND IN MITOCHONDRIAL PREPARATIONS FROM DIFFERENT SOURCES

Source of mitochondria Rat liver Chicken liver Beef heart Mouse L cell Mung bean Tetrahymena pyriformis Sarrharomyrrr rrrevisiae

DNA for gin. Mitochondrial Protein (mg.) Method (1) a

Method (2) a

0,25-0,65

0,5 0,2'1 1,1

0,7, 0,3

0,8

Mitochondria1 DNA per mitochondria (gm.)

references"

x x 1 x 9 x

10-17

10-16 10-16

6 7 8 9 10

0,5-2 7

1.7

1-4

+ -

0,7-1,0

0,7

10-16 10-17

10-17

1-4 5

See the text for an explanation. Keys to the choruses: (1) Schneider and Kuff (1965): (2) S. Nass rt al. (196513); (3) Estimate al. (1964a); (4) P. Parsons and Simpson (1967); (5) Borst ph al. (1967b); (6) M.M.K. Nass (1966); (7) Suyama and Bonner (1966); (8) Suyama and Preer (1965); (9) dear rf a/. (1964b): (10) Tewari et al. (1966). 4p

about 0.5 mg. DNA/gr. Mitochondrial protein for animal mitochondria. The value for yeast mitochondria is controversial, but the higher values given in Table VIII are probably correct in our opinion (see Borst in discussion after Swift et d., 1968a). In the last column, values are expressed as the amount of mitochondrial DNA per mitochondrion. The lowest value for animal mitochondria corresponds to two 5p circles per mitochondrion, the highest values more than 10. Evidence that multiple DNA molecules can be present in a mitochondrion also comes from cytochemical studies. Up to six unconnected regions containing DNA were detected in serial sections of mouse L-cell mitochondria by M.M.K.Nm (1968) and up to 10 unconnected regions were identified by Merker et al. (1967) in giant mitochondria from the rat uterus. There is evidence that changes in the growth rate or physiological state of cells can affect the amount of DNA per mitochondrion. It has been consistently observed in cytochemical studies that mitochondrial DNA is more easily identified in rapidly growing tissue cells (M.M.K. Nass et al.

DNA mitocondrial

145

al., 1965; Swift et al., 1968a) and according to Neubert (discussed after Swift et al., 1968a), there is more DNA per milligram of protein in the mitochondria of embryonic rat tissues than in the mitochondria of adult or weanling rats. Furthermore, Work (discussed after Swift et al., 1968a) reported that injection of triiodothyronine into thyroidectomized rats results in a >fold increase in the amount of mitochondrial DNA. These results suggest that mitochondrial DNA content may increase as the rate of mitochondrial duplication increases. As the amount of DNA per mitochondrion is quite similar for DNA from very different organisms (see Table VIII), it is evident that the relative proportion of the total cellular DNA found in mitochondria depends only on the number of mitochondria per cell and the amount of mitochondria depends on the DNA. D N A per cell nucleus, as long as no other DNA-containing organelles are present. This proportion varies from more than 50% in eggs of giant amphibians (Dawid, 1966) to about 30-40% in haploids Sacrharomyces cerevlsiae (Hollenberg, unpublished observations) with their small amount of nuclear DNA, to about 15% in diploides Sarcharomyces.r (see Section IX), from about 1% in chicken liver (Borst et al., 1967b) to 0.2% in mouse L cells (M.M.K. Nass, 1966). VIII. Mitochondrial DNA Replication1 OF MITOCHONDRIAL DNA SYNTHESIS IN RELATION TO A. TIME WITH THE CELL REPLICATION CYCLE

The relative timing of mitochondrial and nuclear DNA synthesis in the same cell was examined by radioautography of pulse-labeled cells or by following the rate of incorporation of precursors into the DNA of nuclear and mitochondrial fractions isolated from synchronized cells. The results obtained with Tetvuhymenu (J.A. Parsons, 1965) and P. polycephalic (Guttes and Guttes, 1964; Evans, 1966) were essentially identical. Whereas nuclear DNA synthesis was restricted to part of the cellular replication cycle (S phase), mitochondrial DNA synthesis proceeded at an approximately constant rate throughout the replication cycle. In Chang cells synchronized with tissue culture, maximal incorporation of thymidine into mitochondrial DNA occurred between S phase and cytokinesis (Koch and Stokstad, 1967). Apparently, the temporal control of mitochondrial and nuclear DNA synthesis is different. Recently, Mounolou et al. (1968) and Swift o a/. (1968a) reported that exposure of anaerobic yeast to oxygen leads to an immediate and intense burst of mitochondrial DNA synthesis, which stabilizes after 10 minutes. During the period of increased DNA synthesis, mitochondrial DNA is very prominent in yeast cell slices (Swift et al., 1968a). This could represent a promising test

146

S. BORST Y A. M. KRONE

System for studying the control of mitochondrial DNA synthesis. The factors involved in this control are unknown.

B. MITOCHONDRIAL DNA REVOLUTION Mitochondrial DNA turnover in animal tissues in vivo has been studied in detail by Neubert et al. (Bass and Neubert, 1966; Neubert et al., 1968a). Animals were pulsed with radioactive thymidine and the specific radioactivity of DNA isolated from highly purified nuclear and mitochondrial fractions was followed as a function of time. Thymidine incorporation into rat liver nuclear and mitochondrial DNA was linear approximately 6090 min after injection and then plateaued. In the week following the pulse, the specific nuclear DNA activity remained almost constant, indicating the virtual absence of DNA synthesis. In contrast, mitochondrial DNA specific activity steadily decreased after an optimal exponential dilution curve with a half-life of 9 days, as shown in Fig. 8. The half-lives of '000:

.....--._. *-. *......#eu

400200-

\w\x ADN mitocondrial1 \x

a 100: z D

9 Tome

40E

, "

20-

,o ......................I,-* 47

f l > 100 marcas

ADN nuclear I

1 2 I2

sixteen

$ 0

2'8

COWARDLY. 8. Decreased specific activity of nuclear and mitochondrial DNA in rat liver after a single injection of 3H-thymidine. On day zero, adult rats (body weight 300 g) were injected with 1 mCi. HS-thymidine (specific activity, 6 mCi./pmol) per kilogram followed by repeated injections of 40 mg. cold thymidine per kilogram. Reproduced in modified form from Neubert et al. (1968a).

Gross et al. found mitochondrial DNA in the heart, kidney and brain of an adult mouse. (1968) at 5.5, 7.9 and 39 days, respectively. Apparently, mitochondrial DNA is renewed in quiescent cells. A recent work by Neubert et al. (1968a) showed that the

DNA mitocondrial

147

The half-life of mitochondrial DNA depends on the rate of cell division: the higher the rate of cell division, the shorter the half-life of mitochondrial DNA. Indeed, in a rapidly growing Morris hepatoma (naked DNA half-life = 5 days), the decrease in mitochondrial and nuclear DNA specific activity was identical, suggesting that this decrease was entirely attributable to net DNA synthesis without any replacement. Neubert et al. (1968a) suggested that "the increased demand for formation of certain mitochondrial components during rapid growth is met by increased stability of the components rather than an increased rate of synthesis". With higher growth rates, of course, the synthesis rate also increases. An obvious implication of these results is that the relative rates of mitochondrial and nuclear DNA synthesis vary depending on the mitotic index of the tissue under study. Neubert et al. (1968a) showed that after a pulse of 3H-thymidine, the specific activity of mitochondrial DNA is 50 times greater than the specific activity of nuclear DNA in the liver of adult rats. For 200 g rats, this ration was 22 in 130 g. Mice 3.5, in 60 g. in rats 1.4 and in 13-day-old rat embryos 0.7. Preferential incorporation of DNA precursors into adult rat liver mitochondrial DNA has also been observed by others (Schneider and Kuff, 1965; Nass S, 1967; Gross et al., 1968). To date, no attempt has been made to determine which portion of mitochondrial DNA turnover is attributable to replication and which portion is attributable to DNA repair. In the last part of this section, we assume that the contribution of DNA repair is negligible. Based on the results discussed above, it can be predicted that, in the regenerating liver, the ratio of nuclear and mitochondrial DNA-specific activities will drop precipitously after a pulse of H3-thymidine or P32. This was actually observed in two labs. While the H3-thymidine rate (Chang and Looney, 1966) or P:,? (Nass, S., 1967) mitochondrial DNA incorporation increased only 2-3 fold in regenerating liver, nuclear DNA incorporation increased 20-80 fold. According to S. Nass (1967), mitochondrial DNA uptake doubled within 12 h after hepatectomy, whereas nuclear DNA uptake did not increase "until some time between 12 and 24 h". The possibility that this represented an effect on parent pools was made unlikely by Nass's observation that the net increase in mitochondrial DNA in the regenerating liver preceded a net increase in nuclear DNA. Interestingly, the half-life of mitochondrial DNA in the liver of adult mice is of the same order of magnitude as the half-life of other important mitochondrial components1. From a limited body of experimental data, Fletcher and Sanadi concluded in 1961 that soluble proteins, insoluble proteins, cytochrome c and lipids in mass of rat liver mitochondria are renewed with the same half-life of 10 to 11 days. Although a half-life of 9 days was also observed for mitochondrial insoluble protein

148

S. BORST Y D A. M. CROWN

included in a more detailed study by Bailey et al. (1967), the decrease in specific activities of soluble mitochondrial proteins and phospholipids did not follow a simple exponential curve. Soluble protein log plots produced a curve with a continuously decreasing slope, indicating the presence of at least two components with different half-lives (Bailey et al., 1967). Turnover studies of Paβ-labelled mitochondrial phospholipids revealed the presence of two components with half-lives of 1.6 and 10 days, respectively (Bailey et al., 1967; Gross et al., 1968). Furthermore, when phospholipids were labeled with C14-acetate-C14, a half-life of 2 days was found (Bailey et al., 1967), indicating that turnover of the acyl portion of phospholipids is greater than that of the rest. of the molecule. On the other hand, the slow turnover of mitochondrial DNA 1 in the brain mitochondria of adult rats with a half-life of 39 days (Gross et al. are not consistent with the notion that entire mitochondria are renewed as a unit, it would be surprising if the similarity in the half-lives of mitochondrial DNA, mitochondrial membrane-bound protein, and mitochondrial phospholipid in bulk were accidental. mitochondria are degraded as a unit, but that some mitochondrial components are replaced more rapidly, either because they are being exchanged (an example might be the acyl portion of mitochondrial phospholipids) or because they are being degraded within the mitochondria (an example might be mitochondrial-induced 6-aminolevulinate, which breaks down with a half-life of 70 minutes (Marver et al., 1966). have been shown to contain mitochondria at different stages of degradation (see De Duve and Baudhuin, 1966). If this is true, the mitochondrial DNA turnover rate could be an index of the metabolic activity of lysosomes. Extending Neubert's results for mitochondrial DNA to other mitochondrial components will show whether this concept is correct or not. The conclusion that mitochondrial DNA from the liver of adult rats is metabolically unstable was criticized by S. Nass (1967) for three reasons: (1) There is the possibility that thymidine is incorporated into RNA or adsorbed to glycogen. Both RNA and the thymidine-glycogen complex (see Counts and Flamm, 1966) may be present as acid-insoluble impurities in DNA, and turnover of each component may have been responsible for Neubert's decrease in specific activity in mitochondrial DNA. fractions. (2) Although Nass also notes that P32 is incorporated at a much higher rate

DNA mitocondrial

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in mitochondrial DNA than in rat liver nuclear fractions, the degradation of specific A-DNA activity over time suggests that mitochondrial DNA is stable and its turnover is negligible. (3) Nass notes that similar experiments performed by Schneider and Kuff (1965) using H-thymidine as a DNA precursor also show (although the authors do not say so) that stable adult rat liver mitochondrial DNA is, in our opinion, , of great importance. First, several control experiments by Neubert et al. (1968a) and Gross et al. (1968) indicated that acid-insoluble thymidine was indeed present in DNA. Second, the experimental results of S. Nass (1967) do not support his conclusion that mitochondrial DNA is stable. Figure 5 from his work clearly shows that in the period between 4 and 14 days after P32 injection, the specific activity of mitochondrial DNA from normal rat liver is reduced, while the specific results agree well with those of Neubert et al. (1968a) agree when taking into account differences in the turnover of p32 and p32 pools. The turnover of thymidine is very high and 3 hours after injection the incorporation into mitochondrial DNA has completely stopped, whereas the incorporation of P&agr;' is reduced. It should also be noted that S. Nass (1967) does not explain why he also observes a much higher rate of P32 incorporation into mitochondrial DNA than into rat liver nuclear DNA. If mitochondrial DNA were as stable as nuclear DNA, the liver of old mice would be full of mitochondrial DNA. This is not the case. Finally, the incorporation studies by Schneider and Kuff (1965) cannot be interpreted as supporting metabolic stability. of mitochondrial DNA from rat liver, since in this work the specific activity of mitochondrial DNA was monitored only 18 hours after thymidine injection. A half-life of 240 hours cannot be expected to be demonstrated in an 18-hour experiment. We conclude that the concept that mitochondrial DNA renews itself in non-dividing cells is well established, at least in the few cases studied. We pointed out earlier (Borst et al., 1967a) that this metabolic instability does not affect genetic continuity unless all mitochondria in a cell are degraded at the same time. The only known instance where this can occur is in the anaerobic yeast cell. How genetic continuity is preserved under these conditions is discussed in Section IX.

C. THE MECHANISM OF MITOCHONDRIAL DNA SYNTHESIS IN INTACTICAL CELLS The mechanism of mitochondrial DNA replication in the intact cell was studied by Reich and Luck (1966) with N. cradssa using the density staining technique first used by Meselson and Stahl (1958) E. coli. Nezrrospora crassa was cultured using N15 as the sole nitrogen source and the flotation density

150

S. BORST AND AM. Crown

DNA extracted from purified nuclear and mitochondrial fractions was analyzed in CsCl analysis gradients at different time points after changing the culture to N14 medium. The behavior of nuclear DNA was as expected for uncomplicated semiconservative replication. For mitochondrial DNA, most of the DNA synthesized in the first round of replication consisted of N15 DNA undiluted with NI4 DNA. Even after three cycles of duplication, the N15 content of the DNA was 40%, but in this case most of the N15 DNA was diluted with NI4 because most of the DNA coalesced after denaturation at an intermediate position between the N1 DNA * pure and DNS . -NI5. Reich and Luck (1966) conclude from these results: "The nitrogenous precursors for mitochondrial DNA synthesis are drawn from a pool that is effectively large relative to the amount of mitochondrial DNA but slow relative to the rate of mitochondrial DNA synthesis. regenerates and resists dilution from exogenous nitrogen sources. The opposite is true for nuclear DNA. Therefore, replication of the two DNA species is at least metabolically independent and possibly topographically isolated, and a precursor-product relationship between the two is ruled out ." Reich and Luck further conclude that their results are consistent with a semi-conservative replication mechanism for mitochondrial DNA and that pre-existing polynucleotide chains of mitochondrial DNA are conserved during replication.1 In their paper on renaturation of mitochondrial DNA, Corneo et al (1966) briefly mention unpublished results by Grossman and Marmur with yeast that are qualitatively similar to those reported with Nezrrosporu. The nature of the N15 pool from which mitochondria are drawn for DNA replication is not known. Reich and Luck conclude that the flux of 4 in mitochondrial DNA over three replication cycles that the pool of progenitors surrounds must be many times greater than the amount of mitochondrial DNA present. They continue: "Since the existence of such a large pool of soluble deoxyribonucleotides would be surprising, it may be that in mitochondria, as in some other systems, RNA turnover provides the immediate precursors for DNA synthesis." This proposal by Luck and Reich requires four assumptions: (1) Mitochondrial and extramitochondrial deoxyribonucleotide and ribonucleotide pools do not equilibrate. (2) The pool of mitochondrial deoxyribonucleotides is small compared to the number of nucleotides present in mitochondrial DNA. (3) The enzymatic system for converting ribonucleotides to deoxyribonucleotides, presumably at the level of nucleoside di- or triphosphate medium, makes it impossible to conclude from the results of Reich and Luck that the physical continuity of mitochondrial DNA is preserved during replication, since similar results would have been obtained if mitochondrial DNA had been continuously depolymerized and resynthesized.

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1967), is present in the space of the mitochondrial matrix. (4) Mitochondrial RNA turnover is high relative to net RNA synthesis. Some objections can be made to assumptions (I), (3) and (4). The inner membrane of intact and isolated mitochondria has low permeability to nucleotides with the exception of ADP and ATP, which are rapidly transported in and out by a translocase or adenine nucleotide permease (Klingenberg and Pfaff, 1966; Kemp and Groot, 1967; Ohnishi et al., 1967; Greenawalt et al., 1967). Although this permeability barrier has been observed in RNA synthesis experiments in isolated, intact mammalian mitochondria with added UTP, GTP, and CTP as substrates (Neubert et al., 1968b; Saccone et al., 1968), there is no evidence to support this. such Barrier. A barrier has been encountered in in vitro studies of DNA synthesis (see Section VII1,D). More importantly, in the experiments by Neubert et al. no permeability barrier can be seen. (1968a) reported incorporation of thymidine into mitochondrial DNA in vivo, as HS-thymidine co-appeared in mitochondrial and nuclear DNA within 30 minutes of its intravenous injection, whereas incorporation stopped completely 180 minutes after injection. This indicates that in the liver the pool of mitochondrial deoxyribonucleotides is small and that the very low permeability of the inner mitochondrial membrane is sufficient to allow entry of the deoxyribonucleotides necessary for DNA synthesis. Rat liver mitochondria contain about 0.5 pg. Mitochondrial DNA per milligram of mitochondrial protein or 1.5 nmol of DNA per milligram of protein (see Section VII). Assuming a DNA synthesis rate of 1 p per minute, the required input is at most about 0.25 x 0.20 x 1.5 = 75 pmol per minute per milligram of protein from each individual deoxyribonucleotide. That's four orders of magnitude slower than the rate at which ribo-ADP and ribo-ATP exchange, and no in vitro experiment prevents an influx of deoxyribonucleotides at that rate. In contrast, it has been calculated that N A D + , for which the permeability of the mitochondrial membrane is also very low (see review by Borst, 1963), in vivo at a rate of 43 pmol per minute per milligram of mitochondrial protein (Purvis and Lowenstein, 1961). As this rate was a minimum estimate, it seems reasonable to assume that the low permeability of the mitochondrial inner membrane to nucleotides is sufficient to provide the mitochondrial complement of NAD, NADP, ribonucleotides and deoxyribonucleotides in rat liver. Whether this permeability barrier is detected in in vitro uptake studies may depend on the integrity of the mitochondria and the size of the intramitochondrial pool of the nucleotide under study. Nothing is known about the size of deoxyribonucleotide pools in mitochondria. However, it seems reasonable to assume that the concentrations of all deoxyribonucleotides are lower than those of ribouridin and ribocytidine.

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S. BORST Y A. M. KRONE

nucleotides constituting 0.1 and 0.2 nmol per milligram of mitochondrial protein in rat liver (Heldt and Klingenberg, 1965) and 0.1 and 0.6 nmol per milligram of protein in yeast (Ohnishi et al., 1967), provided that no nucleotides are lost during the Since rat liver mitochondria contain about 1.5 nmol of DNA nucleotides per milligram of protein and yeast mitochondria contain 4 to 10 times more, the deoxyribonucleotide pool can be too small to support a 3-fold increase in levels of Explain N15 in mitochondrial DNA during growth in NI4 medium. However, the suggestion that ribonucleotides are converted to deoxyribonucleotides in Neziruspora mitochondria is unsatisfactory for several reasons. First, adenine contributes 38% to the nitrogen of Nerosporu mitochondrial DNA, which has a GC mole percentage of 43% calculated from its buoyant density (Table 11). Since the adenine nucleotide translocase present in NezlroJpora mitochondria (Greenawalt et al., 1967) completely balances intra- and extra-mitochondrial adenine nucleotides, the maximum contribution of NI5 from ribonucleotides is 60%. Second, it is likely that most of the mitochondrial RNA in Nez4roJpor.a is ribosomal RNA and tRNA. It would be surprising if these RNAs were rapidly renewed. Finally, Wintersberger (1966) found that unlabeled GTP, CTP and UTP cannot support dATP uptake by isolated yeast mitochondria, which shows an absolute requirement for the presence of all four deoxynucleoside triphosphates for DNA synthesis. This suggests that no significant conversions of ribonucleotides to deoxyribonucleotides occurred under these conditions. Although none of these considerations are conclusive, we believe that the continued incorporation of N15 nucleotides into Neuro.rporu mitochondrial DNA following a switch to N14 has yet to be satisfactorily explained. Two alternative explanations, equally unsatisfactory, may be mentioned. If newly synthesized mitochondria were preferentially lost during mitochondrial purification, this would lead to a delay in the appearance of -"+ DNA. the bands are only 2 mg/cm 3 above the NIJ nuclear DNA None of these alternatives explain the presence of a large proportion of DNA strands containing N14 and N15.

D. Incorporation of Deoxyribonucleotides into DNA from Isolated Mitochondria Deoxyribonucleotide incorporation into DNA has been observed in mitochondrial preparations derived from rat liver (Schmieder and Neubert, 1966; Parsons, P. and Simpson, 1967, L968), chicken liver (Ter Schegget and Borst, 1968). ), yeast (Wintersberger, 1966, 1968) and P. polycephalum (Brewer et al., 1967). Maximum observed rates of incorporation for a labeled nucleotide are 1.0 pmol of nucleotide per milligram of protein per hour (37° C.) for rat liver.

DNA mitocondrial

I53

Mitochondria, 2 pmol per milligram of protein per hour for chicken liver mitochondria (37OC), nearly 400 pmol per milligram of protein per hour for yeast mitochondria (37OC), and 170 pmol per milligram of protein per hour for P. polyrephalnm ( 25°C.). Although uptake in liver mitochondria is low compared to bacterial systems, it is about 100 times greater than uptake in isolated rat liver nuclei when DNA-based uptake is expressed, as noted by Schmieder and Neubert (1966). ). In experiments by P. Parsons and Simpson (1967), the maximum net DNA synthesis by rat liver mitochondria linearly incorporating dTTP during a 2 h incubation period corresponded to 1'/F of mitochondrial DNA present (based on a mitochondrial DNA content of 0.25 pg per milligram of protein). Although the initial rate of dATP incorporation in yeast mitochondria is 400 times greater than that in rat liver mitochondria, net synthesis is only three times greater than in rat liver mitochondria because yeast mitochondria contain 4 pg. Incorporation of DNA per milligram of protein and dATP ceases after 15 minutes of incubation (Wintersberger, 1966, 1968). The incorporation examined in hepatic mitochondria has the expected properties for DNA synthesis by a DNA polymerase that occurs within the inner mitochondrial membrane (Schmieder and Neubert, 1966; P. Parsons and Simpson, 1967, 1968; Ter Schegget et al Borst, 1968 ): it is insensitive to deoxyribonuclease; unaffected by added DNA; it is inhibited by uncoupling and mitochondrial electron transport inhibitors1; depends only partially on the presence of the four deoxynucleoside triphosphates (probably due to the presence of a small endogenous group); it is inhibited by DNA synthesis inhibitors such as nogalamycin, cinerubin A, phleomycin, or high concentrations of actinomycin D; and the same rate of incorporation is found in semi-sterile and non-sterile mitochondria. TMP-CI4 incorporated into rat liver mitochondria was recovered as 3'-TMP after enzymatic digestion, indicating that most of the incorporation occurred at positions within the DNA strand (P. Parsons and Simpson, 1967). Although the incorporation of dATP into yeast mitochondrial DNA was also insensitive to high concentrations of pancreatic deoxyribonuclease, in this case the incorporation was totally dependent on the presence of dGTP, dCTP and dTTP, and these nucleotides could not be metabolized by GTP, CTP and UTP. replaced (Wintersberger, 1966). Equilibrium density gradient analysis provided further evidence that DNA synthesized in citrus by isolated mitochondrial preparations is indeed mitochondrial DNA. In the case of chicken liver, yeast and P . Policephaly, the fluctuating density of mitochondrial and nuclear DNA differs in CsCl (see Tables I and II), and DNA synthesized in vitro has been shown to have the equilibrium density of mitochondrial DNA (Brewer et al., 1967; Wintersberger, 1968). ; Ter Schegget and Borst, 1968). In rat liver, the density of nuclear cells and

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Chondrial DNA is almost the same, but in this case it was reported that DNA synthesized from isolated mitochondria was renatured under conditions where nuclear DNA was not renatured (P. Parsons and Simpson, 1967). The physical properties of the DNA product synthesized by chicken liver mitochondria in vitro were analyzed by Ter Schegget and Borst (1968). Quite unexpectedly, they found that up to 80% of the embodied radioactivity generated by Borst et al. (1967b), DNA identical to the marker I component was present in sucrose gradients and in CsCl gradients containing ethidium bromide. This result shows that the uptake of deoxyribonucleotides by isolated mitochondria is not the result of aberrant copying of mitochondrial DNA1, similar to that observed with bacterial DNA polymerase and double-stranded DNA in subcellular systems (cf. Schildkraut et al., 1964) . Furthermore, the fact that radioactivity is found in closed circular double-stranded DNA strongly suggests that the enzyme polynucleotide ligase (Gellert, 1967; Becker et al., 1967; Little et al., 1967) is present in mitochondria. Although the results discussed in this section show that mitochondria isolated from various sources can incorporate deoxyribonucleotides into their DNA, two main questions remain unanswered: (1) Is incorporation of deoxyribonucleotides the result of DNA replication or repair? (2) Are all mitochondria in the suspension incorporating nucleotides at the same rate, resulting in a maximum 1-3 year increase in their DNA content, or is all activity a result of a minority of mitochondria replicating their DNA more extensively? Since chromodeoxyuridine is readily incorporated into DNA by isolated chicken liver mitochondria (Ter Schegget and Borst, 1968), an answer to both questions will soon be found.

IX. Effects on Yeast Mitochondria1 Anaerobic DNA, Glucose Repression, and Mutagens Several yeast species have the fortunate ability to grow without functional mitochondria, allowing experiments in mitochondrial biogenesis that are not readily available in obligate aerobes. Three conditions are known in which mitochondrial biosynthesis is altered in yeast: anaerobiosis, glucose repression and in small mutants. The fate of mitochondrial DNA under these conditions has been studied in different laboratories and the results obtained are discussed in this section. A. ANAEROBIOSIS

The synthesis of mitochondrial cytochromes in Saccharomyces occurs only in the presence of oxygen (Somlo and Fukuhara, 1965). If another myth-

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Chondria components are present in anaerobically cultured cells, depending on the chosen growth conditions. When a source of fatty acids and sterols is present in the medium, numerous well-defined mitochondria-like structures are present (Lukins et al., 1966; Swift et al., 1968a). These "promitochondria" consist of a double membrane without cristae and contain typical mitochondrial DNA fibers and ribosome-like particles in electron micrographs (Swift et al., 1968a). When the particle fraction of a homogenate of cells grown under these conditions was centrifuged to equilibrium on a urografin gradient, a band with the density of normal yeast mitochondria was found (Schatz, 1965). Two exclusively mitochondrial enzymes were concentrated in this band, succinate dehydrogenase and oligomycin-sensitive ATPase. It seems likely that the particles purified by gradient centrifugation are identical to the promitochondria seen in electron micrographs. Exact values for the mitochondrial DNA content of these cells have not been reported. Swift and others. (1968a) mention that "DNA isolated from anaerobic cells still showed a satellite band of mitochondrial density, but in reduced quantity", while Fukuhara (1968) concludes that the proportion of mitochondrial DNA in anaerobic cells "does not differ much" from that of cells anaerobic. aerobic cells. A major technical difficulty in these experiments is that respiratory adaptation of anaerobic yeasts occurs at very low oxygen concentrations (Somlo and Fukuhara, 1965). When yeast is cultured anaerobically without a source of fatty acids and sterols, the picture is quite different (Wallace and Linnane, 1964): mitochondrial profiles are completely absent and electron micrographs "only occasionally show a type of vesicle". membrane within the cytoplasm". (Lukins et al., 1966). Furthermore, only traces of succinate dehydrogenase could be detected (Lukins et al., 1966). Using indirect arguments, Wilkie (1963) concluded that, under these conditions, only a genetically active copy of the mitochondrial DNA is preserved It would be very interesting to know what the actual mitochondrial DNA content of these cells is and whether this mitochondrial DNA is present in a cytoplasmic organelle, in the nucleus or free in the cytoplasm About these points no experimental data are available. Also, one wonders how the anaerobic cell ensures that each daughter cell receives a copy of a master template.A physical association with the kernel seems to be the easiest way to achieve this.

B. GLUCOSE REPRESSION When S. cerevisiae is grown at glucose concentrations greater than 6 x 10 -3 M, mitochondrial biosynthesis is repressed (Slonimski, 1956). The degree of repression appears to be strictly dependent on the rate of fermentation (De Deken, 1966a). At very high glucose concentrations, fermentation is maximal and mitochondrial biosynthesis is almost completely suppressed. Fraud-

(ol%),

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S. BORST Y D A. M. CROWN

consequently, mitochondrial profiles in electron micrographs of these cells are poor or absent (Yotsuyanagi, 1962a,b; Polakis et al., 1964, 1965; Jayaraman et al., 1966), all cytochromes are present in very high, low concentrations ( Reilly and Sherman, 1965), and levels of several typical mitochondrial enzymes can fall to less than 5% of unsuppressed aerobic levels (Polakis and Bartley, 1965; Jayaraman et al., 1966). When fermentation is low, either because the glucose concentration is low or because other sugars are used as a carbon source, for example melibiose (Reilly and Sherman, 1965), which cannot be fermented quickly by sacchalomyres, the breathing can be so loud. of cells cultured in lactate. The effect of glucose repression on the proportion of mitochondrial DNA in S . Cerevijiae protoplasts were studied by Moustacchi and Williamson (1966) using preparative CsCl density gradients. In the stationary cell extracts, the proportion of mitochondrial DNA in the total cellular DNA was 20%. After 8 hours of growth in 0.3 M glucose (5.4%), the number of cells increased 30-fold and the proportion of mitochondrial DNA decreased to approximately 3% of the total DNA. This decrease was not due to a transient interruption in mitochondrial DNA synthesis, but rather to a decrease in the rate of synthesis relative to nuclear DNA synthesis, resulting in a gradual decrease in the proportion of mitochondrial DNA. Unfortunately, the authors did not examine the effect of prolonged maximal glucose suppression and it is not known to what extent the proportion of mitochondrial DNA is reduced under these conditions. The decrease in mitochondrial DNA during glucose repression is fully reversible when glucose is removed. Negrotti and Wilkie (1968) reported an interesting exception to the general rule that glucose repression in yeast is fully reversible. They isolated a mutant in which all daughter cells germinated in the presence of more than 0.2% glucose or under anaerobic conditions and were found to have small cytoplasm. The fate of mitochondrial DNA in these mutants has yet to be studied. C. Mutagenic Agents

Slonimski et al analyzed mitochondrial DNA from two mutants and their results are summarized in Table IX. Two main conclusions are evident: (1) A nuclear mutation did not affect either the fluctuating density or the amount of mitochondrial DNA, although functional mitochondria are not produced in this mutant. (2) In the cytoplasmic mutants, the density of mitochondrial DNA changed while the amount of mitochondrial DNA was normal. In previous experiments by Corneo et al. (1966), Moustacchi and Williamson (1966) and Tewdri et al. (1966) found little or no detectable mitochondrial DNA in small cytoplasmic strains. Although this is not the case

chiquita

my tuchondrial

Order No.

Type of genetic determinants Strain

chromosomal

cytoplasmic

QO, (% der Norm I

density

em CsCl

de CsCl (gm./cm.::)

(gm./c1m3)

*I).002

Valor s Re1 (% do total)

Normal gvmd2d~. p7 rho-

Palma 1

Normal

100

1 7 0 1 bis 9.002

1.687

croirisornal pc/I/?

M ti t a k d

Normal

1,701 i O.O(lZ

hW72 0,002

1 I1

used to be

1,701 e 0,001

1.683 =? 0,003

1.792 fc).001

1.695.000 0 001

p7 ro+

Neutral pe/i/r p 7 rho,-

Silencer Pelite p7 rhu,-

recessive

rct-cssivc variable

M e ~ recessive ttd

Modified from hfaunolou t t d., 1966

dominant recessive mutation

7 0

r 0 z B 2 > r G 2

158

P. RORST E A. M. Crown

As shown directly, it seems possible that the prevailing glucose repression in our culture conditions has suppressed mitochondrial DNA synthesis to a level where DNA cannot be detected in density gradients. Carnevali et al. examined other small cytoplasmic mutants. (1966) and Mounolou et al. (1968). Also in these cases, the fluctuating density of mitochondrial DNA was different from that of wild-type yeast, but in one mutant less than 170% of the total DNA was mitochondrial versus 10-14% in wild-type cells. The large changes in the fluctuating mitochondrial DNA density of some of these cytoplasmic mutants can be attributed to a large-scale modification of the bases, for example by methylation, or to a large change in the GC content. Preliminary tests by Mounolou et nl. (1968) indicate that in the mutants studied by this group, the change in DNA density is accompanied by a change in Tn, suggesting that changes in base composition are responsible for the changes in density in these mutants. This was confirmed by direct analysis for the Carnevali et al. confirmed. (1966). Mitochondrial DNA from this mutant has an exceptionally low CsCl buoy density of 1.670 g/cm3 (nuclear DNA 1.699 g m/~m) and contained less than 3% GC (Tecce, personal communication). Since alternating dAT has a density of 1.679 g/cm3 (Schildkraut et al., 1962), while dA:dT has a density of 1.6445 g/cm3 (Wells and Blair, 1967), the mutant DNA must consist of the random A and T sequences that explain the density of 1670 g/cm3 found by Carnevali et al. (1966). The interesting question of how density changes in these mutants arise was discussed at length at the 1967 round table in Polignano. Three mechanisms were considered. (1) Large-area deletions, in which stretches of DNA are removed with a base composition that differs significantly from the rest of the molecule. However, this explanation is highly unlikely, given the narrow unimodal bands observed for yeast mitochondrial DNA on CsCl gradients (Borst et al., 1968). Since these DNA preparations consist of heterogeneous linear DNA, likely derived from larger molecules by random decay (see Section VI), any heterogeneity in the background composition of intact molecules would have manifested itself as fragment density heterogeneity. (2) Slonimski (discussed by Mounolou et al., 1368) raised the possibility that mitochondrial DNA in wild yeast is genetically heterogeneous. According to this hypothesis, most molecules have the same base composition, but a minority, too small to show up in the density gradients, have a base composition very different from most mito molecules.

DNA mitocondrial

159

chondrial DNA. In the presence of agents that induce small mutations, such as acriflavine, mitochondria containing the minority DNA would have an advantage and become larger than normal mitochondria. As Slonimski pointed out, the mitochondrial heterogeneity in normal yeast cells required in this hypothesis to explain the many different mutants already obtained seems impossibly high. (3) Slonimski's preferred hypothesis (discussed by Mounolou et al., 1968) is the "incompatibility hypothesis". Agents that induce small mutations create errors in mitochondrial DNA replication, rendering the newly synthesized DNA non-functional. If all of the mitochondrial DNA replicas in a cell contain errors, the cell is a small piece of cytoplasm. When the dye is removed, the cell initially contains many different molecules of meaningless DNA. In successive generations, the most efficiently replicated nonsense mitochondrial DNA is selected for, eventually resulting in a small clone with one type of mitochondrial DNA. Slonimski apparently assumes that large-scale changes in base composition already occur during the initial incubation with the intercalating dye, since he found that cytoplasmic mutants "result from a change in the buoyant density of their mitochondrial DNA" (Slonimski et al. , 1968). ). This is not very likely, however, since only a few molecules of acriflavine per mitochondrion are sufficient to transform a yeast cell into a small mutant (see Wilkie in the discussion by Mounolou et al., 1968). Therefore, we prefer the idea that the primary mutagenic event can only introduce small errors in mitochondrial DNA. The minimum error required for this is unknown, but it is possible that all mitochondrial DNA gene products are essential for the synthesis of a functioning mitochondrion, so that any mutation that results in a non-functional gene product effectively renders the mitochondrial DNA non-functional. . 2 According to this hypothesis, the altered basic composition of mitochondrial DNA found in small cytoplasmic mutants is attributed to a slow accumulation of additional errors in mitochondrial DNA as the small DNA grows and divides over thousands of generations. To explain the large change in the base composition of 2, this is not unreasonable, given the proposed genetic functions of mitochondrial DNA: specification of mitochondrial ribosomes, specification of inner membrane proteins. and specification of regulatory proteins that coordinate the contribution of nuclear and mitochondrial DNA to mitochondrial biosynthesis (see Section XIII). A non-functional ribosomal protein or ribosomal RNA blocks mitochondrial protein synthesis, which is essential for mitochondrial biosynthesis. A non-functioning membrane protein can prevent normal inner membrane assembly and cytochrome attachment. If the rate of recombination between mitochondrial DNAs is low under mild induction conditions, mutations of a single gene for mitochondria containing the mutated DNA can be lethal.

160

BOOK PAGE . HORST E A.M. KROON

The mitochondrial DNA of some of these mutants compared to wild-type mitochondrial DNA, it is probably necessary to assume that large-scale replication errors contribute to the alteration in the mitochondrial DNA. Selection for replication efficiency will eventually result in a homogeneous population of mitochondrial DNA. According to this hypothesis, the mutation is not caused by the change in buoyancy density, but the change in density is the result of mutation, i.e., the effective loss of genetic information in mitochondrial DNA. The hypothesis implies that errors in mitochondrial DNA replication rarely occur in all yeast cells, but that in normal yeast cells, mitochondria containing "good" copies of mitochondrial DNA grow larger than mitochondria containing "bad" copies. Furthermore, the hypothesis predicts that when examining cytoplasmic mutants as they arise, one will find mutant mitochondrial DNA with the same base composition as wild-type mitochondrial DNA. This mutated mitochondrial DNA will share a high degree of homology with mitochondrial DNA from wild-type cells. Subculturing the mutant over thousands of generations will result in loss of homology and eventually also changes in base composition. These predictions are verified in our laboratory. Since it is inconceivable that proteins with complex biological functions could be encoded by DNA containing only A and T, the results of Tecce and collaborators discussed above demonstrate that all necessary requirements can be met to repair the defective promitochondria of cytoplasmic mutants without the contribution of mitochondrial DNA itself. Thus, mitochondrial DNA polymerase and the proteins needed to make the membrane of defective promitochondria are encoded by nuclear genes (see also Roodyn and Wilkie, 1968). It is not known whether the mitochondrial DNA in other cytoplasmic petites is also completely dysfunctional, but we think this is likely, since mutations of cytoplasmic petites are never reversed, while different mutants of cytoplasmic petites are not complementary (see discussion after Mounolou et al., 1968). Small mutants are known to come in two types: "neutral" and "suppressor" peti1e.r. In crosses with wild-type cells, all daughter cells are normal when crossed with a small neutral type, whereas a variable proportion, depending on the particular mutant selected, of the daughter cells are small when crossed with wild-type silencers. (see Wilkie, 1964). , Mounolou et al. (1968) showed that when crossed with a Petite suppressor, the mitochondrial DNA of the Petite daughter cells (95% in this mutant) had the same density (1.696 g/cm3) as the DNA of the Petite parent, while the E1 mitochondrial DNA of the Petite cells -wild-type daughters had the same density as that of the wild-type parent. So it's clear that when two types of mitochondria are present in a common cytoplasm, one can overgrow. It is not known how this competition occurs at the molecular level. However, if we accept the hypothesis that the '

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The mitochondrial DNA of all cytoplasmic petits subcultured over thousands of generations is completely non-functional, the difference in behavior between neutral and suppressor petits presents an interesting paradox: how is it possible for two non-functional DNAs to be different in the same cell? Obviously, unless other cytoplasmic genetic determinants are present, the cause must be a difference in the chemical or physical properties of the mitochondrial DNA. There is no apparent systematic difference in the mtDNA uplift density of the small suppressors and neutrals studied so far. Therefore, the base sequence or size of the mitochondrial DNA must be responsible for the different behavior, and the rate of DNA replication must be a determining factor in the rate of mitochondrial replication and competition between different types of mitochondria on itself. Cell. Experimental verification of these ideas began in our laboratory. Further speculation on this subject is unlikely to be fruitful until more is known about the devices present in normal cells to regulate the number of mitochondria per cell and the rate of mitochondrial proliferation. It is worth mentioning in this context that Mounolou et al. (1968) found that the proportion of mitochondrial DNA in different yeast strains is genetic and varies between 1 and 15% in diploid cells. Small cytoplasmic amounts are induced with great efficiency by concentrations of acridine dyes that have no apparent effect on yeast nuclear DNA. Two explanations have been proposed to explain the apparent preferential attack of acridines on mitochondrial DNA. (1) Tewari et al. (1966) suggest that the different base composition between mitochondrial and nuclear DNA is responsible for this, since the association constant of the DNA-acridine complex is greater for DNA with low GC content than for DNA with high R content. in GC. This explanation was ruled out by Slonimski eb a/'s proof. (1968) reported that ethidium bromide, which binds low and high GC DNA with equal affinity (Waring, 1965), is an even more specific inducer of cytoplasmic petites than acridine dyes. (2) At low concentrations of ethidium bromide, the dye binds more strongly to closed circular duplex DNA than to open circles or ear DNA (see Section V, B). Given this, Slonimski et al. (1968) proposed that "mitochondrial DNA in yeast may have a native form of supercoiled circles and that changes in supercoiling trigger mitochondrial mutation following dye combination". This proposal implies that yeast nuclear DNA does not consist of closed circular duplexes and is therefore much less dye sensitive. There are two objections to this proposal. First, there is significant doubt about the existence of supercoiling in circular loops.

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Duplex molecules in the intact cell (see Section V, C). Consequently, the (relatively small) difference in affinity for ethidium bromide between open and closed DNA found ipz vitro probably does not exist in the intact cell. Second, the mutagenic effect of intercalated dyes such as ethidium bromide is believed to result from replication errors (Lerman, 1964). Circular DNA replication requires the introduction of a loop into the molecule (see Cairns, 1963), and this loop alleviates the topological constraint on which the preferential binding of ethidium bromide to closed circular duplex DNA is based. We therefore believe that the preferential interaction of acridine dyes with mitochondrial DNA must be a consequence of differential organization between yeast mitochondrial DNA and nuclear DNA, or differential sensitivity of nuclear and mitochondrial polymerase1 to the presence of intercalated dyes. inside the DNA. The different organization can have several meanings in this context: for example, nuclear DNA can be protected from dye intercalation by the presence of histones or divalent cations, or the dye concentration in the nucleus can be much lower than in the mitochondria due to barrier barriers. permeability . . Recently, Slonimski et al. (1968) reported that ethidium bromide also converts non-growing yeasts into small cytoplasmic mutants, in contrast to acridines, which only give rise to mutant daughter cells without affecting parental cells. Conversion of non-growing wild-type yeast cells to small cells by ethidium bromide followed first-order kinetics after a lag phase of approximately 5 hours. Extrapolating the linear part of the induction curve at time zero, it was found that each non-growing aerobic yeast cell contains about six targets that must be "hit" by ethidium bromide before the cell grows. This is at least an order of magnitude less than the number of mitochondria in an aerobic yeast cell, and Slonimski et al. (1968) therefore propose that only a fraction of mitochondria play a role in cytoplasmic character assignment. Although the results of Slonimski et al. are too clear, their interpretation is less convincing. It is generally accepted that intercalated dye mutagenesis is due to copying errors, either in replication or repair (Lerman, 1964). Each of these processes must therefore take place in starving, growing yeast cells. Replication in the presence of ethidium bromide results in a 50:50 mixture of hybrid molecules containing one normal and one mutant strand and molecules containing two mutant strands after two rounds of replication. Hybrids produce normal DNA after removal of ethidium bromide, and therefore conversion of normal cells to small cells represents the loss of the last molecule of hybrid DNA. This follows simple first-order kinetics, but the intersection represents the number of targets x2, i.e. H. the size of the target will be

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three instead of six. However, if recombination and repair occur at significant rates, the target size can be significantly larger, whereas the target size can be smaller if ethidium bromide is slow to enter the cell. Given these complications, it seems premature to draw firm conclusions from the Slonimski ethidium bromide experiment about the proportion of yeast mitochondria involved in cytoplasmic character assignment. If our interpretation of the Slonimski result is correct, the 2-hour half-life is fo; conversion from wild-type to small yeast under no-growth conditions represents the turnover rate of mitochondrial DNA under these conditions. This can be easily verified experimentally.

X. Mitochondrial DNA recombination 1 The work of Sager and others (see Sager and Ramanis, 1965) provided evidence that cytoplasmic determinants can recombine in clunzidomomas. Although the nature of the determinant was not identified in Sager's work, it could be Chlum~~domoma.r chloroplast DNA. Thomas and Wilkie (1968a) have recently reported determinants that affect mitochondrial properties in yeast. In their experiments, they used a series of yeast cytoplasmic mutants in which the mitochondrial biogenesis is resistant to one or the other of the antibiotics erythromycin, spiramycin or paromomycin. In previous experiments by Thomas and Wilkie, discussed in Section XIII, E, it was shown that the resistance of these mutants results from an alteration in a cytoplasmic determinant, probably mitochondrial DNA. Crosses between two strains with a different drug resistance marker resulted in a very high proportion of clones that were not resistant to either or both drugs, as shown in Table X. Thomas and Wilkie conclude that these clones must have arisen from a recombination event1. Some points of interest should be noted. (1) Crosses tabulated in Table X were performed anaerobically under growth conditions where no mitochondrial structure was detected in the cell. This apparently facilitated recombination, as very few recombinants were found after crossings between aerobically grown cells. Therefore, it is still doubtful whether complete fusion of intact yeast mitochondria can occur or not. The recombination event could even involve the "master copy" (nucleus?) of mitochondrial DNA postulated by Wilkie for other reasons (see Section IX). (2) In almost all cases, all cells within a clone derived from a zygote formed under anaerobic conditions were found to contain mitochondria of only one type. Thomas and Wilkie explain this result by assuming that there is only one copy of mitochondrial DNA per cell. ( 3 ) In most of the crosses examined, there is a significant excess

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sensitive to multidrug-resistant clones, as shown in Table X. Thomas and Wilkie propose that this is due to the recessive nature of the mutational change. Circular recombination of DNA results in a circular dimer, and when these dimers are relatively stable, recombinants are predominantly "diploid heterotypes", occasionally producing segregants within a clone. Furthermore, recessiveness in heterozygotes has been observed in other cases of drug-resistant ribosomes.

Child clones Type of clone

Number of clones 25 19 31 5

49 25

14 23 3

4 1

EHSC X ErSN

40 152 1

(Video) Cell Biology: Introduction – Genetics | Lecturio

Adapted from Thomas and Wilkie, 1968a. I?j, sensitivity to 10 pg. erythromycin per milliliter; El', resistance to 3 mg. erythromycin per milliliter; S s , sensitivity at 50 pg. spiramycin per milliliter; St', resistance to 2 mg. spiramycin per milliliter; Ps, sensitivity to 50 pg. paromomycin per milliliter; PV, resistance to 1 mg. Paromomycin per milliliter IL

diploid, apparently because the sensitive ribosomes become trapped in the polyson and block processing of the resistant ribosomes (see Cooper et d., 1967). These very interesting experiments underscore the need for detailed data on the structure, quantity, and intracellular location of yeast mitochondrial DNA under different conditions. They also draw attention to the problem of selection within a heterogeneous population of mitochondria. If the cell could not constantly select the most suitable mitochondria to survive, the recommendation would be useless.

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XI. Mitochondrial DNA renaturation studies1 DNA renaturation, that is, the formation of an ordered double helix from complementary single strands, was discovered by Marmur and Doty and their colleagues (see review by Marmur et al., 1963). They showed that the renaturation reaction follows second-order kinetics and that the renatured DNA had the same melting point as the native starting material before denaturation, indicating that a perfect double helix was formed. Furthermore, they found that the rate of renaturation strongly depends on the DNA source: viral DNA is renatured faster than bacterial DNA and bacterial DNA faster than eukaryotic nuclear DNA. Quantitative aspects of DNA renaturation have been examined more closely by Britten et al. (Britten and Waring, 1965; Britten and Kohne, 1966) and Wetmur and Davidson (1968). They showed that under standard conditions of salt, temperature, and DNA fragment size, the second-order rebirth constant was a linear function of DNA complexity. DNA complexity is defined as the number of base pairs in the genome, ignoring repeated sequences. Since the complexity of mitochondrial DNA is likely to be low, it is not surprising that mitochondrial DNA from birds (Borst and Ruttenberg, 1966a; Borst et al., 1967a,b; Dawid and Wolstenholme, 1968a), rodents (Borst and Ruttenberg, 1966a, Borst et al., 1967a, Corneo et al., 1966, Flamm et al., 1966, Sinclair and Stevens, 1966, P. Parsons and Simpson, 1967), frog eggs (Dawid and Wolstenholme, 1968a,b), and yeast (Tewari et al., 1966; Sinclair et al., 1967a) was completely renatured at a very high rate, comparable to that of DNA from smaller DNA viruses. Although the obvious interpretation of these experiments was that the complexity of mitochondrial DNA is very low, the almost instantaneous renaturation observed under optimal conditions made it necessary to discard the alternative interpretation that complete strand separation during denaturation due to interstrand crossovers was not possible. (Geidushek, 1961, 1962) or the presence of a large proportion of component I. This alternative was proposed by Borst et al. (1967a,b). In their renaturation experiments, they used a mitochondrial preparation of chicken liver DNA containing only the I1 component and showed that renaturation followed second-order kinetics and was strongly dependent on salt. Both results contradict the presence of crosslinks, which lead to renaturation regardless of salt or DNA concentration (cf. Geidushek, 1961, 1962). When comparing the renaturation constants obtained for mitochondrial DNA with the renaturation constants determined by Britten and Kohne (1966) for DNA of different complexities, Borst et al. (1967b) calculated a maximum complexity for mitochondrial DNA of about 12,000 base pairs. Given the correction factors involved, this value could be off by 50%. What is important, however, is that these

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The experiments revealed the maximum amount of genetic information contained in chicken liver mitochondrial DNA and strongly suggest that chicken liver mitochondrial DNA is not only homogeneous in location and base composition, but also in base sequence. Given that chicken liver mitochondrial DNA is packaged into molecules of 15,000 base pairs, it seems reasonable to assume that 15,000 base pairs represent the maximum amount of genetic information available in chicken liver mitochondrial DNA. In this context, the maximal should be emphasized, as the experiments by Borst et al. (1967b) do not rule out redundancy in mitochondrial DNA. Dawid and Wolstenholme (1968a) received an indication that redundancy may be present in the mitochondrial DNA of Xenopus eggs. They examined the renatured mitochondrial DNA and found some open circles with a contour length well below 5 p and no evidence of single-stranded regions. While this may be the result of redundancy in the mitochondrial DNA of Xenopus, Dawid and Wolstenholme (1968a) point out that only a very small fraction of the DNA appeared as small circles and "these may be the product of renaturation of a type". , which contaminated the mitochondrial DNA preparations to a small extent”. Another point to note is that the level of matching accuracy required for renaturation is not complete. Therefore, the renaturation experiments discussed above do not rule out microheterogeneity in the mitochondrial DNA population due to point mutations or small insertions or deletions. Such microheterogeneity can only be ruled out by demonstrating its absence in mitochondrial DNA gene products. Although no quantitative studies of renaturation have been done with mitochondrial DNA from sources other than chicken liver, there is no reason to doubt that the rapid renaturation in all these cases is also a result of the low complexity of these DNAs3. however, there is great interest in using quantitative renaturation studies to determine the genetic information content of mitochondrial DNA from lower organisms such as yeast, and the rather limited renaturation experiments of Borst et d. (1967b) with mitochondrial DNA from higher organisms. 3 DuBuy et al. (1966) reported that DNA from mouse brain nuclei was only 20% renatured and concluded from this result that mouse mitochondrial DNA is comparatively heterogeneous in base sequence. Since in the mitochondrial DNA preparations of Du Buy et al. (1966) has not been proven at all, a more reasonable interpretation of their result is that their "mitochondrial" DNA preparations contained 20% mitochondrial DNA and 80% nuclear DNA. This interpretation is supported by two observations. Mouse mitochondrial DNA is completely renatured (Borst and Ruttenberg, 1966a; Flamin et al., 1966; Sinclair and Stevens, 1966), and DNA preparations extracted from mouse brain mitochondria contain the same circular molecules as mitochondrial DNA in mice. electron micrographs of mouse liver DNA (Sinclair et al., 1967b).

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XII. Evolution of mitochondrial DNA and the relationship between mitochondria1 and nuclear DNA A quantitative analysis of the sequence homology of mitochondrial DNA from different organisms and the homology of mitochondrial and nuclear DNA requires quantitative renaturation experiments. Only one of these studies has been published. Du Buy and Riley (1967) studied the hybridization of C14-labeled mouse brain nuclear DNA fragments with mouse brain nuclear or "mitochondrial" DNA immobilized on nitrocellulose membrane filters. While 23.2% of the nuclear DNA bound to nuclear DNA filters, 12.2% bound to "mitochondrial" DNA filters. Although Du Buy and Riley (1967) conclude from these results that 46% of all nuclear base sequences are represented in mouse brain mitochondrial DNA, we prefer to conclude (cf. Dawid and Wolstenholme, 1968a,b) that 46% of " " The DNA One authors' preparation consisted of nuclear D NH. The renaturation data from Du Buy et al. (1966) suggest an even greater degree of contamination for these DNA preparations, as mentioned in the previous section. Dawid and Wolstenholme ( 1968a,b) performed a series of sophisticated qualitative hybridization experiments using the "coupling" phenomenon first studied by Britten and Waring (1965). These authors showed that renaturation leads to the formation of very high molecular weight DNA complexes over very long periods of time, as stretches of single strand remaining in partially renatured molecules hybridize with complementary stretches in other partially renatured molecules.When two DNAs share sequences, they form a common complex. The minimum degree of sequence complementarity required for a common complex is probably identical to the number of base pairs required for the formation of a stable duplex at the annealing temperature, i.e. about 12 base pairs (Niyogi and Thomas, 1967) . Completely unrelated DNAs, such as nuclear DNA from plants and animals, do not form a common complex during co-annealing (Britten and Waring, 1965). The complexes are detected by analytical centrifugation on a CsCl equilibrium gradient, and as the molecular weight of the complexes is very high, very sharp bands are obtained, allowing discrimination of DNA with small density differences. When this method was used, no inhibition was detected between nuclear and mitochondrial DNA from X. laeuis and Rana pipiens or between Xetzopm and mitochondrial DNA from yeast (Dawid and Wolstenholme, 1968a,b). However, a common complex was found between co-annealed Xennpus mitochondrial DNA (native density = 1.702 gm./cni.3) and chicken liver mitochondrial DNA (native density = 1.709 gm./cm.). These experiments rule out a general large-scale homology of mitochondrial and nuclear DNA of the type

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predicted by Du Buy and Riley (1967), but would be consistent with the presence of one or more master copies of mitochondrial DNA in the nucleus (Dawid and Wolstenholme, 1968a,b). Co-annealing of chicken and frog mitochondrial DNA proves that these two DNAs share at least a 12 base pair sequence if we accept the rather extensive evidence for the specificity of the co-annealing reaction. This sequence complementarity is unexpected, as these DNAs differ by up to 7 mg/cm3 in their CsCl buoyant density, while both are considered homogeneous in base composition due to the sharpness of their thermal transition profiles (Borst et al., 1967b; Tewari and others, 1766).

XIII. Genetic function of mitochondrial DNA1 A. INTRODUCTION The putative genetic function of mitochondrial DNA has been explored in five ways. (1) Quantitative rates of DNA-DNA renaturation were determined by Borst et al. (1967a,b) to determine the information content of mitochondrial DNA. (2) RNA components complementary to mitochondrial DNA have been identified in several laboratories and further analysis of these RNA components has been used to discover whether mitochondrial DNA encodes ribosomal RNA and/or tRNA and whether mitochondrial mRNA is only in matrix space mitochondrial is translated or is also exported to the cytosol. (3) Attempts have been made to identify the products of mitochondrial protein synthesis. alive and that? in vitro, in the hope that proteins synthesized in the mitochondrial matrix space would be encoded by mitochondrial DNA. (4) The range of mitochondrial enzymes that could be specified by mitochondrial DNA has been reduced by the location of the cytochrome c structural gene in yeast nuclear DNA and by the fact that several mitochondrial enzymes are still expressed in cytoplasmic mutants of yeast in This genetic information found in mitochondrial DNA has probably been lost entirely. (5) Attempts have been made to correlate changes in the amino acid sequence of certain mitochondrial proteins with changes in the base sequence of mitochondrial DNA.

The results of DNA-DNA renaturation studies, which indicate that the maximum information content of chicken liver mitochondrial DNA is that contained in a molecule of 15,000 base pairs, were discussed in Section XI. The experiments related to items (2)-(5) are summarized in this section.

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B. DNA-RNA Hybridization Experiments In principle, DNA-RNA hybridization experiments should be able to answer the following questions: (1) Are there RNA species in the cell, inside or outside the mitochondria, with a specific sequence? or fundamentals complementary to it? of mitochondrial DNA? Is the complementary mitochondrial RNA mRNA, ribosomal RNA, or tRNA? (2) Do mitochondria contain RNA species complementary to nuclear DNA? Are these species unique or do they occur both inside and outside mitochondria? The second problem was addressed by Humm and Humm (1966) by hybridizing mitochondrial RNA 1 and nuclear RNA from mouse embryos stained for 20 hours with P3β with mouse nuclear DNA. Both mitochondrial and nuclear RNA combined with nuclear DNA to the same degree, and in competitive experiments, cold mitochondrial RNA competed even more efficiently than cold nuclear RNA for the sites occupied by PZ-tagged nuclear RNA on the DNA. Humm and Humm conclude "that at least a portion of mitochondrial RNA shares base sequences with nuclear RNA". However, the competition experiments, taken at face value, actually show that all major nuclear RNA species labeled in a 20-hour pulse are represented as the major RNA components in mitochondria. We cannot accept this conclusion for two reasons. First, it is highly unlikely that mRNAs for all extramitochondrial proteins are present in the mitochondrial matrix space; Second, Church and McCarthy (1967) showed that large cytoplasmic RNA competes ineffectively with nuclear RNA for sites on nuclear DNA, and that a significant portion of nuclear RNA does not appear to be present in cytoplasmic RNA. It is possible that Humm and Humm's (1966) results are due to false hybridization and false competition, since neither hybridization specificity nor competition specificity has been demonstrated by adequate control experiments. However, other artifacts cannot be ruled out. The issue of the presence or absence of RNA copies of mitochondrial DNA in the cell was investigated in three laboratories. Suyama (1967) isolated two RNA fractions from the mitochondria of Tetvuhymenu. One showed the sedimentation properties of tRNA, the other fraction, named pRNA, was found in the pellet after centrifugation of a mitochondrial lysate for 2 hours at ~0.00.00 x g. pRNA was pelleted through sucrose gradients as a 1:1 mixture of 18-S and 14-S components. pRNA hybridized with mitochondrial DNA up to a plateau of 6.8% RNA/DNA, while the low hybridization of the sRNA fraction with mitochondrial DNA could be explained by the presence of pRNA fragments in the sRNA fraction. It was not possible to detect a competition between post-mitochondrial RNA and pRNA for mitochondrial DNA. Although somewhat binding

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from mitochondrial pRNA and tRNA to nuclear DNA, whose validity was considered doubtful due to high blank values. Two arguments (see Suyama, 1967) indicate that the pRNA is the ribosomal RNA of Tetrahymena mitochondria. pRNA is the main component of RNA and represents about 70% of all mitochondrial RNA. The pRNA does not compete with the RNA synthesized by Tetrahymena mitochondria in vitro, which precedes the absence of viral DNA in this fraction. The membrane is probably fragmented into the mRNA of mitochondrial proteins (see Suyama and Eyer, 1968). Assuming that the pRNA is indeed ribosomal RNA, Suyama (1967) calculates that the mitochondrial DNA of Tetvahymena has a molecular weight of 30 x 106, assuming that the plateau of 6.8% RNA/DNA is correct, that the total molecular weight of RNA in mitochondrial ribosomes is 2 x 10° Daltons and that there is only one copy of this RNA per DNA molecule. In our opinion, Suyama's experiments provide strong evidence that, at least in Tetrahymena, mitochondrial DNA encodes mitochondrial ribosomal RNA. Fukuhara (1967, 1968) investigated the existence of RNA units in yeast that are complementary to yeast mitochondrial DNA. He showed that RNA from aerobic cells labeled with P32 over many generations hybridized to mitochondrial DNA about twice as well as to RNA from anaerobic cells, whereas cold RNA from anaerobic cells competed less effectively with P32 RNA from anaerobic cells. than with cold RNA from aerobic cells. . None of these differences were observed in hybridizations with nuclear DNA. Fukuhara concludes from these results that preferential transcription of mitochondrial DNA occurs during respiratory adaptation. The maximum hybridization achieved in these experiments was approximately 2.5% RNA/DNA and no plateaus were reached in the hybridization or competition experiments. Therefore, it cannot be concluded that aerobic cells contain RNA fractions complementary to mitochondrial DNA that are absent in anaerobic cells. From a membrane-rich fraction containing mitochondrial marker enzymes, Fukuhara (1967) extracted a metabolically stable RNA that hybridized to a maximum of 1.5% (without plateauing) with mitochondrial DNA. On sucrose gradients, RNA complementary to mitochondrial DNA sedimented in a broad band with a maximum sedimentation coefficient of 1 2 S. Although these findings are consistent with the hypothesis that RNA from yeast mitochondrial ribosomes is specified by mitochondrial DNA, more experiments are needed. needed to test this. Recently, Attardi and Attardi (1967) reported the isolation of HeLa cells from an extramitochondrial RNA fraction that specifically hybridized at high levels with the cytoplasmic DNA of HeLa cells. The RNA associated with extramitochondrial cytoplasmic membranes and after a 30 min H-uridine pulse, approximately twice as much newly synthesized RNA appeared in the RNA.

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membrane-associated RNA region as in the free polysome region, indicating their quantitative importance. The authors propose that this RNA is an mRNA synthesized using mitochondrial DNA as a template and exported to the cytoplasm. The obvious conclusion is that the limited amount of information available in mitochondrial DNA also contributes to the synthesis of extramitochondrial components. Unfortunately, the evidence on which this interesting conclusion was based is not complete. The "cytoplasmic" DNA used in the hybridization experiments was not identified as mitochondrial DNA by any criteria, and the absence of viral DNA in this fraction was not verified. The membrane fraction containing the rapidly labeled W I ~ Solo RNA is characterized by its density on sucrose equilibrium gradients. When the cells were homogenized in the absence of Mg++, the RNA fraction was found to have a density of 1.180 g/cm3 versus a density of the mitochondrial fraction (identified by A415) of 1.195 gm. /~m. ~ when; Mg++ was present during homogenization, profiles of Δ260 and acid-insoluble radioactivity were consistent with a peak of 1190 g/cm³. In our opinion, such characterization of mammalian cell fractions is not enough: first, the A of the mitochondrial suspensions is largely attributed to non-specific light scattering and not to the cytochrome c gamma band, as the authors seem to assume. Second, a density of 1180 g m . /~m.no~ agrees very well with the reported density of 1.13 g/cm3 for the smooth endoplasmic reticulum of rat liver (D.F. Parsons, 1966). Thus, even if the band at 1190-1195 is indeed mitochondrial, it remains difficult to rule out that the shoulder at 1180 represents newly synthesized mitochondria with a higher outer membrane to inner membrane ratio than the rest of the mitochondrial population. [The equilibrium density of the pure outer membrane fraction of rat liver mitochondria is 1.13 g/cm3 in sucrose (D.F. Parsons, 1966).] This explanation is consistent with the observation by Attardis (1967) that the "membrane-associated RNA bulge" already sedimented after 10 min of centrifugation at 8100 x 6. Sedimentation of most microsomes under these conditions would be quite unexpected. Finally, it should be noted that the complete and immediate inhibition of uridine incorporation into the membrane-bound RNA fraction by actinomycin D, during incorporation into polysomal RNA, report does not support a mitochondrial origin of this RNA von Neubert et al (1968b) found that intact mammalian mitochondria are completely impermeable to actinomycin, resulting in complete resistance to this inhibitor of mitochondrial RNA synthesis in vivo, but it cannot be ruled out that the mitochondria of HeLa cells differ in this respect. Given these discrepancies, two alternative explanations for the Attardis results should be seriously considered: (1) The rapidly labeled RNA fraction is an mRNA complementary to a DNA virus present in cultured HeLa cells.

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DNA from this virus is present in the cytoplasmic DNA preparations used in the hybridization experiments. (2) The rapidly labeled RNA fraction is present in newly synthesized mitochondria, which are permeable to actinomycin. Although these alternatives have not been ruled out, the concept that mitochondrial mRNA is exported to the cytosol remains unproven. C. THE PRODUCT OF MITOCHONDRIAL PROTEIN SYNTHESIS Incorporation of amino acids into proteins by isolated mitochondria is inhibited by actinomycin (Kroon, 1965; Neubert et al., 1968b) whenever mitochondria are damaged to make them permeable to the drug. This led to the hypothesis that intracellular synthesis of mitochondrial proteins depends on the continuous generation of mRNA synthesized in mitochondrial DNA (Kroon, 1965, 1966a,b). If this hypothesis is correct, identification of the products of protein synthesis by isolated mitochondria will directly provide a list of proteins specified by mitochondrial DNA. Unfortunately, amino acid incorporation occurs through isolated mitochondria into insoluble proteins (Roodyn et al., 1962; Truman, 1964; Wintersberger, 1965; Bronsert and Neupert, 1966; Wheeldon and Lehninger, 1966) associated with the inner membrane (Neupert et al. ., 1967, 1968), and all attempts to achieve incorporation into well-defined proteins have produced ambiguous or negative results. Kalf and GrGce (1964) recovered a large fraction of the amino acids incorporated into a purified fraction of "contractile protein" by isolated calf heart mitochondria. As the existence of the '1 contractile protein in mitochondria is highly doubtful (see Conover and Biriny, 1966), the significance of Kalf's results is unclear. Labeling of protein fractions with the electrophoretic mobilities of mitochondrial F, ATPase, and F4 coupling factor has been reported by Work and co-workers (Haldar et d., 1966; Work, 1967, 1968) (see review by Pullman and Schatz, 1967) . . However, examination of his experimental data shows that the radioactivity has spread throughout the electropherogram. In the absence of clear fractionation, each fraction is labeled and the specificity of the labeling has yet to be demonstrated. Other studies have shown that amino acids are not incorporated into either cytochrome c (Roodyn et al., 1962) or cytochrome aag (Kadenbach, 1968). Although future work in this area may be more successful, two fundamental objections to this approach regarding the genetic function of mitochondrial DNA can be mentioned: (1) There is no evidence that isolated mitochondria synthesize complete proteins. The probability of identifying incomplete proteins is very low when more than one protein is produced. ( 2 ) Given the limited amount of genetic information stored in mitochondria,

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Chondrial DNA Most of the structural genes for mitochondrial protein synthesis must be present in nuclear DNA. Coordination of mitochondrial and nuclear DNA contributions to the synthesis of a mitochondrion requires a system of inducers and repressors. Mitochondria may be lost or inactivated during mitochondrial isolation, and the protein synthesized by isolated mitochondria may not be representative of proteins synthesized under the direction of mitochondrial mRNA in the intact cell. D. IDENTIFICATION OF MITOCHONDRIAL PROTEINS ENCODED BY NUCROSOUS OR SYNTHETIC DNA OUTSIDE THE MITOCHONDRIA In principle, the identification of mitochondrial components encoded by mitochondrial DNA could also be done by deletion, i. H. identifying components specified by nuclear DNA, as structural genes are unlikely to be located in nuclear DNA, they are identically represented in mitochondrial DNA. The only mitochondrial protein for which this has been done is cytochrome c. Sherman et al (1966) showed conclusively that a mutation in the yeast nuclear gene CY1 results in a change in the amino acid sequence of cytochrome c iso-1, the major cytochrome c of yeast. mRNA for cytochrome c is translated outside mitochondria, at least in the rat liver, since the pulse-labeling experiments of Gonzilez-Cadavid and Campbell (1967a,b) showed that cytochrome c from nascent rat liver is found for the first time in the microsomal fraction. and then transferred to the mitochondria. Similar results were reported by Freeman et al. (1967) with cancerous ascitic tumor cells. Following this approach, Beattie et al. (1966) investigated the occurrence of labeled amino acids in the soluble and membrane-bound protein fractions of mitochondria from various rat organs. The increase in specific activity of soluble proteins was somewhat less rapid than that of membrane-bound proteins and, based on this difference, Beattie et al. (1966) concluded that soluble mitochondrial proteins are synthesized in microsomes and then transferred to mitochondria, whereas membrane-bound proteins are synthesized in situ. However, it is unclear how much of this differential labeling is a result of the differential turnover of the two protein fractions, a possibility suggested by Beattie et al. It was excluded. (1966). It should also be emphasized that the site of synthesis of a mitochondrial protein does not necessarily define the site of its structural gene. Os Attardis (1967) asserted that mitochondrial mRNA is exported into the cytoplasm of HeLa cells, nor can it be ruled out by the now available evidence that nuclear mRNA is translated within the mitochondrial matrix space, as we established earlier (Borst et al., 1967a). We conclude that experiments similar to those by Sherman et al. (1966) for cytochrome c may limit the range of proteins that mitochondrial DNA could specify. Studying the intracellular localization of nascent mitochondrial proteins, although less

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concluded that genetic experiments can also provide useful information when studying specific proteins.

E. MITOCHONDRAL ENZYMES FOUND IN CYTOPLASMIC MUTANTS Petite SO F The recent demonstration that in cytoplasmic mutants of yeast the genetic information contained in mitochondrial DNA is completely lost (see Section IX) has experimental support for a different method of identifying the product delivered by deletion. . Mitochondrial proteins still present in cytoplasmic mutants must be specified by nuclear genes. The existence of organized membrane structures containing typical mitochondrial enzymes in cytoplasmic mutants of yeast was first reported by Linnane and Still (1956). Their results were published by Schatz et al. (1963) and others (Mahler et al., 1964; Mackler et al., 1965; Katoh and Sanukida, 1965; Clark-Walker and Linnane, 1967). The membrane structures consist of a double membrane lacking the cristae characteristic of wild-type aerobic yeast mitochondria (Linnane and Still, 1956; Yotsuyanagi, 1962a,b; Schatz et al., 1963). Typical mitochondrial enzymes detected include succinate dehydrogenase (Linnane and Still, 1956; Schatz et al., 1963; Mackler et al., 1965; Clark-Walker and Linnane, 1967), antimycin-sensitive NADH cytochrome C reductase, and D-lactate and lactate cytochronic c reductases (Mahler et al., 1964; Mackler et al., 1965). It was originally reported (Katoh and Sanukida, 1965) that the mitochondrial "structural protein" of small mutants was identical to that of wild-type yeast. However, further investigations (Tuppy and Swetly, 1965) using polyacrylamide electrophoresis and immunological studies revealed that the wild-type yeast structural protein consisted of several components, one of which was absent in small mutant cytoplasmic preparations. The ability of the yeast structural protein to bind ATP in an atractyloside-sensitive process has also been found in yeast mutants. However, the mutant structural protein lost its ability to bind nucleotides when cold extracted. The authors speculate that the small mutation causes the loss of a component present in the structural fraction of the protein and, therefore, induces the cold lability of the nucleotide binding. They refer to unpublished experiments by Schatz showing that ATPase (Fi) from mutated yeast mitochondria is cold-labile in situ, whereas the wild-type enzyme is cold-stable when bound to mitochondria. These experiments demonstrate that the mitochondrial structural protein fraction minus one component is present in yeast cytoplasmic mutants. Since structural proteins and enzymes in the strict sense above are likely to be restricted to mitochondria in yeast, they are likely to be specified by nuclear genes. Similar considerations apply to mitochondrial DNA polymerase and RNA polymerase, which were demonstrated in

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the heavy particle fraction of yeast cytoplasmic mutants by Wintersberger (1968). In addition to the enzymes listed above, aconite hydratase (Schatz et al., 1963), fumarate hydratase (Schatz et al., 1963; Clark-Walker and Linnane, 1967) and malate dehydrogenase (Clark-Walker and Linnane, 1967) have been detected in small cytoplasmic. Since the presence of two malate dehydrogenase isoenzymes in yeast, one restricted to the cytosol and the other found predominantly in mitochondrial fractions, was suggested by Witt et al. (1966) it must be shown that the malate dehydrogenase activity found in the mutant yeast cannot be fully attributed to the cytosolic isoenzyme before it can be concluded (cf. Clark-Walker and Linnane, 1967) that this enzyme is mediated by nuclear DNA is specified . founded. An indication that this conclusion is correct for fumarate hydratase can be found in the observation by Schatz et al. (1963) that the heavy particle fraction of the mutant yeast contains this enzyme (specific activity 24% of the wild-type control). Furthermore, wild-type yeast aconite hydratase activity was recently shown in this laboratory to have the same distribution in cell fractionation studies as cytochrome oxidase. This suggests that yeast aconite hydratase is a unique mitochondrial enzyme and, as the enzyme is found in small amounts in the cytoplasm, it must be specified by nuclear DNA. Only cytochromes aa3, b and c1 were conclusively shown to be absent in Rho cells. However, the conclusion (see Roodyn and Wilkie, 1968; Linnane, 1968) that these cytochromes are encoded by mitochondrial DNA is unwarranted, since a protein encoded by mitochondrial DNA can exert very tight control over the synthesis of these cytochromes. cytochromes. The situation may be similar for succinate dehydrogenase in wild-type anaerobic cells grown in the absence of a fatty acid source. The suppression of succinate dehydrogenase synthesis under these conditions is so effective that less than 1% of the activity found in aerobic cells is present (Lukins et al., 1966). It should be noted in this regard that the synthesis of cytochrome aa3 appears to be dependent on the synthesis and function of other cytochromes (Reilly and Sherman, 1965). For example, the synthesis of cytochrome aa3 is blocked in yeast strains grown in the presence of antimycin A, which inhibits electron transport between cytochromes b and c without affecting the synthesis of cytochromes b or c (Ycas, 1956). Therefore, in principle, the study of mitochondrial proteins in cytoplasmic mutants can only provide information about proteins not specified by mitochondrial DNA. Rabinowitz et al. made an attempt. (1968) and Yu et al. (1968) to obtain direct evidence of the nature of proteins specified by mitochondrial DNA by examining the induction of cytochrome synthesis by oxygen in anaerobic yeast in the presence of cycloheximide. Mitochondrial protein synthesis1 is not

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affected by this drug while effectively inhibiting extramitochondrial protein synthesis. Rabinowitz et al. (1968) did not find cytochrome induction in the presence of cycloheximide, while Yu et al. (1968) found measurable cytochrome oxidase synthesis at cycloheximide concentrations that completely blocked growth and cytochrome c synthesis. However, there are some doubts whether the study by Yu et al. (1968) prior to the addition of cycloheximide during cell harvesting (see discussion following Yu et al., 1968). The investigation of cytochrome synthesis by spectral analysis of cell suspension represents a simple but insensitive test system. . 1:.

CORRELATION OF CHANGES IN MITOCHONDRIAL PROTEINS WITH CHANGES IN MITOCHONDRIAL DNA

Ideally, proteins specified by mitochondrial DNA should be identified by correlating a change in the amino acid sequence of the protein with a change in the base sequence of the mitochondrial DNA. In practice, it can be difficult to select the necessary mutants for this analysis and only two examples have been reported. Woodward and Munkres (1966, 1967) and Munkres and Woodward (1966) studied the amino acid composition of mi-1 and mi-3 mutants of N. crarsn. Both mutants are characterized by respiratory failure due to lack of cytochromes. Both mutations show cytoplasmic inheritance. The mi-1 structural protein contained one fewer tryptophan residue and one fewer cysteine residue than the wild-type structural protein, while the nzi-3 structural protein contained only one fewer tryptophan residue than its wild-type counterpart. wild. Woodward and Munkres (1966) explain the pleiotropic character of the mi-1 and mi-3 mutants by assuming that the structural protein provides the scaffolding to which all membrane-bound mitochondrial enzymes attach. Alteration of this scaffold as a result of an amino acid change in the structural protein results in defective binding or no binding, resulting in non-functional mitochondria. More recently, Woodward and Munkres (1967) extracted structural proteins from cellular fractions other than mitochondria. The surprising result obtained was that all cell fractions contained enormous amounts (40 Cj, of all cytosol proteins) of structural protein of very similar composition. Structural proteins extracted from nuclei, mitochondria, microsomes and cytosol were indistinguishable in amino acid composition and immunological behavior, and their peptide maps were very similar. The structural proteins extracted from all cell fractions of the mi-1 mutants were found to contain one more tryptophan and one less cysteine than their wild-type counterparts. Woodward and Munkres

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(1967) conclude from these results that the structural proteins of all cell membranes are identical and are encoded by mitochondrial DNA. Although the experimental evidence presented by Woodward and Munkres for this view is quite extensive, three points remain doubtful. (1) Recent work in the laboratories of Green (Green and Perdue, 1966) and Racker (Fessenden et al., 1966) suggest that the structural protein is responsible for the reconstitution of subchondrial particles that catalyze oxidative phosphorylation and the nature and function is not Requirement of this protein in mitochondria is currently unclear. ( 2 ) According to Allmann et al. (1967) described the structural protein prepared by the method of Criddfe et al. (1962), used by Woodward and Munkres (1966), is not a homogeneous protein and still contains about 20-2570 contaminating proteins as judged by polyacrylamide gel electrophoresis. It is difficult to see how significant amino acid composition can be achieved with an impure protein. (3) Tuppy and Swetly (1968) recently reported that the structural protein of mitochondria from 5. cerezisiae consisted of several components, one of which was missing in a cytoplasmic petyl mutant thought to lack mitochondrial DNA). No structural protein, defined as a protein capable of binding ATP in an atractyloside-sensitive manner, can be extracted from cellular fractions other than mitochondria in wild-type yeast. As it is difficult to imagine that a fundamental aspect of cell physiology, such as membrane protein synthesis, could be organized differently in related sac fungi, Tuppy and Swetly's results with Saccharomyre.1 are difficult to reconcile with Woodward's. and Munkres. (1967) with Neuro.rporn.

Clarification of these three points of doubt will be necessary before we can accept the conclusion that mitochondrial structural proteins are encoded by mitochondrial DNA. Wilkie et al. and Linnane (Wilkie et al., 1967; Thomas and Wilkie, 1968a,b; Roodyn and Wilkie, 1968; Wilkie, 1968; Linnane, 1968) took a different approach (see also Section XIV). in their yeast studies. They isolated a series of mutants resistant to antibiotics such as chloramphenicol, which inhibit mitochondrial protein synthesis (see Kroon, 1965, 1966a; Huang et al., 1966; Borst et al., 1967a; Clark-Walker and Linnane, 1967). . In the case of erythromycin resistance, mutants were obtained that showed cytoplasmic inheritance, suggesting that resistance was controlled by a gene product of mitochondrial DNA1. In principle, resistance to erythromycin could be due to: impermeability of the cell membrane or mitochondrial membrane, presence of an enzyme that inactivates the drug, or alteration of the mitochondrial ribosome. Thomas has to decide between these alternatives

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and Wilkie (1768b) created erythromycin-resistant mutants in strict anaerobiosis in the absence of fatty acids. Under these conditions, mitochondrial profiles disappear completely or almost completely and mitochondrial membranes apparently cannot be synthesized (see Section IX). Two erythromycin-resistant nuclear mutants completely lost their resistance under these conditions, suggesting that the low erythromycin permeability of the mitochondrial or cell membrane was responsible for the resistance in these cases. On the other hand, the conclusion that resistance to erythromycin in cytoplasmic mutants is due to an alteration in the mitochondrial protein synthesis system itself1 is supported by the observation that the incorporation of amino acids by mitochondria isolated from one of these mutants was also resistant. versus erythromycin in vitro (Linnane, 1968). In bacterial systems, erythromycin is now believed to act on the ribosome at a location close to, but not identical with, the target of chloramphenicol (see Cundliffe and McQuillen, 1967). Resistance to these antibiotics is likely due to a change in a ribosomal protein. Therefore, Wilkie's experiments suggest that at least one of the mitochondrial ribosomal proteins is encoded by mitochondrial DNA. Isolation and characterization of drug-resistant yeast mutants is comparatively straightforward, and analysis of mitochondrial genetics using drug-resistance markers should be one of the most promising avenues for analyzing the genetic function of mitochondrial DNA currently available. see Wilkie et al., 1967; Wilkie, 1968).

G. FINAL CONSIDERATIONS From the experimental results discussed in this section, it is clear that the contours of the genetic function of mitochondrial DNA are beginning to emerge. There are no more than 15,000 base pairs available in mammals, and it is clear that they can only specify a small fraction of mitochondrial components. Suyama's results suggest that these components in Tetrabymena include mitochondrial rRNA but not tRNA, whereas Thomas and Wilkie's genetic experiments in yeast imply a ribosomal protein. Taken together, these results suggest that complete mitochondrial ribosomes can be specified by mitochondrial DNA. E. cnlr ribosomes contain about 5000 nucleotides (see Stanley and Bock, 1765) and at least 50 different proteins (Moore et al., 1766; Traut et al., 1967). Regulated synthesis of these components requires at least 30,000 base pairs, unless ribosomal proteins are extraordinarily small, which builds on the work of Moore et al. it is highly unlikely. (1766) with E. coli. While this amount of genetic information may be present in yeast and other lower organisms, it is already double that found in chicken liver mitochondria. It is possible that some of the mitochondrial ribosomal proteins are specified by

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Nuclear genes, but speculation on the subject is not very useful until it is known whether mammalian mitochondrial rRNA is also complementary to mitochondrial DNA, as is the case in Tetvabytnetza. Other candidates for the role of mitochondrial gene products include mitochondrial inner membrane proteins, similar to a component of the heterogeneous fraction of structural proteins, cytochromes aa3, b, cl, and unknown extramitochondrial proteins specified by mRNA exported to the cytoplasm. In our opinion, none of them are yet supported by conclusive evidence. All other mitochondrial components must be specified by nuclear genes, and although only the cytochrome c iso-1 structural gene in yeast has been identified with certainty as a nuclear gene, good indirect evidence suggests that in yeast the structural genes for succinate dehydrogenase, antimycin Sensitive NADH cytochrome c reductase, D-lactate and L-lactate cytochrome c reductase, aconite hydratase, mitochondrial fumarate hydratase, mitochondrial RNA polymerase, and mitochondrial DNA polymerase also belong to this class. Several recent reviews have considered possible pathways by which these proteins might find their place in mitochondria (Borst et al., 1967a; Roodyn and Wilkie, 1968; Kadenbach, 1968).

XIV Addendum Upon completion of this review, the information summarized below is available. References cited in this appendix appear at the end of the reference list. The two bands found in alkaline CsCl for a variety of mitochondrial DNAs have been identified as complementary strands for mitochondrial DNA from human placenta (Curneo et al., 1968) and rat and chicken liver (Borst et al., 1968). 1969). . Complementary strands also differ in density in neutral CsCl, but quantitatively aggregate when present in the same gradient. The lightest chain in alkaline CsCl of chicken mitochondrial DNA strongly interacts with poly-U and poly-IG; the heavier chain acts exclusively as a messenger chain in the rat liver (Borst and Aaij, 1969). Through mixing experiments, Wolstenholme and Dawid (1968) showed that the mitochondrial DNA circles of two urodele amphibians are 15% smaller than those of two anuran amphibians. The earlier conclusion that most of the DNA in Xeizopus eggs is mitochondrial was supported by Baltus et al. criticized. (1968) who suggest that yolk DNA, unrelated to mitochondrial DNA, is the basis of egg DNA. Suyama and Miura (1968) presented convincing evidence that circularity is not a property of all mitochondrial DNA. They showed that Tetrahymena mitochondrial DNA consists of a homogeneous population of linear molecules of 17.6 p ZL-0.08 (SE). Based on sedimentation studies of

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Sonenshein and Holt (1968) the molecular weight of mitochondrial DNA from slime mold (Physavum) could be of the same order of magnitude. Wolstenholme and Gross (1968) obtained heterogeneous linear DNA up to 60p in length from red bean mitochondria, Pba.reol?Ls udgavis. Different proportions of heterogeneous open circles ranging in size from 1 to 10 p have been observed in yeast mitochondrial DNA in different laboratories, but not in this laboratory (Shapiro et al., 1968; Gukrineau et al., 1968; Avers et al., 1968 ; Bernardi et al., 1968), and the doubts expressed in our article about the reality of these circles proved to be unfounded. Furthermore, Shapiro et al. (1968) suggested that some linear molecules have sticky ends that can interact to create circles with hydrogen bonds. It is not clear how the orderly replication and separation of such a heterogeneous set of molecules is effected in intact yeast cells. Subsequent studies on oligomers of mitochondrial DNA confirmed the absence of circular dimers in normal animal tissues, while more detailed measurements now indicate that 10-16% of total mitochondrial DNA is present in all normal tissues examined, including those from mouse embryos in chain . oligomers (Piko et al., 1968; Clayton et al., 1968; Hudson and Vinograd, 1969). The elusive replicating circles were finally identified by Kirschner et al. (1968) selected a large number of circular DNA molecules from rat liver mitochondria. Approximately 1 in 600 molecules was a replication circle. This proves that mitochondria1 D N A replicates within mitochondria. Synchronized mitochondrial DNA replication, which occurs immediately before nuclear DNA synthesis, was studied by Smith et al. observed in Sachhavomyces. (1968). A mitochondrial DNA polymerase, which differs in properties from the nuclear polymerase, was partially purified from rat liver by Meyer and Simpson (1968), while a mitochondrial DNA ligase from rat liver was detected in this laboratory. Karol and Simpson (1968) reported that the incorporation of deoxyribonucleotides observed in mitochondria isolated from rat liver is attributable to replicative DNA synthesis rather than repair synthesis. Attardi and Attardi (1968) presented additional experiments that they interpret as supporting their conclusion that part of mitochondrial mRNA1 is translated on extramitochondrial ribosomes1 in HeLa cells. Sebald et al. (1968, 1969). Yeast continues to provide important information about mitochondrial biogenesis. Further studies of UV induction of small cytoplasmic mutation in S. cerevisiae by Matoudas and Wilkie (1968) again suggest that in anaerobic yeasts only one hereditary unit is present, whereas in aerobic cells the number of genetically effective copies is greater than one, but many more. less than the number

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Mitochondria present in these cells. On the other hand, studies by Swift and Wolstenholme (1969) and Schatz (personal communication) showed that a large number of mitochondrial profiles containing mitochondrial DNA are present in anaerobic yeasts, regardless of growth conditions. The absence of mitochondrial profiles in Wallace and Linnane's (1964) micrographs appears to be due to the use of permanganate staining, which does not stain mitochondrial profiles1 when cells are cultured in media low in ergosterol and fatty acids. Linnane et al., (Linnane et al., 1968) also presented extensive studies on the cytoplasmic inheritance of erythromycin resistance. Contrary to Thomas and Wilkie (1968), they conclude that the cytoplasmic factor for erythromycin resistance and the Rho factor may not be identical. Wintersberger and Viehhauser (1968) discussed extensively the relationship between mitochondrial RNA and mitochondria1 and nuclear DNA in yeast. They showed that ribosomal RNA components from yeast mitochondria specifically hybridized at a plateau of 0.04 pg RNA per microgram of DNA with wild-type yeast mitochondrial DNA, but not with mitochondrial DNA from a specifically hybridized small RNA component. at a value well above 0.01 pg RNA per microgram of DNA with yeast nuclear DNA and concludes that cistrons for mitochondrial ribosomal RNA are represented in the nuclear genome. ACKNOWLEDGMENTS We thank Professor E.C. Slater for his advice and help with manuscript preparation; to Dr. J. M. Tager for providing proof of articles read in the Round Table on Biochemical Aspects of Mitochondria prior to publication; and several colleagues for permission to reproduce figures or tables from their work. The authors' experimental work was supported in part by grants from the Netherlands Foundation for Chemical Research (SON), with financial support from the Netherlands Organization for the Advancement of Pure Research (ZWO) and the Jane Coffin Childs Memorial Fund for Research.

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FURTHER REFERENCES Attardi, B. and Attardi, G. (1968). Pror. Natl. Air-ad. Si-i. FOR US. 61. 261. Avers, C.J. (1968). Please-. National Academy sr yo FOR US. 61, 90. Baltus, E., Hanocq-Quertier, J. and Brachet, J. (1968). pror. null. Science Academy FOR US. 61, 469. Bernardi, G., Carnevali, F., Nicolaie R.A., Piperno, G. and Tecce, G. (1968). J, Mol. biol. 37, 493. Borst, P.; C. (1969). Biography of Biochemistry. crown of resolution. 34, 358. Borst, P. and Ruttenberg, G.J.C.M. (1969). European Federation of Biotechnology. Sor.. 6to. Reunion, Madrid, summary. In the press. Clayton, D.A. (1968). Nature 220, 976. Corneo, G. (1968). 1 mol. biol. 36, 419. GuPrineau, M., Grandchamp, C., Yotsuyanaji, Y. and Slonimski, P.P. (1968). com/. To loan. 266, 1884, 2000. Hudson, B. and Vinograd, J. (1969). Nature 221, 332. http://dx.doi.org/10.1037/0033-2909.101.2.213 Carroll, M.H. & Simpson, M.V. (1968). Science 162, 470. Kirschner, R.H.. Wolstenholme, D.R. and Gross, N.J. (1968). perc. well Sri Academia. FOR US. 60, 1466. Linnane, A.W. (1968). Itch-. ntl. Arad. science TO US 59, 903, 1288. Maroudas, N. G. and Wilkie, D. (1968). biobrain. Biography. Acid 166, 681. Meyer, R.R. and Simpson, M.V. (1968). pror. And me. and Science FOR US 61, 130. Peak. L (1968). Professional[-. National Academy of Sciences for us. 59, 838. Sebald, W., Beecher, T., Olbrich, B., and Kaudewitz, F. (1968). FEBS Letters 1, 235. Sebald W, Hofstotter T, Hacker D and Beecher T (1969). FEBS Letter 2, 177. Shapiro. http://dx.doi.org/10.1037/0021-843X.116.1.101 Grossman, L.I., Marmur, J. & Kleinschmidt, A.K. (1968). jmol. Tender 33, 907. Smith, D., Taurus, D.. Schweizer, E., and Halvorson, H. O. (1968). perc. Nacional had science for us. 60, 936. Sonenshein, G.E. and Holt, EC (1968). biorhem. Biography. Commons Resolution 33 , 361 . perc. National Academy Science A US 60, 2 3 5 . Swift, H. and Wolstenholme, D.R. (1969). "Handbook of Molecular Cytology", (A. Lima-de-Faria, ed.) North-Holland Publ., Amsterdam. In the press.

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Thomas, D.Y. and Wilkie, D. (1968). Biorema. l3jophy.r. Resolution Maize 30, 368. Wallace, P.G. e Linnane, A.W. (1964). Nature 201, 1191. Wintersberger, E. and Viehhauser, GL (1968). Nature 220, 699. Wolstenholme, D.R. and Dawid, I.B. (1968). J. Célula B i d. 39, 222. Wolstenholme, D.R. and Gross, N.J. (1968). perc. Peru/. Acud. science US 61, 245.

Metabolism of KONRADKECK enucleated cells Srienrer Department of Biological Sciences, University of Avizona. T U C O Arizona? I, I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Introduction of the nuclear state. . . . . . . . . . . . . . . . . . . . . . A. Physical Enucleation B. RNA Syn III Inhibition. mRNA quantification. . FOR . Introductory notes. . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Direct Methods. . . . . . C. Relationship between mRNA and polysomes. . . . . . . D. Relationship between mRNA and protein synthesis. . . . IV. Impairment of mRNA and protein synthesis in anucleated cells A. Prokaryotic organisms B. Eukaryotic organisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Type of mRNA degradation. . . . . . . . . . . . . . . . . . . . . . . . . . . . FOR . enzymatic degradation. . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Beginning of decay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VISA. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . references . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

191 192 102 193 I oh 106

196 198 201 208 208 212 222 222 221 225 225

I. Introduction Studies on the metabolic activities of enucleated cells generally aim to understand the interactions between the cell nucleus and the cytoplasm. The issue has been discussed from this perspective in several reviews (Hammerling et al., 1959; Hammerling, 1963; Prescott, 1960a; Brachet, 1961). This article departs from this conceptual approach and focuses on the nucleated state itself. The term "nucleated" is here defined in the most general sense of the word and applies to all cellular systems in which the flow of genetic information from nuclear genes to the cytoplasm has been disrupted. Thus, the nucleated state can be initiated by physical nucleation and by natural nuclear degeneration, as well as by chemical inhibition of nuclear RNA synthesis. The liberal interpretation of the term "enucleation" avoids limiting the text to a few, often atypical, cells amenable to microsurgical enucleation, and also justifies the inclusion of prokaryotic organisms, which provide a wide range of relevant information on the subject. . The study of the nuclear state, as defined above, essentially involves the characterization of the metabolic changes that occur in cells that are progressively depleting their store of nuclear hereditary genetic information. Dependent on the lifetime of each messenger species and the stability of essential proteins for survival 191

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after complete degradation of their respective mRNA, sooner or later the cellular organization will collapse. This simplified model ignores several additional parameters, namely the replenishment of cytoplasmic ribosomes and tRNA by the nucleus, the nucleus' contribution of non-RNA products, and the existence of independent genetic elements in the cytoplasm. Due to the very low turnover rate of rRNA and the tRNA backbone (Section II, B, I), these RNA components become limiting only in enucleated cells, which contain extremely long-lived mRNA. Furthermore, little is known about the direct contribution of DNA-containing cytoplasmic organelles to overall protein synthesis in the cytoplasm. The chloroplast system was removed from consideration in this article, limiting the discussion mainly to heterotrophic organisms, and few proteins appear to be encoded in mitochondrial DNA1 (Roodyn et d., 1762; Woodward and Munkres, 1766; Kadenbach, 1967). . Despite its somewhat artificial nature, a nucleated cell system can provide valuable information about the post-transcriptional regulation of metabolism in general and protein synthesis in particular. The rate of synthesis of a given protein in the intact cell depends on the available amounts of mRNA, which in turn are determined by the rate of synthesis controlled at the gene level and the rate of degradation. The latter varies greatly between individual mRNA species for reasons that are currently not understood. The nucleated system is ideal for studying mRNA decay and the relationship between mRNA levels and the rate of protein synthesis. The system is equally suited to studying translational control mechanisms of a protein-specific or non-specific nature, without the added complexity of overlapping transcriptional regulation. Some facets of nuclear metabolism also affect cell differentiation. Certain metazoan cells naturally enucleate during their final stages of differentiation. This nuclear phase then represents the final stage in the acquisition of a specialized function. In a broader sense, any differentiated cell can be considered enucleated with respect to multiple repressed genes. The time interval between the onset of gene repression and its subsequent phenotypic expression depends on the half-life of the respective mRNA pools. On the other hand, gene activation during development by suppressing translation or by storing messenger substances in "masked" form may remain without immediate metabolic consequences.

11. Initiation of the anucleation state A. PHYSICAL ENUCLATION Microsurgical enucleation is the most reliable method of eliminating the nuclear genome of the cell. In addition, it allows precise synchronization of the

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Interruption of flow of mRNA into the cytoplasm. In appropriate cells, enucleation can be combined with reimplantation of another cell nucleus containing the same or a different genome. With the help of interspecific nuclear transplants, the experimenter is able to alter just a small part of the genome and examine the nucleated state of individual genes without, at least theoretically, interfering with the cell's basic functions. If there are allelic differences in the molecular structure of a given protein, the depletion of the original protein's specific messenger set and the accumulation of the new homologous set can be followed directly in an unchanged cell. This experimental approach was first tested in the unicellular alga Acetabulavia (Keck, 1960, 1961; Clauss, 1962; Schweiger et al., 1967). Unfortunately, few cell types are amenable to routine microsurgical enucleation. In the case of small cells, insurmountable difficulties arise when a relatively large number of enucleated cells are required for biochemical analysis. In rare cases, large-scale production of enucleated cells or cell fragments can be achieved by collective treatment of cell populations. A well-known example is the enucleation of sea urchin eggs by centrifugation (Harvey, 1956). Enucleation is not limited to eukaryotic cells; Anucleated fragments termed "minicells" were also obtained from an abnormal budding strain of Esrheiichia roli (Adler et al., 1967). Enucleation surgery almost always causes side effects, the consequences of which are difficult to assess. A more or less pronounced traumatic reaction occurs, possibly related to a transient disruption of cell permeability and an inevitable loss of a certain part of the cytoplasm. Other indirect effects could result from the preferential location of organelles or metabolites in the distant part of the cytoplasm. Effects of this type would be more pronounced in highly polar cells. B. INHIBITION OF F RNA SYNTHESIS 1. InhibitorJ

The antibiotic actinomycin D (AD) specifically and at relatively very low concentrations inhibits DNA-dependent RNA synthesis by binding to guanine bases in the minor groove of double-stranded DNA in the B configuration, thereby blocking RNA polymerase activity ( Kirk, 1960). ; Reich et al., 1961; Hurwitt et al., 1962; Goldberg et al.], 1962; Kahan et al., 1963; Hamilton et al., 1963; Rich, 1964). At appropriate concentrations, inhibition of Nozw synthesis of all RNA species is complete, but renewal of the tRNA-CpCpA-terminal group continues at such concentrations (Merits, 1962; Tamaoki and Mueller, 1962; Eason et al., 1963 ; Franklin, 1963 ). . Inhibition of DNA-catalyzed RNA synthesis in eukaryotic cells should

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mimic the effect of enucleation on cytoplasmic metabolic activity. This makes AD a valuable tool for studying enucleated metabolism in all cell types not amenable to direct enucleation, including, more broadly, the enucleation of prokaryotic organisms. As the DNA-specific chemical and biological action of AD is well known and has recently been extensively reviewed (Reich and Goldberg, 1964), the following section is limited to a discussion of the non-specific effects of the drug and the differences between true enucleation and the consequences of AD Inhibition of RNA synthesis by AD. It is often difficult to distinguish between the indirect effects of AD, that is, effects on cell metabolism due to the blockage of RNA synthesis, and the non-specific effects that are a consequence of reactions of the AE with other components such as DNA. The lack of specificity of AD action is sometimes inferred from the observation that a given effect manifests itself in cells long before the overall rate of protein synthesis is significantly reduced as a result of mRNA degradation. However, there is still a chance that some mRNA species live much shorter than average lives and soon stop supporting the synthesis of vital proteins. However, some of the observed effects cannot be easily explained in this way. For example, inhibition of respiration and anaerobic glycolysis in human leukemic leukocytes is caused by AD but not by puromycin, a potent inhibitor of protein synthesis (Laszlo et al., 1966), and in ascites sarcoma cells, inhibition of synthesis. protein breakdown caused by AD can be prevented or, once detected, reversed by the addition of glucose to the medium (Honig and Rabinowitz, 1965). Nonspecific EA toxicity is also indicated when concentrations above those required for complete suppression of RNA synthesis cause additional biochemical damage to cells, such as an accelerated decrease in the rate of protein synthesis (Soeiro and Amos, 1966). ). Another example of AD impairment in protein synthesis, which appears to be unrelated to messenger degradation, was found in the heart of rats, where the rate of protein synthesis declined faster than the polysomal level in vivo. A ribosome defect has been suggested because ribosomes isolated from AD-treated tissue responded much less to polyuridyl acid (poly-U) stimulation than control ribosomes (Earl and Korner, 1966). In contrast to the non-specific effects, some of the indirect effects of AD are also expected to be expressed in physically enucleated cells. This applies, for example, to unstable repressors driven by genes acting at the level of translation (Section III, DJ) or possible mRNA stabilization due to an increased frequency of binding to the ribosome during mRNA depletion (Trakatellis et al. ul., 1965b). ). The observed acceleration of RNA decay in Burilh szlbtilis in the presence of AD was originally interpreted as a non-specific effect (Acs et al., 1963). Subsequently, similar effects in eukaryotic and other cells

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Bacteria have been interpreted differently, namely that the interference of AD with the completion of partially synthesized RNA molecules makes them susceptible to nuclease attack (Girard et al., 1964; Zimmerman and Levinthal, 1967). If this last assumption is correct, the accelerated RNA decay can be classified as an indirect effect of AD, which, however, would not be found after microsurgical enucleation of the cells, as the incomplete RNA would be restricted to the cell nucleus. In eukaryotic cells, after complete inhibition of RNA synthesis by AD, there is a possibility that transient mRNA in the nucleus may still reach the cytoplasm, delaying the time of effective enucleation. Although limited information is available on this subject, it appears that AD interferes with mRNA transport across the nuclear membrane in an unknown manner (Girard et al., 1964, 1965). Proflavin is another compound that binds to DNA (DeMars et al., 1953) and therefore inhibits enzymatic RNA synthesis both in uivo and in utro (Hurwitz et al., 1962). Concentrations of proflavine that potently inhibit DNA primer DNA synthesis in a bacterial cross-linked system have no inhibitory effect on poly-U-directed phenylalanine incorporation into the same system and therefore do not appear to interfere with protein synthesis per se ( Woese et al., 1963). Both proflavin and dinitrophenol have been used to determine the rate of mRNA degradation in E. coli following in vivo inhibition of RNA synthesis (Woese et al., 1963). The usefulness of dinitrophenol for this purpose was recently questioned by Friesen (1966), who cited experimental evidence of a non-specific action of this compound, leading to increased degradation of mRNA and stable RNA. 2. defective mRNA

As an alternative to inhibiting mRNA synthesis, the cell can be induced to produce defective mRNA by administration of certain purine or pyrimidine analogues. Analogs are incorporated into RNA in place of the corresponding natural base and therefore affect the functional properties of RNA. For example, fluorouracil is incorporated into bacterial RNA (Horowitz and Chargaff, 1959) and causes the formation of inactive enzyme proteins, presumably as a result of translation errors resulting from the presence of the abnormal base in the mRNA (Naono and Gros, 1960; Gros et al. al., 1961a; Gros and Naono, 1961; Nakada and Magasanik, 1964). However, the previous interpretation of the effects of fluorouracil on protein synthesis can be reassessed, as Horowitz and Kohlmeier (1967) recently showed that E. coli O-galactosidase synthesis initiated by fluorouracil occurred only in the presence of easily degradable substrates, e.g. example. B. glycerol, while the active enzyme was synthesized during fluorouracil treatment without catabolic repression.

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3. Starvatzon para R N A P i e r u n o r

The availability of cell strains that are auxotrophic for nucleic acid precursors offers the opportunity to inhibit RNA synthesis by depriving cells of the necessary precursor. However, there is a significant time lag between the removal of the necessary precursor and the completion of RNA synthesis. This delay is attributed in part to the time required to deplete the intracellular pool of precursors and in part to precursor recycling of mRNA degradation products into newly synthesized RNA. The recycle period ends when most of the available precursor has been incorporated into stable RNA; its length depends on cellular levels of mRNA and the rate of stable RNA synthesis. The latter is obviously a function of the rate of cell growth. The delay time can introduce significant differences between the actual mRNA decay time and the observed decay time.

111. Quantification of mRNA A. INTRODUCTION Currently, there is no method available that allows routine quantitative analysis of individual species of gene-specific mRNA. However, it has been possible with unique systems to isolate RNA fractions containing only one or very few mRNA species, such as the gramicidin S polypeptide messenger (Hall et al., 1965), hemoglobin messenger (Marbaix et al., 1966; Chantrenne et al., 1967) and messengers from the E. coli luc operon (Hayashi et al., 1963). Consequently, most of our knowledge of mRNA metabolism comes from studies of heterogeneous populations of molecules, which can include hundreds of mRNA species that differ in molecular weight, base composition, and functional lifetime. Adding to this the fact that mRNA constitutes only a small fraction of the total cellular RNA, a small percentage at most, one can easily see the experimental difficulties inherent in this type of research. Therefore, it is not surprising to find significant differences between experimental data obtained from the same biological system by techniques based on different mRNA properties and functions. Therefore, it seems appropriate to give a brief overview of the techniques here.

B. DIRECT METHODS 1.

R N A unstable

Messenger RNA, in contrast to ribosomal RNA (rRNA), which is relatively stable in both prokaryotic (Davern and Meselson, 1960; Meselson et al., 1964) and eukaryotic (Rake and Graham) organisms, is generally characterized by a high turnover. to assess. , 1962; Loeb et al., 1965; Hadjiolov, 1966) and

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Transfer RNA (tRNA) whose turnover is restricted to the pCpCpA terminal group (Franklin, 1963; Merits, 1962; Tamaoki and Mueller, 1962; Eason et al., 1963). Therefore, exposing cells to radioactive RNA precursors for short periods compared to the half-life of mRNA molecules allows for preferential mRNA labeling. When radiolabeled, the mRNA fraction can be isolated and further characterized by base ratio analysis (Volkin and Astrakhan, 1956), sucrose density gradient sedimentation (Nomura et al., 1960), or column chromatography (Ellem and Sheridan , 1964). See Yoshikawa et al., 1964; Yoshikawa-Fukada et al., 1965; Elem, 1966). The rate of mRNA degradation can be determined from the time-dependent loss of acid-insoluble marker after inhibition of RNA synthesis. In exponentially growing cells, relative cellular levels of mRNA can be derived from changes in the labeling distribution between stable and unstable RNA (Levinthal et al., 1962) as well as the labeling kinetics of the precursor pool (Salser et al. , 1968), as discussed in Section IV, A, I have described. The abundance of long-lived mRNA in eukaryotic cells makes the above methods unreliable and assay techniques based on other mRNA properties must be used. two.

stimulating activity

This method takes advantage of the functional properties of mRNA, i. H. its ability to stimulate the incorporation of labeled amino acids into acid-insoluble material in a complete in vitro protein synthesis system (Nirenberg and Matthaei, 1961; Tissicres and Hopkins, 1961). . ). Purified rRNA has very little stimulatory activity or "template activity" whereas mRNA is highly active (Barondes et al., 1962; Brawerman et al., 1963; Hoagland and Asconas, 1963; DiGirolamo rt d., 1964). The stimulatory capacity of a given nucleic acid species probably depends on the lack of secondary structure. Thus, while double-stranded viral RNA is inactive, the same RNA after heat denaturation or native tobacco mosaic virus (TMV) single-stranded RNA are both active (Miura and Muto, 1966). Ribosomal RNA and tRNA have very little stimulatory activity in their native state, but elicit much higher activities after heat destruction of their secondary structure (Holland et al., 1966), and even denatured DNA shows template activity in the system. in vitro (McCarthy and Holland, 1965). The fact that rRNA is methylated but mRNA apparently is not (Moore, 1966) does not seem to explain differences in their template activities, since methyl-deficient rRNA is isolated from so-called "relaxed" E-particles without methionine . coli has the same low stimulatory activity as fully methylated rRNA isolated from "relaxed" particles from arginine or histidine depleted cells (Manor and Haselkorn, 1967; Sypherd, 1967). The molecular weight of the test RNA does not seem to be very critical as long as it remains above a certain value. The RNA-stimulating activity of TMV, commonly used as a

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The "standard" for this assay does not change when its molecular weight changes from normal to approximately 400,000 due to thermal degradation. A further reduction in molecular weight from 300,000 to about 75,000 results in less stimulant activity. This latter value apparently reflects the minimum chain length required for significant in vitro template activity (Boedtker and Stumpp, 1964). Therefore, even fragments of a messenger molecule could function independently in this test. This idea could explain the observation that template RNA levels seem to increase slightly after AD administration (Kennell, 1964). The specificity of cross-stimulation of protein synthesis is still questionable in some cases. For example, a non-specific stimulation of protein synthesis, which is controlled by an endogenous messenger substance, is proposed for an E. coli to which reticulocyte RNA has been added; The synthesized proteins were found to be more similar to bacterial proteins than to hemoglobin (Drach and Lingrel, 1966). 3 . molecular hybridization

The mRNA content of a radiolabeled RNA preparation can be estimated by molecular hybridization with homologous DNA (Hall and Spiegelman, 1961). The distinction between mRNA, on the one hand, and stable RNA species, on the other hand, is based on the realization that normally only a very small part of DNA, less than 1%, encodes rRNA (Yankofsky and Spiegelman, 1962, 1963). and tRNA (Giacomoni and Spiegelman, 1962; Goodman and Rich, 1962). In some cells, slightly greater multiplicities of genetic loci for rRNA appear to exist (Matsuda and Siegel, 1967). In any case, hybridization of labeled rRNA or tRNA can be further suppressed by adding an excess of the respective unlabelled homologous RNA species. RNA-DNA hybrids formed in solution can be separated from uncomplexed RNA by various methods, such as filtration through nitrocellulose filters (Nygaard and Hall, 1963, 1964). Denatured DNA can also be immobilized on agar (Bolton and McCarthy, 1962) or membrane filters (Gillespie and Spiegelman, 1965) to form hybrids. The amount of non-specifically bound RNA background can be greatly reduced by treating the complexes with ribonuclease (Gillespie and Spiegelman, 1965). C. RELATIONSHIP BETWEEN mRNA

POLISOMAS

Cytoplasmic mRNA is associated with ribosomes that form functional aggregates of various sizes called polyribosomes or polysomes (Korner and Munro, 1963; Penman et al., 1963; Staeheh et al., 1963b; Wettstein et al., 1963). A

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The RNA fraction distinct from rRNA has been isolated from polysomes and found to have properties characteristic of mRNA (Penman et d., 1963; Munro and Korner, 1964; Munro et al., 1964; Burny and Marbaix, 1965). . As might be theoretically expected, larger polysomes usually contain heavy messengers and small clusters of light messengers (Staehelin et al., 1964; Trakatellis et al., 1964). However, the distribution is not always clear and, for example, in rapidly growing HeLa cells, up to 50% of the heavy messenger has been recovered from the light polysomes, suggesting that some of the mRNA heavy chains contain fewer ribosomes than their maximum number. (Latham and Darell, 1965). According to the currently accepted model for the translation process, the "binding mechanism" (Gierer, 1963; Gilbert, 1963; Warner et al., 1963; Watson, 1963), ribosomes or their subunits bind to terminal 5 ' of the messenger chain and translate to the 3' end (Salas et al., 1965; Thach et al., 1965; Terzaghi et al., 1966) the genetic message in the correct sequence of amino acids starting with the N-terminus of the polypeptide chain ( Bishop et al., 1960; Dintzis, 1961). At the 3' end of the mRNA molecule, both the terminated polypeptide chain and the associated ribosome are released. Thus, polysomes represent the working unit that connects the growing chain of polypeptides to the mRNA molecule. Consequently, polysome structure and function offers two approaches to mRNA quantification. One approach concerns the quantitative relationship between cellular levels and the "size" spectrum of polysomes, on the one hand, and the overall rate of protein synthesis, on the other. Understanding this relationship would allow us to extrapolate our results using mRNA bulk to individual protein-specific messengers. This possibility is discussed in Section II1,D. The second aspect concerns the quantification of cytoplasmic mRNA in functional form. Assuming that each polysomal aggregate carries only one cistron-specific messenger strand (polycistronic messengers are not considered here), the number of mRNA molecules in each class of polysomes would be proportional to the total number of ribosomes in that class divided by the number of ribosomes adding ribosomes. Estimation of cytoplasmic messenger levels by this method is only meaningful when the following conditions are met: (1) the polysome yield is high and reproducible; (2) there is no extensive degradation or aggregation of the polysomes; (3) the class of monomers in a preparation can be unequivocally assigned a role as free ribosomes or as ribosomes bound to an mRNA strand; (4) there is no substantial amount of cytoplasmic mRNA in free or "masked" form. Although condition (1) may present problems for certain materials, one can estimate the yield of free or membrane-bound polysomes (see Blobel and Potter, 1967a,b) and then refine the techniques until this condition is satisfied. Condition (2) is more difficult to achieve. Endonucleolytic attacks on the messenger chain,

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the "backbone" of a polysome, are probably produced during the isolation and fractionation process. Therefore, large polysomes are split into two or more smaller ones, leading to an overestimation of the relative amount of mRNA. With certain materials, endonuclease activity is difficult to control; Fortunately, however, extensive cleavage of the aggregates can be detected after saturation labeling of the nascent proteins. As predicted from the "band mechanism" of translation, the average polypeptide tag per ribosome increases with increasing length of the messenger strand, assuming approximately equal spacing between the ribosomes along the strand. Consequently, the specific activity of undegraded polysomes (label on nascent protein per RNA unit) increases in a very characteristic way from small to large aggregates (Noll et d., 1963; Kuff and Roberts, 1967). After extensive degradation, all classes of polysomes have approximately the same specific activity due to the random distribution of long and short unterminated polypeptide chains among the polysomal fragments (Warner et al., 1963). There is good experimental evidence that, in certain cell types, polysomes associate and form higher-order aggregates. These polysome pools contain more than one mRNA strand, and therefore the messenger content of the total polysome population would be underestimated. Clusters of polysomes have been observed in certain specialized cells that primarily synthesize collagen (Kretsinger et d., 1964). Treatment with the enzyme collagenase, but not with ribonucleases, was found to break the clusters into smaller units, indicating that originally several polysomes were held together by links spanning between nascent peptide chains (Goldberg and Green, 1967). In another example, stimulated lymph node cells were found to contain a particular class of polysomes called "immune spikes" that are resistant to mild ribonuclease treatment. Administration of puromycin, which should cause the release of nascent peptide chains, does not cause these polysomes to rupture, indicating that they are probably not bound by nascent peptides alone (Manner et al., 1965). Ribonuclease resistance has also been reported for cardiac muscle polysomes, which sediment as large aggregates and are held together by the resulting protein (Rabinowitz et al., 1964). Serious objections can be raised to estimating cytoplasmic mRNA levels from polysome sedimentation profiles based on conditions (3) and (4). Individual ribosomes attached to natural messengers can carry out protein synthesis in vitro (Munro et al., 1964; Dreyfus and Schapira, 1966) and are capable of releasing complete proteins, a criterion for translation of intact messengers (Lamfrom and Knopf, 1964). , 1965). It is difficult to determine whether binding of ribosomes to mRNA strands already existed in vivo or whether it occurred during or after fractionation. Individual ribosomes attached to short fragments of mRNA often result as artifacts of

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endonucleolytic or mechanical degradation of polysomes. Such fragments induce vho it1 amino acid uptake activity, but run-off synthesis does not result in polypeptide release (Noll et al., 1963; Staehelin et d., 1963b; Zimmerman, 1963). Acids are administered ijz in vivo, but isolated monomers do not carry labeled peptides, suggesting that monomeric ribosomes are not involved in protein synthesis in vivo (Noll et al., 1963; Penman et al., 1963; Zimmermann, 1963). does not exclude, of course, that the monomers, although inactive, are linked to the mRNA in Z~VO. Indeed, it has been reported that mRNA is conserved, probably associated with individual ribosomes, when polysomes dissociate under certain physiological conditions. After the cells recover from such a state, the polysomes reform in the absence of de novo RNA synthesis. Reversible polysomal dissociation can be induced in Chang liver cells by omitting glutamine from the culture medium (Elasson et al., 1967) and in rat liver by feeding a low-tryptophan diet (Fleck et al., 1965). Interestingly, reticulocyte amino acid deprivation does not have this effect (Burka and Marks, 1964). Inhibitors that interfere with the energy metabolism of the cell, such as fluorine, dinitrophenol, cyanide or iodoacetate, cause reversible polysonic dissociation as well as anaerobiosis (Marks et nl., 1965; Coconi et nl., 1966; Lin et al., 1966). Originally, the loss of polysomes in rat liver after ethionine administration was thought to be caused by inhibition of mRNA synthesis resulting from reduced cellular ATP levels (Villa-Trevino et al., 1964). Reexamination of the effect clearly demonstrated mRNA conservation after polysome disappearance (Stewart and Farber, 1967). The conserved messenger chain appears to remain associated with individual ribosomes after polysome degradation. Monomeric ribosomes isolated from reticulocytes treated with sodium fluoride were shown to still contain the information for hemoglobin synthesis (Lin et al., 1966). Furthermore, 9-S RNA, presumably the messenger of hemoglobin, can be obtained after polysonic dissociation of the 80-S pellet (Lebleu et al., 1967). Physiological conditions that allow partial or complete dissociation of polysomes, even with subsequent mRNA preservation, would certainly invalidate an estimate of the mRNA content of polysomes at the cellular level. It doesn't matter if the :t11 mRNA remains attached to individual ribosomes, as obviously not all ribosomes can carry a messenger strand.

D. RELATIONSHIP BETWEEN mRNA AND PROTEIN SYNTHESIS 1. Theoretical Citations Estimation of cytoplasmic messenger levels by any of the methods discussed above is limited to bulk messenger or, at best, heterogeneous large messengers.

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generous messenger lessons. However, a theoretical model can be developed that allows the relative cellular levels and half-lives of individual messenger substances to be indirectly determined from the synthesis kinetics of the respective proteins. We assume that at any time after enucleation, the rate of synthesis of a given protein is related to the cellular quantity of its specific messenger by the expression:

where dP/dt is the protein synthesis rate, M is the amount of mRNA, and k is the protein synthesis rate constant. Further assuming that mRNA decay follows first-order kinetics

where k is the mRNA decay constant. Therefore, the amount of mRNA remaining after time point M is

=Moe-bMt

(3)

where rM is the initial cellular amount of mRNA. equation replacement. (3) in Heh. (1) returns

The integrated form of this equation, assuming P = 0 when t = 0

Considering equation (5) when t tends to infinity and rearranges, we arrive at the following approximation:

Therefore, the cellular amount of a given mRNA species at the time of enucleation is proportional to the total amount of the corresponding protein synthesized after enucleation. Since the rate of protein synthesis is proportional to the cellular content of mRNA, the half-life of that mRNA is equal to the period of time during which the rate of protein synthesis drops to half of its original value. In practice, the half-life can be obtained simply from a semi-logarithm

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Graph of experimentally obtained protein synthesis rates versus time after enucleation. This model can easily be extended to suit a hypothetical special case where an inactive set of messengers is needed to replenish the decaying functional messenger and keep the active portion at a constant steady-state level until the entire form is consumed. The remainder of the active portion would drop exponentially again. Based on equation (2), we can write -

A M = kl,briM"At

(7)

where iM is the initial steady-state level of the active fraction during the At "hold period" and AM is the amount of mRNA consumed during this period. Furthermore, according to Equation (I), the amount of AP protein synthesized during the At hold time is AP = kspiMOAt(8), where ksl is again the protein synthesis rate constant. Solve for M and plug it into the equation. ( 7 ) we get h*t Ap -AM = __

(9)

kSP

Adding the amount of mRNA degraded during the retention time [Eq. (9) 1 for the set that the subsequent exponential decay 1 Eq. ( 6 ) ] we get the total value of mail M,l.,,t: MTot

4 II hl ~

kSP

(AP

+P)

(10)

where AP is the protein synthesized during the waiting time and P is the amount of protein synthesized during the exponential decay phase of the messenger. For practical reasons, a period spanning a few half-lives can be infinite time. One approximation is considered sufficient. _The model presented is based on some restrictive conditions. Therefore, it was assumed that during the period of nucleated protein synthesis, mRNA is the only limiting factor and that the concentrations of necessary enzymes, tRNA, amino acids, energizers and other components of the machinery for protein synthesis of proteins are not changing the enough. affecting protein synthesis. In addition to the general shortcomings, however, there are a number of experimental data that indicate that the postulated relationship between the amount of messenger molecule and the rate of protein synthesis, specified by the factor kSP in Eq. (1) must not remain constant throughout the denuclearization period. In particular, three possible causes for a deviation from the ideal system must be considered here: (1) The

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KONRAD KECK

Presence of cellular control mechanisms operating at the level of translation; (2) the effect of the growing cellular pool of monomeric ribosomes on the frequency of polypeptide chain initiation; and (3) the possible occurrence of inactive ribosomes in polysomes. In all three cases, the level of interference, if any, is likely to change during the messenger depletion phase. Another restriction tacitly included in Equation (1) is protein stability. Although the turnover rate of many proteins is negligible compared to their RNA model, there are examples where the half-life of a protein molecule is shorter than that of its messenger, necessitating the addition of a protein degradation term. to Eq. (1). two.

cross border controls

The existence of translational control mechanisms, both protein-specific and non-specific, has been postulated for several systems. A non-specific regulation of protein synthesis is inferred from experimentally induced changes in the functional efficiency of rdtro polysome preparations. Altered polysomal efficiency has been observed in response to certain physiological stresses imposed on cells or animals in vivo, or in response to the addition of certain fractions of cellular homogenates to an in oitro protein synthesis system. Liver polysomes isolated from mice fed a protein-free diet were found to retain much less amino acid uptake in vitro than polysomes from protein-fed animals. Differences in the messenger content or in the composition of the supernatant fraction were excluded as responsible factors, since the experimental and control preparations contained the same ratio of polysomes to individual ribosomes and both supernatant fractions proved to be equally active (Von der Decken, 1967 ). Starvation of mouse ascites tumor cells produces similar effects. Vivo i7z activity of starved cell ribosomal preparations is suppressed, whereas after a brief recovery of cells in supplemented medium, polysomes that support much higher levels of protein synthesis in vitro can be obtained (Kerr et al., 1966). The recovery phenomenon is not prevented by AD treatment and therefore probably does not depend on m'o RNA synthesis. Furthermore, the release and subsequent reinsertion of pre-existing mRNA into ribosomes can be ruled out as responsible factors, since RNA isolated from active and inactive ribosomal preparations has the same stimulatory activity in a vitru reticulocyte system and therefore apparently the work contains . mRNA levels (Kerr et d., 1966). Subcellular fractions of unknown composition have been described that stimulate protein synthesis in homologous cell-free systems in rat liver (Mizrahi, 1965j) and reticulocytes (Beard and Armentrout, 1967j). These preparations, called "Fraction X", were obtained from post-ribosomal supernatants.

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The identity of the responsible factor with messenger RNA, tRNA or activating enzymes has been ruled out. Reticulocyte factor is probably a protein and may be involved in the initiation of polypeptide chains (Beard and Armentrout, 1967), perhaps related to the formation of N-formylated amino acids (Clark and Marcker, 1966). Similar conclusions were also drawn for rat liver factor based on the observed factor-induced acceleration in the rate at which 8 0 3 ribosomes associate with polysomes (Mizrahi, 1965). Membrane-associated ribosomes in normal and regenerating rat liver contained varying amounts of a thermolabile factor of unknown chemical nature that inhibits amino acid uptake in vitro. The factor can be released by sonication of membranes and does not appear to respond by destroying mRNA, interfering with mRNA binding to ribosomes, or releasing nascent protein from ribosomes (Hoagland et al., 1964). Protein-specific regulation at the translational level is thought to involve unstable repressor molecules whose synthesis is initiated at the transcriptional level. In rat liver, such a repressor appears to be responsible for the reduced synthesis of tyrosine-α-ketoglutamate transaminase and tryptophanpyrrolase that normally follows hormone-stimulated increases in these enzymes (Garren et al., 1964). In the rat, specific suppression of hepatic tyrosine transaminase synthesis, but not of all hepatic protein synthesis, can also be induced by the administration of stressors (Kenney and Albritton, 1965). In both cases, treatment of AD cells can block enzymatic repression, suggesting transcriptional initiation of repressor synthesis. Both enzymes appear to be synthesized in stable RNA templates, as AD has no short-term impact on the enzymes' basal synthesis rate. The last observation rules out the possibility that the cellular site of repression is at the gene level. Eliasson (1967a,b) described a system with very similar properties. Arginase translation in Chang liver cells is believed to be controlled by a metabolic repressor, although the messenger of this enzyme appears to be very stable. As in rat liver, arginase repression can be inhibited with DA (Elasson, 1967a,b). -3.

Freynenry de Chair Izitia~ioiz

Under stable conditions of protein synthesis, the number of polypeptide chains released per polysome per unit time is equal to the number of chains initiated. The chain initiation rate, in turn, must equal the frequency of binding of the ribosome to the messenger chain, since each ribosome is active in protein synthesis. For a given translation rate, there may be an upper limit to the number of ribosomes that can bind per unit time, imposed by the requirement of a minimum distance between adjacent ribosomes. Finally, during the germination phase, the breakdown of polysomes leads to an increase in the proportion of free ribosomes. According to the law of mass action

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a high pool of ribosomes might be expected to lead to a higher rate of ribosome binding, thus improving the efficiency of the remaining polysome population (see Williamson and Schweet, 1964). Experimental evidence appears to support this prediction in some but not all systems studied. Trakatelli et al. (1965a,b) observed in two different cell types (reticulocytes and mammary carcinoma cells) that during progressive mRNA depletion the in vivo rate of protein synthesis decreased more slowly than the polysomal level. Similar non-parallel changes, but in reverse order, were observed during reticulocyte recovery from sodium fluoride-induced polysome dissociation. The rate of protein synthesis reached control levels long before normal levels of polysomes were restored (Coconi et al.] 1966; Marks et al., 1965). The presence of inactive polysomes was ruled out because the specific activity of the polysomes (label on nascent protein by ribosome) did not change during recovery from fluoride poisoning. However, alternative explanations for the phenomenon can be given (Coconi et al., 1966). On the other hand, in rat liver, the level of polysomes and the rate of in vivo protein synthesis, corrected for the specific activity of the cellular amino acid pool, decreased at similar rates after AD administration (Wilson and Hoagland, 1967). . As presumably only polysomes containing fewer ribosomes than the saturation number could respond to a high pool of free ribosomes, the observed inconsistencies could simply reflect differences in the degree of polysomal saturation at the time of enucleation. The ribosomal content of polysomes, as indicated by their "size" spectrum, appears to be dependent on the physiological conditions of the cells. Under normal conditions, adult rat liver polysomes appear to have an almost maximal number of ribosomes (Staehelin et al., 1964). Prolonged fasting of mice causes a shift in polysome size distribution to smaller values (Webb et al., 1966), whereas gavage of a threonine deficient diet increases protein synthesis in vivo in mouse liver and results in polysomes. heavier (Sidransky et al., 1964). Polysomes in exponentially growing cells, such as HeLa cells, carry less than the maximum number of ribosomes (Latham and Darnell, 1965). On the other hand, the absence of change in the polysome size spectrum does not necessarily indicate that the rate of ribosome binding is constant, since an associated change in the translation rate could compensate for the change in polysome size.

4. Active ribosome Polysome efficiency would be reduced if the ribosome binding to the mRNA chain was not always attached to the beginning of a polypeptide chain. Sever31's observations support the idea that polysomes can contain varying numbers of ribosomes that are not involved in protein synthesis. Polysomes in maturing reticulocytes gradually lose their efficiency for protein synthesis.

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Thesis, probably due to increased proportions of inactive ribosomes. This is confirmed by the finding that polysomes in aging reticulocytes carry decreasing amounts of nascent protein (Marks et al., 1963a; Glowacki and Millette, 1965) and that poly-U stimulation of such ribosomal preparations resulted in higher rates. Rowley and Morris, 1967). Preparations of polysomes isolated from yeast sampled at different time points in their growth phase differed in their endogenous and poly-U-stimulated ability to incorporate amino acids into proteins. The defect was located on the ribosomes and apparently was not caused by differences in the mRNA content of the preparations (Dietz and Simpson, 1964). It appears that inactive ribosomes can be preferentially separated from mouse liver polysomes by reducing the concentration of magnesium ions (Munro et al., 1964), but it remains to be seen whether this treatment can be used to estimate the proportions of inactive ribosomes in other preparations. . More work is needed before it is reasonable to speculate about the general occurrence of quiescent ribosomes in polysome populations, or the change in their proportions during the nuclear phase. 5 . protein breakdown

The instability of some proteins approaches or even exceeds that of their respective RNA templates (Lin and Knox, 1958; Feigelson et al., 1959; Kenney, 1967; Peterkofsky and Tomkins, 1967). If we assume that protein degradation occurs with first-order kinetics and, in addition, that the protein degradation factor remains unchanged during the germination phase, we can correct for protein degradation by introducing another term into the equation. (1) German CIP

-- - k,,.M - kDpP

where kDP represents the protein degradation rate constant. In many cases, this fix cannot be applied. The relative rates of degradation of some proteins have been found to vary in response to changes in substrate levels (Schimke et al., 1965) and in response to dietary stimuli (Schimke, 1964). There even appear to be genetic factors that control protein degradation, expressed in terms of time and tissue specific patterns. For example, the enzyme UDP-galactose polysaccharide transferase is degraded during a certain developmental stage of the fungus Dictyortelitlm (Sussman, 1965). The process appears to be genetically programmed and initiated by a system that can be inhibited by AD and may involve the synthesis of proteolytic enzymes (Sussman and Sussman, 1965). Recchigl and Heston (1967).

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IV. mRNA DISINTEGRATION AND PROTEIN SYNTHESIS IN PROKARYOTIC A. NUCLEOR CELL ORGANISMS 1. R N Uble

The bacteria contain an unstable RNA fraction, which is preferentially labeled with radioactive RNA precursors after a brief incubation of the cells. This rapidly labeled RNA has been characterized by sedimentation and base ratio analysis, as well as molecular hybridization with homologous DNA, and is believed to consist primarily of mRNA (Astrachan and Fisher, 1961; Gros et al., 1961a,b; Hayashi and Spiegelman, 1961). The turnover rate of this RNA was originally determined by pulse labeling and subsequent "stripping" of the tag with excess cold precursor, but more recent data on its decay kinetics have been obtained after loss of the acid-insoluble tag following inhibition of synthesis. additional RNA by AD (Levinthal et al., 1962). Even after very short labeling periods, rapidly labeled RNA decay in B. rubtilk begins without delay after AD administration and closely follows first-order kinetics with a mean decay time (half-life/ln 2 ) of approximately 2 minutes. (Levinthal et al., 1962). The data strongly suggest that the decay of mRNA molecules is a random process and not the result of functional aging of the molecules. The loss of acid-insoluble RNA initiated by AD parallels the disappearance of a labeled RNA component with an average sedimentation constant of about 15 S and in addition to a decrease in the rate of incorporation of valine into proteins, the latter with a time decay of about 3–4 minutes (Levinthal et al., 1962). When RNA synthesis in E. coli cells, which are not normally susceptible to Alzheimer's disease, was inhibited by dinitrophenol or proflavin, a similar close relationship was found between degradation of rapidly labeled RNA and reduced rate of amino acid incorporation in the observed protein (Woese et al., 1963). Given the possibility that the rate of mRNA degradation in the absence of ~OVORNA synthesis may not reflect the true messenger lifetime, possibly due to indirect or non-specific effects of the inhibitors used, efforts were made to determine the turnover rate. of the mRNA. under stationary conditions of RNA synthesis. E. coli cells were pulsed with radioactive azaguanine and subsequent recycling of the degraded marker into newly synthesized RNA was prevented by the addition of excess cold guanine. The mRNA half-life obtained by this method was not significantly different from that obtained after inhibition of RNA synthesis by DA (Chantrenne, 1965). The decay time of labile RNA under steady-state mRNA turnover conditions can also be calculated from the delay in labeling the pool of cellular guanosine triphosphate (GTP) after administration of radioactive guanine. Degradation of the initially unlabeled mRNA continues to deliver cold guanine to the cell pool, increasing the time it takes to "wash out" everything.

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cold precursor into stable rRNA. Although this method puts more pressure on the longer-lived mRNA species, the decay time for B. snbtilis RNA labile at 37 °C was limited to 3 minutes and about 4 minutes for E. coli at 30 °C . (Salser et al., 1968). The existence of mRNA with lifetimes significantly greater than 1-2 minutes was recognized after careful analysis of RNA decay kinetics in bacteria. Leive (1965b) reports that in E. coli the degradation of pulse-labeled RNA can be represented in a semi-logarithmic graph by two intersecting lines with different slopes. The fastest decaying component had a half-life of 1 1/2 minutes, while the other component with a slower decaying rate had a half-life of 16 minutes or more. When RNA synthesis in an auxotrophic uracil strain of E. coli is inhibited by uracil deficiency, the in vitro stimulatory activity of the isolated RNA, presumably reflecting its mRNA content, is found to decrease in a biphasic fashion; Half-lives of 5 minutes and 42 minutes at 25°C. they were calculated after correcting measurements for uracil recycling (Forchhammer and Kjeldgaard, 1967). In Bacillus megnterium, the presence of mRNA fractions with half-lives of 4 min and 10 min of incorporation of proteins I was inferred from the decrease in rates of in Z ~ Z Y phenylalanine after treatment of cells with AD (Yudkin, 1965) . Furthermore, there is good experimental evidence for the existence of extremely long-lived bacterial messengers; These will be discussed later. The relative proportion of unstable RNA in bacteria can be derived from the time-dependent distribution of radioactive precursors between stable and unstable RNA after saturation of the mRNA pool with marker (Levinthal et al., 1962). Cellular levels of unstable RNA can also be calculated from the kinetics of labeling the GTP pool as previously described (Salser et al., 1968). Reported values are 1.5-376% for E. coli (Leive, 1965b; Mangiarotti and Schlessinger, 1967; Salser et al., 1968) and 7.6-9.0% for B. rubtilis (Levinthal et al. , 1962; Zimmerman and Levinthal, 1967; Salser et al., 1968). two.

levels of polysomes

A high percentage of bacterial mRNA appears to be associated with ribosomes (Mangiarotti and Schlessinger, 1967) and therefore it should be expected that the cellular level of polysomes will reflect the relative amounts of cytoplasmic messenger quite accurately and also that mRNA decay will be narrow, consistent with a corresponding disappearance of polysomes. However, examination of the mRNA associated with the polysome revealed that there were unexpected differences between the half-lives of the unstable RNA and the polysonic mRNA. In E. coli, the chemical half-life of mRNA in polysomes, presumably identical to its functional lifetime, has been estimated to be 11 to 12 minutes (Mangiarotti and Schlessinger, 1967). These values were calculated

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the kinetics of polysomal mRNA labeling under steady-state conditions; are significantly higher than those obtained from the unstable RNA degradation rate. Similar results were obtained with B. meguterizm. Degradation of pulsed labeled RNA and loss of hybridizable RNA occurred at 37°C with a half-life of less than 1 minute. after AD administration, whereas polysome 1 mRNA has a half-life of 3-4 minutes (Schaechter et al., 1965). One of the possible explanations for discrepancies in mean mRNA values is based on the assumption that a significant part of the decaying RNA consists of incompletely synthesized molecules. Incomplete RNA molecules may have been trapped in an unprotected state by the action of AD and thus made vulnerable to ribonuclease attack (Schaechter and McQuillen, 1966; Zimmerman and Levinthal, 1967). After periods of pulsed labeling, which are short compared to the estimated transcription time of 1 to 2 minutes for the average messenger molecule (Alpers and Tomkins, 1965; Goldstein et al., 1965; Leive, 1965a), a significant portion of the full amount is incorporated, the tag would be contained in unterminated RNA molecules and therefore would be preferentially degraded. This interpretation may apply to some, but not all, rapidly decaying RNAs, as studies with inducible enzymes have definitively shown that the functional lifetime of at least some of the bacterial messengers is on the order of 1-2 minutes. 3. Insoluble enzymes

Due to the uniqueness of microbial systems, it is possible to start or stop the transcription of individual operons within a few seconds without significantly affecting the overall metabolism of the cell (Jacob and Monod, 1961). Enzyme induction is immediately followed by the accumulation of a pool of protein-specific mRNA before the appearance of active enzyme (Pardee and Prestidge, 1961). The presence of a specific set of mRNAs is indicated by the acquisition of an "enzyme-forming capacity", which can be defined quantitatively as the total amount of enzyme protein finally synthesized after removal of the inducer (Kepes, 1963; Hartwell and Magasanik, 1963, 1964; Kepes and Begin, 1966). Among the conditions described in Section 111, D,1, [Eq. (6) 1, this amount of protein is proportional to the cellular level of the messenger at the time of deinduction. In support of this assumption, it was observed that the increase in enzyme-forming capacity after induction closely follows a kinetics of 100(1 − e − − k t ), characteristic of a compound synthesized at a constant rate (ignoring growth exponential of a bacterium). culture) and decays with first-order kinetics. Therefore, it is tempting to equate enzyme production capacity with relative cellular mRNA levels at the time of deinduction, but this may also be more appropriate for the following reasons.

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include messenger molecules that have started but not yet completed in the mRNA pool. In bacteria, ribosomes appear to bind to the messenger strand while it is still associated with its DNA template at the point of growth (Schaechter and McQuillen, 1966; Bremer and Konrad, 1964; Byrne et al., 1964; Alpers and Tomkins, 1965 ; Naono et al., 1966; Das et al., 1967; Revel and Gros, 1967). Binding of the ribosome to incomplete mRNA also appears to occur in enzyme-induced messengers (Kepes and Begin, 1966; Kepes, 1967; Leive and Kollin, 1967). There is even evidence for the simultaneous transcription of multiple messenger strands from a given DNA template, that is, new strands are started before one or more previous strands are completed (Zimmerman and Levinthal, 1967). Therefore, if the knockdown blocks the initiation of new mRNA molecules, any partially synthesized strands will still be complete (Alpers and Tomkins, 1965) and the steady state concentration of the functional messenger should not be affected by the knockout until the knockdown is completed in the last topic started. exempt from showing D N A. Apparently AD behaves differently in this system. By combining with DNA, AD not only inhibits further initiation, but also prevents completion of already initiated and partially completed mRNA strands. The delineated difference between AD administration and deduction is clearly demonstrated by experimental data. Addition of AD within 2% minutes after induction of β-galactosidase in E. coli at 30°C. it completely suppresses enzyme formation (Leive, 1965a). This period of time is likely required to complete and release the first initiated mRNA molecule. Elimination of the galactosidase inducer, even within a fraction of 1 minute after induction, does not interfere with the completion of the entire induction process and the so-called "elementary wave" that leads to the production of a small amount of enzymatic protein (Kepes and Begin, 1966; Kepes, 1967). Other experimental data obtained with β-galactosidase in E. coli and with histidase in B. mbtilis confirm this concept. The exponential decrease in the rate of enzyme synthesis begins after a brief delay when deinduction is initiated by rapidly diluting the inducer, but no delay is observed after treatment with AD. Furthermore, slightly higher enzyme levels are obtained after deinduction compared to DA inhibition (Hartwell and Magasanik, 1964; Kaempfer and Magasanik, 1967; Leive and Kollin, 1967). The messenger substances of these two inducible enzymes are very short-lived. The messenger substance pgalactosidase in E. coli is broken down at 30 °C with a half-life of 1.3 to 2.5 minutes. (Nakada and Magasanik, 1964; Leive, 1965b; Kepes, 1967) and the histidase messenger in B. szlbtilis with a half-life of approximately 2.5 minutes at 37°C. (Hartwell and Magasanik, 1963). As in both cases the presence of inductance has no influence on the decay rate

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the respective messengers, and as the rates of messenger clearance after deinduction and after AD administration are not significantly different, we can conclude that the inhibition of RNA synthesis itself has no influence on the clearance of the respective messengers.

4. Enzymatic synthesis of long licensed measurement cellular penicillinase takes 30-40 minutes after AD treatment of fully induced cells of an inducible strain of Bacillus cereus. In the corresponding domestic strain, the RNA template is for the same enzyme with a lifetime of about 2 hours (Pollock, 1963). Increased mRNA stability of constitutive penicillinase compared to its inducible counterpart has also been reported for BacillziJ licheniformis. Yudkin (1966) found that maximally induced penicillinase synthesis stopped 5 minutes after AD addition, whereas in the constitutive strain (which differed from the former by a single mutation in the regulatory gene) penicillinase synthesis continued for 20 minutes. However, both strains of B. licheiziformis responded identically to AD with a biphasic decrease in the rate of total protein synthesis. These observations indicate that the breakdown of the messenger substance may be subject to gene-specific controls. There also appear to be extremely long-lived bacterial messengers. In B. cereus, the sporulation process requires specific messengers that are synthesized about 4 hours before the onset of sporulation. Azaguanine and fluorouracil analogues given at this time prevent sporulation. The same drugs, as well as AD, do not prevent further synthesis of spore proteins when added to the culture at the time of onset of sporulation, although treatment with chloramphenicol at this stage inhibits sporulation (Rosas del Valle and Aronson, 1062; Aronson and Roses del Valle, 1964). Synthesis of flagellin, which is the main protein component of bacterial flagella and does not contain tryptophan, remains unchanged for a considerable period of time when RNA synthesis is severely suppressed by tryptophan or uracil deprivation of a strict B.in/htili strain ~ (Martínez, 1966) or Salmouella typhiimriwn (McClatchy and Rickenberg, 1967). In other experiments, AD inhibited RNA synthesis in S. typhirzirizmz; however, there was no effect on leucine incorporation into flagellin over a period of 90 minutes, although synthesis of β-galactosidase, encoded by a lclc episome, and total protein synthesis were immediately inhibited under these conditions (McClatchy et al. Rickenberg, 1967).

B. ELICARYOTIC ORGANISMS Umtuble K N A Eukaryotic cell mRNA is generally characterized by a much longer functional lifetime compared to bacterial mRNA, ranging from about 1 1 .

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hour to many days. Direct determination of turnover rates of long-lived messengers is very difficult, and indirect estimates based on protein synthesis are often needed. A relatively unstable fraction was found in HeLa cells. After a 30-minute labeling period, up to one-third of the acid-insoluble label was lost from cells within 8 minutes of AD administration. In sucrose gradients, most of this unstable RNA sedimented along with the ribosomal precursor RNA in the region 35 to 4 0 3 . The same region also contained RNA fractions that gave high stimulatory activity in the E. coli system in vitro and, in addition, efficiently hybridized with HeLa cell DNA (Schemer et al., 1963). As in bacteria, consideration must be given to the possibility that the unstable RNA consists in part of unprotected, uncapped molecules which, after brief incubation with radioactive precursors, carry a significant part of the acid-insoluble marker. A portion of the labile RNA in HeLa cells may be identical to the rapidly degraded nuclear RNA described by Harris and Watts (1962). In contrast, cytoplasmic mRNA in polysomes from HeLa cells proved to be more stable. After 3 hours of AD treatment, only 50% of the polysomes were degraded. Isolated polysomal mRNA pelleted as a heterogeneous sucrose gradient fraction peaking at 10-S. region and had a base composition similar to DNA (Penman et al., 1963). Trakatellis and nl. (1965b) determined significant differences in mRNA half-life in breast carcinoma cells, depending on the method of determination. Consistent with the rate of polysomal decay, the loss of labeled RNA in the region 5 to 203, and the decrease in amino acid incorporation activity of ribosomal preparations into zGtw, mRNA appeared to decay in a California lifetime average. 4 hours. A much shorter half-life of only 30 minutes was derived from the kinetics of polymal mRNA labeling under steady-state conditions. On the other hand, from the reduced rate of protein synthesis in vivo after treatment with AD, a messenger half-life of more than 4 hours was calculated. The authors propose that mRNA lifetime is prolonged under transient conditions, possibly as an indirect consequence of an increased rate of ribosome binding to nRNA during the late phase of polysome degradation. A statistically greater occupation of the 5' end of the mRNA by ribosome binding may provide better protection against exonuclease attack (Trakatellis et d., 1965b). Extensive work has been done on the lifetime of mRNA in rat liver. AD must be given to animals in relatively high doses to effectively inhibit RNA synthesis. Under such experimental conditions, the level of liver polysomes decreases by 30-8070 within 4-8 hours (Staehelin et al., 1963a). Revel et al. (1964) also found polysome loss, but could not confirm in vivo IRZ breakdown of polysomes based on electronic-optical observations. They concluded that polysome degradation occurred during isolation, possibly caused by the indirect effect of AD. However, it later turned out that he had ordered

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Arrays of ribosomes attached to the endoplasmic reticulum of the cell persist after mRNA degradation in vivo and therefore their presence cannot be used as evidence for the presence of functional polysomes (Blobel and Potter, 1967a). The extreme stability of most rat hepatic messengers, with a functional lifetime of at least 40 hours, was postulated by Revel and Hiatt (1964) on the basis of sustained protein synthesis after a single AD injection. Applied to the mass messenger, these values are probably too high, as liver cells begin to recover from the effects of AD about 14 to 17 hours after administration of a single dose of the drug (Schwartz et al., 1965). Wilson and Hoagland (1967) also studied the degradation of rat liver mRNA over long periods of time. In their experiments, a second injection of AD was given to prevent cell recovery. A biphasic decay of rat liver mRNA, with half-lives of approximately 3 hours and 80 hours, was derived from the slopes of semi-logarithmic plots of polysome levels versus time. The rate of amino acid incorporation in z ho , corrected for specific amino acid radioactivity in the cell pool, decreased concomitantly with polysome content. A rapidly labeled RNA fraction pelleted on sucrose gradients with a 17-S. Peak e has a G C/AU U ratio of 0.8, it also decays in parallel at the polysomal level. The long-lived population of polysomes appears to be primarily responsible for the synthesis of albumin, the liver's main export protein (Wilson et al., 1967). Korner and Munro (1963), Staehelin et al. (1963a), Villa-Trevino et al. (1964) and Kwan and Webb (1967).

two . Protein-specific differences in model lifetime

Differences in the half-lives of individual protein-specific RNA models are of great interest as they may reveal new and novel mechanisms that regulate protein levels in the cell. Differences in messenger stability occur in unicellular and multicellular organisms; in the second, they appear to be expressed in tissue- and time-specific patterns as part of cell differentiation. The lifetime of a given mRNA species can only be determined indirectly from the synthesis of the respective protein cores and therefore the results are ambiguous as described in Section II1,D. Early work with the unicellular alga Acetabularia showed that the synthesis of various enzymes is affected at characteristically different time points after enucleation, ranging from 1 to 3 weeks (Baltus, 1955; Keck and Clauss, 1958; Clauss, 1959). Since basic metabolic processes were not inhibited during this period, the disruption of enzyme synthesis was thought to be due to depletion of specific protein messengers rather than general biochemical damage (Keck, 1965). Marchis-Mouren and Cozzone (1966) determined the messenger lifetime of six enzymes in the rat pancreas. Enzymatic proteins were pulsed

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labeled for 10 min at various time points after AD injections and then isolated in partially pure form. The amount of marker incorporated into a given enzymatic protein represented the instantaneous rate of synthesis at the time of sampling, as it was found that the specific radioactivity of a given set of amino acids in cells did not change significantly after AD administration, and protein degradation may be due to the brevity of the pulse can be neglected. RNA models for three basic enzyme proteins have been shown to be significantly more stable than models for three acidic proteins; the respective half-lives were 8 hours and 3 hours. No correlation was found between the length of a particular messenger strand, as reflected by the molecular weight of the corresponding protein, and its stability. Pulse-labeling experiments were also extended to the liver of rats, which generally contained more stable matrices than the pancreas (Cozzone and Marchis-Mouren, 1967). However, mRNA encoding basic proteins was again found to be more stable than mRNA encoding acidic proteins. It has been suggested that the net charge of the nascent protein affects the rate of messenger degradation. John and Miller (1966) studied the functional lifetime of the messengers of two liver export proteins. Serum albumin and fibrinogen production of isolated and perfused rat liver was measured chemically and serologically over an 8-hour period after AD infusion. The rapid inhibition of protein synthesis by puromycin prevented the presence of significant amounts of preformed proteins; lack of non-specific toxicity was indicated by normal rates of urea synthesis and changes in α-amino nitrogen. Serum albumin synthesis in rats was reduced with a half-life of 2 to 4 hours and fibrinogen synthesis with a half-life of 1 to 2 hours. The decrease in the incorporation rate of 14C-lysine in total liver protein, which indicates the mean half-life for all liver protein templates, revealed values of 3 to 4 hours, but the existence of templates with significantly longer lifetimes . . Pitot et al. studied inducible enzymes in the liver of rats. (1965). The enzymes serine dehydratase, ornithine transaminase and tyrosine transaminase were induced by feeding casein hydrolyzate in fasting rats. At various times after induction, a second inducing stimulus was administered with or without AD. The following standard lifetimes, defined here as the finite period after AD administration during which the system supports enzyme synthesis, were obtained: G 8 hours for serine dehydratase, 18-24 hours for ornithine transaminase, and less than 3 hours for tyrosine transaminase. The uninduced background level of one of these enzymes, tyrosine transaminase, appears to be maintained by longer-lived molds (Pitot, 1964). The results of preliminary experiments, also reported in the publication, showed mold lifetimes greater than 2 weeks for tryptophan pyrrolase and less than 3 hours for thymidine kinase. It is unlikely that any of the enzymatically inactive precursor proteins existed

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studied enzymes since puromycin could inhibit enzyme induction (Pitot et al., 1965). Contrary to the discussion above, Bloom et al. (1965) suggest that the intracellular environment can determine the lifetime of RNA templates in a given cell type and that the finding of widely different messenger lifetimes in tissues reflects the heterogeneity of cell populations rather than differences. true within a given cell type. They found, in support of their ideas, that AD initiated the exponential decline in Cl4 hydroxyproline uptake, which is responsible for collagen synthesis in their cultured fibroblasts, and in Cl4 proline uptake, which is responsible for all non-protein synthesis. with the same half-life of about 3 hours. However, these experiments do not disprove that a small subset of proteins have matrices with significantly longer or shorter lifetimes. 3. Lorig lived iMe.rJenger in Eiuaryoter

At certain stages of development and differentiation, metazoan cells contain extremely long-lived messenger substances, that is, a lifespan of more than 12 hours. Evidence for the existence of such mRNA species is mostly indirect, derived from continuous protein synthesis or the persistence of a particular population of polysomes without RNA synthesis. The most characteristic examples are cells that have acquired a highly specialized function during their final phase of differentiation, which is often followed by the natural degeneration of the nucleus. Functional specialization is often limited to the synthesis of large amounts of one or very few proteins. Extremely long-lived messengers are produced during the development of mammalian red blood cells. According to cytochemical studies, RNA synthesis does not occur after the basophilic erythroblast stage, while hemoglobin synthesis is more pronounced in later stages, mainly in erythrocytes (Grass0 et al., 1963). The time lag between the terminal period of RNA synthesis and entry into the reticulocyte stage is about 40 hours, as determined by pulse labeling of maturing cells in vivo (DeBellis et al., 1964). Danone et al. (1965) report 48 hours as the time required to complete the developmental process after inhibition of new cell formation by AD. Supporting experiments with in vitro incubated erythrocytes have conclusively shown that reticulocytes do not synthesize significant amounts of RNA (Marks et al., 1962; Burny and Chantrenne, 1964), although the cells maintain high levels of de novo hemoglobin synthesis under these conditions (Kruh and Borsook, 1956; Borsook et al., 1957). In highly specialized lens cells, lens protein synthesis is maintained by stable messenger substances. The tissue-specific localization of the long-lived neurotransmitter has been demonstrated in the 12-day-old chicken lens by radioautographic investigations.

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Amino acid incorporation continued in the highly differentiated lens nucleus for at least 8 hours after DA treatment, while protein formation in epithelial cells was undetectable (Reeder and Bell, 1965). The presence of long- and short-lived mRNA in the 14-day-old lens was inferred from biphasic disintegration of polysomes with half-lives of 3 hours and more than 30 hours after inhibition of RNA synthesis. The functional capacity of the stable polysomal population in the lens was assessed by amino acid labeling (Scott and Bell, 1965). Messenger with a half-life of at least 30 hours has also been found in the calf lens (Spector and Kinoshita, 1965). There, synthesis of a, 1, 1, and y lenses occurs during the transformation of lens epithelial cells into fibrous cells. Although crystalline synthesis remains sensitive to DA during the initial stage of differentiation of epithelial cells into elongated fiber cells, stabilization of messengers occurs during their terminal differentiation into cortical fiber cells (Papaconstantinou et al., 1964, 1966; Papaconstantinou, 1967 ; Stewart and Papaconstantinou, 1967). A very stable population of polysomes believed to be involved in the synthesis of feather keratin has been found in the skin and feather buds of chicken embryos (Humphreys et d., 1964a,b). The stable polysomal population can survive 12 hours of incubation with DA. In 15-day-old skin, at the time when keratin deposition normally occurs, stable polysomes appear more frequently and rapidly incorporate the labeled amino acids into nascent protein (Humphreys et al., 1964b). Subsequent studies provided evidence that the appearance of an inactive class of tetrameric polysomes in embryonic chick skin was the result of an artifact produced by exposure of cells to low temperatures (Humphreys and Bell, 1967) rather than the expression of "masked" messengers. (Spirins, 1966). Short-lived and long-lived messengers appear to support protein synthesis in incubated lamb thyroid slices. Synthesis of some proteins is sensitive to AD, but thyroglobulin labeling lasted from 5 to 21 hours at rates not significantly different from control rates (Seed and Goldberg, 1963). Another example of highly specialized cells are platelets, which incorporate amino acids into proteins for at least 72 hours during incubation, although platelets do not contain measurable amounts of DNA and are therefore unlikely to be able to synthesize mRNA during the process. of incubation. normal lifespan of 3 to 8 days (Booyse and Rafelson, 1967).

4. Masked iMe.iseizger The concept of a masked or inactive messenger was originally developed as a possible explanation for the surprising onset of protein synthesis in sea urchin eggs following fertilization or parthenogenetic activation under synthesis-inhibiting conditions such as pre -treatment with AD (crude and co-

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sineau, 1963, 1964) or enucleation (Tyler, 1962, 1963; Brachet et al., 1963; Denny, 1963). The presence of significant amounts of mRNA in unfertilized sea urchin eggs was later confirmed by two different techniques. Egg RNA preparations have been shown to elicit high template activity in in vitro systems (Maggio et al., 1964; Slater and Spiegelman, 1966a,b) and to hybridize efficiently to homologous DNA (GliSin et al., 1966; Whiteley et al., 1966). A body of research into the nature of biosynthetic blockage in the unfertilized oocyte (see reviews by Spirin, 1966; Nemer, 1967; Tyler, 1967) concluded that suppression of protein synthesis cannot be caused by deficiencies in the general system. protein synthesis machinery, nor could it be entirely the result of incompetent egg ribosomes. Major experimental support for this view came from the discovery that protein synthesis could be induced in an in vitro system from unfertilized eggs by the addition of synthetic polynucleotides (Nemer, 1962; Tyler, 1962; Wilt and Huh, 1962; Nemer and Bard, 1963). The crucial parameter for the suppression of protein synthesis seems to be the unavailability of the mRNA, although a partial alteration of the ribosome function cannot be ruled out. The latter possibility is supported by the observation that gentle pretreatment of egg ribosomes with trypsin further enhances their response to synthetic or homologous mRNA in the in vitro system (Monroy et al., 1965). The proteins also appear to be involved in masking maternal mRNA in the unfertilized egg. In untreated sea urchin egg homogenates, mRNA pellets form relatively quickly and can be located in the 12,000 x 8 pellet due to its template activity. However, after mild trypsin treatment of the homogenate, mRNA appears in the supernatant and ribosomal fraction, possibly initiating spontaneous polysome formation (Mano and Nagano, 1966). Another form of presumably non-functional cytoplasmic mRNA has been detected in early loach embryos (Belitsina et al., 1964; Spirin et al., 1964) and sea urchins (Spirin and Nemer, 1965). This RNA associates with proteins and forms classes of discrete particles with sedimentation coefficients from 20 to 70 S. It hybridizes efficiently with homologous DNA (Spirin and Nemer, 1965; Infante and Nemer, 1968) and causes high template activity (Spirin and Nemer, 1968). et al.al., 1964). There is a clear correlation between the sedimentation value of the particles and that of their RNA component (Nemer and Infante, 1965). Particles containing mRNA can be characterized by density equilibrium centrifugation after formaldehyde fixation (Spirin et al., 1966). The buoyant density of sea urchin embryonic particles is generally less than that of ribosomal subunits, but mRNA particles of relatively high density also exist. However, light ribonuclease treatment of these heavy particles leads to a decrease in their buoyant density, a response not shared by ribosomal subunits bound to mRNA strands (Infante and Nemer, 1968). The mRNA particles were modified by Spirin et al. referred to as "infomosomes".

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(1964) and presumably represent a transient stage of newly synthesized mRNA, possibly providing protection to the mRNA during its transport from the nucleus to the cytoplasm and during its subsequent storage in a masked form (Spirin, 1966). In early sea urchin embryos, the newly synthesized messenger is also found in so-called "light" polysomes, which, in contrast to "heavy" polysomes, do not incorporate labeled amino acids into the nascent protein and therefore appear to be inactive. in protein synthesis (Spirin and Nemer, 1965; Infante and Nemer, 1967). The ultimate fate of informasomes and their possible relationship to non-functional polysomes in early embryos is currently unknown. Attempts have been made to apply the concept of masked messengers to various cellular systems, particularly those that contain long-lived messengers (Spirin, 1966; Tyler, 1967). Whether or not a long-lived messenger passes through a temporal stage as a masked form is based on experimental evidence that there is a significant time lag between the accumulation of a specific set of messengers and the onset of synthesis of the corresponding protein. In some of the examples given, the existence of such a delay has not been clearly established, and other interpretations of the phenomena are possible. Thus, in B. cereuj, the AD-sensitive period for sporulation extends between the culture ages of 8 and 9-4 hours, but the morphological changes that eventually lead to sporulation begin at 12 hours. However, the proteins involved in sporulation may have been synthesized before structural differentiation, and indeed the necessary complement of spore proteins must have been acquired by the cells within 12 hours of adding chloramphenicol to the cell, a culture at that time. it allows the completion of the sporulation process in 10% of the cells (Rosas del Valle and Aronson, 1962). According to Wilt (1965), the delay between the end of the AD-sensitive phase and the appearance of hemoglobin in the chicken embryo may also be due to a delay in the availability of substrate for the synthesis of the prosthetic group. The role of heme in the initiation of new globin chains and in the control of hemoglobin synthesis during cell maturation was discussed by Zucker and Schulman (1968) and Schulman (1968), respectively. The appearance of inactive tetrameric polysomes shortly before keratin synthesis in embryonic chicken feathers, as mentioned earlier in this review, has been shown to be caused by an isolation artifact (Humphreys and Bell, 1967). Finally, the late increase in phosphatase activity in enucleated acetabular cells (Spencer and Harris, 1964) may be the result of cytoplasmic mRNA unmasking, but may also indicate chloroplast control of this enzyme. J

enucleated cells

The single-celled algae Acetabularia is often thought of as an extremely long-lived messenger substance. Originally, this concept was applied to species-specific "morphogenetic substances" of unknown chemical nature that

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It was thought that they were synthesized by the nucleus and remained active in the nucleated cytoplasm for many weeks (Hammerling, 1953). Morphogenesis in nucleated cells is accompanied by a net increase of several hundred percent in the total amount of protein (Vanderhaeghe, 1954; Brachet et al., 1955; Clauss, 1958; Hammerling et al., 1959). Total protein synthesis ceases about 3 weeks after enucleation, at which time basic metabolic processes such as photosynthesis or respiration are little affected (Chantrenne-Von Halteren and Brachet, 1952; Hammerling et al., 1959). Furthermore, since low molecular weight metabolites and protein precursors are still abundant at this point (Clauss and Keck, 1959; Bremer et al., 1962), protein synthesis stops relatively early and is even more restricted to proteins. isolated. enzymes (see Section IV, B, 2), is probably not caused by general metabolic damage but rather by depletion of cytoplasmic RNA templates (Keck, 1965). Estimating the contribution of genetically independent cytoplasmic systems, particularly chloroplasts, to nucleated protein synthesis in Acetabularia is very difficult. The presence of DNA in acetabular chloroplasts was recognized by Baltus and Brachet (1963) and by Gibor and Izawa (1963); Synthesis of chloroplast RNA was reported by Naora et al. (1960), Schweiger and Berger (1964) and Goffeau and Brachet (1965). Stutz and No11 (1967) provided evidence for the occurrence of chloroplast polysomes in higher plant cells. Several attempts have been made to establish the nuclear origin of messengers for various enzyme proteins in Acetabularia. The discovery of specific molecular forms of acid phosphatase in Acetabularia (Keck, 1960) offered the opportunity to determine the location of its structural gene by nuclear transplantation experiments. Buffer extracts from each of the three species examined contained an electrophoretically distinct type of phosphatase. The phosphatase type of one species, Acidaria Schenckii, was shown to be convertible to the Acetabidaria mediterranea type by unknown reactions at the molecular level. The conversion process occurred in vivo in a variety of combinations of cell transplantation between the two species and after injection of A. mediterranea cytoplasm into nucleated or nucleated Acirzla cells. The conversion process can also be started in vitro in a mixture of the respective homogenates (Keck, 1961; Keck and Choules, 1963). Repeated amputations of the cytoplasm of hybrid cells showed that the final electrophoretic character of this enzyme was determined by the remaining nucleus (Keck, 1961). Triplett et al. (1965) subsequently discovered other types of phosphatase in A. mediterranea after detergent treatment of homogenates and examined their specific activities after enucleation. It was found that the activity of one of the types of phosphatase, as well as the total phosphatase activity of the homogenate, increased rapidly after day 12, indicating control of the enzymes by the chloroplast. In another species, Acetabularia rrenulata, phosphatase activity measured at pH 5.0 increased steadily.

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in enucleated cells over a period of 3 weeks (Spencer and Harris, 1964). In contrast to these findings, Keck and Clauss (1958) and Keck (1961) observed that in A. mediterrama the rate of acid phosphatase synthesis persisted at control levels for only 1 week after enucleation, followed by a slow decline thereafter. from then. This residual synthetic capacity is preserved when protein synthesis is inhibited by starved enucleated cells in the dark for periods of up to 3 weeks and is fully expressed after renewed illumination (Keck, 1965; Schlapfer and Keck, 1964, unpublished observations). Although slightly different culture techniques (Keck, 1964) and assays were used, the reason for the discrepancy in results is not easily understood. Schweiger et al. Postulated nuclear control of several specific types of malate dehydrogenase species or isoenzymes in Acetabidaria. (1967). Strictly speaking, however, as with acid phosphatase, it has been shown here that only the ultimate electrophoretic mobility of proteins is determined by the nucleus, and the possibility remains that nuclear control is limited to secondary structural modifications of the enzymatic protein, perhaps similar to these have been reported for cholinesterase (Svensmark, 1961; Augustinson and Ekedahl, 1962). There is good experimental evidence of the occurrence of structural changes in the case of acid phosphatase (Keck and Choules, 1963) and even in the case of malic dehydrogenase a similar transformation may have taken place which has not been demonstrated. Also relevant are the observations (Schweiger et al., 1967) that the cytoplasmic enzyme type mysteriously disappears after implantation of the non-species nucleus, while the same type of enzyme persists in the enucleated cell, which serves as a control. In contrast to Aretabl/larja, the nucleated moieties of Amoeba protezis do not appear to synthesize RNA under tightly controlled conditions. Earlier reports to the contrary (Plaut, 1958; Plaut and Rustad, 1957, 1959) were likely attributed in part to the presence of ingested bacteria. Prolonged starvation of amoebae before surgery greatly reduces the uptake of labeled RNA precursors (Prescott, 1959), although no definitive answer to the question has been obtained. A further difficulty arises from the appearance of self-replicating "DNA bodies" in A. protects the cytoplasm that may be endosymbionts (Wolstenholme and Plaut, 1964; Cummins and Plaut, 1964). This theory has recently been confirmed by the discovery of infectious bacterial strains living as parasites in dircoid amoebae (Jeon and Lorch, 1967). The nucleated halves of Aranthemneba, a small amoeba that can grow in sterile medium, contain no detectable amounts of labeled RNA precursors and are unlikely to be able to synthesize RNA without the nucleus (Prescott, 1960b). Likewise, the lack of measurable cytoplasmic RNA synthesis was inferred from experiments with microsurgically enucleated human amniotic cells (Goldstein et al., 1960) and enucleated Tetruhymena cells (Prescott, 1962). Few studies have been conducted on 1< residual protein synthesis.

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physically enucleated heterotrophic cells. Nucleated fragments of human amniotic cells continue to incorporate amino acids into proteins at controlled rates over a period of 20 to 30 hours (Goldstein et al., 1960). Protein synthesis takes several hours in macronuclear paramecia, which maintain a very low (23% of controls) level of RNA synthesis (Kimball and Prescott, 1964). The protein synthesis capacity of the nucleated tetrabimenu is relatively low; Incorporation of histidine into proteins decreases by 50% within 30 minutes after enucleation surgery (Prescott, 1962).

V. Nature of mRNA degradation A. ENZYME DEGRADATION The nature of mRNA degradation is of great interest, as it may shed light on the molecular events that determine the half-life of individual mRNA species. It may be useful to first distinguish between the inactivation of an mRNA molecule and its subsequent complete destruction (Kivity-Vogel and Elson, 1967). This last process is undoubtedly carried out by enzymes that hydrolyze the RNA. In E. coli, the only system studied in detail, three RNA-hydrolyzing enzymes have been found: RNase I, a potassium-dependent phosphodiesterase (RNase II), and polynucleotide phosphorylase. Of these enzymes, RNase I is the least likely candidate for the proposed function. The end products of RNase digestion, 3'(2')-nucleoside monophosphates, have not been found among the in vitro degradation products of artificial (Barondes and Nirenberg, 1962; Spahr and Schlessinger, 1963) or natural (Andoh et al., 1963a). RNase I degradation of live it2 mRNA can be ruled out based on experiments with E. coli RNase I knockout mutants. Although RNase levels in these strains were less than 1% of wild-type levels, degradation rates are normal for rapidly labeled RNA strains (Gesteland, 1966) and for messenger β-galactosidase (Kivity-Vogel and Elson, 1967). Similar conclusions have also been drawn from experiments with E. cnli spheroplasts that have lost their RNase I activity (Artman and Engelbert, 1965). Polynucleotide phosphorylase, an exonucleolytic enzyme, attacks polyribonucleotides at the 3'-hydroxy end of the chain in the presence of orthophosphate (Lehman, 1963). The end products of this reaction, 5'-ribonucleoside diphosphates, have been identified among mRNA degradation products in some of the in vitro systems (Sekiguchi and Cohen, 1963; Andoh et al., 1963a,b). In other systems, polynucleotide phosphorylase activity is undetectable. The enzyme(s) in the E. coli supernatant fraction hydrolyze mRNA from ribosomal preparations in the complete absence of orthophosphate and without the concomitant accumulation of 5'-nucleoside diphosphate (Spahr and Schlessinger, 1963). Nucleases other than polynucleoside phosphorylase appear to bind

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bind to the mRNA-ribosome complex and degrade the mRNA during incubation of these complexes in phosphate-free buffer. Unfortunately, the end products of the enzymatic reaction were not identified in these experiments (Artman and Engelbert, 1964). Furthermore, inactivation of the 3-galactosidase messenger was shown to be unaffected in vivo in a polynucleotide phosphorylase-deficient E. coli mutant (Kivity-Vogel and Elson, 1967). Evidences that the presence of this enzyme is not necessary for the inactivation nor for the extensive destruction of the mRNA, although obviously the participation of this enzyme in the degradation processes cannot be ruled out. The role of a phosphodiesterase in RNA hydrolysis has been proposed. by several investigators (Spahr and Schlessinger, 1963; Sekiguchi and Cohen, 1963; Spahr, 1964) Phosphodiesterase from E. coli has been purified 600 times and has been found to require the presence of potassium ions and divalent ions (magnesium) for its activity. Diesterase specifically hydrolyzes single-stranded RNA to 5'-nucleoside monophosphates and does not attack RNA in the helical configuration (Singer and Tolbert, 1965). In cellular homogenates, this enzyme binds to some extent to ribosomes and may therefore be present in washed preparations of ribosomal mRNA. It is still questionable whether one of the described nucleases is really responsible for mRNA degradation in vivo. On theoretical grounds, it has been proposed (cf. Kepes, 1967) that mRNA destruction is exonucleolytic and proceeds in the direction from the 5' end to the β/ end of the messenger chain, ie parallel to the process of transcription and translation. . The wave of degradation of a given strand can closely follow the last ribosome. Such a system would prevent abnormal protein formation during mRNA breakdown.

B. INTRODUCTION OF DEGRADATION Knowing that an enzyme or combination of enzymes is responsible for the in vivo degradation of mRNA into reusable end products alone does not explain the initiating event. The often confirmed exponential rate of messenger inactivation indicates the randomness of this event. Their abundance differs greatly among the individual types of messengers in a cell, all presumably exposed to the same concentration of nucleolytic enzymes. Therefore, other parameters that determine the rate of degradation of individual messengers in the cell must be present. Despite the close functional relationship between mRNA and protein synthesis, there is no experimental support for the idea that the lifetime of a messenger molecule is limited by functional "wear and tear". This conclusion is based primarily on the observation that certain agents specifically inhibit protein synthesis without slowing down protein degradation.

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mRNA. For example, puromycin inhibits protein synthesis without prolonging the messenger half-life in B. subtilis (Fan et al., 1964) or the messenger half-life of 3-galactosidase in E. coli (Nakada and Fan, 1964). ). The amino acid analogue methyltryptophan is another inhibitor of protein synthesis in E. coli that does not affect the breakdown of the messenger substance. The rate of decline in pulse-induced 3-galactosidase formation capacity in the presence of methyltryptophan is similar to that of control cells (Kepes, 1963), on the other hand, inhibition of mRNA half-life protein synthesis by chloramphenicol in E. colz it results in a significant increase (Fqriesen, 1966; Woese et al., 1963; Forchhammer and Kjeldgaard, 1967) and in B. subtilis (Fan et al., 1964). Blockade of protein synthesis by amino acid deficiency similarly delays the decrease in stimulatory activity in E. coli RNA (Forchhammer and Kjeldgaard, 1967). Exposure of B. subtilis cells to an anaerobic environment significantly slows the degradation of rapidly labeled RNA; Protein and RNA synthesis is also inhibited under these conditions. Puromycin added during the anaerobic phase neutralizes the protection of the anaerobically labile RNA (Fan et al., 1964). Comparable results were also obtained with the messenger substance 3-galactoside in E. coli (Nakada and Fan, 1964). From the examples given, it can be concluded that it is not the lack of protein synthesis per se, but some of the concomitant circumstances that can protect the mRNA from degradation: (Allen and Zamecnik, 1962; Morris et al., 1962; Gilbert, 1963) and loss of messenger chain ribosomes (Marks et al., 1963b). These reactions likely expose the dysfunctional messenger to nuclease attack, whereas "freezing" the translation process by chloramphenicol (Das et al., 1966) would tend to preserve the messenger due to its protective status. Meaningful interpretation of these experiments is complicated by the fact that neither the identity of the degrading enzyme(s) nor the nature of the hydrolytic attack on the polynucleotide chain is currently understood. The normal relationship between mRNA decay and the rate of protein synthesis is not altered by changes in temperature. The Arrhenius-type plot of the mean decay time of the rapidly labeled RNA, the rate of decrease in leucine incorporation, and the growth constant of B. subtilis gave the same slope over the temperature range of 10° to 40°. w. . (Fan et al., 1964). Therefore, the temperature coefficient for mRNA degradation is identical to the coefficient for protein synthesis and balanced growth in general. This correlation is also confirmed by P-galactosidase induction experiments with E. coli. Although at 25°C. The mRNA decay is significantly slower than at 40oC. In both cases, the same total amount of galactosidase is produced after pulse induction (Kepes and Begin, 1966). consequently, the temperature coefficient for mRNA decay must be very close to that for peptide chain growth.

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VISA. Conclusions Investigation of the nucleated state in prokaryotic and eukaryotic cells has provided strong evidence that different protein-specific messengers differ greatly in their functional half-lives within a cell. Although the half-life of individual messenger species has been indirectly determined from the rate of anucleation of protein synthesis, several factors that may be responsible for significant deviations from the "optimal" ratio between mRNA and protein synthesis must be taken into account. However, it is reasonable to assume that most of these factors affect all proteins more or less equally. The differences observed between different proteins in the decrease of their anucleated synthesis can be interpreted as a reflection of the true differences in the half-lives of the respective messengers, although a strict proportionality does not prevail. The coexistence of short-lived and long-lived messengers in the same cell type precludes the possibility that the mRNA lifetime is determined exclusively by the intracellular milieu, unless the localization of some messenger species in separate subcellular compartments is postulated. , for example. B. Membrane messenger. associated polysomes compared to messengers in "free" polysomes. Such a model would require additional mechanisms to select protein-specific messengers for their respective compartments. More appealing is the hypothesis that the statistical lifetime of a given messenger is determined by the structure of the mRNA itself and therefore encoded in the DNA. Structural differences at or near the 5' end of the mRNA strand may very well control the accessibility of the molecule for exonuclease binding. The concept of a genetically determined messenger lifetime is supported by the observation that a single mutation in the operator region of a repressible gene results in a significant change in the messenger half-life for that enzyme (Yudkin, 1966). Genetic determination of the rate of mRNA degradation involves additional indirect control over the rate of protein synthesis through cellular steady-state levels of the corresponding mRNA species. However, more experimental work is needed before this or any other model can be seriously considered. REFERENCES Acs, G., Reich, E. and Valanju, S. (1963). Bioi-er. Biophy.r. Arthur 76, 68-79. Adler, H.I., Fisher, W.D., Cohen, A. & Hardigree, A.A. (1967). pror. useful Arud. S1.i. United States 57, 321-326. Allen, D.W. and Zamecnik. PC (1962). Biochemistry Biophy J. Artú 55, 865-874. Alpers, D.H. and Tomkins, G.M. (1965). pror. National Academy. Sri. US . 53, 797-803. Andoh, T., Natori, S. & Mizuno, D. (1963a). biological rhyme. Biophysicists Act 76, 477-479. Andoh, T., Natori, S. & Mizuno, D. (1963b). J. Biorhem. (Tokyo) 54, 339-348. Aronson, A.I. and Rosas del Valle, M.R. (1964). Biochemistry Biography. Acla 87, 267-276. Artman, M. and Engelbert, H. (1964). biological rhyme. biophyr. Arthur 80, 517-520. Artman, M. and Engelbert, H. (1965). biobrain. Biography. Law 95. 687-690.

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Stereological principles for morphometry in electron microscopy cytology' EWALD R. WEIBEL Institute of Anatomy, University of Bern, Bern, Switzerland

Introduction . . . . . . . . , . . , . . , . . , , , . . , , . , . . . . . . , , . , , . . .

111.

VI.

VIII.

235

A. Purpose and objectives of morphometric cytology. . . . . . . . 235 B. The problem of surveying structures in sections. . . . 236 C. Classification of Structures . . . . . , . . , . . . . . . . . , . 237 Basic Stereological Principles . , . . , , . . . . . . . . . . . . 238 A. Basic parameters that characterize the structures and their correlates in the sections. . . . , , . . , , . . , . . . . . . . . . . . . . . . 238 B. Terminology and Symbolism, . . , . . . . . . . . , . . . 2 40 C. Stereological evaluation of aggregated structures. . . . . . 242D. Characterization of the size distribution of discrete particles ............................. . . . . . . 257 in sheet thickness. . 261 Application of stereological methods to electron microscopic cytology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261 A. Sample preparation . . . . . , , . . . . . . . . . . . . . . , . . . . . . 261 B. Tissue Collection . . . . . . . , . . , , . . . . . . . . . . . . . . . . . . 263 C. Stereological Analysis of Electron Micrographs . . . . . . . . 273 An example of morphometric characterization of organelles: the liver cell. . . . . . . , . . . . . . . . . . . . . . . . . . . . . 286 A. General Study Concept and Sampling Procedure 286 8. Specific Procedures for Estimating Morphometric Properties of Cells and Subcellular Components . . . . . . . . . . . . . . . . 287 C. Correlation of biochemical data with morphometric data. . . 293 Cytomorphometric methods in experimental pathology. . . . 293 Problems in applying stereological methods to anisotropic systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294 A. Sampling of anisotropic tissue. . . . . . . . . . . . . . . . 294 B. Influence of anisotropy on stereological measurements; Influence of test systems. , . . . . . . . . . . . . . . . . . . . . . . 295 C. Evaluation of structural anisotropy. . . . . . . . . 291 Assessing the Current Situation and Looking at the Possibilities . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 . _ _ . . . . . . . . . . . . 299 references. . . . . , . . . . . . . . . . . . . . .

introduction

A. PURPOSE AND OBJECTIVES OF MORPHOME CYTOLOGY Advances in electron microscopic cytology have shown that the cell consists of a limited and well-defined spectrum of organelles, most of which Funding for Scientific Research and Grant No. 78 by BIGA.

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for biochemical and physiological studies, or h silu can be functionally characterized by cytochemical and radioautographic methods. Functional changes are often associated with enlarged or decreased organelles rather than clear qualitative changes. This is especially true when changes occur in the physiological domain. Cell morphometry is used to provide quantitative information about the fine cellular structure in order to allow a quantitative correlation of biochemical or physiological data with morphological data obtained from structurally intact cells. It is thus evident that the morphometric approach to cytology is not an end in itself, although it can reveal admirable dimensional balances that satisfy our aesthetic needs. It is a means that serves the purpose of structure-function correlation, deriving its rationale from the recognition that any ordered function must have an organized structural base of sufficient but not excessive size. Therefore, in this article special attention is given to the possible applications of morphometric methods to correlative cell biology.

B. THE PROBLEM OF MEASURING STRUCTURES IN INTERFACES The cell is a compact set of structures, that is, three-dimensional objects that we can only resolve if we have the means to penetrate the system. If we want to preserve at least part of the relationship between the structures, we cut the fixed tissue into thin slices; and we know that the best resolution is provided by the thinnest slice or slice. However, an ultra-thin slice randomly slices through the solid organelles, giving us essentially flat profiles. In our experience, we can usually interpret these profiles subjectively in terms of three-dimensional structures, but we also know that this interpretation can be wrong: for example, a circular profile can be derived from a spherical, elliptical, conical or cylindrical structure. Therefore, a single profile does not allow conclusions about the three-dimensional shape of the organelle, unless additional information is available. Likewise, the size of a profile is not representative of the size of the structure from which it was formed. However, there are quantitative relationships between the average dimensions of a large number of organelles and those of their profiles in sections. In this sense, the aggregate of profiles in the unit of area of a slice is quantitatively representative of the aggregate of organelles contained in the unit of volume, therefore the measures obtained in the slices can be interpreted in terms of structural dimensions through stereological relationships. 2 The two terms “morphometry” and “stereopopopia” are closely related but are not synonymous (Weibel and Elias, 1967). Morphology involves the use of quantitative data when describing structural features. Morphometric data can be obtained by a variety of measurement methods performed on any type of specimen, but can also be derived from stereological analysis of tissue sections. Stereology involves a geometric analysis of

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attractive tool for cytologists as its methods allow quantitative analysis of internal cell structure in electron micrographs of tissue sections. The purpose of this article is to review stereological methods that can be useful for morphometric cytology and to provide some general possibilities for their application.

C. CLASSIFICATION OF STRUCTURES Stereological analysis of tissue sections is based on the assumption that the individual tissue components to be examined (I) are present in sufficient numbers; (2) uniquely identifiable within sections; and (3) similar in size and shape from one piece of tissue to another. However, the last condition is not essential for a variety of stereological principles. Fortunately, these conditions are met in most organs and cells, and when differences exist, they are usually of functional importance, allowing structures to be grouped into two or more subclasses, which in turn are homogeneous. However, it must be taken into account that any morphometric investigation must be preceded by a comprehensive qualitative assessment of the structures, which leads to a clear definition of the structures to be measured. Loud (1968), for example, showed marked differences in the structure, size, and shape of mitochondria in different parts of the hepatic lobe. Therefore, when examining liver mitochondria, one must decide whether or not these differences are relevant to the purpose of the study; if so, the tissue should be harvested accordingly. In more recent studies (Weibel et al., 1969; Staubli et al., 1969) this was considered irrelevant, since morphometric data had to be correlated with biochemical information obtained from subcellular fractions of liver homogenates representing random samples from all cells. Another necessary prerequisite for the stereological analysis of tissue sections is the random orientation of the structures in relation to the section plane. This seems to make stereological analysis of biological tissues impossible, since the polarity of structures is one of the basic principles of biological organization. Packing higher order structural units such as g. glandular acini or hepatic lobes, to a composite organ, however, leads to almost unlimited variation in the orientation of the polarity axis of the units relative to a fixed reference plane, as arbitrary. cutting plan. The condition of random orientation of structures in relation to the section plane is thus satisfied. In certain classes of structures, the inherent anisotropy is not eliminated naturally. Skeletal muscle, peripheral nerve fibers, epidermis, and renal medulla are examples. For stereological analysis to be applicable, this anisotropic material must undergo special treatment as described in Section VI. structures and textures; includes methods that allow the direct derivation of metric properties of structures from two-dimensional sections based on geometric-statistical reasoning.

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Another property of the material to be stereologically examined must first be defined, as it determines the measurement method to be used: its size in relation to the field of observation. We have to distinguish objects of finite extent, that is, smaller than the field of view, from objects of infinite size. The liver parenchyma can always be seen as infinitely large compared to an electron micrograph, while the cytoplasm of individual hepatocytes must be seen as a finite entity. The aortic endothelium is incidental: it has finite thickness but infinite width along the vessel wall. These properties must be taken into account when determining the measurement method. Sitte (1967) discussed structure classification and its implications for stereological analysis at length.

11. BASIC STEREOLOGICAL PRINCIPLES A. BASIC PARAMETERS THAT CHARACTERIZE STRUCTURES AND THEIR INTERRELATIONS IN SECTIONS All real structures have properties of three, two, one and zero dimensions; these can generally be described in terms of volume, surface area, length or diameter and number. More recently, the generalization of stereological principles through integral geometry has led to the introduction of the "mean curvature" and the "Eule characteristic" as very general one-dimensional zero parameters (Giger, 1967). For a convex body, the average curvature is directly related to its average width or average tangent diameter; it is, therefore, a one-dimensional parameter that characterizes body size. The Euler characteristic is a parameter that characterizes the topological properties of the structure; Most real tissue structures, which are simply connected bodies, have an Euler characteristic equal to 1. Thus, the Euler characteristic is related to the number of structures. These unknown parameters are currently of mostly theoretical value and are mentioned only when necessary for completeness. 1.

Parameters that characterize the individual 0rganelle.i

Average organelle volumes, surface areas, or diameters can often be derived from parameters that characterize organelle clusters. However, it is usually necessary to make some assumptions about its geometric shape. A better characterization can be obtained by deriving size distributions; Here, too, the shape of the organelles must be known. 2. Parameters that characterize organelle aggregates

However, for most declarations it is sufficient, if not preferable, to directly determine the parameters relative to a well-defined set of

MORPHOMETRIC CYTOLOGY

2 39

organelles. As an example, information on the total volume or total membrane area of all mitochondria in I u n. 3 Tissue may be more relevant to a study of tissue metabolism than a careful deduction of mitochondrial size distribution. Stereological methods that allow the estimation of aggregated parameters are extremely simple, while the determination of size distributions is more complicated. Aggregate parameters are always defined relative to a specific container volume; therefore, they are concentrations or densities5. Thus, we would define the volume of all mitochondria in the tissue unit as their voltage density (V i ), their total surface area in the tissue unit as their main god (Sv ), and so on. Of course, for example, the total volume of mitochondria1 of a liver can be calculated directly from V if the volume of the liver is known. 3 . Section Profile Parameters

Observing more closely the effect of the section on the structures, it can be seen that the dimensions of the profiles are directly related to those of the structures (Fig. 1): the volume V of the structure is related to the AV area

METRO

norte

COWARDLY. 1. Appearance of three-dimensional structures in a two-dimensional slice (from Weibel, 1967a).

of the profile, and the surface area S of the structure depends on the boundary length of the profile B. A linear step of length M appears as point Q in the section. As a general rule, we can deduce that the cut reduces the dimensions do of the structures by 1, thus producing profiles or traces of dimension d i = do - 1. From this we must also conclude that the "point objects" are lost, that is, they cannot be represented in a true two-dimensional slice, because their dimension do = 0.

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EWALD R. WEIBEL

If we consider cutting as a plane probe cutting tissue, then these observations can be generalized and rules can be derived that are of fundamental importance for the theory of stereology presented below (Weibel, 1967a). We will then see that the linear probes placed in the tissue intersect the structures and have traces of dimension o - 2. The surface of the structure intersects the probe at a point I, while its volume is represented by the length L of the line contained in the structure. In general, we can confirm that d-dimensional probes will produce dimensional profiles

d, = c d, - 3 (1) Two consequences follow from this law (Fig. 2a): (1) The track size decreases with the probe size and becomes 0 when d, = 3 - do (2 ) If the dimension of the probe is smaller, there will be no traces, that is, the object will be lost. (2) There is always a probe where d, = 0, and this is convenient because P R O B E ?

VOLUME

LENGTH OF THE TRACK

NUMBER v)

COWARDLY. 2. a, Dimensional relationship between structures, probes and traces of structures in probes, together with systematic symbolism; b, system of basic stereological parameters related to dimensions.

In this case, “measuring” footprints is reduced to simply counting them. All higher dt dimensions require a real, linear, planimetric, or volumetric measurement. We will make extensive use of this consistency below. B. TERMINOLOGY AND SYMBOLICS In recent years, there have been attempts to create a basic set of symbols for stereology that is simple, self-explanatory, and unambiguous (Underwood, 1964; Weibel et al., 1966; Weibel and Elias, 1967). . . . ). ; Elias, 1967). There is general agreement on the use of a double symbol for relative dimensions

241

MORPHOMETRIC CYTOLOGY

mission: The first capital letter defines the Pawmeter and the second capital letter, usually written in subscript, the reference system. For example, the number density of a given structure in volume units is written as

N N,,=-

(3)

The original list of symbols was deliberately limited to a few self-explanatory letters (Underwood, 1964). However, practical application has shown that this limitation can lead to confusion. Therefore, we found it necessary to expand the symbol set to eliminate ambiguity. Table I lists the symbols that LIST

VON

TABLE I BASIC SYMBOLS FOR STEREOLOGY~

Symbol

Definition

dimensions

Structure volume or test volume

cm.3

Frame surface cut surface (flat)

cm.2

METRO

Mean curvature or frame length Limit profile length

Clock:!

cm. cm. cni.

Test line length

norte

number of structures

cm.0

tm.0

Number of crossings in the Number of crossings section

PAG

Number of points (test)

cni.0

cm.

Characteristic length of the structure (diameter, thickness, etc.) Section thickness

Volume density of structures in tissue

cm.O

Surface Density of Tissue Structures Mean curvature density or bulk length of tissue

cm.--l

N" B.4 I, one

Numerical density of structures in the tissue Density of the cut length in the section area Density of the intersection points in the length of the test line

cm.0

cm.

cm-2 cm.-:3 cin.-l

E.--'

Compare Figure 2. (Modified from Weibel and Elias, 1967.)

is used in this review; their relationship to the dimensions of objects, probes and traces is shown in Fig. 2. In practical application it is also necessary to identify the component to which the parameter refers. This was achieved by indexing the stereological

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EWALD R. W E I B E L

Symbol with numbers, Greek letters or lowercase roman letters. We prefer the latter, as again it allows for self-explanatory indexing. The use of computers in data analysis limits the symbolism, using only capital Roman letters and Arabic numerals. We found no problem using a simple string of three or more capital letters to uniquely identify parameters: the first letter identifies the parameter, the second the frame of reference, and the rest the component. Thus, NVMI would mean: "numerical density of mitochondria in tissue volume". Alternatively, the component index can be enclosed in parentheses: NV(M1). C. STEREOLOGICAL EVALUATION OF STRUCTURES TOTAL I. Relationship between structural parameters and profiles in sections

Suppose a tissue model contains a large number of similar bodies. These can be characterized by their volume and surface area, or by their volume and tissue surface density, I/ and Sv, if we consider them as an aggregate. Now the question arises how these parameters appear in a random section of tissue. In 1847, the French geologist Delesse developed the fundamental relationship, often called Delesses' principle, that the area density of profiles in section A is on average equal to Vr7. This equality A, = Vv is now known. ; was empirically derived by Delesse, but proof of it has been made explicit many times since (Smith and Guttman, 1953; Weibel, 1963; Underwood, 1967a; et al.), so it need not be repeated here. The surface of structures appears in section as contour or edge of profiles. Saltykov (1958) proved for the first time that the density of the length of the edge of the profile in the area of the section L 3 is directly proportional to Sir, that is, B.4

(4 1

Note that both BA and Srr have cm.-' dimension. We found that the linear features of the structures appear as intersections when sectioned. Mean curvature density is a linear parameter that characterizes a wide class of structures, but for general structures its meaning is quite abstract. So we have to deal with a special case: "linear" elements that curve in space. They can be thin filamentary structures, edges of polyhedral bodies or axes of tubular structures, and so on. Its total length in unit volume MI is directly proportional to the density of intersections in unit cross-sectional area Q A (Smith and Guttman, 1953; Saltykov, 1958): 1

MORPHOMETRIC CYTOLOGY

243

Aggregates of discrete objects are further characterized by the number of structures per unit volume, which is referred to as their NV number density. In connection with the Euler characteristic, as described in Section II, A, the number of structures is a zero-dimensional property. Therefore, each structure must be represented by a characteristic point, for example, by its centroid. In section I, B, we saw that "point objects" in space are not represented in ideal two-dimensional random sections. Consequently, there are no direct and simple relationships between N V and the number of N profiles observed in the area unit of an ideal section; That is, N A depends on Nv, the shape of the structures and their size. Ideally, N can therefore only be derived from three-dimensional tissue samples, i.e. from sections of known thickness. In quasi-two-dimensional sections, a relationship between Nr and profile density can only be established if some assumptions about the shape and size of the features are introduced. The same limitation applies to attempts to characterize the size distribution of structures where shape and number play an implicit role. 2. MetbodJ to EJtinzatiug Voliimej

The volume density V F of the structures can be estimated directly from the Delesse relation V v = A by planimetry of the combined area A of all profiles, which is then divided by the surrounding test area A . Delesse drew the profiles on thick paper, cut them out and weighed them; This method is still practiced occasionally. Alternatively, A can also be measured with a polar planimeter, but this is quite complicated and, depending on the shape and size of the profiles, can be subject to significant errors. Rosiwal (1898) showed that A, can be obtained by so-called linear integration: if test lines of known length L are randomly placed on the section, it suffices simply to measure the fraction L of these lines included in the profiles linearly means . . measurement to go directly to the desired estimate

This method is much more efficient than planimetry and gives very reliable results if sufficiently dense lines are used. Loud (1962, 1968; Loud et al., 1965) recommends it for volumetric analysis of cellular composition in electron micrographs. Automated scanning devices such as those developed by Lazarow and Carpenter (1962; Clawson et al., 1958; Carpenter and Latarow, 1966) or the Quantimet (see Section III, C, 6) make use of this method. A disadvantage of this method is the need to measure the intersections within the profiles, which is still complicated when automatic recording equipment is not available. It was therefore a breakthrough when the Russian geologist Glagoleff showed in 1933 that A, can be estimated by superimposing a regular point

244

EWALD R. WEIBEL

Grid over the intersection and determine the PI fraction of all points included in the profiles by simple counting; this created a third relationship

V" = P,.

(7)

The theoretical basis of this method was elaborated by Blichfeldt (1914); in fact, it is quite plausible: planimetry can be done by dividing the entire section plane into small squares of unit edge length d (Fig. 3) and determining the number of squares contained in the profile. Squares cut by the edge of the profile should be counted as fractions with respect to the fraction covered by the profile. Any degree of precision can be achieved by making the squares small enough. The rounding problem can be simplified if the midpoint of O

FEIGE. 3.

Planimetry with point counting.

each square is marked: if the center is inside the profile, the square is considered complete, if it is outside, it is discarded. However, this leads directly to scoring flatness when the squares are replaced by the grid of their centers. It is obvious that the marked point does not have to be the center, but it can be one of the corners of the squares. Therefore, the volumetry of the score can be performed using the crossing points of a square grid as markers (Fig. 4). As early as 1943, Chalkley, unaware of Glagoleff's work, proposed an analogous method for the volumetric analysis of tissue sections. He used random, irregularly spaced dots to achieve the same result. In fact, the regular set of test points is devoid of theoretical significance, although in practice it is convenient and can often produce small statistical errors (Hilliard and Cahn, 1961; Hally, 1964; Hennig, 1967). Attardi (1953), Eranko (1955) and Hennig (1956) also recommended the use of these methods in light microscopic histology.

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245

In summary, we note that three related methods are available for estimating

vJ7

v,,

A , = L[ , = P ,

(8)

The question is which one is better. It is immediately evident that the accuracy in estimating individual profile size decreases from area analysis to score analysis. However, Hilliard and Cahn (1961) noted the apparent paradox that volumetric densities obtained by point counting are affected by a smaller overall error than those obtained by area analysis. If the test point grid is set up correctly (see Section III, B, 3), only part of the profile contains a test point. Therefore, the number of profiles in the entire test system is greater than the number of cross points occupied. to a specific number

FIG.4. Count points of difference (P) and points of intersection (I) to estimate the partial area (volume) and boundaries (interfaces) of the composite tissue section. of observations, point counting covers a larger portion of the intersection area than area analysis. The resulting improvement in sample dispersion (see Section II1, B) more than compensates for the apparent inaccuracy of the individual measurement. Also, point counting is many times more efficient than linear and area analysis. Real tissue is made up of a complex series of different components or phases, which we can label as a, b, c, etc. (Fig. 4). The sum of the volumetric partial densities VVa, VVb, Vve, etc. is by definition equal to the unit volume. To determine the relative volumes of these subphases, it is enough to superimpose a section with a grid of measurement points and differentially count and classify the points that are found in the profiles of a, b, c, etc. In a three-phase system, the respective volume densities are obtained from the primary point numbers Pa, PI and Pn as

246

EWALD R. WEIBEL

3. Surface Estimation Methods

Sectionally, the surface density Sv of the fabric structures is quantitatively represented by the contour length density BA of the profile edges as shown in the equation. (4). So just measure BA with a map meter or a wire. However, this is inefficient and also not very accurate. Smith and Guttman (1953) and Saltykov (1958) showed that B A can be estimated by placing a grid of test lines on the section and counting the intersections formed by these lines with the edge of the profile, where ZL is the number of intersections is per unit length of the test leads. the relationship is

It derives directly from the well-known problem of Buffon's needle (Buffon, 1777). As the derivation of this formula is typical for stereological problems with geometric probability and yet easy to follow, it may be appropriate to present it here. A curve of length B drawn on a flat surface can be deliberately divided into n short straight sections of equal length I (Fig. 5). If we superimpose a grid of parallel lines with distance d > I on the curve, a number Z < n of segments will cross the grid lines. The problem is to derive the probability p = Z/n that a segment will intersect a gridline as a function of I and d. This problem was raised by Buffon before the French Academy in 1777. The solution is as follows:

247

MORPHOMETRIC CYTOLOGY

If all segments are perpendicular to grid lines, parallel, p 10. If segments are asymmetric, then x 1 p ( 0 ) = - = - x sin e d d

p = l / d ; yes to the web

son of ello (fig. 5)

If we allow all 0 angles to occur with equal probability, then we have to rotate the segments through a quadrant of a circle and we have to integrate p(e) over the interval 0 < 6 < n/2 and divide by the upper bound to get a average probability of getting:

The integral of the equation. ( 1 2 ) with 1 we get the desired result 1

PAG = - = 12= -

2x1

7lXd

To return to the original situation, we find that the length of our curve was B = n x 1 and we obtain it by rearranging the equation. (1 3 ) 51:

B=-XIXd 2

If both the curve and the test grid are contained within an area A (Fig. 5), the length of the test lines is L = A/d. Substituting this for d in Eq. (14) produces from the equation the relationship between the length density of curve B and the number of intersections per unit length of the test line. (10)

The method thus derived provides an efficient means of determining the edge length of the profile. However, we are generally more interested in directly obtaining the areal density of tissue structures. It is immediately apparent that substituting Equation (10) into Eq. (4) establishes a direct relationship between Sv and I L: sv = 2 11. (15)

This simple formula is one of the basic stereological principles. It has been independently derived at least six times by different authors (Tomkeieff, 1945; Smith and Guttman, 1953; Duffin et al., 1953; Horrikawa, 1953; Hennig,

248

BWALD R. WEIBEL

1956; Saltykov, 1958). The decade covered by these papers also marks the period of increasing awareness of the need for refined methods of morphological measurement in sections, particularly in materials science. During this period, a related principle was proposed by Chalkley et al. (1949), which will be presented below, was developed in the context of biological work. One wonders why it took more than a decade to realize the potential of these simple methods when 20 years later they are still not widely used in biology. We must go into the meaning of S a little further. It is a two-dimensional feature and actually measures the interface between two phases of adjacent components. When we speak of the surface of the mitochondria, what we really mean is "the contact surface between the mitochondria and the cytoplasmic matrix". This notion of interface area has a functional meaning, since interfaces are both boundaries and interchange regions between compartments. In the three-phase a, b, c system discussed above (Fig. 4), we can observe different phase contacts: a-h, a-c, b-c, a-a, b-b, c-c. The relative length of these interfaces can be easily estimated if the intersections are differentially counted with random test lines: I,,.,,, I,,.,., I,,,., etc. To simplify the notation, let's go as follows: In general, use only one index when determining the surface area of a given organelle without considering the phase of the neighboring component. In cytology, the interfaces between cellular components are often marked by membranes, so we can estimate their “membrane surface density” by counting the intersections between membrane strips and test lines. However, we must bear in mind that membranes are not real surfaces, but have layers of finite thickness and, consequently, two (active) surfaces. This can sometimes be important.

4. Methods

FoY

Lithium evaluation parameters

We saw above that curved lines in space or filamentous tissue structures intersect a random cutting plane proportional to their line density M. outside the equation. ( 5 ) we derive the MV relation directly

x S.4

which allows estimating Mv by counting the number of intersections Q of the filamentous structures with the unitary interface. We note the correspondence of this formula with Eq. (15) to derive the area density of the intersections with the test lines. At f x t, these relationships are identical, as these basic stereological principles are independent of the shape and arrangement of the structure and test probe, as long as the structure and probe remain stochastically independent. This method was recently used by Haug (1967) to estimate the length of nerve fiber segments from electron micrographs.

MORPHOMETRIC CYTOLOGY

249

Another parameter that can often be useful for describing the one-dimensional properties of solid bodies is the ratio between mean volume and surface area (v/sj, as introduced by Chalkley et al. (1949) and Cornfield and Chalkley (1951)). (v/s) can be determined by a simple point counting method, which is essentially a combination of point counting volumetrics and intersection estimation. To facilitate this measurement, a set of lines of equal length is used, placed in the section (Fig. 6), each line is identified by its length z and by two extreme points. Obviously, the ends can be used as markers for the volume of the score and the line segment

COWARDLY. 6 Estimation of the volume-surface ratio according to equation. (17) with test lines of length z.

between them for counts of surface intersections. According to Chalkley and Cornfield, the volume-surface ratio is obtained by

where P is the number of ends included in the profiles of structure i and Ii is the number of intersections of the test lines with the boundary of these profiles. It should be noted that Equation (17) can be easily derived from Equation (17). (7 and 15); if test lines of length z are stored in section R, then the total number of test points in Eq. ( 7 j is PTv= 2 ~ y , the total length of the test line is LT r g z . Substituting these two values and dividing Eq. (7) by Eq. (15) gives the result of (17). This also shows that P / S can be calculated from values of I / and Sc independently determined in the same sections is convenient in practice,

250

The geometric meaning of the spheres is found

EWA1.D R. WEIBEL

Z~/J

It depends on the shape of the structures. for U/J

= 2r/6

(18)

= d/6

(19)

and for data, interestingly, V/J

However, it should be noted that the diameter 2r of spheres and the edge length d of cubes do not have the same geometric meaning. However, it is clear that U/J can provide information about certain average linear dimensions of structures if some assumptions can be made about their shape. Two other useful examples: for long cylindrical structures with radius r V/J

= V/2

V/J

= d/2

and for broad leaves

where 2 is the arithmetic mean of sheet thickness (Weibel and Knight, 1964). The latter relationship is useful for estimating the average thickness of cell or cytoplasmic layers (Weibel and Knight, 1964) or endoplasmic reticulum cisterns (Weibel et al., 1969). However, it should be noted that 5 may not be a significant parameter when studying transport phenomena through tissue sheets; for this purpose, the harmonic mean thickness described in Section II, E is more appropriate (Weibel and Knight, 1964). Another useful one-dimensional parameter of structures is their "mean linear cut length" G, as defined by Underwood (1967a).3 It is defined as the average length of random line segments traversing the structure (Fig. 7), is independent of its shape, and can be estimated by stereological methods on random sections according to the following principle. In Section II, C, 2 it was shown that a fraction L L = V v of a random test line passes through the profiles. LI, is the sum of all cut lengths in the unit test line. Consequently, its mean is easily defined as

L, = LL/NL

(22)

where N is the number of profiles intercepted by the test line. On the other hand, we define lLas as the number of intersections of the profile boundary with the test line of unit length. It is easy to see that I L = 2 N L as there are always two boundary intersections per intersection. That's why we got

G = 2 (LL/IL)

(23)

3 The subscript 3 indicates that the intersection of the test line is considered in relation to the three-dimensional structure.

25 1

MORPHOMETRIC CYTOLOGY

of (1 1-

FEIGE. 7.

Relation between the linear intersection point L and the intersection points of the surface I (Eq. 2 3 ).

out of the equation. (8) follows L, = P,. Substituting PI into equation (2 3 ) and using a short-line proof system as in Figure 6, we arrive at the simple relation

The average length of the linear incision is therefore directly related to the volume-area ratio. 5 . MethodJ fov EJtimatiiig the number of StmrtzireJ

Based on theoretical arguments, it was concluded in Section I, C,1 that the number density of N structures cannot be determined directly from a true section analysis by a simple relation. The average number of profiles per unit area N depends not only on N but also on the shape and size of the structures. For randomly oriented particle structures, De Hoff and Rhines (1961) showed that N,zNA/B (25) where is defined as the average diameter of the tangent (or gauge). Whether the shape of all particles is the same depends on their size or size distribution. Methods for determining the size distribution of spherical and globular particles are discussed in Section II, D, 2. When it is justified to assume that all particles are approximately the same size and shape, a calculated theoretical value of 6 can be substituted into the equation. (2 5). Hilliard (1967b) provides formulas for calculating D for various geometric shapes, and De Hoff (1964) has produced a graph from which D can be read for spheroids and cylinders of various axis proportions. It should also be noted that there is a direct relationship between D and V/J for different shapes. for spherical particles

252

EWALD R. WEIBEI.-

D, z ~ ( v / J )

and for cubic particles

0,= 9 ( V / J ) The parameter (v/J) can be estimated by a simple counting procedure through equation (17), but this is not an unbiased estimate when the size and shape of structures vary. However, for many practical purposes it may still be appropriate. Aherne (1967) developed a simple counting method. Introduction of a dependent coefficient of the form k = V')/:~/J. Find the number of structures like

N =vx

kn/(V/J)"

(28)

A similar method to determine the number of cylindrical structures N was proposed by Loud et al. suggested. (1965). Both methods are interesting because they derive NY without counting the structural profiles. An alternative method for N determination was proposed by Weibel and Gomez (1962) and Knight et al. developed. (1963)

where the coefficient f3 is related to the shape and K to the size distribution. Figure 8 is a plot against axial relationships for spheroids and cylinders; for spheres (3 = V G . The coefficient K is defined as

COWARDLY. 8 aspect ratio

for ellipsoids and cylinders as a function of the h-axis ratio

MORPHOMETRIC CYTOLOGY

253

where D and D are the first and third moments of the size distribution; Therefore, it is always greater than 1 unless all structures are the same size. For a normal distribution with a coefficient of variation of +2.5% of the mean, K = 1.07. It has been shown previously (Knight et al., 1963; Weibel et al., 1966) that K for biological objects rarely exceeds 1.1, but is more common in the range of 1.01-1.1. For many practical purposes, the approximate estimated value of K can be entered, particularly when looking for comparisons between control and experimental data. Hilliard (1967a) has recently proposed a new method for counting particles. The section is scanned along a test line of length L1. A very short segment of length AL crosses and moves along the scan line (Fig. 9).

EU,

COWARDLY. 9. Counting of spherical particles in sections from the number of linear intersection points AL with profiles according to Eq. (3 1).

The AN number of scan line cut lengths with particle profiles smaller than AL is counted. For spherical particles we find N V by

The practical application of this method is not easy, but it certainly has advantages, especially when used in conjunction with automatic or semi-automatic scanners. All counting methods described so far are based on real sections with no thickness. For tissue sections of known thickness T, several alternative methods are applicable. These have recently been revised by Haug (1967a,b). Profile numbers counted by test area A should be considered here as "profile" numbers enclosed in a volume type I/ = A T . Aherne (1967) proposed a new alternative method of counting in sections of finite thickness. In summary, we observed that a variety of methods have been proposed to estimate the number of structures per unit tissue volume. They all rely on assumptions about the shape and size distribution of structures; AND

2 54

EWALD R. WEIBEL

everything will consequently be imprecise in practical application. However, if one is aware that these methods provide approximations, one or the other will be helpful. The choice of method depends on the specific circumstances and is also a matter of taste. 6 A coherent set of stereological forms; choice of method

From the above discussion, a coherent system of basic stereological formulas has developed which can be summarized as follows:

vv t

A , = L , = Pp

(32)

It can be seen that this system of formulas is closely related to Figure 2, in which the "trace" dimension of structures in d-dimensional probes was analyzed. Consequently, the list term in each row has dimension 0 and can therefore be determined simply by counting against an appropriate frame of reference. This reference system is in the first line the total number of test points used, in the second line the length of a test line, in the third line the size of the test area and in the last one a test volume. Intermediate formulas require a linear or planimetric measurement. This conclusion influences the selection of suitable methods for practical stereological work and therefore the design of meaningful test systems. Wherever possible we will use counting methods as they are much more efficient than measurements. This formula system is correct for the class of aggregated structures, which may be considered too large in relation to the test track size. Recently, an equally coherent system of stereological equations has been developed by Giger (1967) using methods of integral geometry, which is valid for a very general class of structures in three-dimensional space with no necessary restrictions. This formula system contains what is presented here as a special case. 7. THE EFFECT OF CUTTING FORCE FAILURE

The aforementioned stereological principles are based on the assumption that the examined tissue sections are true two-dimensional sections and therefore have no thickness. This condition is only really satisfied when the polished surfaces of cuts in opaque materials such as stone are carefully examined.

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MORPHOMETRIC CYTOLOGY

incident light. In contrast, so-called "ultrathin" sections of plastic-embedded tissue examined by transmission electron microscopy are always sections that have a real but finite thickness, T > 0, and we can only postulate that T + 0, or in words, that T can be reduced as much as technically possible (see Section 111, A). So how does the remaining slice thickness affect the stereological parameters measured with the above methods? Essentially, opaque structures are overrated at the expense of translucent components. The examined tissue section consists of sections of the individual structures. The light or electron beam projects these sections onto the observation plane (Fig. 10). It can be seen that the outline of the projected image

< .... , . . .: . . ... .. EU :,..... ; .........-

Dri<

COWARDLY. 10. Influence of the thickness of the section T and the diameter of the particle D on the expansion of the projected image, the so-called Holmes effect (from Weibel and Elias, 1967).

an opaque structure represents the widest cross-section of your disk contained in the section. Therefore, a certain proportion of the continuous light transmitting material is covered. Consequently, the "outlines" of opaque structures appear too large and those of translucent structures too small. Obviously, the degree of overestimation of opaque structures depends on their curvature, as measured by their curvature index R = '/ZD, and on the slice thickness. Figure 10 illustrates that spherical opaque structures occupy a lot of D. A larger fraction of a projected slice image is traced when T = D than when T, the apparent fraction of the area of a slice, is covered by opaque structure profiles.

VV” x K,(D,T)

(33)

where the correction coefficient K O is greater than 1 and depends on T and a characteristic diameter D of the opaque structures. This section thickness effect

256

EWALD R. WEIBEL

It was first recognized by Holmes (1927) and is therefore often referred to as the Holmes effect. The value of K also depends on the shape of the structures. If these can be approximated by spheres of mean diameter D, Holmes (1927) and Hennig (1957) found that

Note that K,, + 1 y becomes negligible when T D . If T / D = 0.1, the coefficient K,, x is 1.15, that is, it overestimates the true value at 15:00. This error drops to 5%) when T/D = 0.03. It is a matter of judgment when systematic error due to the Holmes effect can be ruled out. It should be noted that, in practice, part of this effect is offset by a related but inverse effect; that is, not seeing the thin polar portions of the structures or the thin edge of the profiles when the "translucent" phase has some opacity in it, so that the contrast becomes insufficient to distinguish the structures. This is particularly relevant for electron microscopy, where structure recognition is essentially contrast-dependent. Profile loss due to low contrast tends to increase with slice thickness, at least as long as T < D. In practice, it may be appropriate to introduce a Holmes correction when T D /10 . The volume density of opaque structures is then

and the translucent phase

to close

v1.0 + VVR = 1

If the opaque structures are rather granular, the correction factor for spheres according to Eq. (34) can be used as a first approximation. This is sufficient in most cases, as often only rough estimates of T and D are available. Underwood (1967b) has extensively discussed the general case of projection image lysis in the context of tissue thickness studies. A detailed discussion of this work, which provides valuable information for numerous special cases, would be beyond the scope of this article. Some practical applications are discussed below.

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M O RPOMETRISCHE ZYTHOLOGIE

CHARACTERIZATION OF THE SJZE DISTRIBUTION OF

DJSCRETE PARTICLES

Sometimes it may be necessary to estimate the size variation of some particle structures. For example, the size distribution of cell nuclei can give an indication of the frequency of diploidy, tetraploidy, etc. and be relevant to cell kinetics studies (Heiniger et al., 1967; et al.). Or, as another example, the size distribution of lipid droplets, figolysosomes, etc. can provide information about pathological cellular changes. The basic problem in characterizing a particle size distribution from a thin section study can be discovered by first examining a model situation (Fig. 11): If s population of spheres of the same diameter 2R becomes P

0,4

0,2

(Video) Eukaryopolis - The City of Animal Cells: Crash Course Biology #4

COWARDLY. 11. Profile size distribution ( r ) resulting from cutting spheres of equal radius R.

Circular sectioned profiles appear with variable diameter 2~. The radius of the profile r depends on the distance from the center of the sphere to the cutting plane. Larger circles have Y = R; As the slice approaches the tangent plane to the sphere r-0. Figure 11b shows the relative frequencies of the expected cutting radii in this model. Elias and Pauly (1966) state that 86.4% of the diameters of the profiles will be larger and 13.6% smaller than half the diameter of the sphere. If the model contains three sphere size classes, each class results in a profile size distribution similar to Figure 11b, but they overlap to form a composite size distribution (Figure 12). Once the size distribution of the profiles in the sections has been determined, we face the difficult problem of unraveling the particle size distribution. This problem has been widely theorized (Wicksell, 1925; Lenz, 1955; Bach, 1963, 1967; Saltykov, 1967; Hilliard, 1967a; Bockstiegel, 1967; Giger and Riedwyl, 1969). Baudhuin and Berthet (lc)67), Heiniger rt aL (1967) and Couland (1968). A review of all these documents would be very desirable.

2 58

EWALD R. WEIBEL

exceed the available space, but must be handled separately. We limit ourselves to giving some practical methods. In all these methods, three basic conditions must be met if they do not apply: (1) the particles must all have the same shape and can only differ in size (similarity condition); (2) its form must be known; (3) its shape

0,20

0,10

COWARDLY. 12. Profile size distributions (b) generated by three sphere size classes (a). The thick line with open circles marks the composite distribution of profile radii resulting from the intersection of the mixture of sphere sizes.

must be such that a random plane can only intersect each particle once, thus forming a single profile. Furthermore, the usual conditions for using stereological methods must be met, i.e. the particles must be randomly oriented with respect to the section plane and so uniformly dispersed that representative samples can be defined. Basically, there are two approaches: (1) The actual size distribution

MORPHOMETRIC CYTOLOGY

2 59

The particles can be derived directly from the measured profile size distribution. (2) Some parameters characterizing the particle size distribution can be calculated using formulas theoretically derived from the profile distribution parameters. YOU . Direct derivation of size distributions

When a measured distribution of profile radii of spherical structures is given, it is obvious that the largest profile class represents equatorial sections through the largest particles. According to Fig. 1I b we can estimate the number of profiles that these spheres contributed to all minor classes (Fig. 1 2); these can be subtracted from the second largest class, and so on, until the profile distribution is "exhausted". The first method of this type was proposed by Wicksell (1925); Baudhuin and Berthet (1967) recently applied it to characterize the size distribution of mitochondria in subcellular fractions of the liver. It should be noted that small profiles are generally lost the more the smaller they are, mainly due to the lack of contrast of the pole cuts (see Section II, C, 6). Often no profiles are registered in the smaller size classes. The profile size distribution determined is therefore incomplete in small size classes and must be corrected to zero by extrapolation. Schwartz (1934), Scheil (1935), Elias and Hennig (1967) and others proposed methods similar to those of Wicksell. They mainly differ in the methods used to correct small profile losses and in the computational approach. The use of appropriate computer programs (Baudhuin and Berthet, 1967) greatly simplifies the application of these methods. A new method by Saltykov (1967) should be noted. Profile diameters are arranged on a logarithmic scale and are expressed as the profile area divided by the maximum profile area (a/n,,,,). Twelve size classes are usually sufficient. The calculation procedure for this method is very simple. When particles are not spherical, much bigger problems arise. Saltykov (1967) pointed out that the frequency distribution of the profile faces of polyhedra differs significantly from that of spheres, as shown in Fig. 13 for cubes, where the larger class has a lower frequency. Wicksell (1926) gave a method applicable to ellipsoids. 2. Derivation of the size parameter Di.rt~ibi/tio~

Often, it may be sufficient to characterize the size distribution of particles by determining their moments. It is impossible to review all the methods developed for this purpose in this article; instead, we confine ourselves to a general indication of the possibilities. Giger and Riedwyl (1969) developed a semigraphic method to derive the mean diameter D e from the profile diameter distribution

260

EWALD R. WELBEL

Standard deviation 0 of a normally distributed population of (nearly) spherical particles if their size can be considered approximately normally distributed. D can be calculated from the average diameter of profile 2 through the basic relationship

when all particles have an equal probability of being cut by a random section. However, in a randomly distributed population of balls of different sizes, the probability that they will be cut is proportional to their size. According to Eq. (37)

A'Ain,,

COWARDLY. 13. Density of the profile per unit area of the section ( N , ) as a function of the relative area of the profile (,4/,4,,,ax) resulting from the random division of cubes (from Saltykov, 1967).

will therefore be an overestimation of the true mean diameter, depending on the degree of overestimation of ci. Giger and Reidwyl (1969) developed a simple graphical procedure to estimate both ci and the correction factor to be applied to the average diameter calculated according to El. (37). They are estimated from the area under the profile size distribution curve on the right. D. Bach (1963, 1967) proposed a rather complicated method that allows to derive different moments of the particle size distribution from measurements of profiles in sections of finite thickness. . The practical application of this method, which has fewer limitations and provides more information than the previous one, requires the use of a computer. Rather than using profile diameter measurements, Hilliard (1967a) and Bockstiegel (t 967) derived particle size distributions from the distribution of profile cut lengths using random linear probes. These methods have the advantage of being directly applicable to automated linear scan analysis when the sample permits (see Section TII,C,6). Compared to directly measuring profile diameter, these methods reveal a significant amount of information; O

MORPHOMETRIC CYTOLOGY

261

It is only justified when the structures are present in large numbers, as may be the case in the study of mitochondria.

E. CHARACTERIZATION OF LEAF THICKNESS VARIATION A special case of size distribution occurs with variation in the thickness of a tissue layer; This may be relevant when studying the transfer of material across a cell barrier, etc. If passive transport by diffusion can be assumed, the material flux is inversely proportional to the thickness of the local barrier. The mean diffusion resistance is therefore related to the mean reciprocal thickness or harmonic mean thickness D of the barrier. It has been shown (Weibel and Knight, 1964) that D, can be estimated from the distribution of mean linear intersections L, of random lines with the barrier (cf. Section II, C, 4) by

=t(+)1

D,,

is the reciprocal of the harmonic mean of L,. The harmonic mean D is the moment (-1) of the size distribution, while the arithmetic mean thickness D is the first moment and follows from there

Equation (40) follows directly from Eq. (21) and (24); therefore, it can be easily estimated by a point counting method as suggested in the equation. ( 2 4 ) without the need to obtain linear measurements (Weibel and Knight, 1964). 111. Application of stereological methods to electron microscopic cytology

A. SAMPLE PREPARATION

Several reasons require great care in preparing biological tissues for morphometric analysis: (1) dimensions obtained from processed tissue are only meaningful if they are representative of living conditions; (2) Conservation must be equally appropriate for all parts of the tissue, as rigorous sampling does not allow for the selection of "well-preserved" areas. Fixation and embedding techniques used for electron microscopy today.

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EWALD R. WBIHEL

they largely meet these conditions (for references, see Wischnitzer, 1967; Sjostrand, 1967). However, special care must be taken to adjust the osmolarity of the fixation medium to physiological ranges, which for mammalian tissues is 330 millimoles. It should be noted that standard solutions of 6.25% glutaraldehyde in 0.075 M phosphate buffer have an osmolarity greater than 1000 millimoles. For example, approximately isotonic solutions are obtained using 1.5% glutaraldehyde in 0.114 M s-collidine buffer (Gil and Weibel, 1968). Due to considerable fluctuations in stock solutions, it is advisable to check the osmolarity on an osmometer. Starting dehydration with 70% alcohol is most important, as lower concentrations cause swelling of adherent cells (Weibe1 and Knight, 1964). Embedding in epoxy resins appears to give good results with an overall visible tissue shrinkage of about 3-5 cjo. Stark et al. (1965) studied the effect of various preparation methods on liver cells. They observed some buffer-dependent fluctuations in mitochondrial bulk density; However, they comment that the sample fixed with phosphate buffered OsO soaked in Epon was probably defective as this method usually gives good results. In our laboratory, no significant quantitative difference can be observed with fixation of hepatic blocks with OsO or with double fixation with glutaraldehyde followed by OsO, provided that the osmolarity is well controlled (Hess, 1967). How to apply the fixative essentially depends on the organ and the project. For many fabrics such. B. Liver, immersion fixation of small cubes is sufficient. Hess et al. (1968) showed in a dog study that liver needle biopsies yield excellent specimens in which most morphometric measurements are identical to those obtained from paired block biopsies; this opens up the possibility of repeating tissue sampling in laboratory animals as well as humans. The fixation of perfusion vessels, for example, according to the method of Forssmann et al. (1967), can be advantageous in certain circumstances. However, special care is recommended to avoid drastic changes in internal tissue conditions due to insufficient perfusion pressure, flow rate or osmolarity. When the lungs are fixed in the seat, instillation of the fixative through the airway under sufficient pressure gives good results (Kistler et al., 1967). Baudhuin and Berthet (1967) advocated the use of subcellular fractions for the morphometric study of organelles. This is excellent when comparing the data with the biochemical information obtained on the same fractions; however, it does not seem to be very suitable for characterizing cellular or tissue composition, since the preparation processes involved must lead to considerable changes in artifacts and loss of organelles (Weibel et d., 1969). 4 Obtained according to the following formula: 0.2 M s-collidine solution (5 40 ml); 25% glutaraldehyde (60 mL); distilled water to 1000 ml The osmotic pressure should always be checked with an osmometer, as considerable fluctuations are possible.

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The ultrathin section presents the greatest problems because the compression of the section is inversely proportional to the thickness of the section. The basic requirement to reduce the slice thickness as much as possible to avoid the Holmes effect (see section II, C, 7) is therefore limited by the disadvantageous requirement to distort or compress the slice as little as possible. A compromise must be sought to best meet both conditions. Section densification, if the same for all parts, plays no role in volumetric analysis, but does affect the data when linear test systems or calibrated dimensional planes are used, as in surface determinations. Strong et al. (1965) estimated, however, that "normal" transverse compression is at least partially compensated for by electron microscope optical distortions. Freezing methods (Moor, 1964) cannot be used for stereological investigations, since the "intersection plane" does not cut the tissue randomly, but follows predetermined structures, as, for example, B. membranes follows. Currently available sample preparation methods appear to be adequate, but not entirely satisfactory. However, refinement of the cytomorphometric approach requires that systematic studies be conducted on optimal dissection methods.

B. TISSUE SAMPLE 1. Random sampling

Stereological measurements, based on geometric-statistical principles, are derived from the probability that the profiles of the structural sections match an appropriate test system. Therefore, it is imperative that the tissue sample is matched to the test system in a random, distortion-free process. This requires rigorous sampling procedures at every step, from animal selection and tissue block selection to electron micrograph acquisition and analysis. On the other hand, the sample examined must be representative of the material examined. Therefore, the electron micrographs must be well distributed in the area in question, for example, in an entire organ. To ensure sufficient variance in the sample, a simple random sample, in which the selection of each subsample depends on chance, can be replaced with a systematic random sample. Here, the dispersion of the subsamples is ensured by their regular spacing within a sampling grid (Fig. 14). Sampling remains random if (1) the network is randomly applied to the material and (2) the material has no inherent periodicity that would disturb that of the sampling network. Good variability is also ensured by stratified random sampling (Fig. 15): a series of evenly spaced slices are taken from the organ; each is cut into cubes, and the resulting set of fabric blocks is processed separately; One or more blocks are randomly selected from each group.

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EWALD R. WEIREL

Ebbesson and Tang (1967) experimentally compared simple random and systematic random sampling for counting nucleoli in the superior cervical sympathetic ganglion. They found that the systematic sample gave the lowest standard error, while the stratified sample was quite similar. single random

COWARDLY. 14. Comparison of sample distribution in simple (a) versus systematic (b) sampling of the pituitary gland.

COWARDLY. fifteen . stratified random sample of rat liver; Separate the sample into regions (layers) A, B, C, and D.

are randomly drawn

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Sampling proved to be the worst method (Fig. 16). Hennig (1967) arrived at the same conclusion through theoretical reasoning. Typically, each block provides a single section for further analysis. If several sections are to be derived from a block, they must be separated by 1000 r

\,-Syatemaliano \

- random number. 16. Precision comparison of simple, stratified. and systematic sampling to estimate the mean number of nucleoli per section N=60; X = 510.3; S = 329.6. (from Ebbesson and Tang, 1967).

Intermediate cut of the block to avoid double measuring of the same structures, as this can lead to distortions. It is much more difficult to avoid bias when taking electron micrographs, as bias can cause the microscopist to focus his micrographs on "interesting".

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EWALD

R. WEIBEL

Characteristics. Forgery can only be ruled out if the position of the micrographs is fixed in relation to a structure-independent reference system. A simple method places the fluorescent screen tangent to a given corner of the supporting copper grid squares, as shown in Figure 17 (Weibel et al., 1966). Using suitable mesh grids, any distance between micrographs can be achieved. It should be noted that this is a systematic random sample. If for some reason this procedure is not suitable, latex particles can be sprayed over the incision; micrograph is taken with fixed latex particle

COWARDLY. 17. Practical method for systematic scanning of electron microscopy fields (de Weibel et al., 1966).

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Reference to a reference point on the screen (Fig. 18). This creates a simple random sample; Proper distribution can be ensured by recording a fixed number of micrographs within each square of the supporting copper grid, equivalent to one stratification.

COWARDLY. 18. Simple random sampling with bias; the position of the screen is placed in a predetermined relationship with the latex particle (L) deposited on the section. The lines mark the etched crosshairs on the screen (white) and the position of the frame (black).

268

EWALD B. WEIBEL

For light microscopy, a motorized automatic sampling table was developed to allow systematic sampling of fields in histological sections (Freere and Weibel, 1967; Gander, 1967); A similar drive would be conceivable with pre-selectable splitting distances for the electron microscope tables. This can be useful, for example, in sampling polarized structures such as vascular endothelium, when it is important to obtain a sample of equidistant micrographs along the vessel wall (Burri et al., 1968). In his stereological studies of liver cells, Loud (1962; Loud et al., 1965) concentrated his micrographs on equatorial slices of hepatocytes. His frame of reference was therefore the cell nucleus and therefore cannot be considered independent of the material studied. This method may be suitable for examining some cytoplasmic organelles, but it will not produce a representative sample of the cell or even the tissue. If certain organelles show a preferred orientation towards the cell nucleus, an unbiased sample is not provided. In his most recent article, Loud (1968) modified his sampling method, but still did not use an independent reference system to position the microimages. The last scanning step includes randomly comparing the micrographs with the stereo test system. Again, test points or lines are best grouped into a regular grid, which is then arbitrarily superimposed on the micrograph; details are discussed in Section 111, CJ. The simplest method is to draw the test system onto a sheet of transparent celluloid which is placed over the paper impressions. Strong et al. (1965) incorporated a fine wire mesh into the structure of his extendable easel; This proofing system appears on prints as a square grid of white lines. It is often possible to work directly from the negative, projecting it onto white cardboard using the system of ink-drawn proofs. For efficiency reasons, we prefer that tQ register the micrographs in 35mm. The films (s; positive contacts printed on the film) are visualized and analyzed on a compact tabletop projector, which has an interchangeable screen on which the proofing system is located (Weibel et al., 1966). Details are discussed in Section III, C, l 2. Reasons why you might choose

the kingdom

Random sampling of an entire organ may not always be appropriate. For example, the glomeruli are concentrated in the labyrinthine region of the renal cortex; When examining the glomeruli, it seems wasteful to examine the medullary regions in detail. This is avoided by collecting separate samples from the cortical and medullary regions after determining the relative volumes of the two regions. This leads to multistage sampling, as described in Section IV, A. Likewise, in cytological studies, it may be sufficient to determine the dimensions of a given organelle in relation to a specific cell type, or even just in relation to its cytoplasm. The tissue sample must be randomly obtained again; however, the parts of the micrographs that do not contain the

MORPHOMETRIC CYTOLOGY

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the specified scanned cell can be skipped. One example is the study of the volume density of a specific organelle in the cytoplasm of endothelial cells (Fuchs and Weibel, 1966; Burri and Weibel, 1968); of course, smooth muscle or intimal cells, although present in the micrograph, can be excluded from the analysis. As described in Section III, C, 2., the test system should be defined in terms of the volume it contains, in this case the endothelial cytoplasm. 3 . sample size

The larger the sample examined, the more reliable the information and the greater the effort. Therefore, the definition of the sample size depends on the necessary precision and the time available for the study, but also on some properties of the structures themselves. Unfortunately, there are no generally applicable rules for determining sample size in advance. Determining the sample size is clearly a statistical problem. For example, Giger (1967) showed that the basic stereological formulas of Eq. ( 3 2 ) Pp, I and Q provide unbiased estimates of the structural parameters Vv, Sr and Mv, respectively. Giger and Erkan (1968) studied the relationship between the structural parameters denoted by X and the corresponding unbiased stereological estimate x and showed that X can be estimated by the mean gn of observations j z and that the mean variance a2zn is estimated by (xi

- .,)X

This result is in agreement with general statistical theory (Cochran, 1953), Eq. (41) representing the square of the standard error of the mean. It is valid regardless of the geometric properties of the structures, as long as the system is isotropic. If samples x are normally distributed, then 2 2 0 ; defines the "95% confidence interval") (cf. Cochran, 1953). Under this assumption, De Hoff (1967) proposed to estimate the number n of observations needed to obtain a result with a 95% confidence interval of +y percent of the mean E

where 2 and S, can be estimated from a comparatively small sample. In many cases, however, a normal distribution cannot be assumed. Giger and Erkan (1968) proposed an alternative method, independent of the type of distribution, to estimate the variance of point counting volumetry,

270

BWALD R. WEIBBL

or better, score the planimetry as a true observation on average. When a grid of points is laid out on the section, an average of 7 points are added to individual structural profiles. With d as the area of the fundamental parallelogram of the dotted grid (Fig. 21), the area of profile A is estimated by

A=aXF

(43)

and the variance of P as

S',5F (P*

-F)

(44)

where P* is the maximum number of points included in the profiles. A rough preliminary estimate of 7 and P* provides an estimate of expected variation; from this one can derive the point density needed to obtain satisfactory accuracy. Giger and Erkan (1968) also discussed a similar procedure for selecting the appropriate test line density for intersection counts to estimate Sv with specified accuracy. of the mean In biological work, a 95% confidence interval (y) is usually sufficient. If the application of equation (4 2 ) requires examining an excessively large sample, it may often be acceptable, depending on requirements and time availability, to reduce the accuracy requirement. Note that increasing the confidence interval to *15% halves the number of observations required. The variation between individual observations crucially depends on whether the field of observation covers a representative fraction of the tissue. The "representation", that is, the degree of representativeness of a sample for the population, in turn, must be defined based on the accepted confidence interval. Giger (1969) developed a general procedure for deriving the representative size of tissue samples for stereological analysis. By this method it can be shown that the area of the representative section A is inversely proportional to the volumetric density Vn of the structures and also depends on a dispersion measure not yet defined. An examination of the liver revealed that the A for hepatocyte nuclei is about 10 times that of mitochondria, although the volume density of mitochondria1 exceeds that of nuclei by a factor of only about 2.5; this is due to the greater degree of distribution of mitochondria compared to thick and widely spaced nuclei (Weibel and Gnagi, 1968). This problem is also discussed by Chayes (1965). It can thus be seen that the cross-sectional area refers to a “fundamental area” of the material, that is, the minimum area or volume that contains a representative set of all structures. In cytology, the fundamental domain seems to coincide with the single cell. However, defining the area of a cross section in terms of the fundamental is not straightforward, as a random section through a fundamental is not necessarily representative.

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positive and, in fact, it probably isn't. The theoretical basis for a solid practical solution to this problem is still lacking.

4. Various

Standard

sampling

From the above, it became evident that the wide range of sizes and degrees of distribution of cellular components introduces considerable problems in defining a representative but reasonable sample size when applying stereological techniques to microscopic cytology. For example, does it make sense to measure mitochondria in a much larger sample of micrographs than is needed just to meet the accuracy requirements of nuclei? Or should nuclei accuracy requirements be reduced to a level where the data becomes meaningless just to keep the sample size within a reasonable range in terms of mitochondrial measurements? The solution to this dilemma is multistage sampling, which offers the possibility of meeting identical accuracy requirements for all components while keeping the analytical effort within a reasonable range. The details of the multistage sampling are essentially determined by (1) the minimum magnification needed to clearly identify a specific constituent and identify its intersections with test lines, etc. and (2) the rationale and purpose of the study. . Figure 19 shows a sequence of incremental increases in rat liver cells over test lines. Distinguishing test spots falling into mitochondria and microbodies is easy at 10,000X magnification, but at least 40,000X magnification is required to identify intersections of lines with rough and smooth ER membranes or mitochondrial cristae. In the practical application of multilevel sampling, a hierarchical sequence of reference systems is defined, through which detailed but equally efficient measurements can also be obtained on rare subcellular components, relating them to the whole organ or even the organism. . . This sequence is defined for the liver as an example, where the sequential reference systems are body weight (W), liver weight and volume (VL), liver parenchyma volume (V,) and hepatocyte cytoplasmic volume (V, ) with the following relationship:

VL==fXW V , = vv, x V , = vv,

= vv, xf

x v,= vvc x vv,,x VIA= V v c x VvII x f x w

The coefficient f indicates the proportion of body volume occupied by the liver. It is evident that the determination of the corresponding volume fractions V v is sufficient to allow the transformation of all measurements obtained with respect to a given subspace to any other reference frame. Needless to say

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EWALD R. WEIBEL

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273

Similar sequences of reference spaces can be defined for any other organ or tissue.

C. STEREOLOGICAL ANALYSIS OF ELECTRONIC MICROGRAMS 1.

Electron Recording Mirropzphs

iiz

Formflexibel

Stereological work requires recording and evaluating a comparatively large number of micrographs. Usual engraving on film or large plates with subsequent printing on 8 x 10 inches or 18 x 24 cm. therefore, the paper feels heavy and expensive, although certainly acceptable. However, printing on paper suffers from the poor dimensional stability of photographic papers, which when dry can shrink up to 10% in one direction, causing noticeable distortion. Loud (1968; Loud et al., 1965) eliminated this difficulty by simultaneously printing the proof system and the micrograph. If the paper shrinks, the micrograph and proofing system are distorted as well. However, care must be taken when calibrating the test system. We found 35mm photos. More efficient films such as 40 to 50 micrographs can be obtained in a single microscopy operation. The quality of the micrographs is sufficient for this type of work. Unfortunately, some microscopes that would be great for efficient detection, like the Zeiss EM 9, still lack 35mm capabilities. Recording, but I hope this is fixed. In addition to efficiency and low cost, 3j-mm. Microsections have the advantage that they can be evaluated on compact projection systems without the need for paper printing. In the device shown in Fig. 20, the recording is projected through two mirrors onto a screen on which the test system is located (Weibel et al., 1966); Film transport is within the operator's reach. While it is possible to project the negatives, we prefer to contact print them onto a strip of the same film in a long light box. For large negatives, a similar setup can be done with a photo enlarger projecting the negatives or contact prints onto a whiteboard with the test screen. We recently modified the device shown in Figure 20 to accommodate the two 35mm devices. and 70mm. Film; can also be made to accommodate lantern slide plates. 2. S ~fems J Stereological Assays for Stiidj, by Stt.iictureJ Aggregators

It follows from Section II, C that the efficient application of stereological methods requires the following basic proof checks: (1) a set of points for volume estimation; (2) Test line j of known length to estimate surface or boundary FIG. 19. Increasing the magnification decreases the amount of material covered by the patch, but improves the ratio between stroke size and test line thickness. Magnifications: a, 10,000~; 2 0 , 0 0 0 ~ ;( , 4o,(loX; d, 8u.(~(~ox.

274

EW'A1.DR R. WELBEL

areas; (3) a test area of known size to estimate the length of curvilinear features and particle counts (profiles). These probes are carried simultaneously in a simple square grid of lines, as shown in Fig. 21: the cross

FIG.20. Stereological laboratory equipment for the analysis of electron micrographs. A projector (P) with a 35mm film slide (P) projects the micrograph through two mirrors onto a screen (S) equipped with a suitable test grid. The data acquisition unit consists of a keyboard (K) which supplies count values to a data accumulator (D) with 10 counters. This data is automatically transferred to tables (T) and optionally to a card punch, not visualized.

The points on the lines serve as markers for measuring point counts, the lines for counting points of intersection and the area between the outermost lines, and the dashed lines for counting profiles and points of intersection of linear features with the cutting plane . This test system is consistent in the sense that there is a precise relationship between the number of points, the length of the test lines and the size of the test area. Its unit is a square with area n = &, of which two sides and a point are available as probes. With IZ unit squares forming the test network, the system is defined as follows: Test points: P, = 12 Test line: L, 12 n x d 1P, z 12 x dz = P, Test area:

x 2d x dz

The use of coherent test systems is advantageous because it allows the study of structures contained in relatively small compartments. As mentioned above, it can often be preferable to estimate endoplasmic reticulum surface density in the cytoplasm of a given cell type rather than across the entire tissue. However, the cytoplasmic outline does not always fill the entire screen (Fig. 2 2 ). The length

MORFOMI'IRI(.

275

(.YTOLOGIA

of test lines in the cytoplasm Li can be easily estimated by counting the number of test points P contained in the cytoplasm: Li = Pi x 2d; and the test area for counting organelles in the cytoplasm is A i = P x da. It can also be seen that the

for example, the bulk density of mitochondria in the cytoplasm is Yp:i = Pnki/Pe. The optimal density of test points depends on the size of the constituent unit.

' D

= d2

COWARDLY. twenty-one

--____.EU

Coherent test system with square unit

With point-number volumetry, the measurement points must be so far apart that at V = 0.5 no more than one point is included in the single profile; Under these conditions, the sampling error is minimal for a given total number of test points (Hilliard and Cahn, 1961). In the case of a square network, the distance d between the measurement points depends on the maximum area of the din profile:

d2 > 4 1 ,

(45)

On the other hand, the total number of test points needed to achieve a prescribed accuracy is inversely proportional to the square root of V c - (Hennig, 1957; Weibel, 1963). A point network constructed according to Eq. (45) may therefore still be too dense when looking at 3 common components, as for reasons of representative sampling it is better to distribute fewer points over a larger number of microscopic images than many points over just a few test wipes. rehearse

From the above, it was clear that, when defining a method for multistage sampling, we must consider the following conditions: (1) optimal magnification; (2) the minimum number M of photomicrographs at that magnification necessary to form a representative sample of the total area A; (3) the total number of test points P,,? required to obtain a result with specified accuracy; (4) the optimal distance d of the measurement points according to Eq. ( Four five ) . With these

COWARDLY. 2 2 . Application of the coherent test system of Fig. 2 1 to a liver cell to exclude the cell nucleus from the measurement (data related to cytoplasmic volume),

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277

under the specified conditions, the number of test points P to be applied to each photomicrograph of the area A AI,/rM is fixed between the following two limits (PT//+f)

< p-1 < ( A / d 2 )

(46)

P i should therefore be as close as possible to P i /M for efficiency reasons. However, we would like to deviate from this rule if the examined organelle has the volume or less. In this very rare case, i.e. it only occupies 2-5 cases, we want to ensure that virtually all profiles present in the section are covered by the test system and therefore contain approximately one grid point. In these cases, we would choose the grid period so that d 2 is approximately equal to the average area of the profile. The previous argument related to the volumetry of measuring points. Identical regulations can be given for test system construction for area density measurements. According to tc Hilliard (1965), the total number of intersection points needed to achieve a given accuracy is

AND

7==3

0,4

[ASK FOR HELP,.)]'

(47)

From this we can estimate the total length of test line L distributed over M micrographs as

Therefore, it suffices to perform a limited number of counts on any test line to approximately estimate i, and a(lr,) to fix L,. The test line density L,4 to be applied to each micrograph is then obtained from

Using a simple grid of parallel, equidistant test lines, their spacing becomes d = l / L , , ; with square grid d = 2/L.,,. The above reasoning showed that the spacing of the test points must be adjusted to the size and frequency of the structures under investigation. Given the large differences in the properties of cytoplasmic organelles, one test system is certainly not suitable for all structures. However, coherent multi-plant test systems allow simultaneous estimation of very different structures (Weibel et al., 1966). Such test systems (Fig. 23) consist of a square grid with a distance d, in which every g-th line is stronger. With P, thick line crossing points, the total number of points in the test system is P', 1g2 x P,. The advantages of multi-net test systems, which in a sense represent a kind of multi-stage sampling, are obvious: When estimating the bulk density of a dispersed organelle, or in the cytoplasm, c, it is only necessary to count everything.

278

EWALD R. WEIHEI.

Po points included in the o profiles and the number of heavy P points included in the cytoplasm. Then V y , = P,//jj' x P, follows. A double-grid test system with g=5 has been used successfully to study a sparse organelle of the V.ISCUlar endothelium (Fuchs and Weibel, 1966; Burri and Weibcl, 1968). In a liver cell morphometric study, a double grid with g = 3 is allowed

FIG.23. Dual coherent grid test systems with a grid point ratio of 1:4 (a) and 1:9 (b).

simultaneous and efficient estimation of the volume density of nuclei, cytoplasm and mitochondria with the coarse grid and of microbodies and lysosomes with the fine grid (Weibel et al., 1969). It should be noted that dual grid coherent test systems avoid the need to scan every point on the fine grid and are therefore very efficient. The main disadvantage of the square grids discussed so far is the excessive density of test lines in relation to test points. It was explained earlier that, for example,

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For statistical reasons, the test system should not have more than one intersection with the features (Hilliard and Cahn, 1961). When determining areas and volumes at the same time, we would configure the inspection system so that the profile does not contain more than one inspection point and its boundary forms one or two intersections. (Note that test lines will make at least two sharp intersections if their endpoints are outside the curve.) With the square line grid this cannot be achieved, as the boundary of any profile containing a point will have at least four intersections with test lines. Furthermore, for all profiles that are entirely within the test area, there will always be an even number of intersection points, at least two, as there will be an “out point” associated with each “in” of the line. The number of crossings counted is therefore at least twice as high as necessary. To overcome this difficulty, a test system consisting of short test lines of equal length z was developed (Weibel et al., 1966); arose from a suggestion by Chalkley et al., (1949) to use short "needles" to estimate volume-surface relationships. The test systems shown in Fig. 24 are all consistent in the sense mentioned above. Using the endpoints of the n test lines as markers for titration, the test system is defined as follows

PZ = 271

For n = 21 or 84, the test area is approximately one square; if PT = 100 is desired, the test area is rectangular (Fig. 24b). Arranging the lines in a triangular grid results in a homogeneous distribution of test points and lines; it also provides easy working conditions in which lines of lines can be conveniently scanned. Especially with regard to special applications, an unlimited number of alternative test systems can be designed. When studying anisotropic structures, Merz's (1968) curvilinear proof system can be useful (Fig. 25); It basically consists of semicircles arranged in such a way as to facilitate scanning. The use of circular test lines eliminates directional errors resulting from structural anisotropy. Sitte (1967) proposes the use of triangular test lines for this purpose. It should be noted that a square lattice also at least partially eliminates the effects of structural anisotropy. The problem of anisotropy is discussed further in Section VI. In practical application to electron micrographs, we found a screen size of around 30 x 30 cm. to be more convenient, whether for desktop projector

280

EWALD R. WEIBEL

Unit of Fig. 21 or to project larger negatives onto a whiteboard. The thickness of the lines significantly affects the error; They should be thick enough to be easily recognized, but as thin as possible to clearly identify intersections. The ideal thickness is between 0.2 and 0 . 3 mm.

............. .............. .............. ........ ..... ................ .................. ......... ......... ............... .............. ,;;;;;;A;;;; ;:'

COWARDLY. 24. Multipurpose coherent test system with 42 test points (a) points (b).

and 100 heads

3. Krrovditig de P r i n i q Duta

Primary stereological data are graded counts; Therefore, they can be counted on any counting device, e.g. B. Mechanical or electrical blood cell counters are recorded. These counters must have a capacity of 5 to 10 classes, each with a minimum of two or three digits. Counts are made after reading each micro-

MORPHOMETRIC CYTOLOGY

28 1

Graphic. This is complicated and error-prone; For stereological work to become a serious research tool, a more efficient recording system is needed. To this end, we have recently developed a 10 meter electricity meter with selectable meters (Fig. 20) that automatically prints tables and punch cards (Weibel, 1967c, cf. Section III, C, 6).

FIG.2 5 . Coherent semi-circular test grid to eliminate test line anisotropy Lde Merz, W.A., Mikioskopie 22, 1 3 2 (1968)i.

4 . Calculation

von

Aggregat Morphonzetric Puvameterj

The compiled raw data is entered into the appropriate formulas to generate the desired morphometric information. To obtain volume densities, difference scores are introduced into the equation. (9). The surface densities of the membranes result from Eq. (1 5 ) by entering the counted intersections and the total length of the test line. To estimate the number densities based on Eq. ( 2 9 ) the number of profiles must be divided by the test area (N,) and defined in relation to the estimated spatial density by counting points; shape and size distribution coefficients must be determined independently. It is evident that the combination of the basic formulas of Section II, C can provide information on an unlimited number of specific parameters that characterize each cell type. This must be left to the researcher's ingenuity; a specific example relating to the liver cell is explicitly discussed elsewhere (Weibel et al., 1969). It is also evident that the calculation of all these data can easily be done with the help of a computer (see Section III, C, 6). Note that the primary data must refer to a

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fine transfer system; For example, it is important to decide in advance, that is, before starting the examination, whether the volume of a given organelle should be related to the volume of the entire tissue or to the cytoplasm of a given cell. Morphometric parameters can be expressed as relative or absolute quantities, the latter also having to be defined in relation to a reference system such as body weight. For practical purposes, we find the following types of parameters useful: (1) tissue concentrations or amounts (X, ); these are derived directly from stereological formulas. (2) Absolute dimemion per organ or animal, obtained by multiplying X by the volume of organ V. (3) “Specific” dimemion relating amount of structure to body weight unit W; this is obtained by multiplying X by the "specific" volume of the organ V i /W and is particularly useful for eliminating variations in body size of animals in experimental studies (Weibel et al., 1969; Staubli et al., 1969). (4) Relative D i m e m i o m in the strict sense expressing one parameter in relation to the other, eg surface area of mitochondrial cristae1 in relation to matrix volume. 5 . Statistical data processing

In discussing sample size (Section III, B, 3), we point out some of the statistical problems inherent in stereological analysis. In summary, it can be said that well-founded statistical methods have not yet been established to verify the validity of the data. Hennig (1957) and Hally (1964) used the notion that the expected error in the count point volume estimates should be inversely proportional to the square root of the number of test points applied to the sample and should also depend on the sample volume density. the component under study. For now, we can use standard statistical methods and determine standard errors of means or 95% confidence intervals; or in experimental studies, group means can be compared using, for example, Student's t-test (Staubli et al., 1969), assuming that the estimated parameters are normally distributed for individual animals. More suitable methods should be available soon. One of the unresolved problems is defining the appropriate sampling unit for statistical analysis. Should morphometric parameters be calculated for each microscopic field, with these estimates averaged and subjected to statistical analysis? Or should the raw data be subjected to statistical testing using the morphometric parameters calculated from the average of the raw data? This can lead to different results, especially when the size of the reference system varies from field to field, as is the case when determining relative size. Currently, we have more confidence in relative parameters derived from average primary data; However, no method is yet available to express the statistical validity of these parameters.

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von

antomation

Several devices have been introduced in recent years to automate stereological analysis; these have been verified by Iischmeister (1967). Probably the most advanced instrument is the Quantimet Image Analyzing Computer from Metals Research, Ltd., Cambridge, England (Fisher, 1967). With this device (Fig. 26), the image is fragmented into dense lines with the aid of a television camera.

MONITOR

CAMERA

EPIDIASCOG

I?;-I measure

MICROSCOPE

PROBE

PRODUCTION

COWARDLY. 26. Functional diagram of the Quantimet automatic stereo image analyzer. The epidiascope provides electron microscopy images to the camera, which can also be connected directly to the electron microscope. (From instrument documentation from Metals Research, Ltd., Cambridge, England).

Changes in beam intensity resulting from variations in microimage contrast are analyzed on a small computer that converts these primary signals into stereological information. For example, the fraction of the LL linear scan that passes through regions with a given contrast level is used to estimate the volume fraction V v of the corresponding component [Eq. (6) 1; or the number of steps or sudden changes in contrast level per unit scan length I, is used to estimate surface density or interface Sv according to equation (15). The device can also generate profile size distributions and can be programmed to calculate particle size distributions directly. As a more recent development, the Quantimet can be connected directly to the AEI EM 6 electron microscope, eliminating the need to take micrographs. Although this excellent tool can distinguish between five levels of contrast, it requires as essential conditions for its applicability that the components of interest are uniquely characterized by well-defined contrast differences and that the contrast does not vary significantly within each profile. In biological electron microscopy, these conditions are obviously not met. many organelles

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they are only recognized on the basis of a characteristic configuration of membranes or associated structures. It currently seems pointless to have a computer automatically distinguish mitochondria or rough cisterns from the endoplasmic reticulum outside the labyrinth of the cytoplasmic membrane system. It is therefore unfortunate that most biologists cannot benefit from automated image analysis; the decision to classify subcellular structures should be left to the experienced examiner. Even Moore's extremely advanced SADIE (Scanning Analogue to Digital Input Equipment) computer (1967) does not help in our case. However, semi-automation is of great help to improve the efficiency of stereological analysis. Lazarow and Carpenter (1962) developed a semiautomatic scanning instrument for electron micrographs. A point marker scrolls through the micrograph while the researcher presses a button associated with the component traversed; the duration of the trip is recorded automatically. This device has many interesting features which, with further development, could make it very versatile, leaving the identification of structures to the researcher. It should be noted that the recording of individual transverse lengths or intersections in such a device can be used to derive particle size distributions according to the method of Hilliard (1967a) cited above. In our lab, we use point counting methods whenever possible. The reilsons are many. Above all, it is the most efficient and least strenuous of all methods. Hilliard and Cahn (1961) also showed that point counting is statistically superior to linear analysis for titration, despite the large imprecision in estimating the contribution of a single organelle. Furthermore, point counting leaves fewer uncertainties than linear analysis: ambiguities always arise at transition points from one component phase to another; in linear analysis, as in automatic systems, such ambiguities must constantly occur, whereas in point counting they are limited to the comparatively rare points that lie "exactly" on the phase boundary. To identify the location of these points, the investigator may pause while examining the devices and is pressured to make a quick decision. Point counting does not require automation of the analytical part, as measurements are not required. However, the need to tabulate counts at short intervals made it desirable to automate the entire process of transferring primary data to spreadsheets and punched cards. This is possible with the device shown in Fig. 20 (Weibel, 1967b,c). The 10 tokens are fed from a keyboard, with the key assignment of the tokens being pre-selected via a breadboard. Each counter can be paired with one of the two totalizers or neither; this offers, for example, the possibility to check the total number of test points counted. At the push of a button, meter readings are automatically written onto a table with an IBM 72 typewriter, and the data can be transferred to an IBM card punch at the same time.

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The final step in automation involves data analysis. A complete computer program was developed for the automatic calculation of a variety of morphometric parameters from primary data obtained by point counting (Gnagi and Weibel, 1968); is available in FORTRANlayout from the authors. The supporting program consists of several basic and special subprograms listed in a catalogue; they can be combined at will through entry cards. The first step of the program calculates the basic morphometric parameters needed for all the micrographs that make up the primary specimen representing, for example, an experimental animal, and determines the standard error of the mean for each parameter. A second step calculates the means of the groups and a third performs their statistical comparison using Student's t test (Weibel et al., 1969; Staubli et d., 1969). Introducing this limited level of automation into data acquisition and calculation has increased the efficiency and performance of our stereology laboratory many times over. 7. Determination and registration of profile size

Di.\ tvibzrtion

The derivation of the grain size distributions and their parameters from the sections requires the determination of the grain size distributions of the profiles. The dimensions of individual profiles are measured directly, for example the (average) diameter of circular profiles derived from approximately spherical particles. It is more convenient to estimate the profile diameter by fitting circles to the profile (Fig. 2 7 ). This is easily done using a clear plastic graduated circle stencil, such as used for graphic work, or by drawing a set of concentric circles on a sheet of clear plastic. Proper choice of ranges allows direct classification into several size classes, finding 1 2 to 1 5 classes

is FIG. 27. Plastic template with graduated circles to estimate the average diameter of the profiles.

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it is usually sufficient to characterize the size distribution of the profile (Saltykov, 1967; et al.). The Zeiss Particle Size Analyzer (Endter and Gebauer, 1956) is a sophisticated tool for analyzing electron micrographs printed on paper: the size of a circular disk of light projected onto the photograph approximates the profile; the disk size is automatically read and, by the action of a switch, a count is added to the appropriate class registered in one of the 50 counters. The intervals between classes can be chosen between linear or logarithmic. This device was, for example, by Heiniger et al. (1967) and by Staubli et al. (1969) to determine the size of nuclear profiles of lymphocytes and hepatocytes in electron micrographs and by Haug (1967) to derive the size distribution of nerve fibers.

IV. An Example of Morphometric Characterization of Organelles: The Liver Cell A. GENERAL CONCEPT OF

AND

STUDY AND SAMPLING METHODS

To illustrate the application of stereological methods to morphometric cytology, the essential steps in a recent systematic study of the subcellular composition of rat hepatocytes are briefly described (Weibel et al., 1969). This study was carried out with the aim of obtaining a quantitative correlation between morphological and biochemical changes after treatment with phenobarbital (Staubli et al., 1969). Therefore, morphometric data should be obtained according to biochemical studies. These were performed on subcellular fractions of liver homogenates, which are essentially random samples of components from all parts of the organ; Therefore, assuming that hepatocytes form a unit, the morphometric sampling also had to be random. This contrasts with a more recent study by Loud (1968), who examined regional variations in the morphometric parameters of liver cells in different zones of the lobule and, consequently, had to examine a sample of cells selected from three different zones of the lobule, in accordance with a In line with the biochemical practice of relating data to liver tissue unit weight, basic morphometric data were expressed in terms of 1 ml. tissue,5 that is, as structural densities. Due to the wide range of structures - from about 1 mm lobes. in diameter to endoplasmic reticulum smooth tubules with a width of 300 A. The following multi-step sampling procedure was applied: (I) Goldner-stained paraffin sections were evaluated automatically at 200X magnification using a WILD sampling table microscope for systematic sampling 5

Liver tissue specific gravity was determined to be 1 0 6 7 on average.

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Campos (Freere and Weibel, 1967; Gander, 1967). Using a 100-point square grid test system on the projection head screen, the volume density, V, of the lobular parenchyma throughout the liver was estimated. (11) A random sample of five blocks per animal was taken from a mixture of tissue cubes fixed in osmium and soaked in epon. Micrometer-thick slices were used for microscopic evaluation of the number and size of core profiles at 1000x magnification; this allowed an estimation of the number of cells in the tissue volume unit.

(H I ) Ultrathin sections (600–900 Å, ) were cut from sdme blocks and mounted on 200 mesh copper grids for electron microscopy. Six electron micrographs, systematically scanned by placing the viewing screen at specific corners of the support grid (Fig. 17) (Weibel et al., 1966), were taken at 2500X magnification. Fields without lobular parenchyma were discarded; the data obtained in this sample were therefore related to the lobule parenchyma and had to be multiplied by Vv to establish its relationship to the entire liver tissue. The final magnification of the photomicrograph projected onto the projector unit's test screen was 2 2.5 0 0 ~ By . Using a 9:1 dual grid test screen (Fig. 28), the cytoplasmic volume density of hepatocyte nuclei V v pof and of larger cytoplasmic organelles (mitochondria, microbodies, lysosomes) in the lobular parenchyma was estimated using only the fine mesh for microbodies and lysosomes. (IV) A second sample of six micrographs from each of these five sections per animal was obtained at a primary magnification of ~0.000El~. The rules for placing the screen were like at level 111; However, fields containing less than 50% cytoplasm were discarded, as all measurements at that level were related to cytoplasm volume. Stereological analysis was performed at 90,000x final magnification using an 18-line multipurpose test screen (Fig. 29). It included an estimate of the volume and surface area of the rough and smooth membrane of the endoplasmic reticulum and the envelope and cristae of mitochondria1. To relate it to the total liver tissue, these data had to be multiplied by (V,,, x Vvc.) obtained in levels I and III.

B. SPECIFIC METHODS FOR ESTIMATION OF THE MORPHOMETRIC PROPERTIES OF CELLS AND SUBCELLULAR COMPONENTS When providing specific methods for the morphometric characterization of cell structure from electron micrographs, we refer mainly to the liver cell as a model (Weibel et d., 1969). Obviously, some of these methods need to be slightly modified when applied to other cells. In addition, we will consistently propose scoring methods for the reasons set out in Section II, C, 2.

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COWARDLY. 2 8 . Double grid test system (9:1) superimposed on liver section (111 sample plane) with sinusoids (S), biliary capillary (B) and hepatocyte nucleus (N). Note that bold dots are useful for estimating mitochondrial volume (MI), while more sparse organelles (lysosomes, 1.Y) should be scored using a fine grid (de Wribel CI al., 1969). 12,500x magnification.

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1 . Zellgroße bin! i\.l~ilorCell Compcirtmeizts

The number of cells per unit volume was estimated from the number of N,-,, hepatocyte nuclei counted in the stage II sample; Binucleated cells were not considered separately. At level I1, the volume density of cytoplasm V,.,. y of Vvn cores was estimated using a 99-point square grid; To point

COWARDLY. 29. Electron micrograph of liver cells (sample level IV) with full-size ratio test system (de Weibel et al., 1969). 37,500x magnification.

the distance was 2.5 cm. on the screen. The average volume of a mononuclear hepatocyte was found by TIL

= (V,.,. + VL~ll)jNl~lL

(50)

Cell surface was measured in 111-level photomicrographs with a 2 cm square grid. Distance, where intersections with sinusoidal, biliary and juxtacellular surfaces were recorded separately to estimate cell polarity. An estimate

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of the average diameter of cell 2 results from the volume/surface ratio (v/.r), since a= 6 (2 ~/5) = 6 (V v/S v). two . mitochondrial

Mitochondria are quantitatively characterized by their volume density VVrmi, their number density Nvmi, and the outer (Svmo) and inner membrane surface density of cristae (Sr,nc.). The relationship between the surface of the cristae membrane and the volume of the matrix can, of course, be derived from these basic parameters. VVllli and Nvmia are better bypassed at a lower power like level 111 ( 2 2 , 500x); the coarse grid of 99 points was considered sufficient to determine Vvlli. Calculation of Nvmi by equation. (29) requires knowledge of mitochondrial shape; Based on an analysis of the axis ratio of approximately elliptical profiles, it was considered elliptical with an average axis ratio of 4:1, giving a value of p = 2.35. Obviously, this can only lead to rough estimates, as significant variations in mitochondrial shape are to be expected. Ndmi was used to count mitochondrial profiles, a 103 p2 frame; The profiles that are entirely inside the frame and those that cross its left and upper sides were counted, while those that cross the right and lower sides were discarded; this is analogous to the usual red blood cell count. Counting intersections of test lines with mitochondrial membranes requires higher magnification for better resolution of intersections; 9 0 .0 0 0x was considered appropriate, although 50,000X is still appropriate (Fig. 19). For convenience, intersections with the cristae (ILmc) and the innermost outer membrane (ILmo) were counted as one point, even though two membranes were involved (Fig. 30). While the area density of the envelope membrane is given directly by Eq. (15) that of the inner membrane with cristae was obtained through

where an asterisk indicates that the surface density and test line length refer to the cytoplasm and not the entire tissue (see Section III, C, 2). Note that the thickness of the Hnit section causes the extent of the ridges to be underestimated by 20-400/0, while the surrounding membrane area is not much affected by this error. 3. Eidoplasmic Keticiltim

Measuring this finely structured component requires a high magnification of 50,000-90,000x (Fig. 19 and 30). Again, an 18-line multipurpose test screen is adequate (Fig. 29). When determining the volume and surface density of the endoplasmic reticulum, it is often critical to distinguish between its rough and smooth form. To avoid uncertainties and arbitrary judgments

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COWARDLY. 30. Actual magnification of a portion of the electron micrograph used for Stage IV stereo analysis. The intersections of the test lines with the mitochondrial membranes1 are circled, a circle represents a count. Note that double intersections of ridges and "contour membranes" are counted as one point for simplicity (cf. Eq. 5 0). 80,000x magnification.

At the transition points, it has been useful to define the endoplasmic reticulum (ER) to include all membrane-bounded cytoplasmic cisternae, tubules, and vesicles (Weibel et al., 1969). In particular, the perinuclear cisternae were considered to be part of the rough ER space, their ribosome-studded outer membrane part of the rough ER membrane. Likewise, all Golgi elements were assigned smoothly

ex. It is obvious that this simplification will not be appropriate for all theorems. The number of membrane-bound ribosomes can be determined by an indirect method: in high-power electron micrographs (-9 0.0 0 0 ~), irregular ER profiles with clearly recognizable membranes are randomly selected (Fig. 30). . The length of the contour b of the membrane trace is measured with a wire or map gauge and the number of ribosomes attached to this contour is counted. It must be assumed that these ribosomes are attached to a membrane strip that extends across the entire thickness of the section, which in our study was 600 to 900 Å. The range can be extended to about 1.2 times the section thickness, or about 850 Å to appreciate. The number of ribosomes can thus be related to a range of membrane area (b x 850 Å), which allows calculating the number of p ~ particles per 1 piece of membrane. The number of ribosomes per unit cell or tissue volume is obtained by multiplying this number by SVrer. As with mitochondrial cristae, section thickness leads to an underestimation of Vv,,rm d Svrr by 20-30% because the profiles formed by grazing sections cannot be discerned. Loud (1967) further estimates this underestimation, but this could also be due to the lower powers used in his studies.

4. Cytop1u.r mir Gruiz/les Cytoplasmic granules or paraplasmic organelles are generally rare constituents; therefore, estimation of their apparent density requires a relatively dense dot grid applied to low-power, ie, large-field, electron micrographs. In liver cells, microbodies (peroxisomes) and lysosomes are easily identified in micrographs at 2 2, 5 0 0 magnifications. Its bulk density was determined using fine dots 891 (99 x 9) of the 9:1 dual grid test system level 111 (Fig. 28); the thick dot grid proved insufficient (Weibel et al., 1960). To estimate the number density of peroxisomes according to Eq. (29) Profiles were counted within the frame used for mitochondrial counts1; as peroxisomes are short ellipsoids, the shape factor 13 was assumed to be 1.45. When investigating rod-shaped granules of endothelial cells (Fuchs and Weibel, 1966; Burri and Weibel, 1968), higher powers were required, as the specific organelle examined could only be identified by means of a fine internal structure. To relate their bulk density to cytoplasmic volume, a 25:t double grid test system was used (Weibel et al., 1966); the thin tips were used to estimate the volume of the organelles, the thick tips to assess the extent of the cytoplasm. It should be noted that this was a difficult case in terms of sampling, as endothelial cells are thin and highly polarized cells and the organelles are not evenly distributed throughout the cytoplasm but are found in clusters (Burri et al., 1968).

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morphometric data

As one of its main possibilities, morphometry offers the possibility of correlating quantitative data from biochemical and morphological studies of the same material. So far, only limited use has been made of this option. In their morphometric study of mitochondria, Baudhuin and Berthet (1967) determined cytochronic oxidase activity, but used this information mainly to size their sample relative to the whole liver, as they were working with subcellular fractions. Kimberg and others. (1968) investigated mitochondrial respiration1 and oxidative phosphorylation of mitochondria after cortisone treatment in conjunction with a cytomorphometric study ('Wiener et B., 1968). Mitochondria were found to be larger and fewer in number than controls, but the surface density of cristae membranes per cell remained normal. These morphological findings did not allow direct interpretation of the observed decrease in oxygen consumption and uncoupling of oxidative phosphorylation based on structural changes. It is known that treatment with phenobarbital induces smooth ER proliferation in liver cells and increased activity of several microsomal enzymes. In a recent study, Staubli ef a/. (1969) attempted to quantitatively correlate morphological changes with biochemical changes. Rats received phenobarbital for up to 5 days. From 16 hours after the first dose, the animals were sacrificed at different times; Each liver was divided into two samples: one for morphometric electron microscopy, another for fractionation and biochemical analysis of the microsomal fraction. This study established a linear relationship between proliferation on the ER smooth membrane surface and increased activity of three microsomal enzymes involved in drug metabolism. Furthermore, the thick ER was shown to increase initially with a sharp increase in the number of ribosomes, followed by regression to control dimensions after 2 days; This was interpreted as possibly related to the synthesis of enzymes and structural membrane proteins in the rough ER that were secondarily translocated to the smooth ER. This correlative approach to quantitative cell biology can be expected to play an increasing role as molecular biology concepts move from the microbiological level to that of the animal cell with its complex organization.

V. Cytomorphometric Methods in Experimental Pathology Pathological histology is increasingly using electron microscopy for the precise localization of tissue damage. Again, cytomorphometric methods can be very helpful. Some investigations into small structural changes in the lungs under the influence of pure oxygen may serve as an example. with microelectrons

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morphometric-scopic methods, Kistler et al. (1967) showed that rat lung alveolar tissue responds to oxygen breathing with initial formation of edema, resulting in a doubling of the thickness of the air-blood barrier. Kapanci et al. (1969) showed that similar events occur in monkey lungs, although there are different species in the cellular response mode. In both studies, the quantitative changes in the tissue could be correlated with the functional decline observed in the animals. Kapanci et al. (1969) also quantitatively defined restoration of tissue structure during convalescence in oxygen-poisoned monkeys. It turns out that this type of study has great potential to establish quantitative structure-function correlations in human pathology as well. Two other examples should be mentioned. Hollmann (1968) compared the cytomorphometric data of mouse breast cancer cells with those of normal lactating tissue. He found a significantly lower density of organelles in cancer cells. Poche et al. (1968) stereologically studied the volume ratio between mitochondria and myofibrils in the myocardium of rats undergoing experimental hypertension. Myocardial hypertrophy reduced this ratio to less than 50% of the control value. These studies show that stereological methods can lead to significant results in many projects that have not yet been investigated.

VISA. Problems that arise when applying stereological methods to anisotropic systems

Cells are anisotropic in nature, as their function requires an orientation to related functional spaces: glandular cells extend between the interstitial and luminal spaces, muscle cells between the origin and insertion. However, the addition of many anisotropic cells is often isotropic in the stereological sense, because the preferred orientations of individual elements with respect to the shear plane cancel each other out. Indeed, this is an essential prerequisite for the applicability of stereological methods to biological tissues. If anisotropy is not eliminated naturally, as is the case with surface epithelium, muscle, etc., this must be explicitly taken into account when establishing stereological methods. These concern sampling as well as the selection and use of test systems.

A. ANISOTROPIC TISSUE SAMPLE 1.

source distribution

It is plausible that all parts of an anisotropic system are represented in the sample in proportion to their frequency, that is, in proportion to their proportional volume. For example, surface epithelial cells exhibit a functional and structural polarity whose axis is perpendicular to the surface. In

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In a first stage of the stereological work, the density gradient of the structures along the polarity axis must be evaluated. Note that a line coinciding with the polarity axis crosses the different layers in relation to their relative volumes; this follows from equation (6). For this, the sample must be extended throughout the depth of the cell layer; If high magnification is required so that a field cannot span the full depth, a sequence of adjacent frames should be examined. It is important to ensure that the frames do not overlap. If possible, point counting volumetry should be used to determine the structural gradient, as this method is not affected by additional internal anisotropy of cells resulting from preferential orientation of membranes or organelles relative to the surface. Test points can be conveniently arranged in lines aligned parallel to the polarity axis. These 'column' sample units should be collected repeatedly across the surface, preferably at equal intervals to ensure systematic sampling, unless inherent periodicity requires random sampling to avoid bias. If tissue anisotropy results from the sequential arrangement of clearly differentiated units of specialized structure and function, multi-stage sampling can be efficient: in a low-power first stage, the fractional volume of the different structural units is estimated, while in the second, higher power stages, it is possible to focus on specific units. Units to get a more accurate assessment of your composition. The procedure for weighting the detailed results against the total tissue depends essentially on the specific conditions of the study. two.

alignment of sections

Flat sections used for microscopic examination are anisotropic; This requires careful alignment of the section with respect to the axis of tissue anisotropy to ensure an unbiased sample. In general, the clipping plane must contain the axis of anisotropy; Therefore, the epithelium must be cut perpendicular to the surface. Of course, deviations from this rule may be indicated under special circumstances. If the tissue has multiple axes of symmetry, as is the case with skeletal muscle, the sections should be oriented parallel to each of them; in the case of skeletal muscles, longitudinal and cross sections are sufficient.

B. EFFECT OF ANISOTROPY ON STEREOLOGICAL MEASUREMENTS; ON THE INFLUENCE OF PROOF SYSTEMS It has already been pointed out that anisotropy causes problems only if it somehow interferes with an anisotropic proof system. Therefore, point-of-difference counting volumetry is not problematic when the section is an unbiased tissue sample. However, it may be appropriate to use random point arrays to avoid interference with periodic tissue structures. Grids of random points with even dispersion can be obtained, for example, by marking a random point within each of the

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25 squares from a grid as shown in Fig. 31. This also facilitates exploration of the field. The usual grids of parallel lines used to estimate area by counting intersections are highly anisotropic. Therefore, special care must be taken when using it if preferential orientation of structural surfaces is expected. There are several remedies. One is to use an isotropic test line, represented by a circle. Merz (1968) proposed an isotropic test system consisting of a sequence of semicircles that is easy to scan (Fig. 25). Triangular test lines (Sitte, 1967) or short test lines arranged in equal numbers in three directions at a distance of 60' (Weibel and Knight, 1964) have comparatively low anisotropy, as do square line grids. Sitte (1967) pointed out that successful compensation of aniso-

COWARDLY. 31. Grid with 25 random dots. In each square, the position of a point was determined based on two-digit random numbers read from a table.

Tropic effects in oriented structures can be obtained when the lines of the triangular or square lattice are oriented at fixed angles to the axis of structural anisotropy. When in doubt, multiple readings can be taken in different orientations. It should be noted that estimating Sv on sections cut parallel to the orientation axis, as recommended above for sampling reasons, leads to a biased estimate. It is often necessary to take measurements in several sections with different orientations. Hilliard (1967c) discussed this problem at length; he also showed that using a special elliptical test figure (Fig. 32) gives an unbiased estimate of Sv via Eq. ( 1 5 ) counting the intersections with the surface trace in a single cut parallel to the orientation axis, regardless of the degree of anisotropy. In the case of cylindrical or prismatic structures, Sv can also be estimated from exact cross sections. Counting the intersections I of the surface trace per unit length of a suitable line grid, we obtain

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where h is the length or height of the cylinders. It can be seen that here the surface density is directly proportional to the contour length density of profile B,4 as defined in the equation. (This). Similar restrictions and rules apply to the evaluation of the length density of the A4 curve and the numerical density N,. oriented structures. Hilliard (1 9 6 7 ~ ) also discussed the problem of diseases, ... C. ASSESSMENT OF STRUCTURAL ANISOTROPY

It can often be of functional importance to assess the degree of anisotropy of the cellular structure. The most sensitive measurements are Sv or Mv. any surface

COWARDLY. 3 2 . Test figure to estimate 5,. of anisotropic structures. Built after Hilliard (1 9 6 7 ~).

can be divided into very small flat elements; the orientation of these elements is defined by two angles. In isotropic systems all angles are equally frequent, but in anisotropic systems there is a distribution function that depends on the degree of anisotropy (Hilliard, 1 9 6 7 ~ ) Da . The probability of intersection of a straight test line with a plane depends on the angle between the test line and the normal to the plane, the anisotropy also leads to a distribution function of the number of points of intersection depending on the angle of orientation between the lines test - test and axis of anisotropy. The number of intersections is small (0 for perfectly parallel structures) when this angle is 0° and greater when the angle is 90°. This can be used to assess the degree of anisotropy within a structure. A rough estimate of the degree of anisotropy can be obtained by counting the I section densities. The number of intersection points on the lines parallel to the axis of anisotropy (IL o) and the perpendicular lines (/L9, ,) are entered separately in the grids . The smaller the ~ L O / I L Q O ratio, the greater the degree of anisotropy.

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The pollination of cells or subcellular troorganelles is often defined by the proximity of parts of their surface to other structures. In the liver cell example discussed above, the polarity was determined by the fraction of the cell surface adjacent to the sinusoids, bile duct, and adjacent cell; was estimated from the ratio of the respective intersections with a square grid (Weibel et al., 1967). Sitte (1767) estimated the proportions of the basal, lateral and apical surfaces of renal tubular cells by the same method. Dorfler (1967) introduced the term “proximity parameter” to describe the degree of contact of one structure with another; This parameter is estimated by the relative size of the immediate context surface.

VIII Assessment of the Current State and Prospects for Future Possibilities Orderly progress in recent years has made stereological techniques applicable to electron microscopic cytology without great effort. In fact, there are now several methods that allow the quantitative study of almost all structural properties of cells and tissues at any necessary magnification. Error sources are defined and widely workable methods are available to eliminate them. The specific methods highlighted in this overview are also quite efficient when used correctly. In our experience, estimation of up to 10 parameters in a sample of 30 to 40 electron micrographs can be performed in about 2 hours using point counting methods. This is a small effort compared to the information produced. It should therefore be demanded that the pseudo-quantitative descriptions still common among morphologists be replaced by real morphometric data that can be statistically verified. This is particularly important when morphological findings must be correlated with biochemical or physiological information or pathological changes at the cellular level must be interpreted quantitatively in terms of a functional impairment of the organism. Stereological methods can also be applied to histochemical or cytochemical studies. As the group of Carpenter and Lazarow showed in several papers (1966, 1967; Lazarow and Carpenter, 1962) and Leibnitz (1964), the amount of histochemical reaction product can be quantified very easily by point counting or linear analysis. Ross and Benditt (1965) successfully used volumetric spot counting in conjunction with autoradiography to derive a specific labeling index of cellular components. Williams (1768) has recently developed a related method. The use of stereological methods in this area is still in its infancy. The coming years are expected to bring new and expanded approaches. It may seem regrettable that stereological methods still require the examiner's active participation in the interpretation of micrographs and in the decision-making process.

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about sorting points or measurements. The error is not in the stereology, but in the properties of the electronic image of the cut biological material, in which the organelles are recognized only by their configuration and context. However, it is still not possible to clearly distinguish cellular components using electron contrast. If cytochemical methods are available that can specifically and quantitatively enhance the contrast of certain organelles, so that contrast discrimination is sufficient for their detection in electron micrographs, then automated scanning techniques, as currently used in materials science, can be applied to biological electron microscopy. It is very likely that such methods will become available for some organelles in the not too distant future. For now, however, efficient point counting methods and semi-automated analysis methods should suffice. However, these methods are valuable tools that deserve a broader introduction to electron microscopic cytology.

ACKNOWLEDGMENTS In concluding this report, I would like to acknowledge the help and encouragement I have received over the last few years in developing the concepts presented. Without the mathematical collaboration of Drs. D.M. Gomez, €3. W. Knight and H. Giger would have remained rudimentary for a long time; and dr I must thank G. E. Palade for a challenging introduction to cell biology. Much encouragement also came from interdisciplinary discussions within the framework of the International Society of Stereology. I thank Dr Hans Giger for critically reading this article.

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Some possible roles for isothmic substitutions during hardening in D. W. A. ROBERTS plants Regression: Station h. Caiiada Depattmeti~of Agri: ultuie Lethbiid, te, Alheitn, Canada I. Introduction . . . ... ...................... 103 Effects and Prevention of action . . . . . . . . . . . . . . . . 304 A. Extracellular ice. . . . . . . . . . . . 304 B. Intracellular ice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 111. The effect of low temperature on proteins. . . . . . . . . . . . . . 309 A. Description of Effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 B. Protection of Proteins from Cold Damage 311 IV. Metabolic imbalance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313 A. Effects of Temperature Changes on Crll Regulatory Machinery 113 . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Effects of Temperature on the Rate of Enzymatic Reactions 314 C. Prevention of Metabolic Imbalance. . . . . . . . . . . . . . . . . . 31 6 318 V. The isoenzyme substitution hypothesis. . . . . . . . . . . . . 318 A. Nature of the Hypothesis . . . . . . . . . . . . . . . . . . . . . . . . B. Isoenzyme substitutions and strain hardening. . . . . . . . . . 319 C. Problems Related to Testing the Isoenzyme Substitution Hypothesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 D. Implications of the Isoenzyme Replacement Hypothesis 121 VI. Final remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 References . . . . . . . . . . . . . . . . 323 11

I. Introduction As noted above (Levitt, 1962), there is an urgent need for new approaches to the problem of robustness and robustness in higher plants. This article addresses the issue of robustness to show where and how replacing the modified form of a protein with the form normally present at higher growing temperatures would be beneficial to the plant at lower growing temperatures. A plausible mechanism for performing such substitutions is presented. Some of the few known possible examples of such substitutions are critically discussed to show the experiments that need to be performed to prove that the proposed hypothesis works. As very little work has been done in this regard with higher plants, the plausibility of many of the proposals is supported by evidence from microorganisms and poikilotherms. It is assumed that the same basic mechanisms of resistance and adaptation to cold occur in plants as in microorganisms and poikilotherms. The relative importance and details of these mechanisms are expected to vary from one group of organisms to another. 301

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Plants face a number of difficulties when the temperature drops. Ice forms when the temperature drops low enough. Proteins undergo reversible or irreversible conformational changes at low temperatures. A metabolic imbalance can arise and can be harmful or lead to a remodeling of the biochemical system. Hardening is proposed as one of these retrofits, allowing the plant to partially neutralize some of the adverse effects of low temperatures. Part of this remodeling may consist of replacing a modified protein with the form of the protein that performs the same function at higher growth temperatures. These substitutions can result from the masking and unmasking of different parts of the genome. 11. Effects and Prevention of Ice Formation

Ice formation can occur in plant tissues in two types of locations, extracellular or intracellular. Extracellular freezing is not necessarily fatal, whereas intracellular freezing is usually fatal (Asahina, 1956; Levitt, 1958). Although intracellular ice formation has rarely been observed in nature (Levitt, 1956), it can occur in nonresistant cells that are frozen very slowly (Asahina, 1956) and even in cells resistant to low temperatures after extracellular freezing (Tumanov and Krasavtsev, 1959). . Since ice that forms inside the cell wall but outside the protoplast and vacuole is likely to produce effects similar to those of extracellular ice and intracellular ice, this will not be discussed. Ice formation in living organisms has recently been reviewed (Levitt, 1956, 1966; Mazur, 1966). A. EXTRACELLULAR ICE Extracellular ice forms in the intercellular air spaces of higher plants (Levitt, L956). When the temperature drops sufficiently below the freezing point, extracellular water freezes, but intraprotoplasmic water usually does not. As long as the vapor pressure of water in the protoplasts is greater than that of ice, the water will leave the protoplasts, if possible, and then freeze in the intercellular spaces and initiate ice growth there. This ice growth has two effects, the desiccation of the protoplasts and, if the crystals grow large enough, the mechanical deformation of the cells (Meryman, 1957). The drying effects of extracellular ice formation may partially explain the correlation between drought and cold hardiness observed in many, but not all, plants (Levitt, 1956). Desiccation of the protoplast will greatly increase the concentration of solutes in the aqueous phase within the plant cell. Such changes can produce large increases in chemical reaction rates, as well as in partially frozen solutions (Pincock and Kiovsky, 1966). Some of these reactions can be harmful to the plant.

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The sulfhydryl disulfide hypothesis (Levitt, 1962) may illustrate a special case of the more general situation considered here. The concentration changes themselves increase the salt concentration and can lead to precipitation of some substances and possible changes in pH. Proteins can be denatured by high salt concentrations or pH changes, if large enough. Certain types of mechanical deformation of plant tissue are known to increase respiration rates (Audus, 1935, 1939; Roberts, 1951). The mechanism of this effect is currently under investigation (Bagi and Farkas, 1967). Mechanical deformation can damage the protoplast. Such damage could contribute to the formation of intracellular ice. Plants could protect themselves from damage caused by extracellular ice formation by blocking water loss. Such an effect would increase the possibility of intracellular ice formation and would be deleterious. Furthermore, there is much evidence in the literature that strain hardening is accompanied by increased rather than decreased membrane permeability (Levitt, 1956). Plants could protect themselves from the effects of high salt concentrations caused by desiccation, replacing normal proteins with slightly modified proteins that are more resistant to this type of denaturation. These modified forms of proteins were discovered in animals (Warren and Peterson, 1966). Protection against other harmful effects of dehydration such as B. increased concentrations of other metabolites and soluble proteins may also be possible through proper protein replacement.

B. INTRACELLULAR ICE Intracellular ice is generally considered fatal, although the reasons are unknown. Possible causes are (1) protein denaturation (see Section 111); (2) higher rates of chemical reactions as they occur in frozen living systems; (3) mechanical damage resulting from the breakdown of sophisticated structure within cells by expanding water during freezing. Since little is known about mechanical damage, they will not be discussed further. Large increases in chemical reaction rates have been observed in frozen solutions. Extensive kinetic studies have shown that some of these rate increases are the result of increased concentrations of reagents in small pockets of fluid on ice (Wang, 1961; Kiovsky and Pincock, 1966; Pincock and Kiovsky, 1966). The possibility remains that some rate changes result from other factors (Grant et al., 1966). This possibility requires a very thorough kinetic study, as it is important to know whether factors other than the concentration of reagents in the liquid bags and the formation of dry surface films (Wang, 1961) are involved in increasing the reaction speed in the frozen state.

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Solutions The observed slopes are large enough that reactions that take place at negligible rates in the liquid before freezing can take place at appreciable rates in the frozen liquid. Therefore, it is likely that damage to frozen tissue is the result of chemical reactions that are too slow at higher temperatures to produce deleterious effects. This is a reasonable explanation for the damage that occurs when frozen tissues are maintained under equilibrium conditions (Levitt, 1956, 19%).

Intracellular freezing can be avoided if the water in the cell can be (1) kept in a supercooled state, (2) prevented from freezing at the temperatures encountered, or (3) freely leave the cell. Keeping water in the supercooled state at subzero temperatures indefinitely requires preventing nucleation. Nucleation can occur outside or inside the protoplast. Extraprotoplasmic ice will only form by extraprotoplasmic nucleation if there is a barrier preventing extraprotoplasmic ice from forming intraprotoplasmic water. The plasma membrane can provide such a barrier (Chambers and Hale, 1932). It is also likely that there is a barrier preventing the formation of ice in one nuclear protoplast by water in adjacent protoplasts. The maintenance of these barriers at low temperatures along with changes in their permeability will be discussed later. The possibility of nucleation within the protoplast could be reduced if the protein and lipid membranes within the cell were modified to reduce the efficiency of the nucleation sites. One modification method would be to exchange a new form of a protein at low temperatures for the form that is normally present at higher temperatures. Another method would be to alter the fatty acid composition of membranes with changes in growth temperature (Howell and Collins, 1957; Marr and Ingraham, 1962). Either or both of these methods would alter the properties of the membranes by altering, perhaps only slightly, the surface conformation of the membrane and, consequently, its effectiveness as a nucleating agent. The fact that the surface films of some long-chain compounds, including some proteins, are relatively inactive as nucleators (Evans, 1966a) may partially explain the supercooling capacity of protoplasts. Unfortunately, the identity of the nucleating agents that can be found in cells is unknown (Salt, 1958). The freezing point of water can be lowered by increasing the concentration of substances dissolved in it or by binding the water. Plants often exploit the first of these phenomena by increasing levels of protective proteins (see Section III, B) and possibly other low molecular weight compounds in their cells during hardening. Considering the role of bound water in resistance to cold requires an understanding of the structure and role of water in the living cell. Unfortunately, the structure of liquid water and the mechanism involved in it

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Nucleation and freezing in vitro, let alone in vivo, are still debated (Frank and Wen, 1957; Bernal, 1965; Frank, 1965; Evans, 1966b; Falk and Ford, 1966; Wicke, 1966). Important advances in understanding the behavior of water in cells must be made before these problems can be resolved. Water in the cell probably exists as free water, a thin layer that extends across the macromolecular surface and into fine pores. Water may be involved in maintaining some membrane layers within a cell (Hechter, 1965a). Some of this water, present as thin films or capillary columns, can have drastically altered properties, including a lower freezing point (Hori, 1956; Mazur, 1960, 1966; Derjaguin, 1966; Meryman, 1966). Some of this water may be bound and present as water in tissues down to a temperature of -60" or -70°C (Wood and Rosenberg, 1957; Sussman and Chin, 1966). If this is the case, its structure and properties remain uncertain are the environmental factors responsible for their existence at the molecular level things Nearby cells (Klotz, 1958; Vasil'eva ef ul., 1964; Ling, 1965; Schwan, 1965) sensitive plants Some attempts have been made to use some of these methods for studying water in living cells (Hopkins, 1960; Cerbon, 1964; Verzhbinskaya and Sidorova, 1964; Koga et al., 1966. Kowlasky and Cohn, 1964) are great and so far there are no simple and absolutely reliable methods to study bound water in living cells. It has been postulated that the ability of tissues to store water is correlated with their resistance to cold (Levitt, 1956, 1966). Proteins and conjugated proteins may be responsible for binding some of this water It has been suggested that proteins with very different structures carry very different amounts of bound water with them (Berendsen and Migchelsen, 1965). When this is the case, small changes in a protein's structure can lead to changes in its ability to bind water. Replacing a modified protein with high water-binding capacity at low growth temperatures with one with lower water-binding capacity, normally present at higher temperatures, can reduce the amount of water that freezes. If this development were taken to an extreme, all the water needed for viability could be in bound form. This may be the case for dry viable seeds. Therefore, it can be beneficial for a plant to lose all free water in its cells and retain just enough bound water to maintain viability. This loss can occur during extracellular ice formation when the plasma membrane is sufficiently permeable to water. This could explain why many cells show a correlation between increased permeability and resistance to cold (Levitt, 1956). When increased permeability of the plasma membrane to water is associated with cold

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hardening and serves to protect the cell from intracellular ice formation, and if the plasma membrane prevents extracellular ice from nuclear water in the protoplast, then factors that affect the structure and stability of the plasma membrane must be important in cold hardening. Apparently, the plasma membrane consists of proteins, lipoproteins and lipids. Unfortunately, the details of the arrangement of these components within the membrane are still controversial (Hurry, 1964; Hechter, 196513; Korn, 1966; Wallach and Zahler, 1966). It is not yet possible to relate the structure of the plasma membrane to its water permeability or cold stability. Evidence is accumulating that some of the plasma membrane proteins are involved in the transport of inorganic ions, such as Na+ and K+, across the membrane (Skou, 1965; Baker, 1966), while others are involved in the transport of sugars, such as galactose. (Fox and Kennedy, 1965). The identification and isolation of these proteins opens the possibility of studying their stability at low temperature and the effects of concentrated solutions of other metabolites (including salts and protective substances) on their stability. In some cases, presumably intact membranes can be isolated and examined. Comparative studies of the properties of such proteins and membranes of hardened and cold-sensitive organisms can be instructive. While it has long been known that injury and death at low temperatures results in the loss of semipermeability of living cell membranes, recent studies have provided evidence that the plasma membranes of some organisms are not stable at low temperatures and that the protein complements of the membranes plasma cells of other organisms can penetrate through the environment are modified. Even a partial loss of membrane semipermeability would allow partial mixing of metabolites with each other or with enzymes from which they are normally separated by membranes. This mixture can cause additional damage to the cells involved. Changes in cell permeability have been observed after cooling (Strange, 1964; Ring, L965a,b). Cold shock in bacteria may be an example (Strange and Postgate, 1964). Sometimes cold shock can be reduced by the presence of a protective protein, such as sucrose, in the external environment (Meynell, 1958). The protective effects of some non-penetrating compounds may serve to reduce this sensitivity to cold. If rigorous evidence can be provided that these compounds do not enter the cells they protect, then the plasma membrane is a site of injury. This doesn't have to be true across the board. Membrane properties of isolated erythrocytes are altered by freezing (Scharff and Vestergaard-Bogind, 1966). ATPase isolates involved in the transport of Na+ and K+ across rat brain microsomal membranes are sensitive to cold (Gruener and Avi-Dor, 1966). A similar enzyme in bovine heart mitochondria is protected from cold inhibition in vivo by its association with another protein (Pullman and Monroy, 1963). Little data is available

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enzyme or comparable enzymes in plant cells (Dodds and Ellis, 1966; Pitman and Saddler, 1967). Evidence for changes in the protein complement of membranes in organisms comes from studies of galactose induction in bacteria. These studies indicate the existence of a galactose permease in the plasma membrane of induced cells, but not in the plasma membrane of non-induced cells. Evidence for the association of a protein with galactose permease in bacteria has recently been obtained (Fox and Kennedy, 1965). This result shows that proteins present in the plasma membrane can change depending on the medium used to support growth. If plasma membrane protein complement can change with the medium used for growth, it is reasonable to postulate similar changes at modified growth temperatures and comparable changes in other cell membranes. Such changes can occur in higher plants during cold hardening and provide the basis for increased permeability and cold stability of the plasma membrane during cold hardening. Some of these changes might involve replacing one protein with another that does the same job but has a slightly different structure. Changes in the ouabain-sensitive ATPase properties of goldfish intestinal mucosa during cold acclimatization suggest that they undergo such a substitution (Smith, 1967). 111. The effect of low temperature on proteins

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Although many proteins appear to be unaffected by cold temperatures, some proteins denature when frozen in solution, while others denature when refrigerated in solution, even without ice formation. Examples of proteins that denature when their solutions are frozen in vitro are known (Nord, 1936). Some lipoproteins show this effect (Bornstein, 1953; Lovelock, 1957). This may be important, as lipoproteins are believed to be important components of cell membranes. Other types of proteins also denature when their solutions are frozen (Leibo and Jones, 1964). The chemical changes resulting from freezing red blood cells have been studied (Chanutin and Curnish, 1966). The erythrocyte protein denaturation products were electrophoretically separated from the undenatured protein. These changes were not observed after freezing at -75°C. although they occurred after freezing from -13" to -20 °C. Storage time increased amounts of denatured protein in red blood cell hemolysates. Some of the proteins in winter wheat clot at subzero temperatures (Heber, 1959). Sucrose (Ullrich and Heber, 1958 ) and other protective proteins may have a protective effect against such phenomena. The mechanism of these denaturation phenomena is unknown. The phenomenon

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Not only the increase of reaction rates in frozen solutions may be involved (see Section 11, B). Contact of the protein with hexagonal ice can be sufficient to cause denaturation (Shikama, 1963), since the native configuration of a protein is strongly influenced by the structure and composition of the surrounding medium (Kauzmann, 1959). ; Reitel, 1963). On the other hand, changes in weak bonds (eg hydrogen bonds) may be involved (Leibo and Jones, 1964) and cause changes in protein conformation. Some enzymes reversibly dissociate and lose activity at low temperatures in the absence of ice (Penefsky and Warner, 1965). Other enzyme proteins apparently undergo reversible or partially reversible conformational changes when their solutions are cooled (Numa and Ringelmann, 1965; Jarabak et al., 1966; Massey et al., 1966). These changes probably explain the significant increase in activation energy that occurs in several enzymes (Roberts, 1967b) at low temperatures. Changes in the activation energy of enzymatic reactions at low temperatures would alter metabolic equilibrium at low temperatures. Such changes can be detrimental or beneficial to the metabolic adaptation of the plant. Another result of conformational changes is reversible or irreversible inactivation induced by low temperatures. In some cases, reversible inactivation is followed by slow, irreversible changes that result in loss of enzyme activity with prolonged exposure to low temperatures (Penefsky and Warner, 1965; Jarabak et al., 1966). In the case of the obligate psychrophile Vibvio marinus, purified malate dehydrogenase undergoes reversible low- and high-temperature inactivation in vitro, although no evidence of low-temperature inactivation was found in vivo (Langridge and Morita, 1966). The presence of ammonium sulfate in vitro stabilizes this enzyme. The environment of this protein strongly influences its stability. About 2 OT. this enzyme is inactivated and the organism does not grow. Mutant and isozyme forms of enzymes and proteins are known that differ in their ability to withstand low temperatures (Fincham, 1957; Zondag, 1963; Hultin et al., 1966). In Neurosporu, a cold-sensitive mutant form of glutamic acid dehydrogenase disrupts metabolic processes below 20 °C. (Fincham and Pateman, 1957). Although the examples of enzymes shown have reduced stability at low temperatures, there may be enzymes with increased stability. These modified enzymes can be used by cold-resistant plants to help them withstand low temperatures. One facet of cold hardening would then be the replacement of a cold sensitive form of an enzyme with a cold resistant form. A similar theory involving thermolabile enzymes has been postulated to explain the behavior of Avubidopis thulium strains at high temperatures (Langridge and Griffing, 1959). In this species, enzyme replacements are more likely to occur in different breeds of a species than in the same breed of a given species in response to modified growth conditions. Support for this theory comes from

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Working on temperature-sensitive mutants of tobacco mosaic virus. Mutants with known differences in the amino acid sequences of their coat proteins differ in both i-zuitro and vjvo thermal stability (Jockusch, 1966). OF PROTEINS AGAINST LOW TEMPERATURE INJURIES B. PROTECTION

In vivo studies suggest that enzymes can be protected from inactivation at low temperatures using appropriate protective agents such as glycerol or sucrose (Ullrich and Heber, 1958, 1961; Shikama and Yamazaki, 1961; Chanutin and Curnish, 1966; Jarabak et al. , 1966). ). Glycerol, sucrose, and other nontoxic polyhydroxy compounds also protect proteins from heat denaturation (Beilinson, 1929; Kiermeier and Koberlein, 1957; Jarabak et al., 1962; Yasumatsu et al., 1965) and urea (Jarabak et al. ., 1966). Glycerol protects isolated lactic acid dehydrogenase from radiation damage (Lohmann et al., 1964). Glycerol (Sumner and Somers, 1947; Meyerhof and Ohlmeyer, 1952; Langer and Engel, 1958) and sucrose (Potter, 1955) are widely used in enzyme extraction and in the preparation of enzymatically active particle fractions (Axelrod, 1955; Gorham, 1955; Hogeboom, 1955). They are presumably used to create isotonicity, although it is not certain whether this is the only or even the main method of protecting enzymes and particulate proteins from denaturation. Some of the cited cases suggest that glycerol and sucrose act as protein protectors independently of their osmotic effects. Unfortunately, the mechanism of these effects is unknown (Jarabik et al., 1966). The same compounds in uiuo protect organisms from damage caused not only by low temperatures (Polge et al., 1949; Luyet and Keane, 1952; Lovelock, 1954; Perkins and Andrews, 1960; Trunova, 1964), but also by high temperatures (Molotkovskii and Zhestkova, 1964) and radiation (Vos, 1965). The protective effect of these compounds may explain the advantage that insects have in glycerol accumulation (Salt, 1961) and plants in sugar accumulation (Levitt, 1956; Parker, 1963). Modifying an organism's metabolism at lower temperatures to increase the accumulation of an appropriate protective protein is certainly another facet of cold hardening. Such modifications can be caused by isocynic substitutions that enhance the natural effect of temperature. Accumulation of low molecular weight compounds would also result in a lower freezing point of cell fluids in which such accumulation occurs. I think this freezing point depression is of secondary importance for protein protection. This would explain why relatively low concentrations are more effective in some cases than might be predicted from the ability of such concentrations to lower the freezing point. In living vehicles, dimethylsulfoxide appears to have protective effects similar to those of glycerol and sugars (Lovelock and Bishop, 1959; Sherman, 1964; Bouroncle, 1965). In the few cases studied, there was dimethyl sulfoxide

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It was found to be able to protect proteins from low temperature inactivation (Chilson et al., 1965; Graves et al., 1965; Chanutin and Curnish, 1966) and radiation damage (Lohmann et al., 1966). It also protects animals from radiation damage (Vos, 1965). However, dimethyl sulfoxide is metabolized in animal tissues (Williams et al., 1966) and therefore may not be the active compound to protect proteins in vivo. Furthermore, it may increase membrane permeability in vivo (Kligman, 1965; Altland et al., 1966; Fowler and Zabin, 1966; Hellman et al., 1967) and this would help to prevent intraprotoplasmic freezing. There are examples where glycerol, sucrose or dimethyl sulfoxide do not protect against cold (Hollander and Nell, 1954; Taylor and Gerstner, 1955; Terumoto, 1965; Wang and Marquardt, 1966). Differences in protective activity do not invalidate the concept that these compounds act as antifreezes when (1) the non-functioning compounds are toxic while the active compounds are non-toxic, or (2) the active compounds are permeable to cells but not ineffective. If such evidence fails, the concept of frost protection must be rejected in individual cases. Based on their protective effects at different freezing rates, it has been suggested that the mechanism by which glycerol and dimethylsulfoxide work is different from that of sugars (Rapatz and Luyet, 1965). Perhaps further studies on the protein-protective effects of these compounds could explain these discrepancies. The various sugars differ in their protective effects against higher plants (Tumanov and Trunova, 1957; Perkins and Andrews, 1960; Trunova, 1964). Only sugars that enter plant cells and are further metabolized are effective in increasing frost resistance (Trunova, 1964). If true, this result shows that sugars do not protect higher plants by lowering the freezing point of cell fluids or by serving as protein shields. More detailed information is needed on the ability of various sugars to enter plant cells, act as protein protectants, and serve as metabolic regulators through catabolic repression (Magasanik, 1961; Maas and McFall, 1964) and other induction and repression phenomena. metabolism (Glasziou et al., 1966; Marri et al., 1965). Several other compounds are protective when alive. These compounds seem to be characterized by their high affinity for water (Nash, 1966). They form strong hydrogen bonds with themselves and with water (Mazur, 1966). This group of compounds, which includes dimethylformamide, dimethylacetamide, and N-methylpyrrolidinone, does not appear to have been tested to protect against iiz vho proteins. They deserve to be tested with cold-labile enzymes. A second group of safeners that function at fairly low concentrations include maleic hydrazide (Sxkai, 1957; Gaskins, 1959; Stewart and Leonard, 1960; Hendershott, 1962), 2-chloroethyltrimethylammonium chloride (CCC or CYCOCEL) (Wiinsche, 1966) and N-dimethylaminosuccinic acid (B9 or B995) (Marth, 1965) and Dormin (Irving and Lanphear, 1968). The capacity

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These compounds that increase cold resistance are poorly studied and their mode of action is currently unknown. This group of compounds is effective at concentrations so low that they can behave like hormones, either triggering the production of proteins that support cold resistance or stopping the production of proteins that are inimical to cold resistance. Another possibility is that gibberellins are the hormones and that a reduction in their concentration or potency is associated with cold hardening. This possibility is suggested by the natural dwarfism often associated with stress (Levitt, 1956) and the known physiological effects of Dormin and CCC (Anderson and Moore, 1967; Khan and Faust, 1967).

4. Metabolic Imbalance Changes in temperature can affect the relative rates of turnover of different enzyme pathways through at least two mechanisms, namely, changes in allosteric inhibition and changes in the rate of individual enzyme reactions with temperature. A consideration of these disturbing effects suggests possible mechanisms for overcoming them. One mechanism is to substitute one form of protein for another. The final section of this article analyzes this possibility in terms of work hardening and cold resistance. CHANGES IN A. EFFECTS OF CELLULAR TEMPERATURE REGULATION MACHINES

AND

Allosteric inhibition (Monod and Jxob, 1961; Jacob and Monod, 1963; Monod et al., 1963) is believed to be an important factor in regulating metabolism in bacteria through feedback inhibition of metabolic pathways (Umbarger, 1961) and through induction is - phenomena of repression (Jacob and Monod, 1961). Both mechanisms are probably at work in higher plants (Umbarger, 1963). Some enzymes involved in feedback inhibition show changes in sensitivity to inhibition with changes in temperature (Taketa and Pogell, 1965; Bailin and Lukton, 1966). Mutant forms of enzymes subject to feedback inhibition are produced that differ not only in sensitivity to allosteric inhibition, but also in the effect of temperature on that sensitivity. This phenomenon has been proposed to explain the need for histidine at low temperature for the growth of Escheril hid r di mutants (O'Donovan and Ingraham, 1965). Such a situation could lead to a serious metabolic disorder that would lead to slow death in higher plants. This phenomenon suggests the possibility of changing an isozyme form of an enzyme subject to feedback inhibition to another, in order to adjust the metabolic balance with changes in temperature. Comparable phenomena can occur in the regulatory machinery of induction and repression, since temperature-sensitive alleles of regulatory genes are known.

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(Horiuchi and Novick, 1961; Gallant, 1962; Sussman and Jacob, 1962). Some of them are constitutive at high temperature and inducible at lower temperatures (Horiuchi et al., 1961; Udaka and Horiuchi, 1965). Others are inducible or constitutive at low temperatures (Gartner and Riley, t965). Changes in apparent inducibility can also result from changes in the amount of repressor present at different temperatures (Halpern, 1961). These phenomena may explain the disappearance of dextransucrase and its replacement by invertase in a strain of Luctobucilw when cultivated at temperatures above 37°C. (Dunican and Seeley, 1963). The modified structure of the polysaccharides that accumulate in O~cill'ztoru grown at temperatures below 5°C. it may be the result of modified relative rates of phosphorylase and branching enzyme synthesis (Fredrick, 1953). The failure of some strains of Newo.rpom to produce tyrosinase at 35°C. when prepared at 25°C. (Horowitz and Shen, 1952) may be another example of the effect of temperature on the regulatory machinery of the cell. The genetics of this effect are complex and multiple regulatory phenomena may be involved (Horowitz and Fling, 1953). B. EFFECTS OF TEMPERATURE ON THE RATE OF ENZYME REACTIONS The rate of enzymatically catalyzed reactions decreases with decreasing temperature. The knockdown rate is different for each enzyme (Sizer, 1943). Consequently, as the temperature decreases, the size of the pool of metabolites that accumulate changes. Consequently, a drop in temperature can (1) slow the rate of a reaction so much that the organism is harmed or killed by the scarcity of reaction products, (2) induce the accumulation of toxic amounts of substrates for some enzymes, (3) the size of the pool of metabolites changes, which serve as metabolic regulators or protein protectors. Unfortunately, cases of cold sore injuries have not been analyzed in enough detail to place them in one or the other of these categories. Therefore, injuries caused by cold should be treated in general. However, as changes in pool size can have interesting theoretical consequences, this topic will be discussed separately. Since the continued life of a cell or an entire organism depends on the proper functioning of a very complex and finely balanced integrated network of chemical reactions, this becomes more likely as the temperature moves away from the network's normal operating range. of chemical reactions that is changed. Therefore, metabolic imbalance should become more important with decreasing temperatures. There are a number of examples of cold damage occurring above freezing, but very few examples of such damage below freezing. Since this doesn't seem to make sense, one should look for damage caused by a metabolic imbalance below the freezing point. It is very likely that a metabolic imbalance is one of the factors involved in field death, even in temperate plants.

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The specific low-temperature responses of tomatoes to nicotinic acid, Kosmos to B vitamins, and eggplant to mixed ribosides (Ketellapper, 1963) suggest that the rate of some responses, relative to others, may be slowed sufficiently by low temperature to interrupt the growth. The lowest temperature used in these studies was 10°C. Experiments with lower temperatures should be carried out. Several examples of cold injuries above freezing are known (Sellschop and Salmon, 1928; Pentzer and Heinze, 1954; Lieberman et al., 1958; Harrington and Kihara, 1960; Youngner, 1961; Kislyuk, 1964a,b). Although its mechanism is not fully documented, these effects are likely due to a metabolic imbalance. For example, low-temperature spoilage of apples has been linked to the accumulation of oxaloacetic acid (Hulme et al., 1964). Some of these processes can be reversed in their early stages by increasing the temperature. Fatalities or injuries that occur above freezing can likely be explained on the basis of this type of metabolic imbalance, the effects of low-temperature conformational changes on proteins, and disruption of the regulatory machinery of protein inhibition, feedback, or induction-repression mechanisms. (Ng et al., 1962). Some experiments with bacteria indicate that metabolic damage can occur at subzero temperatures (Straka and Stokes, 1959; Macleod et al., 1966; Moss, 1966). Among higher plants, tobacco callus provides another example (Das et d., 1966) of subfreezing damage that does not appear to be caused by ice formation. In this case, fatal injury required considerable time at -10°C. and it manifested itself only after reheating and partial regeneration of cytoplasmic filaments that had disappeared with cooling. Observations of cellular destruction in this tissue at temperatures above freezing can be easily explained on the basis of a metabolic imbalance that occurs in the temperature range of 0-12°C. Death is likely with prolonged exposure to lower temperatures. The size of the panel of compounds that act as metabolic regulators would lead to feedback inhibition and the entry into action of induction and suppression mechanisms (Dennis and Coultate, 1966). Such changes could also allow the operation of other regulatory mechanisms (Monod and Jacob, 1961; Korner, 1966), such as those that act at the ribosomal level (Kerr et al., 1966). Induction and repression seem to work in higher plants (see, for example, Roberts, 1967b). pathway enzymes that produce excess relative products. Despite these mechanisms, changes occur in the concentration of cumulative metabolites. The temperature-dependent starch sugar (Levitt,

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1956) and conversions of glycogen to glycerol (Asahina, 1966) in plants and insects, respectively, are relevant examples. Ascorbic acid accumulates in plants at low temperatures (L'vov and Altukhova, 1951; Areshidze and Podrazhanskaya, 1956; Franke, 1957; Andrews and Roberts, 1961). The increase in the value of the proportion of unsaturated and saturated fatty acids in plants grown at low temperatures is another example (Howell and Collins, 1957; Marr and Ingraham, 1962; Gerloff, 1966). There are many other examples (eg Schwemmle, 1953; Hasegawa et al., 1966). When the size of hormone-like regulator pools is changed, large changes in synthesized proteins are expected. For example, gibberellin induces the formation of amylase and other hydrolases in the endosperm of barley and wild oats (Simpson and Naylor, 1962; Briggs, 1963; Varner et al., 1965). Indoleacetic acid modifies the peroxidase isoenzyme pattern of pea stalks (Ockerse et al., 1966).

C. PREVENTION OF METABOLIC IMBALANCE The deleterious effects of metabolic imbalance caused by the thermally induced differential reduction in enzyme reaction rates can be overcome by using isozyme forms of enzymes with lower activation energies for the enzyme form, normally at higher temperature present should be replaced. This substitution appears to occur by invertase (Blagoveshchenskii and Gavrilova, 1954; Roberts, 1967b) and possibly catalase during cold hardening of cold hardened wheat cultivars, however, the lower activation energy for sucrose hydrolysis in hardened Kharkov wheat Cold weathering has not been shown to actually be caused by isoenzyme substitution. There are other possible explanations (Roberts, 1967b). Several cases are known in which psychrophilic bacteria appear to contain forms of enzymes with lower activation energies than those present in related forms of mesophilic and thermophilic bacteria (Brown, 1957; Sultzer, 1961; Langridge, 1963). These cases need to be thoroughly investigated to show whether this phenomenon is caused by differences in primary structure or conformation between enzymes. These conformational changes have been proposed as the basis for changes in wing nerves with changes in temperature in Dro.rophila (Milkman, 1963; Milkman and Hille, 1966). Conformational changes and monomer-dimer conversion seem to be involved in the loss of thermal stability of glucose dehydrogenase in Bacillus cevei4.r spores during germination (Sadoff et al., 1965). Conformational changes may explain the changes in the thermostability of aldolase from Bucillus rtearothermophilzis, since the treatment of this enzyme with sulfhydryl compounds causes a loss of thermal stability (Thompson et al., 1958). A slow conformational change at low temperatures could explain the development of freeze-thaw resistance reported for purified catalase (Shikama and Yamazaki, 1961). An investigation into this

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if the material is suitable, two options should be possible. Both can actually operate on ,iv+o. A temperature-sensitive mutant of bacteriophage T4 has a deoxycytidylate hydroxymethylase that has a lower temperature coefficient than the wild-type. This mutation maps to the region thought to control the structure of this enzyme (Wiberg and Buchanan, 1964). This observation supports the concept that enzymes with altered temperature coefficients differ from their normal counterparts because of small changes in their amino acid sequences (ie, they are isoenzymes). Examples of what also appears to be the substitution of one form of protein for another during cold acclimation are known in cold-blooded animals (Prosser, 1963; Smith, 1966). Electrophoretic patterns generated by goldfish liver milk dehydrogenases show that the pattern changes with acclimatization. The change indicates a relative increase in the production of one type of subunit at lower temperatures (Hochachka, 1965). Changing a protein made up of several subunits in this way could allow for almost continuous adaptation to changes in temperature. Changes occur in the amino acid composition of the protein synthesized by the goldfish intestinal mucosa as temperature changes (Morris and Smith, 1967). Upon acclimating GoIdfish, the inhibitory effect of actinomycin D on the production of some of the new proteins produced in response to an increase in temperature suggests that it is produced after new mRNA has been synthesized (Smith and Morris, 1966). This observation is consistent with the hypothesis of substitution of different isoenzymatic forms of proteins functionally similar to each other when the ambient temperature changes. At the upper end of the temperature range, there is evidence of similar isozyme substitutions. Both Bciri1lii.r coagr/lan.s and B. stearothermophilz/.s produce amylases with greater thermostability when grown at 55°C. than when grown at 35°C. (Campbell, 19541). These enzymes were crystallized (Campbell, 1954b). Unfortunately, since the amino acid composition of only one of the amylases involved has been determined, it is impossible to be sure that they differ in primary structure (Campbell and Manning, 1961). Changes in pyrophosphatase thermostability with changes in growth temperature have been observed in 6. stearothermophiles (Brown et al., 1957). Two additional trains in salivary chromosomes (Clever, 1964) can be induced in Drosophilu larvae by exposing the larvae to 37°C. (Ritosa, 1963). This suggests that the production of at least two new proteins is induced by exposing Dro.rophilu larvae to high temperatures. Some thermophilic bacteria grown at higher temperatures are known to produce more heat-stable enzymes and proteins than related mesophilic forms (Koffler et d., 1957; Purohit and Stokes, 1967). In one case (Loginova et al., 1967), these differences in thermal stability

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appear to be accompanied by changes in amino acid composition. None of them have been fully investigated. Among higher plants, heat-hardened cucumber leaves appear to contain a more heat-stable urease than non-hardened leaves (Fel'dman, 1966). Leaf protein Fraction I from heat-cured beans appears to be more heat stable than uncured beans (Sullivan and Kinbacher, 1967).

V. The isoenzyme substitution hypothesis A. NATURE OF

AND

HYPOTHESIS

Some of the harmful effects of low temperatures on plants have been considered. It is suggested that these adverse effects may be partially compensated for by substituting one isoenzyme form of a protein for another form or by altering the relative proportions of the isoenzymes present. As the temperature drops, such substitutions or changes may result from changes in plant composition caused by a drop in temperature. The present hypothesis proposes that such substitutions or changes are an important part of the cold hardening process of plants. To support such a hypothesis, it is necessary to show not only that substitutions and isozyme changes occur, but also that those that do have adaptive robustness benefits. Experimentation is required to distinguish between substitutions of proteins with different amino acid sequences and proteins with the same amino acid sequence but different conformations. While there is evidence that both types of substitution occur naturally, this review focuses primarily on the substitution of proteins with different amino acid sequences. A possible metabolic mechanism to bring about isoenzyme substitutions and changes has already been considered along with some relevant examples such as invertase in wheat and thermostable amylases in bacteria. Differences in the isoenzyme forms of lactic acid dehydrogenase in different tissues of higher animals (Markert, 1963) are another type of example of isoenzyme alteration. This enzyme consists of four subunits that can belong to two types. There are five isozyme forms. Animal cells continually vary the relative proportions of these isozyme forms by changing the proportions of the two types of subunits they produce. Differences in temperature coefficients and optimal substrate concentrations for heart and liver lactic dehydrogenases likely result from differences in the isocyne composition of these two types of lactic dehydrogenases (War et al., 1967). For enzymes composed of more than one subunit, such a mechanism would be invaluable for an organism to make gradual adaptations to changes in temperature. To a researcher, this appears to be an enzyme whose properties continually change with changes in the organism's growth conditions.

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Data on duplicated maize alcohol dehydrogenase genes (Schwartz, 1966) suggest that the postulate of isoenzyme substitution is reasonable for higher plants. One of these two genes specifies an enzyme with greatly reduced activity. This gene is normally repressed but can be derepressed in the scutellum. In this tissue, anaerobic conditions can alter the relative proportions of the two isoenzymes. Therefore, it appears that such substitutions and changes are possible when cells are exposed to modified environments. There is evidence that such substitutions also occur in geographic races. They likely confer adaptive advantages on organisms. An example of isoenzyme substitution is known among the organic races of Typha latifolian. The thermal stability of apple dehydrogenase from different breeds varied (McNaughton, 1966), but these breeds did not differ in the thermal stability of their aldolases or glutamic acid-oxaloacetic acid transaminases. Isozyme or similar substitutions between structural proteins of the plant photosynthetic machinery could explain changes in the effect of temperature on photosynthetic rates that occur as a result of acclimatization (Semikhatova, 1960; Mooney and West, 1964). Data on the oxidation rate of mitochondria from different strains of Sitanioiz hy.Ihix (Klikoff, 1966) suggest a similar possibility of respiration. Isoenzyme substitutions can result in a variety of subtle changes in the properties of enzymes present in a given tissue. Isoenzymes can differ in response to inhibitors (Wieland et d., 1959), temperature, different substrates and different concentrations of the same substrate (Plagemann et al., 1960; Plummer and Wilkinson, 1963), as well as in the properties listed next. in terms of resistance to cold. Therefore, isoenzyme substitutions can be used to perform many types of delicate metabolic adaptations. The present hypothesis will require proof of this.

B. ISOZYME SUBSTITUTIONS

AND COLD CURE

Isoenzyme substitutions during cold hardening can help the plant resist low temperatures by (1) increasing the tolerance of its proteins to high concentrations of salts and other metabolites, including other proteins; (2) reduce the sensitivity of its proteins to low temperatures; (3) Increase the ability of your proteins to bind water; (4) reduce the likelihood of intraprotoplasmic ice nucleation; (5) change the metabolic balance. Changes in metabolic balance can help the plant withstand low temperatures by (1) increasing the buildup of protective compounds; (2) reduce the accumulation of toxic metabolic intermediates; (3) mitigate the effects of differential reductions in enzyme reaction rates caused by temperature drops. All of these changes can occur without serious changes in a tissue's overall metabolic schedule. Comparable small modifications in structural proteins may also be involved.

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cold hardening. Such changes can modify the permeability and stability of cell membranes or alter the properties of ribosomes to increase resistance to cold. Significant changes in plant proteins may be related to robustness (Terumoto, 1957; Heber, 1959; Hodges, 1964; Pauli and Zech, 1964; Vasil'eva et al., 1964; Meador, 1965; Simura and Sugiyama, 1965). ; Coleman et d., 1966) and Preparing for Fall (Siminovitch, 1963). These fall preparations have been associated with increased RNA synthesis. Such a synthesis is required by the proposed hypothesis, as it postulates that some distinct proteins are synthesized. Work on peroxidase isoenzymes indicates qualitative and quantitative changes in isoenzymes with changes in growth temperature (Gerloff, 1966; Olson et al., 1967; Roberts, 1967a). Changes in the concentration of repressor proteins induced by changes in growth temperatures in bacteria (Marr et al., 1964) contribute to complications. It has been difficult to correlate or explain many of the observed changes in protein content robustly. The hypothesis presented here suggests that in order to understand the adaptive advantage conferred by specific protein changes during cold hardening, we need to study in detail the properties of the specific proteins involved and, if applicable, the metabolic pathways in which they reside and develop. work in vizm. The details involved are probably species-specific. I do not mean to claim that the isoenzyme substitution hypothesis explains all facets of strain hardening, but I suggest that it is part of the phenomenon. The hypothesis concerns the metabolism of nucleic acids, since changes in the primary structure of the proteins produced require changes in the mRNA base sequence. In this area, there is already evidence that low temperatures are uncomfortable. For example, the relative rates of enzyme production under the control of a single operon in bacteria can be altered by changing the growth temperature (Nishi and Zabin, 1963). Such a phenomenon, if verified, could create or prevent a metabolic imbalance. In Drosophila, it seems that exposure to low temperatures (14°C) can induce or potentiate chromosome heterochromatinization, which induces the inactivation of the affected genes (Hartmann-Goldstein, 1967). In vitro tests indicate the possibility of errors in the translation of the genetic code at low temperatures (Szer and Ochoa, 1964). It is not known whether this harmful possibility exists in Z E z h o . After cold shock, some authors (Byfield and Scherbaum, 1967) observed that RNA, although stable, loses its ability to synthesize proteins, while other authors (Strange and Postgate, 1964) observe that cooling causes subsequent RNA degradation caused by . There is evidence that propulsion does not function properly at low temperatures (Marr et al., 1964). At the high temperature end of the growth temperature range, there is evidence that the

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Ribosomes stabilize RNA. This may be the result of differences in protein and RNA packaging or differences in the primary structure of ribosomal proteins (Saunders and Campbell, 1966). Such changes may be due to the type of protein replacement proposed in this article. C. PROBLEMS RELATED TO THE ISOZYME SUBSTITUTION HYPOTHESIS TEST

If the proposed hypothesis correctly describes an important facet of cold-hardening, it is necessary to compare the properties of specific proteins from cold-hardened plants with those of comparable proteins from similar tissues of cold-sensitive plants of the same species or cultivar. Attention should be paid to the possibility that the behavior of proteins in Ziroma may differ from that of Oizw because other proteins and metabolites are present in the living organism, but not present in utro. Gel electrophoresis may not separate the isozyme forms involved if the molecules of the two forms do not differ sufficiently in shape, size, or electrical charge. Enzymes are the easiest proteins to work with because they have catalytic properties that serve to identify them. Little work has been done to compare in detail the properties of cold-resistant and cold-sensitive plant proteins from the same species. Furthermore, it is necessary to work on the relative turnover rates of metabolites in the metabolic pathways of cold-resistant and cold-sensitive plants of the same species at different temperatures. Such experiments can help identify some of the proteins that require detailed study.

D. EFFECTS OF

AND

ISOZYME REPLACEMENT HYPOTHESIS

There are several implications of this hypothesis. First, the phenotypic expression of the genome must depend on the temperature at which the plant is grown. Consequently, plants must be cold hardy before testing for hardiness. This principle is now generally accepted. The morphogenetic effects of low temperature (Resende, 1951; Levitt, 1956; Roberts, 1967b) and the temperature sensitivity of some of the rust resistance genes in cereals (Waterhouse, 1929; Gordon, 1933; Martens et al., 1967) are the terms associated with the temperature of the genome are sensitive. Second, the genetics of hardiness must be very complex, as subtle changes in many proteins are likely to be involved. There is already evidence of this complexity. Transgressive segregation for robustness occurs in oats (Finknec, 1966). The F generation of a cross of wheat plants that differ in hardiness tends to be intermediate in hardiness to the parents (Martin, 1927). Third, there is unlikely to be an exact match between the accumulation of specific metabolites and cold resistance, except for protein protectors and metabolic regulators that trigger the production of those proteins involved in cold resistance.

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Exceptions Again, exceptions to the general pattern are to be expected in these cases, particularly when comparisons are made between taxonomically distant species. If it is true that a lower growth temperature can lead to isozyme substitutions, a corresponding phenomenon must be expected for plants grown at higher than "normal" temperatures. Other environmental influences should generate other isoenzyme substitutions whenever they change the size of the pool of regulatory compounds. The production of nitrate reductase (Afridi and Hewitt, 1964) by plants supplied with nitrogen in the form of nitrate is a simple example of this phenomenon. Genetic studies of maturity in Sorghum vulgare, onset of flowering in Lolium rigidam, and flowering date in strains of Potentilla glandidosa (Clausen, 1959) suggest that other environmental factors induce isozyme substitutions and unmasking of normally latent parts of the genome. There is no reason why poikilotherms cannot behave in a similar way. Isozyme substitutions may explain part of the acclimatization phenomenon in animals.

VISA. Concluding Remarks A speculative hypothesis was described about one facet of cold hardening in plants. This hypothesis is based on the substitution of a modified form of a protein at curing temperatures for the functionally identical form of protein present at higher temperatures. It is assumed that such substitutions are triggered by increases or decreases in the size of the pool of metabolites with regulatory functions. These increases or decreases in pool size are believed to be induced by the environmental factors that cause hardening. This hypothesis suggests two lines of research that have received little study until recently. One line is the search for one or more cold hardening hormones. In such a quest, the possibility that healing is partially triggered by a decrease rather than an increase in the concentration of a compound with regulatory activity should not be ruled out. The other line of research is a detailed comparison of the properties of specific cold-hardened plant proteins with the properties of their non-hardened plant counterparts of the same species or variety. In cases that suggest that isoenzyme substitution is effective, purification of the protein is required, followed by detailed structural studies to determine whether the substituted protein differs from the normal protein in amino acid sequence, degree of polymerization, conformation, or a combination of these distinct types of differences. . With the techniques now available, it appears that some of the processes associated with hardening and acclimatization to other environmental influences are amenable to experimental attack.

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328

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Author index numbers in italics refer to pages where full references are listed.

A Aaji, C., 179, 184. 189 Acs, G., 194, 225 Ada, G. L.. 81, 99 Adams, J. A.. 31 1, 325 Adamson, A. W.. 34. 60 Adiga, P. R., 196, 228 Adler, 1999. 1999. H. I., 193, 225 Adye, J., 317, 325 Afridi, M. M. R. K., 322. 323 Afzelius, B. A., 109, 143, 145, I85 Aherne, W., 252, 253, 299 Ajtkhozhin, M. A ,, 218 . , 219, 226, 2317 Akers, C. K., 70. 103 Herberge. F., 312, .?26 Albritton, W . L., 205. 2-79 Alburn, H. I., 305, 324 Alexander, M., 193, 195, 228 Allen, D . W., 224. 225 Allmann, D. W., 75, 101. 177, 181 Alpers, D. H., 210, 211, 225 Altamirano. M., 35, 5 9 Altland, P. D., 312, 323 Altukhova, L. A,, 316, 326 Ambrose, E. J., 79. 91, 97. 98, 99, 103 Ames, A., 96, 100 Amos, H . . . . . . . . . . . . . . . . . . . . . ., 194, 231 Anderson, J.D., 112, 323 Anderson, T.F., 95, 100 Andoh. T., 222, 225 Andrews, J. E., 311, 312, 316, 323, 3-76 Antonini, E., 51. 60 Areshidze, I. V..316, 323 Arlinghaus, R., 224. 230 Armentrout. S. A, 204, 205, 126 Aronson, AI, 212, 219, 225, 231 Artman, M., 222, 223, 225 Asahina, E., 304, 316, 32.3 Asakura, S., 56, J8 Asconas , 225; 225; BA , 197 , 228 Astbury , WT , 11 , 36 , 58 Astrachan , L , 197 , 208 .

Attardi, B., 170, 171, 173, 180, 181. 189 Attardi, G., 170, 171, 173, 180, 181, 189, 195, 208, 228, 244, 299 Audia. WV. Y.. 308. 32-5 Axelrod, A. E.. 194, 199, 206, 215, 232 Axelrod, B., 311, 323

B Bass, S. S. 137. 184 Babcock. K. L. 93, 102 Rach. G.. 257, 260. 299 Bach, J. A.,. 316, 3-37 Bachmann, E.. 75, 101 Barlund, H., 2. 58 Body, G.. 305, 3173 Bailey, €..148, 181 Bailin, G., 313, 323 Baker. J.R., 68. 9 9 Baker, P.F., 308, 323 Bakerman. S. 71, 74, 76, 99 Ballentino. R., 71, 103 Baltus, E., 179, 189, 214, 220. 226, 232 Bangham, A. D., 65, 67, 85, 86, 94, 97, 99, 262, 263, 268, 273, 302 Bardo . S. G., 218. 230 Barker, S. L., 312, 325 Barnard, E. A,, 83. 100. I01 Barnard, P. J., 88, 100 Barondes, S. H., 197, 222, 226 Bartley, W., 148, 156, 181 , 186 Basford, R. E., 173, I 8 2 Bass, R., 146, 147, 149, 151, 181, 185 Baudhuin, P., 148, 183, 257, 259, 262, 293, 299.

320

330

AUTHOR INDEX

Bauer, G. E., 201, 227 Bauer, W., 120, 121, 124, 125, 126, 131, 133, 181, 186 Baum, H., 75, 101 Baumgarten, D., 121, 130, 188 Bear, R. S. , 69, 104 Beard, N. S., Jr.. 204, 205, 226 Beattie, D. S., 173, 182 Becker, A, 154, 182 Becker, F. F., 95, 102 Becker, Y., 198, 199, 201, 213 , 230 Beebe, S . P., 96, 100 Beguin, S., 210, 211, 224, 229 Beilinsson, A, 311, 323 Belitsina, N. V., 218, 219, 226, 232 Bell, E., 217, 219, 228, 231 Beller, B., 200, 231 Bello, J., 86, I05 Benditt, E. P., 298, 301 Bennett, H. S., 4, 58, 94, 100 Ben-Or, S., 87, 88, 100, 101 Benson, A A, 73, 74, 100 Berendsen, H. J. C. . , 307, 323 Berger, S., 220, 231 Berlin, C. M.. 207, 231 Berman, G. R., 216, 226 Bernal, J. D., 307, 323 Bernardi, G., 180, 189 Bernstein, J., 28, 33, 58 Berthet, J., 257, 259, 262, 293, 299 Berwick, K., 96, 100 Berwick, L., 86, 96, 100, 101, 103 Betzinger, R. J., 56, 58 Beutner, R., 35 , 58 Bianchetti, R., 312, 326 Biggs, D. R., 177, 184 Birnstiel, M. L., 114, 188 Bishop, J., 199, 226 Bishop, M. W. H., Jr., 311, 325 Bitensky, L., 91, 92 , 100 Bladen, H. A, 211, 226 Blagoveshchenskii, A. V., 316, 323 Blair, J. E., 158, 188 Blair, P. V., 75, 101 Blichfeldt, H. F., 244, 299 Blobel, G . , 199, 206, 214, 226, 232 Bloom, S., 216, 226 Bock, R. M., 74, 75, 100, 101, 177, 178, 182, 187

Bockstiegel, G., 257, 260, 299 Bode, V. C., 130, 136, 182 Boedtker, H., 198, 226 Bogorad, L., 114, 184 Bolton, E. T., 198, 226 Bond, H. E., 165, 166 183 Bond, S. B., 165, 166, 183 Bonner, J., 3 15, 323, 324 Bonner, W. D., Jr., 110, 114, 143, 144, 187 Booth, F., 79, 100 Booyse, F. M., 217 , 226 Bornstein, J., 309, 323 Borsook, H., 216, 226, 229 Borst, P., 109, 110, 112, 115, 116, 118, 120, 122, 124, 125, 126, 127, 128 129 130 131 136 138 139 140 141 143 144 145 149 151 152 153 154 158 165 166 168 173 177 179 182 1x4, 186, 188, 189 Bouroncle, B. A., 311, 323 Bowers, M. B., 91, 100 Boyle, P. J., 3, 3, 4, J8 Brachet, J., 191, 218, 220, 226, 227, 230 Bradley, S., 20, 58 Bragg, J. K., 42, 61 Bragg, W. L., 42, 58 Branton, D., 96, 100 Bratton , C. B., 21, 58 Brawerman, G., 197, 226 Brdiczka, D., 172, 186 Bregman, J. I., 14, 58 Breidenbach, R. W., 114, 182 Bremer, H., 211, 226 Bremer, H. J., 220, 226 Brenner, S., 196, 230 Brewer, E. N. 152, 153, 182 Briggs, D. E. 316, 323 Briggs, D. R., 53, 60 Brinton, C . C . , 79, 100, 198, 201, 232 Britten, R. J., 165, 167, 182 Bronsert, U., 172, 182 Brooks, D. E., 98, 104 Brown, A. D., 316, 323 Brown, D. K., 317, 323 Brown, D. M., 83, 100 Brunschwig, A., 96, 100 Buchanan, J. M., 317, 3–78

AUTHOR INDEX

Buck, C. A., 197, 228 Bücher, T., 172, 186 Bütschli, 0.. 11, 58 Buffon, G., 246, 299 Bula, R. J., 320, 323 Bulger, J., 310, 324 Bull, H. , 20, 21, 58 Bull, T. A., 312, 324 Bullivant, S., 96, 100, 104 Bungenbeg de Jong, H. G., 64, 105 Burdon, M. G., 138, 183 Burge, R. E., 11, 59, 69, 101 Burgoyne, L. A, 115, 182 Burka, E. R., 85, 100, 201, 206, 216, 224, 226, 230 Burn, G. P., 5, 59 Burnet, F. M., 81, 100 Burny, A, 196, 199, 201, 226, 229, 230 Burr, H. E., 165, 166, 183 Burri, P., 268, 269, 278, 292, 299 Burstein, S. H., 312, 328 Byheld, J. E., 320, 323 Byrne, R., 2 11, 226

C Cahn, J. W., 244, 245, 275, 279, 284, 301 Cairns, J., 142, 162, 182 Caldwell, P. R., 262, 294, 301 Campbell, L. L., Jr., 317, 321, 323, 327 Campbell , P. N., 173, I83 Campbell, W., 173, I87 Caputo, A., 51, 60 Carnevali, F., 112, 158, 182 Carpenter, A . M., 243, 284, 298. 299, 301 Carruthers, C., 96, 100 Carstensen, E. L., 80, 100 Casby, J. U., 14, s und Casley-Smith, J. R., 68, 100 Castelfranco, P., 114 , 114; 182 Catalano, P., 96, 100 Cecere, MA , , 199, 232 Cerbon, J., 307, 323 Cereijido, M., 26, 60 Chalkley, H.W.

3 00 Chambers, R., 7, 21, 31, 58, 91, 100, 306, 323 Chandra, G. R., 316, 328

331

Chang, L. O., 147, 182 Changeux, 1. P., 49, 51, 52, 18, 60, 313, 326 Chantrenne, H., 196, 208, 216, 220, 226, 230 Chantrenne-Van Halteren, M. B., 220, 226 Chanutin, A, 309, 311, 312, 323 Chapman, D., 67, 100 Chapman, G., 7, 21, 23, 58 Chargaff, E., 136, 188, 195, 228 Chaudhuri, S. , 82, 98, 100 Chayes, F., 270, 299 Chen, P. Y., 74, 101 Chilson, 0. P., 312, 323 Chin, L., 307, 328 Choules, E. A., 220, 221, 229 Chrispecls, m J., 316, 328 Chun, E. H. L., 114, 116, 182 Church, R., 169, 182 Clark, B. F. C., 205, 226 Clark, D. E., 305, 324 Clark, G. L., 69, 104 Clark-Walker, G. D. , 110, 174, 175, 177, 182, 184 Clausen, J., 322, 3-33 Clauss, H., 191, 193, 214, 220, 221, 226. 228, 229 Clawson, C., 243, 299 Clayton, D. A., 115, 120, 122, 126, 128. 133, 134, 143, 180, 182, 189 Clever, U., 317, 323 Clifford, J., 67, 100 Cline, M. J., 193, 197, 226 Clowes, G.H.A., 96, 100 Cobble, J.W., 34, 60 Cochran, W.G., 269. 300 Coconi, F.M., 201, 206, 226, 230 Cohen, A, 193, 221 Cohen, J.A., 136, 137, 186 Cohen, N. , 204, 229, 3 15, 325 Cohen, S. S., 222, 223, 231 Cohn, M., 307, 325 Cohn, W. E., 83, 104 Cole, K. S., 3, 35, 58 Coleman, E. A.,. 320. 323 Collander, R., 2, 3, 58 Collins, A, 148, 185 Collins, F. I., 306, 316, 324 Coman, D. R., 95, 97, 100

332

AUTHOR INDEX

Conover, T. E., 172, 182 Conway, E. J., 3. 4, 5, 58 Cook, G. M. W., 73, 82, 86, 87, 88, 100, 104

Cook, W. H., 72, 100 Coombs, R. R. A.. 82, 92, 101, 105 Cooper, D., 164, 182 Cope, F., 26, 27, 58 Cordes, S.. 117, 185 Cornaggia, M. P., 312, 326 Corneo, G., 110, 112, 116, 150, 156, 165, 179, 182, 189 Cornfield, J., 248, 249. 279. 299, 300 Costello, LA, 112, 323 Cotman, C., 156 , 184 Cottier, H., 257, 286, 300 Coultate, T. P., 315, 323 Counts, W. B.. 115, 148, 182 Coupland, R. E., 257, 300 Cousineau, G . H., 218, 228 Cowan, S. L., 35. 5 8 Cozzone, A,, 214, 215, 226, 230 Craft, c. C., 315, 325 Crane, F. L., 67, 100 Crawford, L. V., 1 2 5 , 126, 129, 182 Crestfield, A. M.. 83, 100 Criddle, R. S., 74, 75, 100, 101, 114, 177, 182

Crocco, R. M., 205, 227 Crocker, T. T., 221, 222, 227 Croft, J. H., 159, 161, 162, 187 Cudney, T. L., 86, 10.5 Cummins, J. E., 117, 182, 221, 326 Cundliffe, E., 178, 183 Cunningham, W.P., 67, 100 Curnish, R.R., 309, 311, 312, 323 Curti, B., 310, 326 Curtis, H.J., 35, 58 Curtis, P.J., 138, 183 Cuzner, M.L., 148, 183

D Damadian, R., 54, 58 Danielli, J, F., 3, 58, 64, 65, 66, 93. 100 Dannenberg, M. A., 177, 183 Danon, D., 207, 216, 224, 226, 230 Darnell, J.E., 195, 198, 199, 201, 206, 213, 227, 229, 230, 231

Das, HK, 211, 224, 226 Das, TM, 315, 323 Dashman, T, 207, 227 Davern, C, 196, 230 Davern, CI, 196, 227 Davidson, N, 165, 188 Davis, RL, 323 Davison, A. . N., 148, 183 Davson, H., 3, 58, 64, 100 David, 1. B, 166, 167, 168, 179, 183, 188, 189, 190 Dawson, D. M., 4, 58 Dawson, R. M. C. 94 , 99 , 101 Dean , R.B. , 3 , 58 DeBellis , R.H. , 216 , 227 de Boer , J.H. , 20 , 58 DeDeken , R.H. , 155 , 183 Duve , C. , 91 , 101 , 148 , 183 Defesa , V . 98, 101 DeGier, J., 67, 100 DeHoff, R. T., 251, 269, 300 DeJong, D. W., 320, 326 de Cloet, S. . W., 82, 101 DeLander, A.M., 54, 59 Delesse, MA. , 242. 300 Delius, H., 157, 178, 185, 188 DeMars, R.I., 195, 227 de Mello Mattos, C.M., 294, 301 Dennis, D.T., 315, 323 Denny, PC, 218, 227 Derjaguin, B.V. 307 , 323 De Salle, L., 110, 186 Deutsch, D., 93, 101 De Vries, A . , 152, 151, 182 De Vries, H., 2 , 58 Diana, A . L., 65, 104 Dickson, R.C., 115, 152, 186 Dietz, G.W.. 207, 227 DiGirolamo, A,, 197, 227 DiMargio, E.A,, 42, 59 Dingle, J.T. 10-7, 105 Dingman, C.W., 197, 226 Dintzis, H.M., 199, 227 Dodds, J.J.A., 309, 323 Dorfler, G. , 298 , 300 Doljanski , F. , 82 , 87 , 88 , 100 , 101 , 103

AUTHOR INDEX

Doty, S. 41. 59, 109. 111, 158, 185, 187, 199. 218, 227, 232 Douglas, H . C.. 174, 181, Drach, J.C., 198, 227 Dreyfus, J.C., 200, 227 Du Buy. HG, 166, 167, 168, 183 Duffin, RJ, 247. 300 Dumonde, DC, 92

And Earl, D.C.N. 194, 227 Eason, R. 193, 197, 227 Eastman, N.J., 54. 59 Easty, G.C. 96, 101 Ihbesson, SO, 264, 265, 300 Echigo, A,. 307, 324 Edelmann, M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112, 183 Edwards, D.L., 74. 100 Eisenbeg, S., 82, 87, 88, Jon, JOI, 103 Ilisenman, G., 14, IS, 17. 59 Eisenstadt, J.. 197, 226 Ekedahl. G., 221, 226 Elbers, P. F.. 67* 78. 101 Elias, H., 236, 240, 241, 255, 257, 259. 300, 302 Eljasson. ICH!. E., 201, 205, 227 Ellem. K. A. 0.. 197, 227 Ellis, R. J., 309, 323 Elson, D., 222, 223. 229 Emrich. 199, 232 Endter, F., 286, 300 Ingel, L. L., 311, 323 Ingelbert, H., 222, 223, 22J Epstein, E., 28, 5 9 Epstein, H. T., 112, 183 Eranko, O., , . . . . . E., 112, 117, 145, 182: 183 Eyer, J.. 170, 187 Eylar, E. H., 82, 89, 104 J.?

Falk, G., 35, 59 Falk, M., 307, 324

333

Fan, D. P. 224, 227, 230 Farber, I:., 201, 209, 214, 232 Farkas, G. L.. 3 0 5 , 323 Farquhar, M. G . , 91, 101 Farrelly. J. G, 312, 324 Faust, M. A, 312. 321 Favelukes, S., 224. 230 Fawcett, D. W., 91, 101 Feigelson, P., 207, 227 Fel'dman. N. L., 318, 32.r' Fell, H. B.. 91, 92. Klo, 101, 102 Fenichel, 1. R.. 7, 9. 5 9 Fernindez-Moran, H.. 75. 101 Fessenden, J. M., 177, 183 Fiq. A, 218, 226 Fincham, J. R. S., 310, 324 Finean. J. B. 69, 101 Finkner. V.C., 321, 324 Fischer, H., 12. 1.5, 59. 1'95. 227 Fischnieister, H.F. 283, 300 Fisher, C., 283, 300 Fisher, E.H., 216. 226 Fisher, T.N., 208, 226 Fisher, W.D., 193, 2 2 j Flamm. W. G., 1 1 5 . 148, 165, 166, 182, 183 Fleck. A , , 201, 227 Fleischer, B., 68. 74, I 0 1 Fleischer, S., 68. 74. 101 Fletcher, M. J.. 147, 183 Fling. M., 314, 324 Folch-Pi, J. 77, 101 Forchhammer, J., 209, 224, 227 Ford, T. A., 107. 324 Forssmann. W. G., 262, 300 Cazador. A . V., 312, 324 Fox, C. F.. 308. 309, 324 Fraenkel-Conrat, H., 86, 101 Frank, H. S.. 107. 324 Franke. W.. 316, 324 Franklin. N. C..196 228 Franklin, R. M.. 193, 1'97, 227, 231 Fredrick. J. F., 314. 324 Freeman, J. A, 66. 101 Freeman, K. B., 172. 173, 183 Freere, R. H.. 268, 287, 300 Freese, E.. 136, I86 Freifelder, D . , 131, 283

334

AUTHOR INDEX

French, E. L., 81, 99 Friesen, J. D., 195, 224, 227 Frisbie, W. S., 96, 100 Fritz, 0. G., 7, 21, 36, 59 Fuchs, A, , , 269, 278, 292, 300 Fuhrmann, GF. 80, 88, 98, 100, 101, 103

Fukada, T., 197, 233 Fukuhara, H., 154, 155, 170, 183, 187 Fuller, W., 193, 228 Furth, J. J., 193, 195, 228

G Gadaleta, M. N., 151, I87 Gallant, J. A., 314, 324 Galston, A. W., 316, 326 Gander, R. H., 268, 287, 300 Ganther, H., 310, 326 Garbus, J., 312, 323 Garofalo, M ., 214, 231 Garren, L. D., 205, 227 Gartner, T. K., 314, 324 Gary-Bobo, C . M., 26 Gasic, G.J., 86, 98, 101 Gaskins, M.H., 312, 324 Gavrilova, L.P., 218, 226, 316, 323 Gebauer, H., 286, 300 Gefter, M., 154, 182 Geidushek, E.P. , 165, 183 Geiling, E . MK, 54, 59 Gelinas, R, 315, 326 Gellert, M, 154, 183, 185 Georgi, C . E., 316, 317, 323, 327 Gerard, R. W., 35, 59 Geren, B. B.. 69, 101 Gerloff, E. D., 316, 320, 324 Gerstner, R., 312, 328 Gesteland, R. F., 222, 227 Getz, . . . . . G. S., 140, 144, 145, 146, 147, 148, 149, 155, 175, 176, 183, 186, 187 Giacomony, D., 198, 227 Gibbs, J. H., 42, 59, A. 1, 183, 220 , 220 . 220. 227 Gier, A., 199, 227 Giger, G., 257, 286, 300 J., 262.

Gilbert, W., 195, 199, 208, 224, 227, 228 Gilden, R. V., 315, 323 Gillespie, D., 198, 227 Gillespie, M. E., 210, 231 Gimigliano, A. F., 151, 187 Ginelli, E., 110, 116, 182 Girard, M., 195, 227 Girardier, L., 262, 300 Gittens, G. J., 80, 101 Glagoleff, A. A., 243, .?OO Glasziou, K. T., 312, 324 Glauert, A. M., 67, 102 Glick, M. C., 84, 104 Glick, P. M., 83, I 0 1 Gligin, M. V., 218, 227 Gliiin, V. R., 218, 227 Glover, J. C., 85, 99 Glowacki, E. R., 207, 227 Gluck, N., Goffeau, A, 216, 227, 220, 227 Gold, L., 197, 226 Goldberg, B., 200, 216, 226, 227 Goldberg, 1. H., 193, 194, 217, 227, 231 Goldstein, A ., 210, 211, 224, 226, 227 Goldstein, L., 221, 222, 227 Gomez, D. M., 252, 253, 301, 302 Gonzilez-Cadavid, N. F.. 173, 183 Goodman, H. M., 198, 227 Gordon, M. P., 114, I83 Gordon, W. L., 321, 324 Gordy, W., 38, 59 Goren, H. J., 83, 101 Gorham, P. R., 31 1, 324 Gorter, E., 64, 101 Gorton, S., 318, 325 Gottschalk, A, , 81, 101 Gould, B. S., 200, 229 Gould, W. A, 316, 324 Graham, A. F., 196, 231 Grandchamp, S., 174, 185 Granick, S., 109. 132, I83 Grant, N.H., 305, 324 Grasso, J.A., 216, 228 Gratzer, W.B., 41, 59 Graves, D.J., 312, 324

AUTHOR INDEX

GrPce, M.A., 1 1 5, 172, 184 Verde, B. R., 114, 183 Verde, D. E., 68, 73, 74, 75, 100, 102, 103, 177, 182 Verde, G. J., 321. 326 Verde, H. , 200, 216, 226, 227 Greenawalt, J. W., 151, 152, 183 Gregory, K. F., 319, 327 Gregson, N. A, 148, 183 Grendel, F., 64, 101 Griffing, B., 310, 325 Groot , G. S. P., 151, 184 Gros, F., 195, 208, 211, 224, 228, 231, 233 Gross, N. J., 120, 130, 131, 141, 143, 147, 148, 149, 166, 180, 183, 187 ,

101,

230, 146, 189,

190

Gross, P. R., 218. 228 Grossman, L. J., 110, 112, 150, 156, 165, 182 Gruber, M., 118, 120. 188 Gruener, N., 308, 324 Grundfest, H., 35, 59 Guerineau, M., 180, 189 Guttes, E., 145, 183 Guttes, S., 145, 183 Guttman, L., 242, 246, 247, 301 Guzhova, E. P., 317, 325

H Haber, J. E., 51, 59 Hadjiolov, A . A . , 196, 228 Hammerling, J., 191, 220, 228 Hagen, C. E., 28, 59 Haldar, D., 172. 173, 183 Hale, H. P., 7, 21. 58, 306, 323 Hall, B. D., 197, 198, 228, 230 HaII, D.O., 151, 152, 183 Hall, J.B., 196, 228 Hallett, J., 21, 22, 59 Hally, A . D., 244, 282, 300 Halpern, Y. S., 314, 324 Hamburger, H. . J., 2, 5 9 Hamilton, L. D., 193, 228 Hammett, L. P., 38, 59 Hanawalt, P. C., 112, 139, 186 Hanig, M., 81, 101 Hardesty, B., 201, 229

335

Hardigre, A. A, 193, 225 Harkins, W. D., 12, 59 Harrington, J. F., 315, 324 Harrington, W. F., 41, 59 Harris, E. J., 5, 5 9 Harris, H., 213, 219, 221, 228 , 232 Hartley, G. S., 93, 101 Hartman, J. F., 243, 299 Hartmann-Goldstein, I. J., 320, 324 Hartridge, H., 93, I 0 1 Hartwell, L. H., 210, 211, 228 Harvey, E. B., 193 , 228 Harvey, E. N., 64, 65, 100 Hasegawa, S., 316, 324 Haselkorn, R., 110, 116, 186, 187, 197, 229 Haslbrunner, E., 140, 144, 187 Haug, H., 248 , 253, 286, 300 Haughton, G., 98, 102 Hauschka, T., 99, 102 Hawker, K. M., 320, 326 Hawthorne, D. C., 174, I85 Hayashi, M., 196, 198, 208, 228 Hayashi , M. N., 198, 228 Haydon, D. A,, 65, 67, 79, 80, 81, 99, 102 Heard, D. H., 70, 79, 81, 82, 85, 88, 100, 102, 104 Hearst, J. E. 114 , 128, 188 Heber, LI., 309, 311, 320, 324, 327 Hechter, O., 307, 308, 324 Heckmann, K., 34, 59 Heiniger, H. J., 257, 286, 300 Heinle, E ., 206. 232 Heinze, P.H., 315, 326 Heldt, H. W., 151, 152, 184, 186 Helge, H., 151, 171, 172, 185 Helinski, D.R., 135, 186 Hellman, A, 312, 324 Hellmann, W., 186 Hendershott. C. H., 312, 324 Hennig, A.. 244, 248, 256, 259, 265, 275, 282, 300 Henniker, J., 80, 102 Hennix, LJ., 144, 185 Henriques, V., 56, 59 Henrique , J. B., 318, 32 J Henshaw, E. C., 197, 227 Heppel, L. A,, 3, 59 Herbst, C., 97, 102

336

AUTHOR INDEX

Herbst, R., 144, 185 Hermann, L., 32, 59 Hess, F. A., 237, 250, 262, 278, 281, 282, 285, 286, 287, 288, 289, 291, 292. 298, 300, .301, 302 Hess, R., 237, 282, 285, 286, 293, 301 Heston, W.E., 207, 231 Hewitt, E.J., 322, 323 Hiatt, H., 208, 228 Hiatt. H. H., 195, 197, 208, 213, 214, 227, 228, 231 Hickler, S., 140, 186 Higa, A, 197, 208, 209, 224, 227, 229 Highman, B., 312, 323 Hildebrandt, A.C., 315, 323 Colina, A. V., 32, 50, 56, 59 Colina, H. z, 214, 233 Hille, B., 316, 326 Hilliard, J. E., 244, 245. 251, 253, 257, 260, 275. 277, 279, 284, 296, 297, 301 Hinke, J. A. M. 26, 59 Hoagland , M.B., 197, 205, 206, 214, 228, 232, 233 Hochachka, P.W., 317, 324 Hochstein, P., 194, 229 Hodges, H. F., 320, 324 Hodgkin, A.L., 3, 6, 32, 33, 35, 36, 59 Hoffman, C. 294, 301 Holmes, A. . H., 256, 301 Holt, C. E., 180, I89 Holter, H., 4, 59 Holtzer, H., 4, 59 Holwill, M. E. J., 11, 59 Holzer, H., 175, I88 Honig, G. R., 194, 228 Hoover, E.F., 20, 60 Hopkins, A.L., 21, 58, 307, 324 Hopkins, J.W. , 197, 232 Hori, T., 307, 324 Horiuchi, S., 314, 324 Horiuchi, T., 314, 324, 328

Horowicz, P., 6, 35. 59 Horowitz, J., 195, 228 Horowitz, N. H., 314, 324 Horowitz, S. B., 7, 9, 59 Horrikawa, E., 247, 301 Howell, R. R., 196, 205, 227, 229 Howell, R.W., 306, 316, 324 Hradecna, Z., 118, 184 Huang, M. 177, 184 Huang, R.C., 315, 323, 32.1 Huberman, J.A., 142, 184 Hudson. B., 120, 126. 133, 134, 135. 180, 184, 189 Huez, G.. 196, 201. 229, 230 Hulcher, F. H.. 76, 102 Hulme, A. C., 315, 324 Hultin, H. 0. 74, 103, 310, 324 Hultin, T., 201, 218, 227, 2.33 Humm, D.G., 169, 184 Humm, J.H., 169, 184 Humphreys. T.. 217, 219, 228 Datum prisa, S. W.. 308, 325 Hunvitz. J., 154. 182, 193, 195, 228. 229 Huxley, H. E., 33, 53

Oi Infante. A. A., 218, 219, 228, 230 Ingraham, J. L.. 306, 313, 315. 316, 320, 326 Inouye, M.. 199, 2.?2 Irving. r M., 312, .?25 [a. s., 94, 103 Izawa, M., 220, 227

J Jackson, R. J., 199, 200, 207, 230 Jacob, F., 210, 228, 313, 314. 315, 325, 326, 327 Jacobson, L., 4, 60 Jacques, M., 28, 59 Jahrisch, S., 93, 102 Jakob, H., 112, 145, 358, 159, 160, 161, 181, James, A. M., 79, 80, 101, 102 Janin, J,, 197, 209, 231 Jansen, E. F., 320, 326 Jansz.H . S., 136, 137, 184, 186 Jarabak, J., 310, 311, 321,

AUTHOR INDEX

Jayaraman, J., 156, 184 Jensen, W. A., 4, 60 Jensen, W. N., 206, 232 Jeon, K. W., 221, 228 Jinks, J. L., 109, 184 Jockusch, H., 311, 325 John, D. W., 215, 228 Johnson, R.M., 316, 324 Jones, A. , 54, 59 Jones, RF, 309, 110, 325

K Kadenbach, B., 172, 179, 184, 192, 228 Kaempfer, R. 0. R.. 21 1, 229 Kagawa. Y, 76, 102 Kahan, I., 193, 2-79 Kahan, F.M., 193, 229 Kalant, H..97, I02 Kalf, G.F., 115, 172, 184 Kamat, V.B., 67, 82; I 0 4 Kanner, L. C., 224, 226 Love, Y . , 96 , 102 Kao , C.Y. , 33, 3 5 , 58. 59 Kapanci, Y., 294, 301 Kaplan, H. P., 294, 301 Kaplan, N. O., 312, 323 Karol, M. H., 180. 189 Karpukhina, S. Y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317, 325 Karreman, G., 7, 9, 54. 59. 60 Karrer, H. E., 67. 102 Kartha, G., 83, 102 Katchalsky, A., 97, 102 Katoh, T., 174, 184 Katz, 184 . 184. 184. 184. J.H., 82, 103 Katz, M. 98. 104 Katz, R.D. 3 3, 36, 59 Kauzrnann, W., 310, 3-35 Kavanaugh. J.L., 65, 66, 10-3, 197, 233 Keane, J.F., 311, 325 Keck, K., 191, 193. 21.1. 220 , 221 , 226 , 228 , 229 Keighley , G. , 216 , 226 Kemp , A. , Jr . , 198. 229 Kenney, F.T.

337

Kepes, A, 210, 211, 223, 224, 229 Kerr, IM, 204, 229, 315, 325 Ketellapper, HJ, 315, 325 Keynan, A, 197, 208, 209, 229 Keynes, 229, 29. . D. V., 291, 300. 302 Kimura, Y., 35, 59 Kinbacher, E. J., 318, 327 Kinoshita, J. A,, 217, 232 Kiovsky, T. E., 304, 305, 32>, 326 Kirk, J. T. O., 114, 327; 327; 114. 184, 193, 229 Kirschbaum, J. B., 210, 227 Kirschner, R. H., 180, 189 Kislev, N., 114, 184 Kislyuk, I. M., 315, 32g Kislyuk, N. M., 315, 325 Kistler, G. S. , 0, , . 24. 56.2 22. 266, 268, 273, 277, 279, 287, 292, 294, 301, 302 Kittel, C., 52, 58 Kitzinger, C. 131, 183. 186 Klenk, E., 81, 102 Kligman, A . . . . . . . . . . . . M., 312, 325 Klikoff, L. G., 319, 325 Clima, J., 174, 175, 187 Klingenberg, M., 151, 152, 184, 186 Kloss, K., 144, 185 Klotz, I. M., 307. . 187; 3257 Knight, B. W., 250, 252, 253, 261, 262, 296. 301, 302 Knijnmburg, C.M., 136, 137, 186 Knopf, P.M , , 325 Koehn , P. V. , 217 , 230 Koffler , H. , 317 , 325 Koga , S. , 307 , 325

338

AUTHOR INDEX

Kohlmeier, V., 195, 228 Kohne, D. E., 165, 182 Koketsu, K., 35, 59 Kollin, V., 211, 229 Konrad, M. W., 211, 226 Kook, J. W., 316, 327 Kopaczyk, K., 75, 101 Koritz, S.B., 173, 182 Korman, E.F., 75, 101 Korn, E.D., 67, 68, 102, 307, 325 Kornberg, A,, 117, 154, 187 Korner, A,, 194, 198, 199 , 200, 207, 227, 229, 230, 315, 325 Koshland, D. E., 51, 5 9 Kowalewski, V., 26, 60 Kowalsky, A., 307, 32 A., 304, 328 Kretsinger, R. H., 200, 229 Krieg, A.F., 318, 325 Kroger, R.A., 151, 152. 186 Krogh, A., 3, 19 Kronau, R., 175, 188 Kroon, A.M., 109, 110, 112, 115, 120, 122, 124 125, 127, 128, 136, 138, 139, 140, 143, 144, 149, 154, 165, 166, 168, 172, 177, 179, 182, 184 18.4 Kruh, J., 216, 229 Kubinski, H . , 118, 184 Kuff, E. L., 110, 144, 147, 149, 187, 229 Kurland, C. G., 208, 228 Kwan, S. W., 214, 229

L Lachmann, P. J., 82, I05 Laipis, P., 120, 127, 129, 188 Lamar, C., Jr., 215, 216, 232 Lamfrom, H., 200, 229 Lane, B. P., 95, 102 Langer, L. J., 311, 321 Langmuir, I., 19, 5 9 Langridge, J., 310, 316, 325 Langridge, P., 310, 325 Lanphear, F. O., 312, 321 Lansing, A. I., 82, 85, 102 Lark, KG, 136, 184 Larsson, A., 151, 184

214,

118, 131, 145, 173,

200,

Laszlo, J., 194, 229 Latham, H., 195, 199, 206, 213, 227, 229, 23 1 Lauffer, MA, 79, 100 Lauwers, A., 177, 181 Layne, DS, 312. 328 Lazarus , A, , 243, 284, 298, 299, 301 Leahy, J,, 199, -326 Lebedeva, L.A,, 307, 320, 328 Lebleu, B., 201, 229 Lebowitz, J., 2011, 120, 121 , 125 , 127 , 129 , 188 Lehman , I. R. , 222 , 229 Lehninger , A. L. , 132 , 172 , 184 , 186 Leibnitt , L. , 298 , 301 Lenard, J. , 72 , 73 , 102 , Lenz1 0 7 . Lenza , G. , 177 , 181 Leonard , C. D. , 312 , 327 Lerman , L. S. , 162 , 184 Lerman , 184. MI , 218 , 232 Levi , H. , 5 , 19 Levin , J. G. , 211 , 22G Levinthal , C . , 195, 197, 208, 209, 210, 211, 224, 227, 229, 231, 233 Levitt, J., 303, 304, 305, 306, 307, 31 I. 313, 316, 321, 321; 325 Lewis , G. N. , 79, 102 Lewis, I. C. 38, 61 Lewis, M. S., 26, 5 9 Lieberman, I., 82, 98, 100 Lieberman, M., 315, 3-35 Lin, E. C. . C., 207, 229 Lin, S., 201, 229 Lindegren, C. C., 140, 186 Lindegren, G., 140, 186 Ling, G . N., 2, 3, 4, 5, 6, 7, 8. 9, 11, 12, 14, 15, 16, 20, 21, 24, 25, 26, 27, 28, 29, 30, 31, , . . . . .

AUTHOR INDEX

Little, J. W., 154, 185 Loeb, A. L., 79, 105 Loeb, J. N., 196, 229 Loewenstein, W. R., 88, 96, 102 Loginova, L. G., 317, 325 Lohmann, W., 311, 312, 325 Looney, W. B. , 147, 182 Lorch, I. J., 221, 228 Loud, A. V., 237. 243, 252, 262, 263. 268, 273, 286, 292. 293. 301, 302 Lovelock, J. E., 309, 311, 325 Lowenstein, J. M. , 151, 186 Lowney, L. I., 211. 226 Luck, D. J. L., 109, 112, 142, 149, 150, 185, 186 Lucy, J. A., 67, 91, 102 Lukins, H. B., 155, 175, 176, 185, lR9 Lukton, A., 313, 323 Lundsgaard, E., 56, 59, 60 Luria, S. E. 195, 196, 227, 228 Luyet, B. J., 7, 21, 22, 60, 311, 312, 325, 327 Luzrati, V ., 67, 70, 102 L'vov, S. D., 316, 326 Lyklema, J., 79, 102 Lyn, G., 154, 182

M Maas, W. K., 312, 326 McBain, J. W., 34, 60 McCarthy, B. J., 169, 182, 197, 198, 218, 226, 228, 229, 232 McCarthy, K. S., 194, 229 McCarty, M., 4, 58 McClatchy, J. K., 212, 229 McCluskey, R. T., 91, 104 McConnell, D. G., 75, 101, 102 McCrea, J. F., 81. 100 McDonald, J. S., 35, 60 McFall, E., 312, 326 McHattie, L. A., 130, 182 McKenzie, R. I. H., 321, 326 Mackler, B., 112, 141, 144, 156, 168, 174, 185, 188 McLaren, A. D., 4, 60, 93, 102, 103 McLauchlan, K. A., 7, 21 , 23, 58 MacLennan, D. H., 75, 101, 102 MacLeod, C. M., 4, 18 Macleod, R. A,, 315, 326

339

McNaughton, S.J., 319, 326 McQuillen, K., 178, 183, 210, 211, 231 Maddy, A.H., 76, 103 Magasak, B., 195, 210, 211, 228, 229, 230, 326 Maggio 195; R., 218, 229, 230 Mahler, H. R., 112, 141, 144, 156, 168, 174, 18$, 185, 188 Mahr, S. C., 68, 104 Maisel, G. W., 5, 59 Malamy, M., 193. , 195, 228 Malcolm. B. R., 76, 103 Mallett, G. E., 317, 325 Malmgren, H., 92, 104 Mangiarotti, G., 209, 229 Manner, G., 200, 229 Manning, G. B., 317, 323 Mano, Y . , 218, 229 Manor, H., 197, 229 Marbaix, G., 196, 199, 201, 226, 229, 230 Marchis-Mouren, G., 214, 215, 226, 230 Marker, K. A,, 205 , 226 Marcus, P. I., 82, 98, 103 Margoliash, E., 173, 187 Margolis, F., 207, 227 Markert, C . L., 318, 326 Mark, P. A,, 201. 206, 207, 216, 224, 226, 227, 230 Marble, J,, 109, 110, 111, 112, 117, 150, 156, 158,165,182,185 , 186 Maroudas , N.G. , 180 , 189 Marquardt , W.C. , 312 , 328 Marr , A.G. , 306 , 315 , 316 , 320 , 326 Marre , E. , 312 , 326 Martens , J.W. , 321 , 326 Marth , 1999 ; P. C., 312, 326 Martin, D. G., 312, 324 Martin, J. H., 321, 326 Martin, W. G., 72, 100 Martinez, R. J., 212, 230 Marver, H. S., 148, 185 Massey, V., 310, 326 Master, . RWP, 193, 221, 231 Matsuda, K, 198, 230 Matsumura, C, 311, 328 Mattern, CTF, 166, 167. , 18 H5 reif , 18 H5 reif

340

AUTHOR INDEX

Mayhew, E., 82, 84, 87, 88, 89, 90, 103, 105

Mazur, P., 304, 307, 312, 326 Meador, D.B., 320, 326 Meek, G. A , . 156, 186 Mellanby, E., 91, 101 Mellon, S. R., 20, 60 Mercer, E. H., 95, 96, 101. 103 Merishi, J. N., 89, 103 Fund, I., 193, 197, 230 Merker, H. J., 197, 144, 151, 171, 172, 185 Meryman, H.T., 304, 307, 326 Merz. W.A., 279, 281, 296, 301 Mesleson, M. 149, IRS, 196, 227, 230 Meussner, R.A., 247, 300 Meyer, R.R., 180, 189 Meyerhof, O., 311, 326 Meynell, G.G., 308 , 326 Micou, J., 221, 222, 227 Migchelsen, C., 307, 323 Militzer, W., 316, 317, 323, 327 Milkman, R., 316, 326 Miller, A., 82, 103 Miller, D.S. , 194, 229 Miller, L.L., 215, 228 Millette, R.L., 207, 227 Miura, K.I., 179, 189, 197, 230 Mizrahi, I.J., 204, 205, 230 Mizuno, D., 222, 225 Molotkovskii, Y G ., 311, 326 Mond, R., 3, 60 Monod, J., 49, 51, 52, 60, 210, 228. 3 1 3 , 315, 325, 326 Monroy, A, 218, 229, 230 Monroy , G.C. , 308, 327 Montjar, M., 194, 199, 206. 213, 232 Mooney. JA, 319, 326 Moore, H., 263, 301 Moore, C., 110. 112, 150, 156, 165, 187 Moore, G. A., 12. 14, 59, 284, 301 Moore, P. B., 157, 178, 1 8 5 , 188, 197, 230 Moore, S., 83, 100 Moore, T. C., 312. 323 Morales, M. F., 56, 197. Ij8 Morita, R. Y., 310, 325 Morris, A,, 224, 230 Morris, D., 317, 326, 327 Morris, J. A,, 207, 231 Moss, A. J., Jr., 311, 312, 32s Moos, C. W., 315,

Mosteller, R. D., 201, 229 Mounolow, J. C., 112, 145, 158, 159, 160, 161, 1x5 Mous, W., 97, 102 Moustacchi. E., 112, 140, 156, 185 Mueller, G. C., 193, 197, 232 Mueller, P., 65, 103 Munkres, K. D., 176, 177, 1x5, IXX, 189, 192, 232 Munro, A . J., 198, 199, 200, 207, 214, 229. 230

Munro. H.N., 201, 227 Murray, K., 315, 324 Murray, R.K., 97, 102 Mustacchi, H., 67, 102 Muto, A . , 197, 230 Mysels, KJ, 34, 60

N Naaman, J., 82, 103 Nase. H. , 218 , 229 Nakada , D. , 195 , 211 , 224 , 230 Naono , S

Naota, H., 220. 230 Nash, T.. 312, 326 Nass, M. M. K., 84, 104, 109, 110, 118, 131, 140, 1 4 1 , 144, 145, 185 Nass, S., 109, 110, 132, 143, 144, 147, 148, 149, 1x5 I!. 3, 60 Neuberto. D., 146, 147, 149, 151, 152, 1 5 3 , 171, 172, 181, 185, 287 Neupert, W., 172, 182, 186 Ng, H., 315, 32G Nichols, S., 96 , 100 Nielsen, J.M., 34, 60 Nirenberg, M.W., 197. 211, 222, 226, 230 Nishi, A, , 320, 326 Niyogi, S . K., 167, 186 Noll, A,, 213, 214, 232 Noll, H., 198, 199, 200, 201, 206, 209, 214, 220, 2.30, 232

ALJTHOR INDEX

Noller, H., 157, 178, 185. 188 Nomura, M., 196, 197, 230 North, F.F., 309, 326 Nordling, S., 82, 89. 10.3 Novick, A., 314, 321 Nowell, P.C. , 96, 100, 10.3 Numa, S., 310, 326 Nygaard, A . S., 198. 2.30

0 Oberdisse, E., 146, 147, 1-19. 151 , 185 Ochoa , J. , 199 , 231 Ochoa , S. , 320 , 328 Ochsenfeld , MM , 3 . 7, 9, 25. 26, 27, 28, 29, 30, 31, 32, 60 Ockerse, R., 316, 326 Order, T., 75, 101 O'Donovan, G. A,, 311. .326 O'Grady, E. A., 88. I03 Ogur, M., 140, 186 Ohlmeyer, P., 3 11 326 Ohnishi, T., 1 5 1 , 1 5 2 , I86 Ohno, M., 311, 328 Okada, Y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , 199, 232 Oki, T., 307, 325 Olcott, H. S., 86, 101 Oldenziel, H., 137, 181 Olivera, B. M., 121. 130, 188 Olson, A. C., 3 2 0, 3-76 Oncley, J . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L., 72, 103 Opara-Kubinska, 2.. 118. I84 Orci, L., 262, 300 Ortanderl, F., 319, 328 Oshinsky, C. K., 154, 18J Osterhout, W. J. V., 28, 60, 70, 103. .Oura, H., 199. 206. 232 Overbeek, J.T.G.

Packets P, BA , , 241, 2 5 2 , 262. 263, 268, 273, 301

Palade, G. E., 91. 101 Pallansch, M. J.? 53. 60 Papa, S., 109, 187 Papaconstantinou, J.. 2 17, 230. 232 Pardee, A. B., 210, 230 Park, H., 248, 249, 279, 299 Parker, J.. 173, 187. 311. 326

34 1

Parkes, A.S., 311, 327 Parpart, A.K., 71, 103 Parsons, D.F., 67, 70, 74, 76. 77. 103, 171, 186 Parsons, J.A,, 115, 145, 152, 186 Parsons, P ., 144, 1 5 2 , 153, 174, 165, 186 Pasternak, G., 98, 103 Patrman. JA, 310, 324 Pauli, A . W., 320, 326 Pauly, J.E. . 257 , 300 Peaker , C. R. , 34 , 60 Pearson , P. , 157 , 178 , 185 Penefsky , H. S. , 310 , 326 Penkett , S. A. , 67 , 100 Penman , S. , 195 , 198 , 199 Pentzer , W. T. ino , 315 , Perdue, 231 , J. F., 73, 75, 101, 177, 183 Perez-Esandi, M. V., 82, 104 Perkins, H. J., 311, 312, 326 Perkins, . W. H., 311, 325 Perl, W., 201, 206, 230 Perodin, G., 159, 161, 162, 187 Prrutz, M. F., 70, 103 Peterkofsky. € 3. . ., 1 5 1 , 152, 184, 186 Pfeffer, C. R., 140, 181 Pfeffer, W., 2, 60 Pfefferkorn, L. C., 2 0 5 . 228 Pfleiderer, G., 319, 328 Pictet, R., 262, .?00 Pico, L,. 110 , 120 , 230, 231 Plagemann, P. G. W., 319, 327 Plaut, W., 221, 226, 231, 233 Plummer, D. T., 319, 327 Poche, R., 294, 301 Podolsky, R. J. 56, 60 Podrazhanskaya, 2 L. . , 316 , 323 Pogell , B. M. , 313 , 328 Polakis , E. S. , 156 , 186 Polge , C

342

AUTHOR INDEX

Polli, E., 110, 116, 182 Pollock, M. R., 212, 231 Porter, K. R., 91, 103 Postgate. J. R., 308, 320, 327 Potter, J. L., 91, 104 Potter, V. R., 199, 206, 214, 226, 232, 311, 327 Pouwels, P. H., 136, 137, 184, 186 Preer, J. R., Jr., 112 , 144, 187 Preisig, R., 262, 301 Prescott, D. M., 191, 221, 222, 229, 231 Prestidge, L. S., 210, 230 Previc, E. P., 210, 231 Prince, L. M., 70, 104 Prose, P. H., 92, 101 Prosser, C. L., 317, 327 Pullman, M. E., 172, 186, 308, 327 Pulvertaft, R. J. V., 85, 103 Purdom, L., 98, 103 Purohit, K., 317, 327 Purvis, J. L., 151, I86

Q. Quagliariello, E., 109, 187

Rabinowitz M 227, 23 1 Racker, E., 76, 102, 177, 183 Radloff, R., 120, 124, 126, 127, 129, 133, 186, 188 Rafelson, M. E., 217, 226 Rafikova, F. M., 307 , 320 , 328 Rastrillo, A . V., 196, 231 Ramanis, Z., 163, 187 Rampersad, O., 200, 231 Rancourt, M. W., 140, 181 Randall, M., 79, 102 Rapatz, G., 7, 21, 22, 60, 312, 327 Rapp, F., 98, 104 Ratcliffe, T. M., 88, 89, 91, 100, I05 Ray, D. S., 112, 139, I86 Rechcigl, M., Jr., 148, 185, 207, 231 Reeder, R., 217, 231 Reich, E., 112, 142, 149, 150, 185, 193, 194, 225, 227, 228, 231

140, 147, 176, 228,

131,

186,

Reichard, P., 151, 184 Reilly, C., 156, 175, 186 Reinders, 93, 103 Reiss-Husson, F., 67, 70, 102 Reithel, F. J., 310, 327 Rembarz, H. W., 294, 301 Rendi , R., 77, 78, 103 Resende, F., 321, 327 Revel, J.-P., 94, 103, 213, 231 Revel, M., 211, 213, 214, 231 Rhaese, H . J., 136, 186 Rhines, F. N., 247, 251, 300 Rich, A, 114, 116, 182, 198, 199, 200, 227, 229, 232 Richardson, C. C., 154, 187 Richardson, S. H., 74, 103 Richter, G., 191, 220, 228 Rickenberg, H. V., 212, 229 Riedwyl, H., 257, 259, 260, 286, 300 Rieske, J. S., 75, 101 Rifkind Riker, A. J., 315, 323, 229, 230 Rinaldi, L. M., 97, 103 Ring, K., 308, 327 Ringelmann, E., 310, 326 Risebrough, R. W., 208, 228 Ritossa, F., 317, 327 Roberts, D. W. A,, 305, 310, 315, 316, 320, 321, 323, 327 Roberts, N. E., 200, 229 Robertson, J. D., 66, 69, 101, 103 Robinson, F. R., 294, 300 Roe, J. W., 93, 101 Rogers, H. J., 94, 10.3 Roman, A,, 210, 227 Roodyn, D. B., 92, 101, 109, 132, 160, 172, 175, 177, 179, 186, 192, 231 Rosas del Valle, M. R., 212, 219, 225, 231 Rosenberg, A. M., 307, 328 Rosenthal, T. B., 85, 102 Rosiwal, A,, 243, 301 Ross, R., 298, 301 Ross-Fanelli, A., 51, 60 Roth, T. F., 91, 103, 135, 186 Rotunno, C. A. , 26, 60

AUTHOR INDEX

Rouiller, C., 262, 300 Rouvitre, J. 2 1 1, 230 Rowley, P. T., 207, 231 Rownd, R., 165, 18T Rubin, H., 98, 104 Rudin, D. O., 14, 59, 65, 103 Ruhenstroth-Bauer, G., 88, 103 Ruhland, W., 3, 60 Runner, C. M., 110, 118, 120, 131, 139, 140, 143, 288 Rusch, H. P., 117, 152, 153, 1x2 Rush , M. G., 135, 186 Rustad, R. C., 221, 231 Ruttenberg, G. J. C. M., 109, 110, 115, 118, 120, 122, 124, 125, 127, 128, 129, 130, 131, 136, 139, 1 4140 , 143, 144, 145, 154, 158, 165. 166. 168, 173, 179, 182, 184. 186, 188, 1x9 Ryser, H. J. P., 4, 60

343

Schellman, J.A., 41, 42, 59, 61 Scherbaum, 0.H., 320, 323 Scherle, W.F., 240, 253, 266, 268, 273, 277, 279, 287, 292, 302 Schemer, K., 198 , 199, 201, 213, 230, 231 Schiff, J.A., 112, 183 Schildkraut, CL, 111, 154, 158, 165, IRT, 186

138,

Schimke, R. T., 207, 231 Schlessinger, D., 196, 209, 216, 222, 223,

112, 126, 138, 149, 177,

Schmidt, W. J., 70, 103 Schmieder, M., 152, 153, 187 Schmitt, F. O., 69, 97, 103, 104 Schneider, W. C., 110, 144, 147, 149, 187 Schreml, W., 85, 100 Schulman , H. M., 219, 231, 233 Schulman, J. H., 70, 104 Schuurmans Stekhoven, F. M. A. H., 110, 118, 120, 131, 138, 139, 140, 143,

Saccone, C., 151, 287 Saddler, H. D. W.. 309, 327 Sadoff, H. L., 316, 327 Sager, R., 163, 187 Sakai, A,, 312, 327 Salas, M., 199, 231 Salb, J. M., 82, 103 Salmão, S. C. . 315, 327 Salser, W.. 197. 209, 231 Salt, R. W., 306, 3 1 1 , 327 Saltykov, S. A.. 242, 246, 248, 257, 259, 260, 286, 301 Sanadi, D. R., 110. 112 , 147, 150, 156, 165,

Schwan, H. P., 307, 327 Schwartz, D., 319, 327 Schwartz, H. A,, 259, 301 Schwartz, H. S., 214, 231 Schwartz, VG, 82, 103 Schweet, R., 199, 206, 224, 226, 230, 232 Schweiger, E., 220, 226, 231 Schweiger, H. G., 193, 220, 221, 226, 231 Schwemmle, B., 316, 327 Schwindenvolf, U., 34, 61 Scornik, O. A, , 205 , 228 Scott, R.B., 217, 231 Sealock, R.W., 312, 324 Seaman, G.V.F., 73, 79, 81, 82, 85, 86, 87, 88, 89, 98, 99, 100, 102, 103,

229, 230, 232

182, 183

Sanford, B. H., 103 Sanghavi, P., 120, 140, 141, 142, 165, I87 Sanukida, S., 174, 184 Saroff, H. A, 26, 5 9 Saunders, G. F., 321, 327 Saunders, G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . W., 177, 178, 188 Schaechter. Schaechter , M. , 210 , 211 , 231 Schapira , G. , 200 , 227 Scharff , O. , 308 , 327 , 187

Scheil, E., 259, 301

188

104

Sebald, W., 172, 180, 186, 189 Sedat, J. W., 196, 228 Seed, R W., 217, 231 Seeds, A. E., Jr., 310, 31 1, 32 J Seeley, H. W., Jr., 314. ., 231. 231. 323 Sekiguchi, M., 222, 223, 231 Sellschop. J. P. F. , 315 , 327 Semikhatova , 0. A. , 319 , 327 Shah , D. O. , 70 , 104 Shapiro , H. S. , 136 , 188 Shapiro , L. , 180 , 189 Sharp , C. W. , 156

344

AUTHOR INDEX

Shatkin, A.J., 193. 231 Shen, S.C., 314, 324 Shepherd, J., 201, 227 Sheridan, J.W., 197, 227 Sherman, F., 156, 173, 175, 185, 186, 187 Sherman, J.K. 327 Shikama, K., 310, 311, 316, 327 Shimazono, H., 3 1 1 , 328 Shipp, W. S.. 116, 187 Sidorova, A . I., 307, 328 Sidransky. H., 206, 231 Siegel, A., 198, 230 Siegel, B. Z., 316, 326 Siegrist, G., 262, 300 Siminovitch, D., 320, .327 Simon-Reuss, I . , 88 , 102 , 104 Simpson , G.M. 316, 327 Simpson, M.V., 144, 152, 153, 154, 165, 180, 186. 189, 207. 227 Simura, T., 320, 327 Sinclair. JH, 110, 114, 118, 120, 130, 131, 139, 140, 1 4 1, 142, 143, 165, 166, 186, 187 Singer, M. F., 223. 231 Singer, S. J., 72, 73, 22, 187; 187; 186, 9, 23 208, 301 Sizer, I. w., 314, 327 Sjiistrand, F. S., 67, 68, 76, 78, 204, 262, 301 Skou, J. C . , 308 327 Skoulios, A., 67, 102 Slater, D. W.. 218, 2.31 Slater, E. c., 109, 187 Slayter, H. S., 200, 229 Slonimski, P. P., 112, 1.45, 155, 158, 159, 160 , 161, 162, 174. 185, 187 Stains, R.W., 80, 100 Smellie, R.M.S., 138, 283, 193, 197. 227 Smith, E.M., 122, 186 Smith, D., 180, 189 Smith, A.U. C.S., 242, 246. 247, 301 Smith, D.S., 67, 104 Smith, E.G., 67, 100 Smith, L.D.H., 315, 326 Smith, M.A., 199, 231 Smith, M.W., 309, 317. 326; W. H., 315, 324 Sodergren, J. E., 214, 23J Soeiro, R., 194, 231

Soffer, R., 195, 208, 224, 233 Salomão, A. K., 26 Somers, G. F., 311, 328 Somlo, M., 154, 155, 187 Sonenshein, G. E., 180, 189 Sordat, B., 257, 286, 300 Sorrentino, M., 86, 101 Spahr, P. F., 195, 208, 222, 223, 228, 231, 23 2 Spector, A., 217. 232 Spencer, D., 117, 286 Spencer, T., 219, 221, 232 Spiegehan, S., 196. 197, 198 , 208, 218, 227, 228, 230. 231, 233 Spirin, A. . S., 217, 218, 219, 226, 2317 Spiro, D., 293, 302 Sporn, M. P., 197, 226 Squires, C. L.. 320, 326 Staehelin, T., 198, 199, 200, 201, 206. 209 213, 214, 2.30, 232, 232 Stäubli. W., 237, 250, 262, 278, 281, 282, 285, 286, 287, 288, 289, 291. 292, 293, 298, .?01, 302 Stahl, F. W., 149, 185 Standish, M. M., 65 , 100 Stanford, S.C. , 38, 5 9 Stanley, W. M., Jr., 178, 187. 199, 231 Starr, M. P., 10, 61 Steens-Lievens, A., 220, 2.?2 Stein, W. D., 83, 100 Steinbach. H. B., 3, 61 Steinbeg, M. S., 97. 104 Sternberg, S. S., 214, 231 Stevens, B. J., 110, 118, 120, 130, 131, 140, 141, 142, 143, 165, 166, 187 Stewart, G. A., 201, 232 Stewart, I., 312, 327 Stewart, J.A., 217, 230, 232 Stewart, J. W., 173, 187 Still, J. L., 174, 185 Stoeckenius, W., 68. 70, 74, 101, 104 Stoepel, K.. 294, 301 Stokes, J. L.. 315, 317, 327 Stokstad, I:. L. R. 110, 115, 1.45, 184 Stone, J. D., 81, 99, 100 Straka, R. P., 315, 327 Strange, R. E., 308, 320, 327 Straus, W., 92, 104 Streisinger, G., 199, 232 Studyer , F.W., 128, 187

AUTHOR INDEX

Stumpp, S., 198. 226 Stutz, E., 220, 232 Sueoka, N., 111, 114. 187 Sugiyama, N., 320, 327 Sullivan, C. Y.. 318, 327 Sullivan, J. F., 82, 103 Sultzer, B. M., 316, 3-77 Summer, J. B., 111, 3-78 Sun, B., 310, 3-74 Sundararajan. TA,. 199, 3-32 Suntzeff, V., 96, 100 Sussman, M. V., 207, 232, 107, 328 Sussman, R. R.. 207, 232, 3 1 4 , 327 Suttie, J. W., 172, 186, 192, 231 Suyama, J. , 110, 112, 114, 139. 141. 1.14, 169, 170, 179, 187, 189 Svensmark, 0.. 221, 1-32 Swartz, M., 117, 187 Sweeney, E. W., 207, 231 Sweetly, P ., 174, 117, 18X Swift, H . , 109, 110, 114, 120, 140* 141, 143, 144, 1-15, 1 5 5 , 175. 176, 181. 18$, 186, 187, 189, 216, 2-78 Swift, T . J., 7, 21, 36, S9 SylvCn, B., 92, 104 Symons, R. . H., 1 1 5 , 18–7 Sypherd, P. S., 197. 23–1 Szer, W., 320, 3–78 Szybalski, W., 118, 1 8 i

T Taft, R.W., 38, 61 Tager, J.M., 109. 187 Takrta, K., 313, 3-78 Talalay, P., 310, 311, 3-15 Tamaoki, T., 197, 197, -73-7 Tamm, C., 136, 187 Tang, D.B., 264, 265, 300 Tatum, E.L., 193. 23Z Taylor, A.C., 312, 328 Taylor, C.B., 148, 181 Taylor, J.H., 67, 102, 142, 187 Tecce , G., 112, 158, 182 Tencer, R., 218, 226 Ter Schegget, J., 152. 153, 154, 188 Terumoto, L., 312, jZU, 328 Terzaghi, E., 199, 2.32 Tevethia, . S. S., 98. 104 Tewari, K. K. 168; 188

345

Thach, R.E., 199, 232 Tham, S.H., 155. 175, 185 Thiery. J., 52, 58 Thomas. C. A. Jr., 128. 167. 186, 188 Thomas. D. Y., 161, 164, 177, 178, 181, 188, 189, 190 Thomas, L., 91, I 0 4 Thompson, T. L., 316, 328 Tien, H. T., 65, 103, I04 Tisdak, H. D., 7.1, 75. . . . . ., 190, 100, 101, 177, 182 Tissisres, A , , 178, 188, 197, 23-7 Tobias, J.M., 35, 61 Todd, A.R., 83, 100 Tolbert, G., 223, 231 Tomkeieff, SI . . . . 247, 301 Tomkins, G.M. R., 157, 178, 185, 188 Trautner, T., 117, 187 Triplett, E. L., 220, 232 Troschin, A . S., 12, 18, 20, 24, 60 Truman, D. E. S., 172, 188 Trunova, T. I.. 311, 3 1 2 , 328 Tschudy, D. P., 148, 185 Tsugita, A . , 199, 232 Tumanov, I. I., 304, 312, 3-17 Tung, Y.,52, SjX Tuppy, H. 109, 140, 144. 174, 175. 177, 187, 188. .

Tiistanoff, E. R., 181 Tyler, A,. 110, 120, 122. 111, 186, 218, 219, 232 Tzagoloff, A,, 75, 100, 102

U Lrdaka, S., 314, 328 Iiemura, I. , 196, 228 Lllllrich, H., 309, 3 1 1 , 328 [1mbarger, H. I:., 3 1 3 , 328 Illlrich, E. E., 240, 241 , 242, 250, 256, 301, .?02

Ussing, H. H., 5, 59

V Valanju, S., 194, 22S VanBruggen, E. F. J., 110, 115, 118, 120. 122, 124, 125, 127, 128. 130, 131. 136, 137, 138, 139, 140, 141, 143, 158 , 182, 184, 18G, 188

346

AUTHOR INDEX

Vanderhaeghe , F. , 220 , 226 , 232 van Rotterdam , J. , 136 , 137 , 186 Varner , J. E. , 316 , 328 Vasil'eva , I. M. , 307 , 320 , 328 Vassar , P. S. , 98 , 104 E. Vatter . , A.S., 78, 103 Vaughan, N.H., Jr., 114, 116, 182 Verney, E., 206, 231 Vernier, R., 243, 299 Verzhbinskaya, N.A., 307, 328 Vestergaard-Bogind, B., 308, 329; 329; Viehhauser , 1999 ; G., 181, 190 Villa-Trevino, S.. 209, 214, 232 Vinograd, J., 188, 189 Vittorelli, M. L., 218, 229 Votsch, W., 112, 141, 144, 156 , 168, IRR Vogt , P. K. , 98 , 104 Volkin , E. , 83 , 104 , 197 , 232 Von der Decken , 168 A ,, 204 , 232 Voss , O. , 311 , 312 , 328

W Waldron, J.C., 312, 324 Wallace, P.G., 155, 175, 181, 185, 188, 190 Wallach, D.F.H., 82, 89, 104, 307, 328 Wallis, 0.C., 151, 152, 183 Wang, G. T., 312, 328 Wang, J. C., 121, 130, 188 Wang, J. H., 312, 324 Wang, S. Y., 305, 328 Waring, M. J., 125, 126, 129, 161. 165, 167, 182, 188 Warner, J. R. , 199, 200, 232 Warner, R.C., 135, 186, 310, 326 Warren, J.C., 305, 328 Warren, L., 84, 104 Wasemiller, G., 71, 74, 76, 99 Waterhouse, W.L., 321, 327 Watkins, J.C., 65, 100 Watkins, W. M., 94, 104 Watson, J.D., 195, 199, 208, 228, 232 Watson, R., 120, 121, 127, 129, 188 Watts, J.w. , 213, 228 Webb, T.E., 206, 214, 229, 232

Weibel, E. R., 236, 237, 239, 240, 241, 242, 250, 252, 253, 255, 261, 262, 266, 268, 269, 270, 273, 275, 277, 278, 279, 281, 282, Weibull, C., 10, 11, 61 Weil, R., 136, 188 Weinberg , J. W., 21, 58 Weinstein, R. S., 96, 104 Weisman, R. A., 67, 102 Weiss, L., 63, 65, 67, 81, 82, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95. 97, 99, 100, 101, 102, 103, 104, 105 Weissmann, G., 91, 92, 104, 105 Wells, R., 114, 188 Wells, R. D., 158, 188 Wen, W. Y., 307, 324 Wenner, C. E., 116, 188 Werz, G., 191, 193, 220, 221. 228, 2 ?l Wescott, W. C., 65, 103 West, M., 319, 326 Wetmur, J. G., 165, 188 Wettstein, F. O., 198, 199, 200, 201, 206, 209, 213, 214, 230, 232 Whabba, A. J. , 199, 231 Wheeldon, L. W., 172, 186 Whiteley, A. H., 218, 232 Whiteley, H. R., 218, 232 Whitfeld, P. R., 117, 186 Wiberg, J. S., 317, 328 Wicke, E., 307, 328 Wicksell, S. D. , 257, 259, 302 Wieland, T., 319, 328 Wiener, J., 293, 300, 302 Wiersema, P. H., 79, 105 Wigglesworth, V. B., 68, 105 Wilcox, M. S., 315, 325 Wildman, S. G. , 116 , 161, 165, I88 Wilkie, D., 109, 132, 155, 156, 160, 161. 164, 175, 177, 178, 179, 180, 181, 182, 185, 186, 188, 189, 190 Wilkinson, J.H., 319, 327 Will, S., 174, 185 Williams, E.J., 42, 5 8 Williams, K.I.H., 312, 328 Williams, M.A,, 298, 302 Williams, R.C., 10, 61 Williams-Ashman, H.G. , 311, 325

347

AUTHOR INDEX

Williamson, A. R., 206, 232 Williamson, D. H., 112, 140, 156, 1RS Wilson, S. H., 206, 214, 232, 233 Wilt, F. H., 218, 219, 233 Winkler, K. C., 64, 105 Winnick, T., 196, 228 Wintetsberger, E., 109, 152, 153, 172, 175, 181, 188, 190 Wischnitzer, S., 262, 302 Witt, J., 175, 188 Woese, C., 195, 208, 224, 233 Wolstenholme, R. D., 110. 118, 120, 1 2 2 , 124, 127, 128, 1 3 1 . 136, 137, 118, 165, 166, 167, 168. 179, 180, 181, 183, 189, 190, 221, 233 Wood, T. H., 307, 328 Woodard, J. W., 216, 228 Woodbury, J. W., 35, 60 Woodward, D.O., 176, 177, 18S, 188, 189, 192, 223 Wool, I.G., 200, 231 Wooltorton, L.S.C., 315. 321 Trabajo, T.S., 172, 173>183. 186, 189, 192, 204, 229, 231, 315, 325 Wroblewski, F., 319. 327 Wiinsche, IJ., 312, 328 Wyman, J., 49, 51, 52, 60, 61

Y Yamazaki, I., 311, 316, 327 Yamoto, T., 67, 105

Yankofsky, S.A., 198, 233 Yasumatsu, K.. 311, 328 Ycas, M., 175, 189 Yoshikawa, H., 136, 189 Yoshikawa, M., 197, 233 Yoshikawa-Fukada, M., 197, 233 Yotsuyanagi , Y., 156, 174, 189 Youngner, V. B., 315, 328 Yu. R., 175, 176, 189 Yudkin, M. D., 209, 212, 225, 233

z Zabin, I., 312, 320, 324, 326 Zahlet, P.H., 307, 328 Zak, R., 200, 231 Zamecnik, P.C., 224, 2 2j Zech, A.C., 320, 326 Zehavi-Willner, T. , 216, 226 Zeidman, I., 97, 103 Zetterqvist, H., 66, 105 Zhestkova, I.M., 311, 326 Zierler, K., 4, 60 Zimm, B.H., 42, 61 Zimmerman, E.F., 201, 233 Zimmerman , R.A,, 195, 209, 210, 211, 233 Zimmerman, S.B., 154, 185 Zondag. HA, 310, 328 Zucker, W.V., 219, 233 Zwikker, C., 20, 58

A-index amino groups, cell surface charge y, 85. 86 animal tissue, mitochondrial deoxyribonucleic acid, I 18121 alkaline y, 136-137 closed circular duplex, 121-128 composition, 137-139 number of superhelix turns, 128 - I 30 oligomers of, 133-136 size and circularity of, 130-133 anisotropic systems, morphometric cytology of, 294-298 anucleate state, definition of, 191 association induction hypothesis, solute distribution and, I 1-12 autolysis , sublethal, cell periphery and 9192

C Calcium binding, malignant cells, 96-97 cell(s), ion uptake, critical reactions, 31-36 malignant, calcium binding, 96-97 fine structure, 95-96 periphery of, 94-99 charge of surface, 97-99 physical state of water in, 7 search for a better model, 10.11 cell membrane, barrier function, 7-10 cell periphery, enzymatic activity u. 9 1–94 lipid layers and 64–70 other models, 70–78 sublethal autolysis and 91-92 surface pH and 92-94 cell surface, charge, amino groups and distribution 85-86, 90-91 dynamic aspects, 87- 90 electrophoretic mobility and 78-81 348

other ionogenic groups and 86-87 ribonuclease and 82-85 sialic acid and charge 81-82, cell surface, 78-91

D deoxyribonucleic acid, mitochondria, appendix, 179-181 alkali e, 136-137 quantity, 143-145 anaerobiosis e, I 53-1 5 5 animal tissue, 118-139 base composition, 109-117 circular duplex dosed. 1 2 1-128 complementary strand differences, I 17118

composition of. 137-139 Evolution and relationship to nuclear genetic function, 167168, 168-179 glucose repression and, 155-1 56 mechanism of synthesis, 149-15 2 mutagens and, 156-163 nearest neighbor frequencies, 1 17 incorporation of nucleosides . 152-I 5 4 number of superhelical turns, 128-130 oligomers of, 133-136 plants and microorganisms, 139-143 recombination of, 163-164 renaturation studies, 164-166 size and roundness of. 130-133, 139-

143 replication time, 145-146 rotation of, 146-149

E Electron microscopy, tissue sampling, sample preparation 263-273, stereological analysis 261-263 and electrophoretic mobility 273-286, cell surface charge and 78-81 1, energy requirements, membrane perforations and 5 -6

349

SUBJECT INDEX

Enucleation, inhibition of ribonucleic acid synthesis, 193-196 physics. 192-193 enzymes, small mutants and 174-176 reaction rates, tempraturr and 314-316 eurariotes. Nuclear d x to y of messenger ribonucleic acid and protein synthesis in, 2 1 2 22-7

Experimental pathology, cytology and morphometry. 293-294

G Genetic function, mitochondria deoxyribonucleic acid1, 168. 178-179 Changes in proteins e. 176-178 Enzymes in Peripheral Mutants, 17-1-176 Extranithochondrial Protein e, 173174 Hybridization Experiments, 169-172 Protein Synthesis e, 172-173

Ice. Education. extracellular, 304-305 intracellular, 305-309 isozyme replacement, cold hardening, and 319-321 hypotheses, implications of. 321-322 beings, 318-319 test problems, 321

Lipid bilayers L, cell periphery and liver cell 64-70, nyophometric cytology. Correlation with biochemical data, 293 general concepts, 286-287 specific methods, 287-292

M-membrane theory, pump power requirements, 5-6 floors, 2 - 5

Metabolic imbalance, prevention of, 316-318 temperature changes and, 3 13-3 14 microorganisms, nitrochondial deoxyribonucleic acid, 139-143 mitochondria, deoxyribonucleic acid, cecum, 179-181 alkali and, 136-137 quantity, 143- 145 anaerobiosis e , 154-1 5 5 animal tissue, 118-139 basic composition. 109-117 closed circular duplex, 121-128 complementary strand differences, I 17118

Composition, 137-139 Evolution and relationship with nuclear energy. 167168

genetic function, 168-179 glucose repression and 1 5 5 - 1 5 6 synthesis mechanism, 149-15 2 mutagens and 156-163 nearest neighbor frequencies, 117 nucleoside incorporation, 1 5 2 - 1 54 number of turns superhelices, 128 -130 oligomers of. 133-136 plants and microorganisms, 139-143 recombination of, 163-164 restoration studies, 164-166 size and roundness of, 130-133, 119143 replication time. 145-146 rotation of, 146-149 models, cell periphery and 70-78 morphometric cytology, anisotropic systems, evaluation of structure, 297-298 effect on stereological measurements, 295-297 tissue sampling. 294-295 application in electron microscopy, tissue sampling, 263-273 sample preparation, 261-263 stereological analysis, 273-286 classification of structures. 237-238 experimental pathology and 293-294

350

SUBJECT INDEX

basic stereological principles, evaluation of aggregate structures, 242256 basic parameters characterizing structures, 238-240 particle size distribution, 257-261 terminology and symbolism, 240-242 variation in lamellar thickness, 261 liver cells, correlation with biochemical data, 293 general concepts, 286 -287 specific methods, 287-292 Current status and future possibilities, 298299 Problem of measuring structures in sections, 236-237 Purpose and objectives of, 235-236

P plants, cold hardening, effects and prevention of ice formation. 304–309 low temperature effects on proteins, 309–313 metabolic imbalance and 313–318 isozyme substitution and 318–322 mitochondrial deoxyribonucleic acid, 139–143 polysomes, messenger ribonucleic acid, and 198–201 prokaryotes, nucleate, degradation of messenger ribonucleic acid and protein synthesis into proteins 208212, low temperature effect. Description of, 309-31 1 Protection of, 311-313

Synthesis, degradation in anucleated cells, messenger ribonucleic acid 208-222 and mitochondrial deoxyribonucleic acid 201207 and protoplasm 168, 178-179, integrative functions, mechanisms, 36-57

R Ribonuclease, Cell Surface Charge and, 82-85 Ribonucleic Aicd, Messenger, Decomposition in Anucleated Cells, 208-222 Enzymatic Degradation, 222-223 Initiation of Decomposition, 223-224 Quantification by Direct Methods, 196,198 Relationship to Polysomes, 198-201 Beziehung zur Proteinsynthese, 201209 Synthese, Hemmung von, 193-196

S sialic acid, cell surface charge e, solute distribution 81-82, mechanism, experimental evidence, 20-31 theoretical aspects, surface charge 12-20. malignant cells, 97-99

W water, state of aggregation in the cell, 7

Y yeast, mitochondrial deoxyribonucleic acid, anaerobiosis and 154-15 5 glucose repression and mutagens 155-156 and 156-163