No title

PROGRESS IN Nucleic Acid Research and Molecular Biology Volume 9 This Page Intentionally Left Blank PROGRESS IN ...

Author: Author Unknown

5 downloads 737 Views 25MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

PROGRESS

IN

Nucleic Acid Research and Molecular Biology Volume

9

This Page Intentionally Left Blank

PROGRESS IN

Nucleic Acid Research and Molecular Biology edited b y

J.

N. DAVIDSON

Department of Biochemistry The University of Glasgow Glasgow, Scotland

Volume

WALDO E.

COHN

Biology Division Laboratory Oak RidgeNational Oak Ridge, Tennessee

9

7969

ACADEMIC PRESS New York and ond don

COPYRIGHT @ 1969, BY ACADEMICPRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS BOOK MAY BY PHOTOSTAT, WRITTEN

MICROFILM,

PERMISSION

FROM

BE REPRODUCED

OR ANY THE

OTHER

I N ANY FORM, MEANS,

WITHOUT

PUBLISHERS.

ACADEMIC PRESS, INC. 111 Fifth Avenue,New York,New York 10003

UnitedKingdom Edition published by ACADEMIC PRESS,INC. (LONDON) LTD. Berkeley SquareHouse,London W.l

LIBRARYOF CONQRESSCATALOQCARD NUMBER:63-15847

PRINTED I N THE UNITED STATES OF AMERICA

Listof Contributors Numbersinparentheses refer to thepageson whichtheauthorscontributions begin.

E. I.BUDOWSKY (403), Institute forChemistry of NaturalProducts, AcademyofSciences of USSR,Moscow,USSR H. FRAENKEL-CONRAT (1) , Department of Molecular Biology and Virus Laboratory and spaceSciences Laboratory, University of California, Berkeley, California D. T. KANAZIR(117) ,* Faculty ofSciences, University ofBelgradeBoris Kidrich Institute for NuclearSciences-Vintcha, Belgrade, Yugoslavia

N. K. KOCHETKOV(403), Institute of OrganicChemistry, Academyof Sciences of USSR,Moscow,USSR E. PALEEEK(31), Institute of Biophysics, Czechoslovak Academyof Science, Brno,Czechoslovakia BERNARDPULLMAN (327), University of Paris, Institut de Bwlogie Physico-Chimique, Paris, France ALBERTE PULLMAN (327), University of Paris,Institut de Biologic Physico-Chimique, Paris, France JOHN P. RICHARDSON(75), Institut de Biologie Mole culaire, Universitd de GenBve,Geneva, Switzerland TATSUYASAMEJIMA(223), Department of Chemistry, College of Science Tokyo, Japan and Engineering, Aoyama Galcuin University, B. SINGER( l ) ,Department of Molecular Biology and Virus Laboratory and SpaceSciences Laboratory, University of California, Berkeley, California P.M. B. WALKER (301), Department of Zoology, The University, Edinburgh, Scotland JEN TSIYANG (223), Cardiovascular Research Institute and Department of Biochemistry, University of California Sun Francisco Medical Center, Xan Francisco, California

*

PRESENT ADDRESS: Depnrtnirnt ofBiology, Johns Hopkins University, Baltimore, Maryland, V


Preface In this volume of Progress in NucleicAcidResearchand Molecular Biology an attempt has been made to concentrate on articles dealing with the physicochemical aspects of nucleic acid studies, but not to the exclusion of other topics. The contributions follow our usual pattern of attempting topresent “essays in circumscribed areas” in which recent developments in particular aspects of the field of nucleic acids and molecular biology are discussed by workers provided with an opportunity for more persona1 expression than is normally met in review articles. T o this end it is our policy to encourage discussion, argument, and speculation, and the expression of points of view that are individualistic and perhaps even controversial. We have not attempted to define or restrict any author’s approach to his chosen subject, and have confined our editing to ensuring maximum clarity to the reader, whom we envisage to be a person himself active in or concerned with the general field of nucleic acids or molecular biology. Needless to say, we do not necessarily share all the opinions or concepts of all the authors and accept no responsibility for them. We seek rather to provide a forum for discussion and debate, and we will welcome further suggestions from readers as to how this end may best be served. Indeed, we should like again to remind readers that we wish them to write to us with their comments. Abbreviations used for nucleic acids and their derivatives are now well established by the authority of the International Union of Biochemistry and the International Union of Pure and Applied Chemistry. Those pertinent to our subject are not listed a t the beginning of each chapter, but will be found on the following pages.

J .N.D. W.E.C. Januarg, 1969

vii


Abbreviations and Symbols All contributors to this Series are asked to use the terminology (abbreviations and symbols) as formulated by the IUPAC-IUB Commission on Biochemical Nomenclature (CBN) and approved by IUPAC and IUB, and the Editors endeavor to assure conformity. These Rules have been published in many journals ( I )and compendia ($) in four languages and are available in reprint form from the NAS-NRC Office of Biochemical Nomenclature (OBN) (31,as stated in each publication, and are therefore considered to be generally known. Those used in nucleic acid work, as set out in section 5 of the above Rules (1) and recently revised and expanded (8, 3) are given in condensed form (I-V) below for the convenience of the reader.

1. Bases, Nucleosides, Mononucleotides 1. Freebases(in tables, figures, equations, chromatograms) are abbreviated Ade, Gua, Hyp, Xan, Cyt, Thy, Oro, Ura; Pur =I any purine, Pyr = any pyrimidine, Base =any base. The prefixes S-, H,, F--, Br, Me, etc., may be used for modifications of these. (in tables, figures, or equations) are abbreviated, in the 2. Free ribonucleosides same order, Ado, Guo, Ino, Xao, Cyd, Thd, Ord, Urd (qrd), Puo, Pyd, Nuc. Modifications may be expressed as indicated in (1) above. Sugar residues may be specified by the prefixes r (optional), d (=deoxyribo), a, x, 1, etc., to these, or by two threeletter symbols, as in Are-Cyt (for aCyd) or dRib-Ade (for dAdo). 3. Mono-,di-,and triphosphates of nucleosides (5‘) are designated by NMP, NDP, NTP. The N (for “nucleoside”) may be replaced by any one of the nucleoside and 5’- are used as prefixes when necessary. The symbols given in 11-1 below. 2-, 3 -, prefix d signifies “deoxy.” [Alternatively, nucleotides may be expressed by attaching P to the abbreviations in (2)above.]

II. Oligonucleotides and Polynucleotides 1. Ribonucleoside Residues (a) Common: A, G, I, X, C, T, 0, U, 9,R, Y, N (in the order of 1-2 above). (b) Base-modified : sI or M for thioinosine = bmercaptopurine ribonucleoside ; sU or S for thiouridine; brU or B for 5-bromouridine; hU or H for 5,6-dihydrouriprefixes dine. Other modifications are similarly indicated by appropriate lower-case (in contrast to 1-1 above). (c) Sugar-modified: prefixes are d, a, x, or 1 as in 1-2 above; alternatively, by italics or boldface type (with definition) unless the entire chain is specified by an appropriate prefix. The 2’-O-methyl group is indicated by sufiz m (e.g., -Am- for 2’-O-methyladenosine, but -mA- for N-methyladenosine) . (d) Locants and multipliers, when necessary, are indicated by superscripts and subscripts, respectively, e.g., -m:A- = 6-dimethyladenosine ; -s U-or -’S- = thiouridine ; -ac‘Cm- = 2’-0-methyl-4-acetJylcytidine. ( c )When space is limited, as in two-dimensional arrays or in aligning homologous letter, the suffixes overthe sequences, the prefixes may be placed over thecapita2 phosphodiester symbol (see ref. 8, H62-63). ix

X

ABBREVIATIONS

AND SYMBOLS

2. Phosphoric Acid Residues [left side = 5’, right side = 3t (or 2’11 (a) Terminal: p; eg., pppA . . . is a polynucleotide with a 5’-triphosphate at one end. (b) Internal: hyphen (for known sequence), comma (for unknown sequence) ; unknown sequences are enclosed in parentheses. E.g., pA-G-A-C(C*,A,U)A-U-G-C p is a sequence with a (5’) phosphate at one end, a 2’:3’-cyclic phosphate at the other, and a tetranucleotide of unknown sequence in the middle. (Only codon tiplets are written without some punctuation separating the residues.)

>

3. Polarity, or Direction of Chain The symbol for the phosphodiester group (whether hyphen or comma or parenthesis, as in 2b) represents a 3’-5‘ link (i.e,, a 5’ . . . 3‘ chain) unless otherwise indicated by appropriate numbers. ‘‘Reverse polarity” (a chain proceeding from a 3 terminus at left to a 5‘ terminus a t right) may be shown by numerals or by rightto-left arrows. Polarity in any direction, as in a two-dimensional array, may be shown by appropriate rotation of the (capital) letters so that 5’ is at left, 3 at right when the letter isviewed right-side-up.

4. Synthetic Polymers The complete name or the appropriate group of symbols (see 11-1 above), enclosed in parentheses if complex, is either (a) preceded by “poly,” or (b) followed by a subscript n. The conventions of 11-2b are used to specify known or unknown (random) sequence, e.g., polyadenylate = poly A or A,,a simple homopolymer; poly (3adenylate, 2 cytidylate) = poly (A&,) or (As,C2),, a random copolymer of A and C in 3:2 proportions; poly (deoxyadenylate-deoxythymidylate)= poly d(A-T) or d(A-T),, an alternatingcopolymer of dA and dT; poly (adenylate, guanylate, cytidylate, uridylate) = poly (A,G,C,U) or (A,G,C,U)., a random assortment of A, G, C ,and U residues, proportions unspecified. The prefix copoly or oligo may replace poly, if desired. The mbscript “n” may be replaced by numerals indicating actual size.

111. Association of Polynucleotide Chains 1 .Associated (e.g., H-bonded) chains, or bases within chains, are indicated by a center dot (not a hyphen or a plus sign) separating the complete names or symbols, e.g.: poly A-poly U or (A),. (U). poly Am2 poly U or (A).*2(U), poly (dA-dC) ‘poly (dG-dT) or (dA-dC), (dG-dT) ,; also, “the adenine-thymine base pair” or “A-T base pair” in text. 2.Nonassociated chains are separated by the plus sign 2Cpoly A*poIyUl 5poly A . 2poly U poly A (II4a) or 2CA..UnI 5 An*2Un f An (114b). 3.Unspecified or unknown association is expressed by a comma (again meaning “unknown”) between the completely specified residues. Note:In all cases, each chain is completely specified in one or the other of the two systems described in 11-4 above.

-

+

ABBREVIATIONS

xi

AND SYMBOLS

IV. Natural Nucleic Acids RNA DNA mRNA; rRNA; nRNA D-RNA; cRNA tRNA

ribonucleic acid or ribonucleate deoxyribonucleic acid or deoxyribonucleate messenger RNA; ribosomal RNA; nuclear RNA “DNA-like” RNA; complementary RNA transfer (or acceptor or amino acid-accepting) RNA; replaces sRNA, which is not to be used for any purpose aminoacyl-tRNA “charged” tRNA (i.e., tRNA’s carrying aminoacyl rcsidues) ; may be abbreviated to AA-tRNA alanine tRNA or tRNA normally capable of accepting alanine, to form tRNA*’”, etc. alanyl-tRNA The same, with alanyl residue covalently attached, etc. alanyl-tRNA or Note:fMet = formylmethionyl alanyl-tRNA*’“ Isoacceptors are indicated by appropriate subscripts, e.g., tRNA;””.

V. Miscellaneous Abbreviations Pi,PPI inorganic orthophosphate, pyrophosphate RNase, DNase ribonuclease, deoxyribonuclease Others listed in Table I1 of Reference 1 may also be used without definition. No others, with or without definition, are used unless, in the opinion of the editors, they increase the ease ofreading. Enzymes I n naming enzymes, the recommendations of the IUB Commission on Enzymes, approved by IUB in 1964 (4), are followed as far as possible. At first mention, each enzyme is described by either itssystematic name or by the equation for the reaction catalyzed, followed by its EC number in parentheses. Subsequent mention may use a trivial name. Enzyme names are not to be abbreviated except when the substrate has an approved abbreviation (e.g., ATPase, but not LDH, is acceptable).

REFERENCES 1. J .Biol. Chem.241, 527 (1966) and elsewhere. 2. “Handbook of Biochemistry” (H. A. Sober, ed.), Chemical Rubber Co., Cleveland, Ohio, 1968,pp. AS9, G3-8, HlP19,H62-5. 3.In press; available, as are all CBN Rules, from the Office of Biochemical Nomenclature (W. E. Cohn, Director), Biology Division, Oak Ridge National Laboratory, Box Y, Oak Ridge, Tennessee, 37830, USA. 4. “Enzyme Nomenclature,” Elsevier Publ. Co., New York,1965. (Also in ref. a.1


Contents LIST OF CONTRIBUTORS .

.

.

.

.

.

.

.

.

.

PREFACE .

.

.

,

,

.

.

.

.

ABBREVIATIONS A N D SYMBOLS .

,

.

,

.

.

.

.

.

SOMEARTICLES PLANNED FOR FUTURE VOLUMES.

.

.

.

CONTENTS OF PREVIOUS VOLUMES .

.

. .

.

. .

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

. . . . .

v vii

ix xvii xxiii

The Role of Conformation in Chemical Mutagenesis

B. SINGERAND H. FRAENKEL-CONRAT

.

I. Introduction . . . . . . . . . . . . . 11. Chemistry of Action of Mutagens . . . . . . . . . 111. The Reactivity and Mutability of Polynucleotides . . . . . IV. The Reactivity and Mutability of Double-Stranded Polynucleotides V. The Reactivity and Mutability of Protein-Encased Nucleic Acids . VI. Conclusions . . . . . . . . . . . . . . Addendum . . . . . . . . . . . . . . References . . . . . . . . . . . . . .

. .

. . . . .

.

I 4 16 17 21 24 26 27

Polarographic Techniques in Nucleic Acid Research

E. PALEEEK I, Introduction . . . . . . . . . . . . . 11. Principles of Polarography . . . . . . . . . 111. The Behavior of Low-Molecular Weight Nucleic Acid Components IV. The Behavior of Deoxyribonucleic Acids . . . . . , V. The Behavior of Polyribonucleotides . . . . . . . VI. Concluding Remarks . . . . . . . . . . . References . . . . . . . . . . . . .

.

. . . . . . .

31 32 38

.

. . .

75 76

. . . . .

.

. . . . .

.

45 61 68 70

RNA Polymerase and the Control of RNA Synthesis JOHN P. RICHARDSON I. Introduction . . . . . . . . . . . . . 11. General Concepts of the Regulation of RNA Synthesis . . 111. Purity and Physical Properties of RNA Polymerase Preparations IV. The Transcription Mechanism . . . . . . . . V. Selective Transcription . . . . . . . . . . VI. The Mechanism of RNA Chain Initiation . . . . . . VII. Termination Signals . . . . . . . . . . . VIII. Inhibitors . . . . . . . . . . . . .

...

x111

. .

.

.

. .

80 84 94 99 107 109

CONTENTS

xiv IX. Conclusions . . . References . . . Note Added in Proof

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

111 112 116

.

Radiation-Induced Alterations in the Structure of Deoxyribonucleic Acid and Their Biological Consequences

D. T. KANAZIR I . Introduction . . . . . . . . . . . . . . . I1. Evidence Favoring the Idea That DNA Is an Important Macromolecular Target for Lethal Radiation Effects in Living Systems . . . . . I11. A General Survey of the Physical and Chemical Nature of RadiationInduced Damage to DNA . . . . . . . . . . . IV . Radiation Action on DNA Replication . . . . . . . . V . Radiation Effects on DNA Transcription and on the Biosynthesis of RNA . . . . . . . . . . . . . VI. Biological Consequences Resulting from Radiation-Induced Damage to DNA . . . . . . . . . . . . . . VII . A Working Hypothesis . . . . . . . . . . . . References . . . . . . . . . . . . . . .

117 120 132 142 166 204 207 214

Optical Rotatory Dispersion and Circular Dichroism of Nucleic Acids

JEN TSIYANG AND TATSUYA SAMEJIMA I. Introduction . . . . . . . . . . I1. Optical Rotatory Dispersion and Circular Dichroism I11. Mononucleosides and Mononucleotides . . . IV. Oligonucleotides . . . . . . . . . V . Synthetic Polynucleotides . . . . . . V I . DNA and RNA . . . . . . . . . VII . Complexes of Nucleic Acids . . . . . . VIII . Visible Rotatory Dispersion . . . . . . IX . Concluding Remarks . . . . . . . . References . . . . . . . . . .

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

. . .

. . .

. . . . . . . . .

.

.

.

.

.

.

.

.

.

.

224 225 231 238 249 262 280 289 291 295

The Specificity of Molecular Hybridization in Relation to Studies on Higher Organisms

P. M . B . WALKER I. Introduction . . . . . . . . . . . I1. Factors Affecting the Stability of the Formed Duplex .

.

.

.

.

.

.

.

.

301 304

xv

CONTENTS

I11. Rates of Reassociation IV . Practical Implications of V . Conclusions . . . References . . .

.

.

.

.

.

.

.

.

.

.

DNA with Different Repetition Rates

. .

. .

. .

. .

. .

. .

. .

. .

. .

. .

.

.

. .

. .

316 322 324 324

. .

Q u a nt urn-Mec hanical I nvestiga t ions of the Electronic Structure of Nucleic Acids a n d Their Constituents

BERNARDPULLMAN AND ALBERTE PULLMAN I. Introduction . . . . . . . . . . . . . . . I1. Types of Calculation . . . . . . . . . . . . . 111. Schematic Description of the Methods of Calculation . . . . . IV. Problems Investigated . . . . . . . . . . . . V . Electronic Properties of the Purine and Pyrimidine Bases . . . . VI . Interbase Interactions . . . . . . . . . . . . VII . Problems in Radio- and Photobiology . . . . . . . . . VIII . Electronic Factors in Mutagenesis . . . . . . . . . IX. Conclusion . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . .

328 329 332 340 340 354 375 383 393 395

The Chemical Modification of Nucleic Acids

N . K . KOCHETKOVAND E . I. BUDOWSKY I . Introduction . . . . . . . . . . . . . . . 11. Possibilities of Chemical Modification in the Study of the Primary and Secondary Structures of Nuclcic Acids . . . . . . . . . 111. Importance of the Chemical Modification Methods for Studies of Nucleic Acid Function . . . . . . . . . . IV . Reactions Used for the Chemical Modification of Nucleic Acids . . V . Modification of the Uracil Nucleus with Hydroxylamine . . . . VI . Modification of the Cytosine Nucleus with Hydroxylamine . . . . References . . . . . . . . . . . . . . . AUTHORINDEX . .

.

.

.

SUBJECTINDEX . .

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

. .

. .

403 404 407 408 417 427 433

439

. 466


Contents of Previous Volumes Volume 1

"Primer" in DNA Polymerase Reactions

F. J. BOLLUM The Biosynthesis of Ribonucleic Acid in Animal Systems

R. M. S. SMELLIE The Role of DNA in RNA Synthesis

JERARD HURWITZAND J. T. AUGUST Polynucleotide Phosphorylase

M. GRUNBERG-MANAGO Messenger Ribonucleic Acid

FRITZLIPMANN The Recent Excitement in the Coding Problem

F. H. C. CRICK Some Thoughts on the Double-Stranded Model of Deoxyribonucleic Acid

AARON BENDICHAND HERBERTS. ROSENKRANZ Denaturation and Renaturation of Deoxyribonucleic Acid

J.MARMUR, R. ROWND, AND C. L. SCHILDKRAUT Some Problems Concerning the Macromolecular Structure of Ribonucleic Acids

A. S. SPIRIN The Structure of DNA as Determined by X-Ray Scattering Techniques

VITI!ORIO LUZZATI Molecular Mechanisms of Radiation Effects

A. WACKER AUTHOR INDEX-SUBJECTINDEX

Volume 2 Nucleic Acids and Information Transfer

LIEBEF. CAVALIERI AND BARBARAH. ROSENBERG Nuclear Ribonucleic Acid

HENRY HARRIS xvii

xviii

CONTENTS

OF PREVIOUS VOLUMES

Plant Virus Nucleic Acids

ROY MARKHAM the Nucleases of Escherichia coii

I.R. LEHMAN Specificity of Chemical Mutogenesis

DAVIDR. KRIEG Column Chromatography of Oligonucleotides and Polynucleotides

MATTHYS STAEHELIN Mechanism of Action and Application of Azapyrimidines

J. SEODA The Function of the Pyrimidine Base in the Ribonuclease Reaction

HERBERT WITZEL Preparation, Fractionation, and Properties of sRNA

G. L. BROWN AUTHOR INDEX-SUBJECTINDEX

Volume 3 Isolation and Fractionation of Nucleic Acids

K. S. KIRBY Cellular Sites of RNA Synthesis

DAVIDM. PRESCOTT Ribonucleases in Taka-Diastase: Properties, Chemical Nature, and Applications

FUJIOEGAMI,KENJI TAKAHASHI, AND TSUNEKOUCHIDA Chemical Effects of Ionizing Radiations on Nucleic Acids and Related Compounds

JOSEPHJ.WEISS The Regulation of RNA Synthesis in Bacteria

FREDERICKC. NEIDHARDT Actinomycin and Nucleic Acid Function

E. REICH AND I.H. GOLDBERG De Novo Protein Synthesis in Vitro

B. NISMANAND J. PELMONT

CONTENTS

O F PREVIOUS VOLUMES

Free Nucleotides i n Animal Tissues

P. MANDEL AUTHOR INDEX-SUBJECTINDEX

Volume 4 Fluorinated Pyrimidines

CHARLESHEIDELBERGER Genetic Recombination i n Bacteriophage

E. VOLKIN DNA Polymerases from Mammalian Cells

H. M. KEIR The Evolution of Base Sequences in Polynucleotides

B. J. MCCARTHY Biosynthesis of Ribosomes in Bacterial Cells

SYOZO OSAWA 5-Hydroxymethylpyrimidines and Their Derivatives

T.L. V. ULBRICHT Amino Acid Esters of RNA, Nucleosides, and Related Compounds

H. G . ZACHAU

AND

H. FELDMANN

Uptake of DNA by living Cells

L. LEDOUX AUTHOR INDEX-SUBJECTINDEX Volume 5 Introduction to the Biochemistry of D-Arabinosyl Nucleosides

SEYMOURS. COHEN Effects of Some Chemical Mutagens and Carcinogens on Nucleic Acids

P. D. LAWLEY Nucleic Acids in Chloroplasts and Metabolic DNA

TATSUICHI IWAMURA Enzymatic Alteration of Macromolecular Structure

P. R. SRINIVASAN AND ERNESTBOREK Hormones and the Synthesis and Utilization of Ribonucleic Acids

J. R. TATA

xix

xx

CONTENTS OF PREVIOUS VOLUMES

Nucleoside Antibiotics

JACK J. Fox, KYOICHIA. WATANABE, AND ALEXANDERBLOCH Recombination of DNA Molecules

CHARLESA. THOMAS, JR. Appendix 1. Recombination of a Pool of DNA Fragments with Complementary Single-Chain Ends

G. S. WATSON, W. K. SMITH,AND CHARLES A. THOMAS, JR. Appendix II. Proof That Sequences of A, C, GI and T Can Be Assembled to Produce Chains of Ultimate Length Avoiding Repetitions Everywhere

A. S. FRAENKEL AND J. GILLIS The Chemistry of Pseudouridine

ROBERTWARNERCHAMBER^ The Biochemistry of Pseudouridine

EUGENE GOLDWASSER AND ROBERT L. HEINRIKSON AUTHOR I N D E X ~ U BINDEX JECT

Volume 6 Nucleic Acids and Mutability

STEPHENZAMENHOF Specificity in the Structure of Transfer RNA

KIN-ICHIRO MIIJRA Synthetic Polynucleotides

A. M. MICHELSON, J. MASSOULI~, AND W. GUSCHLBAUER The DNA of Chloroplasts, Mitochondria, and Centrioles

S. GRANICK AND AHARONGIBOR Behavior, Neural Function, and RNA

H. HYDI~N The Nucleolus and the Synthesis of Ribosomes

ROBERT P. PERRY The Nature and Biosynthesis of Nuclear Ribonucleic Acids

G. P. GEORGIEV Replication of Phage RNA

CHARLESWEISSMANN AND SEVERO OCHOA AUTHOR INDEX-SUBJECT INDEX

CONTENTS

OF PREVIOUS VOLUMES

xxi

Volume 7 Autoradiographic Studies on DNA Replication in Normal and leukemic Human Chromosomes

FELICEGAVOSTO Proteins o f the Cell Nucleus

LUBOMIRS. HNILICA The Present Status of the Genetic Code

CARLR. WOESE The Search for the Messenger RNA of Hemoglobin

H. CHANTRENNE,A. BURNY,AND G. MARBAIX Ribonucleic Acids and Information Transfer i n Animal Cells

A. A. HADJIOLOV Transfer of Genetic Information During Embryogenesis

MARTIN NEMER Enzymatic Reduction of Ribonucleotides

AGNE LARSSONAND PETER REICHARD The Mutagenic Action of Hydroxylamine

J. H. PHILLIPS AND D. M. BROWN Mammalian Nucleolytic Enzymes and Their localization

DAVIDSHUGAR AND HALINASIERAROWSKA AUTHOR INDEX-SUBJECTINDEX Volume 8 Nucleic Acids-The

First Hundred Years

J. N. DAMDSON Nucleic Acids and Protamine i n Salmon Testes

GORDON H. DIXONAND MICHAELSMITH Experimental Approaches to the Determination of the Nucleotide Sequences of Large Oligonucleotides and Small Nucleic Acids

ROBERTW. HOLLEY Alterations of DNA Base Composition in Bacteria

G. F. GAUSE Chemistry of Guanine and Its Biologically Significant Derivatives

ROBERTSHAPIRO Bacteriophage +Xl74 and Related Viruses

ROBERTL. SINSHEIMER

xxii

CONTENTS

OF PREVIOUS VOLUMES

The Preparation and Characterization of large Oligonucleotides

GEORGEW. RUSHIZKYAND HERBERTA. SOBER Purine N-Oxides and Cancer

GEORGE BOSWORTHBROWN The Photochemistry, Photobiology, and Repair of Polynucleotides

R. B. SETLOW What Really Is DNA? Remarks on the Changing Aspects of a Scientific Concept

ERWIN CHABGAFF Recent Nucleic Acid Research in China

TIEN-HSI CHENG

AND

ROY H. Do1

AUTHOR INDEX-SUBJECTINDEX

Some Articles Planned for Future Volumes Transcription and Translation i n Mitochondria

W. E. BARNETT The Ribosomal Cistrons of Higher Organisms

M. BIRNSTIEL N"-A’-lsopentenyladenosine: Biosynthesis, Metabolism, and ItsSignificance to theStructure of tRNA

R. H. HALL DNA Ligases

J. HURWITZ,M. GEFTER,AND A. BECKER X-Ray Diffraction Studies of Nucleic Acids

R. LANGRIDGEAND M. SUNDARALINGAM Induced Activation of Amino Acid-Activating Enzymes by Amino Acids and tRNA

A. H. MEHLER Natural and Artificial Regulation o f Purine Salvage

A. W. MURRAY,DAPHNEC. ELLIOTT, AND M. R. ATKINSON Synthetic Nucleotidopeptides-Models for Possible linkers in Nucleic Acids and Nucleotide Transferring Enzymes

Z. SHABAROVA Modifications of tRNA and Protein Synthesis

N. SUEOKA Species Specificity of Protein Synthesis

0. CIFERRIAND B. PARISI

xxiii


The Roleof Conformation in Chemical Mutagenesis B. SINGER AND H. FRAENKEL-CONRAT Department of Molecular Biology and Virus Laboratory and Space Sciences Laboratory, University of California., Berkeley, California

I. Introduction . . . . . . . . . . . . 11. Chemistry of Action of Mutagens . . . . . . . . A. Nitrous Acid . . . . . . . . . . . . B. Hydroxylamine . . . . . . . . . . . C. Alkylating Agents . . . . . . . . . D. Nitrosoguanidine . . . . . . . . . . . E. Photochemistry . . . . . . . . . . . F. Brominating Agents . . . . . . . . . . G. Other Reagents . . . . . . . . . . . 111. The Reactivity and Mutability of Polynucleotides . . . . IV. The Reactivity and Mutability of Double-Stranded Polynucleotides . V. The Reactivity and Mutability of Protein-Encased Nucleic Acids . VI. Conclusions . . . . . . . . . . . . . Addendum . . . . . . . . . References . . . . . . . . . . . . .

1

4 4 5 8 9 10 12

16 16 17 21 24 26 27

1. Introduction Recent reviews of the field of niutagenesis have confined themselves to selected aspects of this very extensive area of research (1-6). The limitations of the present article, derived from our chemical backgrounds, are to concern ourselves only with definitive in vitro chemical point modifications of nucleic acids, and the mutations detectable after such treatments. It then appears that the entire field, onIy 10 years old,' shrinks to a manageable volume of published data, particularly if one omits from consideration those papers in which the presumed reactions are derived from dogma, or from genetic evidence unsupported by chemical data. It is now becoming evident that reactions observed with one type of material (mononucleotide, singlestranded nucleic acid, etc.) do not necessarily occur, or represent the mutagenic event, in another type of material (double-stranded nucleic 1 We will not discuss the early isolated observations of mutants r e s d t h g from chemical treatments, such as the mustard gas studies of Auerbach etal., as reviewed recently by Auerbach [Science 106,243(1947); 168,1141(1967)l.

1

2

B. SINGER AND H. FRAENKEL-CONRAT

acid, virus, etc.). Our experimental approach to the problem of chemical niutagenesis has impressed on us the importance of the conformation and milieu of a nucleic acid for two of its not necessarily related properties, namely its chemical reactivity and its mutability. It is these aspects of the field we particularly emphasize in this review. Before entering into a discussion of specific reagents, a few general comments on mutagenesis are in order. According to our present concept, all biologically active nucleic acids are templates, be it for replication or for transcription (DNA) or translation (RNA).Thus each change of a nucleotide in a part of the molecule carrying information represents a potentially mutagenic event. In such terms, mutagenesis can be studied without reference to biological materials. If poly C can be transformed chemically to poly U, and if in the process it loses its ability to bind poly G and instead binds poly A, then mutational events have been observed. Similarly, the ability of polynucleotides to act as templates for RNA polymerase has been used to detect chemical changes-the replacement of a C residue in poly C by a U (or T) leading to the incorporation of some A (from ATP), besides the predominant G (from GTP). Finally, the ability of polynucleotides to act as messengers and to direct the formation of polypeptides can be used, and changes in the nucleotide composition resulting from chemical mutagenesis can be deduced from the incorporation of a new amino acid. Since the base-pairing (coding) properties of each nucleotide are largely determined by the tautomeric state of the atoms bound to the 1 and 6 positions of the purines and the 3 and 4 positions of the pyrimidines2 (-N=C-XH t--) -NH-C=X), reactions that do not directly affect this

I

I

part of the molecule but that favor the unusual tautomeric form must also be regarded as mutagenic in the statistical sense, their effectiveness depending on the extent to which they favor the unusual tautomeric statea3 Other chemical modifications of the bases render them unable topair 2 Two different numbering systems have been used forthe pyrimidines, even by the 6ame authors and occasionally even in the same paper. We have translated all data into the official IUPAC-Chemical Abstracts-Ring Index system, even though this has the disadvantage, compared to the original Fischer system, that the corresponding positions on purines and pyrimidines carry different numbers. Thus the N-1 and C-6 positions of the purines correspond to the N-3and C-4 positions on the pyrimidines.

8

See also the article by Pullman and Pullman in this volume.

ROLE O F CONFORMATION

I N CHEMICAL

MUTAGENESIS

3

with any other natural base, and these reactions can be defined as inactivating, broadening the meaning of this word from the customary biological to the chemical level, as we have already done with the term “mutagenic .” Finally there may exist harmless chemical changes in the bases, changes that neither interfere with base pairing nor alter its nature in either the absolute orthe statistical sense. The more classical manner of defining mutation is naturally derived from the action of mutagens on biologically active nucleic acids and the appearance of variants detected by biological or biochemical methods. The latter techniques have supplied by far the most data now at hand, but the use of simpler nucleic acids and of the isolated functions discussed above may become the more fruitful method for the study of mutagenesis. Whenever in the present discussion we use the term mutational or inactivating event we may be presumed to be thinking either in these molecular terms, or in those of the classical biologist. According to presentIy prevailing concepts, all the base triplets in information-carrying nucleic acids have phenotypic counterparts in amino acid sequences. Thus the ideal method for detecting all nucleotide replacements resulting in mutations, barring complete nucleotide sequence analysis, is to isolate all proteins coded by a nucleic acid, and determine their amino acid sequences. This has not yet been achieved for even the simpIest RNA-virus systems. However, the isolation of one gene product, namely the virus coat protein, is often easy, and the analysis for amino acid replacements in this protein has been a useful tool for the characterization of mutants in the TMV system. Unfortunately, considerable numbers of mutants must be isolated and studied before a sufficient number of amino acid exchanges is found to validate or establish the probable mutagenic event. This has been achieved only for nitrous acid deamination where by far the greatest number of amino acid exchanges support the established mutational mechanisms (C -+. U, A -+ hypoxanthine G G) (see Fig. I). The same types of exchanges have been detected after nitrosoguanidine treatment of TMV-RNA. Mutants produced by NHzOH and NH20CH3have, unaccountably, shown hardly any exchanges in the viral coat protein. The other reactions discussed in this essay present a random pattern of exchanges, suggesting the replacement of any base by any other. Since most of these reagents are rather ineffectual mutagens, the attribution of a given mutational event to the chemical employed is obviously quite dubious, and only when amino acid exchange data are available in statistically significant numbers for a given mutagen can one deduce the nature ofthe reaction from the analytical results. Such numbers of exchanges have notyet been obtained with any mutagen except nitrous acid. Such data as are available have recently been summarized (7).

4


One aspect of mutagenesis that is generally not appreciated is the fact that not the absolute frequency of mutational events, but rather the ratio of mutational to inactivating events represents the true measure of mutagenesis. It must further be stressed that the observed chemical reactions need not necessarily be responsible for either the inactivating or the mutagenic events. Thus an average nucleic acid molecule may be modified in 100residues in a manner that has no biological consequence, and at the same time undergo one mutational and from 1 to 10 inactivating reactions. It would be then the level of the latter side reaction that would determine how powerful the reagent was as a mutagen. In discussing specific mutagens, we will point out examples in which such situations may exist. It should also be noted that mutagenic events are potentially coupled with lethality, since many mutations must be phenotypically lethal by producing inactive orincomplete enzymes. Thus all claims of having detected a noninactivating mutagen should be a priori discounted. Furthermore, the use of inactivation as a supposed measure of the mutagenicity of a reagent in a system where such mutagenicity has not been clearly demonstrated is obviously unjustified.

II. Chemistry of Action of Mutagens A. Nitrous Acid The dea.minating action of HNOt on adenine, guanine, and cytosine, under conditions where polynucleotides would not be degraded, was first studied by Schuster and Schramm (8). It appears that in TMV-RNA the three amino bases are deaminated at similar rates. Adenine and cytosine react normally, yielding only hypoxanthine and uracil, respectively. But guanine yields xanthine in poor yield (9), an additional unexpected minor product being 2-nitrohypoxanthine (10, ll), the others remaining unidentified. The reaction rate is a function of the concentration of undissociated HN02, and thus is greatly dependent on the pH of the solution. The discovery of the mutagenic action of HNOt by Gierer and Mundry (12) represents the beginning of our understanding of the nature of chemical mutagenesis. That deamination of cytosine t ouracil is a mutagenic event is selfevident (Fig. 1). The deamination of adenine to hypoxanthine is also mutag-enic, since hypoxanthine resembles guanine rather than adenine in its substituents at N-1 and C-6 and thus in its base-pairing properties (Fig. 1).The deamination of guanine does not affect these critical positions and thus would not be expected to be mutagenic. The data of Schuster and Vielmetter ( I S )suggest that the demination of guanine is actually inactivating. The findings that poly X is unable to hind polyC (I,$), that

ROLE

O F CONFORMATION

Inactivating events

Mutagenicevents

-

5

I N CIIEMICAI, MUTAGENESIS

HNO,

alsocrosslinking, depurination HNH

0

FIG.1. Reactions of nucleatides (anddeoxynucleotides) withnitrous acid.

xanthine (X) cannot replace guanine in reactions catalyzed by DNA polymerase (15), and that the deamination of guanine-containing polynucleotides renders them inactive as templates (16), all suggest trhat the presence of a carbonyl group in position 2 actually interferes with the binding of cytosine and therefore represents an inactivating event.

6. Hydroxylamine Hydroxylamine and its derivatives appear to react only with the pyrimidines. These reactions have been studied quite intensively. It appears very probable that only the reaction with cytosine is mutagenic. However, the nature of the chemical reaction that accounts for the mutagenicity of hydroxylamine is still somewhat in dispute. The reaction proceeds optimally and rapidly about pH 6.As shown by Fig. 2,NHzOH Probable mutagenic event

Probably inactivatine events

FIG.2.Reactions of nucleatides (anddeoxynucleotides) withhydroxylamine and methoxyamine.

6

B. SINGER AND

H. FRAENKEL-CONRAT

tends to add t.0 the 5,6 double bond of cytosine2 (I), and to replace its 18).The first reaction path (reaction a and b) is amino group (11,111) (17, the faster, by a factor of about 4 (19),and reaction a had been thought until recently to be the obligatory primary reaction, as well as the mutagenic event on the basis of studies of the ability of hydroxylaminetreated poly C t o incorporate ATP along with the GTP into mixed polymers (20-2%)>. However, the recent finding of two laboratories that reaction c occurs directly and concurrently with reactions a and b (23, 24) has been confirmed by Brown and Hewlins, who determined the relative rates as about 1:4.These authors have also recently demonstrated that the preferred tautomeric form, by a factor of 10,of the hydroxyaniinocytosine is that corresponding formally to uracil oxime (111) (25). They also indicate that experimental evidence hasbeen obtained showing that this reaction product of NH20H with cytosine has the hydrogen bonding properties of uracil (25). In contrast, Janion and Shugar (26) report that neither polymers of N-4-hydroxyaminocytidine (111)nor of the dihydroxyamino compound (11) bind poly A, poly U, poly I, or poly C. However, they stress the importance of testing the activity of copolymers of cytidylic with a little N-4-hydroxyaminocytidylate as templates and, we might add, also as messengers. These authors also point out that the formation of I11 predvminates in acid solution. The conclusion, that reaction c represents the mutagenic event, is supported by the fact that in 5-substituted cytosines, in which reaction a does not occur under the usual reaction conditions (27), NHzOH reacts readily according to reaction c, and is an excellent mutagen in T-even phages (28). This reaction had been overlooked in earlier studies in which it was concluded that 5-methylcytosine does not react with NHzOH because no decrease in the typical absorbancy of the pyrimidine waa detected. While Phillips and Brown (6)are willing to accept reaction c as the mutagenic event for the T-even phages, they maintain their belief that, for nucleic acids containing unsubstituted cytosine, reaction a represents the mutagenic event. This preference is based in part on the results obtained in collaboration with Grossman (21, 29) on the effects of hydroxylamine and [W]NH20CH3on poly C. These studies showed a correlation between the extent of reaction a and the template-mutational data. However, the high and variable background of I4C-binding weakens the quantitative reliability of these data. Furthermore, since in poly C, as contrasted to monomeric cytosine compounds, reaction b isslow and product I, the “mutated” species, is believed to accumulate, one would expect this reaction to be reversible in both the chemical and biological sense, and yet

ROLE OF CONFORMATION IN CFIEMICAI> RIUTAGEKESIS

7

neither Phillips and co-workers (21) nor Wilson and Caicuts (22) were able to decrease the relative incorporation of ATP and GTP after acid treatment designed to reverse reaction a. Although Brown and Hewlins (19, ,L6)have obtained evidence that reduction of the 5,6double bond of cytosine greatly decreases the predominance of the amino form in the tautomeric equilibrium, there is no evidence nor reason to suppose that compound I would be mainly in the imino form, as would be required to account for the high mutagenic efficiency of NHzOH on the basis of reaction a. The availability of the recent data and the advantages of a unified hypothesis for all cases of hydroxylamine mutagenicity make us confident that reaction c leading to compound 111represents the mutational event. We have favored this hypothesis in discussing NHzOH mutagenesis at scientificmeetings since 1966, and we are happy to observe recent accumulation of experimental support coming from most laboratories engaged in the study of this r e a ~ t i o n We . ~ believe that our presentation of the mutagenic and inactivating events resulting from NHzOH treatment of RNA, as given in Fig. 2,has the support also of Kochetkov and his collaborators (24) (see page 403 of this volume). The action of NHzOH on uracil is most pronounced a t pH 9-10, although it occurs also at pH 6 (17,31). It results in an opening of the ring (Fig. 2) and must thus be presumed to be inactivating rather than mutagenic. There is no evidence that the reaction with uracil represents a mutagenic event. Poly U loses template activity for ADP upon NH20H Thus, the mutants treatment, but gains no new template specificities (21). found by us (32) when TMV-RN-4 was treated with NHzOH even at pH 9 are probably due to the cytosine reaction. Methoxyamine (HzNOCH3) reacts considerably more slowly with cytosine than does NHzOH (29), but it does not react with uracil (33). The mechanism of its action appears to be the same as that of NHZOH. N-Methylhydroxylamine (CH3NHOH) resembles NH20H in its reactivity (6) and its inactivating activity, but gave mutants only in one case and under exceptional conditions (see further discussion in Section IV) (34). Preliminary data indicate that this agent is not mutagenic on TMV-RNA (32). Hydroxylamine, as well as N-methylhydroxylamine, but to a much lesser extent methoxyamine, decomposes in aqueous solution and yields radicals that cause inactivation of biologically active nucleic acids (34,SS). These oxygen-dependent reactions become particularly significant a t low concentrations of NHZOH. They can be suppressed by addition of radical trappers (34, 36), but are best avoided by using fresh NHzOH solutions at

8


high concentratioris and low temperature for niinirnal time periods. It appears that these side reactions cause mainly iiinctivating events (34).

C. Alkylating Agents Many reagents can introduce alkyl groups into nucleic acids in neutral aqueous solution. The extensive literature is reviewed comprehensively by Lawley in this series (4). The primary and often the only noticeable event upon treatment of nucleic acids near neutrality with dimethyl and diethyl sulfate, methyl and ethyl methane sulfonate, epoxides, mustard gas, etc., is the alkylation of guanine at N-7.Secondary sites of alkylation are adenine at N-1, N-3,and N-7 (the relative amounts of which differ for different types of nucleic acids and reagents, and are discussed in later Presumedmutagenic event

Probably inactivatlng events

J

no

R

Eventswithunknown effects: 3-alkyl-A ’I-alkyl-A Depurinatfon

FIG. 3. Reactions ofnucleotides (anddeoxynucleotides) withalkylrtting agents.

sections) and cytosine at N-3.Diazomethane a t the ether-water interphase inethylates preferentially the N-3of uracil and the N-1 of guanine, but in the squeous phase the saine products may result as with dimethyl $8).Whether diaaomethane or any of the allrylating agents sulfate (37, causes appreciable phosphate esterification remains uncertain, but doubtful. Alkylation might be expected to be, a t best, a low-level mutagen when causing substitution a t the 3 or 7 position of the purines, and to inactivate when involving their 1 position or the 3 position of the pyrimidines (Fig. 3). In studies concerning the effects of alkylation (with dimethyl sulfate, diethyl sulfate, and mustard gas) on the messenger activity of poly A

ROLE O F CONFORMATION

I N CHEMICAL

MUTAGEXESIS

9

(39-41) (see Addendum), the only effect noted was inactivation. The fact that poly A containing 14% of N-l-methyladenine can still bind one, but not two, strands of poly U (42)may supply a hint as to a, mechanism of inactivation of nucleic acids by alkylation. I n the case of TMV-RNA%,methylation was poorly mutagenic and all other substitutions tested (--CzF15,-CH2C€I,OH, -CH&OO-, -CH,CH,SCH&H?,CI), were nonmutagenic (43). Working with Newcastle disease virus, Thiry (&) reported dimethyl sulfate as well as ethyl ethane sulfonate to be mutagenic. When acting on T-even phsges, ethylating agents generally proved more mutagenic than methylating 4G), a fact further discussed in Section IV.With TMV and agents (45, TMV-RNA diethyl sulfate was found nonmutagenic but ethyl methane sulfonate was mutagenic (Table I), a finding that requires further study (32).

D.

Nitrosogomidine

The action of nitrosoguanidine on RNA has onIy recently been elucidated. Data primarily derived from the template activity of polynucleotides who suggested have been tentatively interpreted by Chandra et a2. (47), that alkylation occurs and that the order of susceptibility is G > A > C. Simultaneous and independent work in our laboratory has clearly demonstrated that 7-methylguanine is the main reaction product, and that much less of a methylated adenine \\;as also formed. The latter showed a white fluorescence as the nucleotide, but the site of the substitution has not yet been definitively identified ( 4 8 ,49)(see Addendum). Only under special conditions, discussed below, does nitrosoguanidine react with 48). 1-Methyladenine has since cytosine, methylating the 3 position (32, also been detected as B product of this reaction. The fact that alkylation of guanine by nitrosoguanidine is appreciable only in polynueleotides is a strong indication that its mechanism of action is not the same as that of typical alkylating agents, which readily alkylate mononucleotides. The relative levels of methylation of guanine and adenine also differed for these reagents (see Addendum) (48). The action of nitrosoguanidine on TMV-RNA caused a similarly low ratio of mutational to lethal events (@) as did dimethyl sulfate (32) (Table I), quite in contrast to the effectiveness of nitrosoguanidine when acting on metabolizing On the other hand, the finding that the latter was bacteria or cells (5U-52). a very good mutagen when acting on intact TMV rather than on its RNA represented a major stimulus to our interest in the topic of conformation dependence ofmutagenesis, and this aspect of the action of nitrosogunnidine is discussed in more detail in Sectioii V.

10

B. SINGER AND

H. FRAENKEL-CONRAT

TABLE I RELATIVEMUTAQENICITY OF MUTAGENS ACTINGON TMV-RNA IN VARIOUSCONFIQURATIONS (3.8) Mutagenicitya -~~~

Reagent Nitrous acid (pH 4.5) Hydroxylamine (pH 6) Methoxyamine (pH 6) N-Methyl hydroxylamine Dimethyl sulfate Diethyl suIfate Methyl methane sulfonate Ethyl methane sulfonate Ethyl ethane sulfonate Nitrosoguanidine Bromine Thiopyronin light Ultraviolet light

+

RNA in water

RNA in formamide

34 (8) 11 (8) 12 (8)

1 (2) 3.5 (7) 1 (5) 5 (6) 4 . 5(3) 2.1(5) 2 (6)

7 (3) 5 (3) 2 (16)

RNA in virus 110(5) 1 . 3(2) 1 . 6(1)

3 (4)

2 . 5(6) 1 (5)

4 (6) 7 (6) 9 (3)

8 (3) 3.6(3) 60 (15) 1 (2)

3 (2)

The following test was used: After reaction with the mutagen, at neutrality unless stated otherwise, the RNA was reisolated, or in the case of virus, separated from the protein, and tested for residual infectivity (with or without reconstitution with TMV protein) on the quantitative local lesion host, N . tJmcum var. Xanthi nc. Both the mutated sample and a mock-treated control were applied at equal concentration of infective particles (0.1-1 p g / d for fully active reconstituted TMV) to N . sybestris on which only certain TMV mutant strains produce local lesions (in numbers approximately proportional to concentration). The ratio of the local lesions found with the mutated RNA to the control (spontaneous or endogenous mutants) is termed “mutagenicity.” Such data have relative quantitative significance, but obviously do not measure all mutational events. The figures in parentheses are the number of samples, each ofwhich has been assayed 3 or more times on 9 leaves of N . sylvestris.

E.

Photochemistry3

Many dyes, particularly flavines, are effective mutagens for living bacteria or their phages, but these effects, largely deletions and insertions, are not within the scope of this review, since their mode of action is not clear, beyond the concept that many of the flavines may be readily intercalated between the bases of double-stranded DNA (53). Thiopyronin, methylene blue, and to a much lesser extent the flavines, serveas photocatalysts and sensitize nucleic acids toward visible light (54-68). These photoreactions are specific for guanine residues, causing the 68422).Illumination of various polynucleotides imidazole ring to open (65, leads to losses in amino acid incorporating activity only in the case of

ROLE OF CONFORMATION

IN CHEMICAL

RlUTAGENESIS

11

polymers containing guanine, but not specifically for amino acids requiring guanine in their codons (61, 62). However, these techniques would not necessarily reveal rare mutational changes in guanine residues. No degradation products are released from RNA even after extensive treatment (57). Since this reaction is somewhat mutagenic for TMV-RNA (57) (Table I) one may assume that the tautomeric state at N-1 and C-6 can be affected by this reaction, unless another, not yet detected, reaction actualIy represents the mutagenic event. It should be noted that the number of guanine residues lost exceeded the number of inactivating events in these experiments. The action of light in the presence of iron (Fe3+) also causes rapid inactivation and the appearance of some mutants (Sa, 63). I n chemical terms, no clear specificity of action has been detected, although the pyrimidines are more affected than the purines. Release of small-molecular material, partly in the form of bases, has been noted (63). It is possible that the mutants in this case should be attributed to base deletion although another possibility is considered below. The effects of Fe3f plus light could be greatly augmented by low levels of HzOz and are probably due to the formation of free radicals. These reactions do not seem to be of particular interest, because of their nonspecificity and the high ratio of lethal to mutagenic events. The action of ultraviolet light on nucleic acids, viruses, and microorganisms has been studied very intensively and has been reviewed in detail by J. K. Setlow (64). (See also the article by R. Setlow in Volume 8 of this series.) As far as its chemistry is concerned, the hydration of the 5,6double bond of the pyrimidines is one predominant reaction (6548), and the dimerization of neighboring pyrimidines another (69,70). Grossman (?I,72)studied the effect of UV irradiation on poly U and that on poly C with particular reference to the coding and Ono et al.(73) template activities of these polymers, respectively. As a first consequence of irradiation, the polymers lose much of their coding as we11 as their template activities, i.e. , they are inactivated. Surprisingly, irradiated poly U acquired a limited capability to stimulate the incorporation of serine, as that of phenylalanine declined. Conditions favoring the reversal of the reaction, dehydration of the 5,6 double bond (pH 8.3,85"C1 15 min) partly reversed both phenomena. No serine incorporation was observed in similar experiments by Logan and Whitmore [quoted by Johns et al.(SS)], and Setlow (64)suggests that the results might be due to the action of undesirable enzymes in the incorporation system. When the template activity of UV-irradiated poly C was tested, it appeared that the decrease in polymerization of GTP was followed, a t higher UV doses, by increased incorporation of ATP. This new capability was reported to be completely abolished by heating. The latter results

12


would seem to givestrong support to the authors’ preferred interpretation of the data, namely that the hydration of the 5,6 double bond of cytosine represents the mutagenic event, possibly by causing the tautomeric shift at N-3,C-4. However, if hydration of the double bond caused the tautonieric shift of amino to imino in cytosine, one could hardly expect the same reaction to cause the opposite shift, keto to enol, in uracil to account for the serine coding activity detected by Grossman in irradiated poly U. Deamination has definitely been shown to occur upon UV irradiation of cytosine compounds and would be regarded as the most likely explanation for mutations resulting from such treatment (68, 74). On0 et al.(73)found no evidence for the formation of uracil under their conditions of inactivation of template activity, but it is not clear whether the formation of uracil had been eliminated a t the level of irradiation required to obtain “mutagenesis” (ATP incorporation). The deamination of cytosine hydrate or of cytosine dimers, which occurs very readily, was definitely not eliminated, and it would seem preferable to attribute the mutagenic events caused by UV to the known ready deamination of these photoproducts, were it not for the remarkable claim of Ono et a2. (73)that the mutation of poly C is completely reversed by dehydrating conditions. Since there is no evidence that the particular heated samples that had lost their ability to incorporate adenylate continued to act as template as far as guanylate is concerned, one wonders whether they could possibly have been overheated (70 , 15 min), and inactivated by extensive dimer formation, or degraded to a point where they lost all detectable activity. This question relates to that previously discussed, concerning the mutagenic event after NHzOH treatment. Since both UV and KHzOH readily came the saturation of the 5,6double bond, one would expect them to be equally good mutagens if this reaction were the mutagenic event. Since NHzOH is an excellent mutagen (IS, 28, ?5), whereas UV is a quite poor one in phage (64) and TMV-RNA (32) and has been found not to mutate DNA ( 76, (Tables 77) I and II), this consideration seems to supply additional support for the belief that reactions at (2-4 that occur readily with NHzOH (-NH? -+=NOH) and only as occasional side reactions after irradiation (-NH2 =O), represent the actual mutagenic events. An interesting combination of reactions was recently reported by Small and Gordon (SO), in that the UV product of cytidine, 5,6-dihydro-6-hydroxyoxycytidine1 reacts much more rapidly with hydroxylamine than cytidine, to yield the N-4-hydroxylamino derivative. KO tests of the mutagenesis of this reaction have as yet, been reported. --j

F. Brominating Agents Bromi~iation of TMV-RKA

was fourid to result in mutants (78). According to Rrammer ($9)the most clear-cut results were obtained with

ROI'E O F CONFORMATION I N CHENrCAL

MUTAGENESIS

13

bromine, which a t pH 7 reacts r:q)idly md, in limitiiig amounts, almost exclusively with cytosine, while around p1-i 9, it affects mainly guanine. The first reaction is nil addition of BrOII to the 5,6double bond (Fig. 4). This product loses water at acid pH's, yielding, 5-bromocytosine which in turn can be further reacted to yield 5,.5-dibromo-6-hydroxy-hydrocytosine. In alkali, 5-bromo-0-hydroxy-hydrocytosine may lose HBr, forming 5-hydroxycytosine.

?

,

R

FIG.4. Reaction of cytosiiie with 1)romirie-water at pH 7.

A current comparative study of the mutagenicity of various agents on TMV-RNA indicates that broniinatioti is more mutagenic than all other reactions with the exception of HNOz and NHzOH (32)(see Table I).It remains to be established whetJherthis is due to tautomeric shift or ionization resulting from reaction a, b, or d, or t o other causes. Studies concerning the mutational specificity of these reactions are in progress. An extensive literature exists on the incorporation of Fi-deoxybromouridine into DNA and of 5-fluorouracil into RNA (1-,5). The substitution of thymidine by bromodeoxyuridine leads to many mutants while the sub-

TABLE 11

MUTAGENICITYAND Mutagen Nitrous acid

Hydroxylamine PH 6

ACTION

h f O D E OF CHEMICAL

TXV-RIiA

Denaturedb transforming DNA

Alkylation

TMV

Very strong (19) Deaminat ion G.A > C

Strong (88-91) Deamination G>>C > A cross-liking

Deamination C >A

Strong (76) C 4 substitution C-5.6addition

Very strong

Negative (SI)

Strong (52) C-4 substitution C-5,6addition Very weak (43,S.S) (metbylation) G-7 > A-1 > (2-3.A-7 > A-3

Verystrong (13)

3

,;:;~itu;m Methoxyamine

OF V A R t O U S

Strong (34, 96, 96) C-4substitution C 5 , 6addition

c

1

-

Negative (88)

MUTAGENS ACTINGON 813, +X174 Phage

VARIOUS NUCLFJC ACIDS IN VARIOUS STATESa

T2,T4 Phage

Positive (113,126) Strong (ffa,f23) [G,A > Cl Deamination G H M C >>A

>>

positive ($6, 116) C-4 substitution G5,6addition

c

1

Positive ($6) C-4 substitution C-5.6 addition

1

c

Very weak ($1) Weak (116) (methylation) (ethylation) G [G-7> A-1)

Vary strong (IS,Z8) C-4 substitution

Bacteria Positive (ISO)

(46, 46, 116)

(ethylation) G-7 > A-3

Denatured poly d(A-T) (template) positive (81), deamination. croas-linking Polynuoleotides (messenger) positive (16)

-

PoiyC (template) positive ($1, 32) C-3 substitution C-5,6addition

-

Poly C (template) positive (29) C-4substitution C-5,6addition

[C-4 substitutionl Strong (114) Strong

Polynucleotida

Positive (121)

Poly A (messenger) inactivated (39,41) (see Addendum) Poly U, G (messenger) inactivated (40) G-7 Poly C (template) positive (see Addendum)

Nitrosoguanidine Very weak (48,32) Positive (96) G-7 > A-1 > C-3, A-7 > A-3 Dyes and light

Weak ( 6 7 )

-

Strong (@.as) G-7,C-3 > A

-

Negative (32)

-

Negative (111)

Weak (218)

-

Very strong (60-68)

Polynucleotides (messenger) inactivated except poly A mutated (47) IG > A,Cl

Weak (116, 117)

Positive Polynucleotidea (messenger) (122-134) inactivated (67, 61,62,127)

Weak (64, 110)

Weak (64) Poly U (messenger) positive (71, 78), negative (68). hydration. dimeriaation Poly C (template) positive (78) hydration [deaminationl

G

G Ultraviolet

Very wcak (SI)

c.u

Negative (76. 77)

a The adjectives to denote mutagenic efficiency are based on our attempt to evaluate quite heterogeneous data [except for TMV-RNA4and TMV. where comparative data by one method are available (see Table I)]. The references refer only to the use of each reagent as a mutagen. The chemical reactions given are the major chemical events observed, a8 reported by various investigators, and not necessarily the mutational events. When no chcrnical data are available, we have used our judgment in giving the c1 probable reactions in brackets. 01 b The data quoted in this column refer to DNA that is more or less denatured by the use of low pH (4.2) or high temperature (60"-75OC). For reasons discussed in the text, no data on the modification of truly native DNA seem to exist in the literature, since the reaction conditions are generally not favorable for the maintenance of complete double-strandedness, except for the alkylation and UV irradiation of transforming DNA at neutrality and a t ambient temperature (91). Under these conditions, methylation is considerably more mutagenic than UV irradiation.

16

B. SINGER AND 13. FRAENKEL-CONRAT

stitution of much of the uracil of viral nucleic acids by fluorouracil produces very doubtful mutagenic results. Since these effects axe produced in vivo, with the analog usually being present during the intracellular replication of the DNA or RNA, these studies do not fall within the domain of in vitro mutagenesis to which we are confining this discussion. On the other hand, the study of the template activity of a poly d(A-BrU) cannot here be disregarded. Trautner etal.(80) found that DNA polymerase copying such a polymer caused the incorporation of a sniall but definite amount of deoxyguanosine. Howcver, nearest neighbor analysis did not show the expected scquences, with the deoxyguanosine being regularly adjacent to broinodeoxyuridine residues, since it was fourid next t o guanosine and adenosine in 42 arid 17y0of the cases. The interpretation of the nature of mutants resulting from DNL4 containing bromodeoxyuridine is similarly complex. It seems that a high coritent of the latter favors nonspecific errors in replication, and the same may be the case, with much lesser frequency, for fluorouracil in RNA. It appears reasonable that halogenation of cytosine gives it more of a uracil-like character, without the reverse (bromodeoxyuridine approximating cytjdine) being true or expected.

G. OtherReagents A considerable number of reagents used as mutagens have not yet been discussed. The chemical events for some of these have been elucidated. All of them seem to be less effective mutagens in vitro than those discussed in detail in the preceding sections. They all may be surmised to act mutagenically either by favoring the rare tautomeric form of the bases or by effectively deleting a base. Since none of these reagents have been found to show unexpected conformation dependencies, we list only some of the better known ones and their probable mode of action, without discussing them in detail. Most of these reactions have been discussed in other reviews (1-5). These agents are: peroxides and peracids (giving purine N-oxides) ;weak acid and high temperature (depurination, C -+ U) ; X-rays (pyrimidines); hydraeine arid other amines (cytosine); nitroso compounds (alkylation?); formaldehyde, manganese and other metal ions.

111. The Reactivity and Mutability of Polynucleotides The preceding discussion of potentially mutagenic reactions is in part based on model experiments performed with the four nucleotides, nucleosides or bases. Obviously the resct,ivities of tdie free bases, lnckjngthe pentose substitution at N-1 or N-9, respectively, can differ from those of the nucleosides and nucleotides, but these iieed riot coiiccrn us here.

ROLE

OF CONFORMATION

I N CIIEMICAL MUTAGENESIS

17

One clear exception to the usefulness of nucleosides and nucleotides as models for nucleic acids was our finding that nitrosoguanidine does not react to a detectable extent with guanosine, guanylic acid, GpU, or CpG, We will but does react readily with guanine in polymer linkage (48). therefore now consider the reactivity of the polynucleotides with various reagents. Polynucleotides are both larger and different in character from their monomers. Their long-chain nature with multiple diester bonds introduces chain breakage as a potential side reaction, which can complicate the interpretation of inactivation data. But more important is the fact, which until recently has been often overlooked, that the bases in all oligoand polynucleotides tend to stack themselves and interact with their neighbors by pi electron interaction.3 Thus all single-stranded homo- or heteropolynucleotides have more or less “structure.” The result is that most modification reactions are slower in the polymer than in the monomer. For instance, NHzOH causes no detectable loss in UV absorbance of poly C in several hours (20). Even though this was shown to be in part due to the fact that the initial loss of absorbance is compensated by the hyperchromic effect resulting from “denaturation” of the polymer by the reagent, the determination of the amount of product 111 (Fig. 2)after acid treatment confirmed the fact that the reaction rate is actually lower in the polymer. Other instances of this were reported by Kotaka and Baldwin (81) with HNOz and by Pochon and Michelson (8.2) for various alkylating agents. Quite in contrast to the generally slower response of polynucleotides, the action of nitrosoguanidine leading to methylation of guanosine was The fact that the initial observed, as stated, only in polynucleotides (48). rate of inactivation by nitrosoguanidine shows a maximum a t about 20°C and sharply declines at 37 ,as it does in dispersing solvents such as formamide and particularly dimethyl formamide, suggests that a binding of the reagent between the stacked bases is a prerequirement for the reaction (49). The question whether base-stacking favors or affects other reactions of nucleic acids is under continuing study. The observation that alkylation of adenosine and adenylic acid may yield products substituted at 1, 3,and 7,but that in poly A only 1-methyladenine has been reported to be formed in appreciable quantities, indicates that this reaction is also affected by base stacking (see Addendum).

IV. The Reactivity and Mutability of Double-Stranded Polynucleotides The most significant and singular structural feature in the field of nucleic acids is the double-strandedness resulting from the interaction of

18

B. SINGER AND

H . FRAENKEL-CONRAT

the complementary pairs of purine and pyrimidine bases (A with T or U, G with C or hydroxymethyl-C). The multiple bonds resulting from the interaction of two complementary strands, as it occurs in typical DNA and some viral RNA's, gives this structure considerable stability. The same type of interaction occurs in random fashion over short complementary segments of single-stranded RNA or DNA but it then has considerably less stability. As expected, the interaction of the substituents on the 1 and 6 positions of the purines with those on the 3 and 4 positions of the pyrimidines interferes markedly with all chemical reactions involving these atoms. This is most clearly demonstrated by a reaction we have not previously discussed because of its dubious mutagenic significance, i .e., that with formaldehyde. This reagent adds readily to all amino groups of RNA but fails to react with double-stranded DNA (83, 84) unless very high concentrations are employed (85).Thus reaction with formaldehyde has frequently been used as a means of ascertaining the extent of double-strandedness of polynucleotides, as well as for the purpose of preventing renaturation of complementary chains (86, 87). The most effective mutagen for RNA, HN02, has proved less useful for DNA because of the obvious fact that the amino groups of double-stranded molecules are not available to the reagent. However, HNOz is necessarily used in acid solution, i.e., under conditions where DNA begins to lose its structure. Thus the published results of nitrous acid treatment have been somewhat ambiguous. It seems that those who found HNOz not to cause many mutants in transforming DNA (88, 89) used a slightly higher initial pH and lower buffering capacity than customary, and noted that the reaction mixture became less acidic. On the other hand, HNOz was found to be highly mutagenic when a lower pH of 4.25 was maintained; these are conditions where the reaction may be demonstrated analytically (90, 91). I n relative reactivity, guanine is much more extensively deaminated than cytosine, and adenine is most resistant under conditions of incipient acid denaturation (90). Similar results were obtained by Schuster and Vielmetter (13) with calf thymus DNA. They are also in line with the finding of Kotaka and Baldwin (81) that poly d(A-T) could not be noticeably deaminated at pH 4.25 until the temperature was raised to over 60O"C.Free guanosine has recently been reported (11 )to react faster with HNO2 then does adenosine, while free cytidine reacts more slowly (A.M. Michelson, private communication). In contrast, the relative rates observed with transforming and phage DNA were G >> C >> A. This suggests that the third hydrogen bond of the G . C pair (Bainino .2-carbonyl) is much the most sensitive toward acid denaturation, followed by the typical G . C bonds, and that the A ' T linkage is definitely the most stable under these conditions.

ROLE O F CONFORMATION IN CHEMICAL MUTAGENESIS

19

However, it should again be stressed that native DNA is quite resistant to nitrous acid and that side reactions, such as depurination (81), chain breakage, and cross-linking, as first pointed out by Geiduschek and coworkers (92,93), probably account for most of the biological effects resuking from nitrous acid treatment of DNA. This was also evident from the study of the nitrous acid reaction on poly d(A-T) (81). The frequently mentioned hydroxylaniine reaction is also greatly affected by double-strandedness. Thus, DNA reacts quite slowly (18). The single-stranded phage DNA 513 reacts much more rapidly than the double-stranded replicative form (9Sa). Hydroxylamine shows a very low level of mutagenesis on transforming DNA, unless conditions are chosen that tend t o eliminate base pairing in the DNA (94,91). Although this can be achieved by low ionic strength or dispersing solvents (ethylene glycol), heat is most frequently used t o render double-stranded DNA susceptible to hydroxylamine mutagenesis. This is an unfortunate choice, since obviously side reactions due to hydroxylamine decomposition, as well as deamination and depurination, may be greatly favored at 7O-8O0C,and may account not only for the observed inactivation but also for part or all of the mutagenesis observed under such conditions. This fact probably accounts for several surprising findings of Freese and Freese (34), which in our eyes have clouded the interpretation of the action of hydroxylamine. One of these findings, singular to heated DNA, is that NHzOH is most effective as a mutagen at pH 4.2(34). The other is that CHsNHOH is mutagenic, actually showing a greater frequency of mutation than does the generally accepted good mutagen, NHZOCHZ (methoxyamine), in these 95). It must be recalled that the latter reagent, lacking experiments (34, the free -OH group, is the only one of this group that does riot decompose to form radicals (35, 96). Since CH3NHOK has recently been found not to be mutagenic, while rapidly inactivating TMV-RNA (32)(Table I), it would appear advisable to omit its effects on DNA a t 70"from considerations of the mechanism of NHzOH mutagenesis. Another striking example for the protecting action of double-strandedness, in this case clearly affecting the "backside" of the pyrimidine molecule (the 5,6double bond) is the much slower rate of ultraviolet hydration of poIy C . poly I, or t,he double-stranded deoxy polymer, as compared with the corresponding single-stranded polymers (97, 98). As previously mentioned, transforming DNA is also not very susceptible to mutation by UV irradiation (76, ’27, 91). Another class of reactions showing a qualitative difference resulting from double-strandedness is alkylation. While the N-7 of guanine is always similarly highly reactive, the most reactive position on adenine in RNA and in poly A is N-1 and only that position is said to react with diniethyl sulfate (41) and methyl methane sulfonate (go), although the very similar

20


chromatographic properties of 1-methyladenine and 7-methyladenine make it difficult to rule out the formation of the latter (4).When increasing amounts of poly U are added to poly A, complementary binding prevents the N-1 methylation of the adenine residues (99). In comparative experiments of the reactivity of different nucleic acids toward dimethyl sulfate by Lawley and Brookes ( I O O ) ,these authors found appreciable amounts of 3-methyladenine and only traces of 1-methyladenine in DNA, whereas in denatured DNA, like RNA, it is the N-1 of adenine and to a much lesser extent the N-3 of cytosine that is methylated. Why the 3 position of adenine is alkylated in DNA but not in poly A . poly U remains to be determined, but it must be noted that dimethyl sulfate was used for the DNA experiments, while methyl methane sulfonate was used with poly A . poly U (see Addendum). As previously mentioned, methylation of adenine at N-1 may be presumed to be an inactivating rather than a mutagenic event, since the part of the bases protected by double-strandedness is obviously also the template-active part during the replication of both single- and doublestranded nucleic acid. One is tempted t o use these facts to explain the observations that alkylation of RNA is weakly mutagenic compared to that of DNA. Thus one may assume that the alkylation of the N-7 of guanine is not inactivating but occasionally mutagenic, presumably by an increase in the unusual enol tautonier; however this mutagenic effect is depressed in RNA by the concurrent inactivatirig alkylations of the positions critical for replication In contrast, the latter on adenine and cytosine (N-1 and N-3,re~pectively).~ reactions do not occur in double-stranded DNA and thus the occasionally mutagenic effect of the guanine methylation becomes the predominant result of alkylation. On the basis of these considerations, one would predict alkylation to be the only effective mutagenic reaction for transforming DNA under conditions where it is completely double-stranded. This deduction has now been experimentally established by Bresler etal.(91), although it semis riot to be supported by the data of Wilhelm and Ludlum (40) , who found no significant incorporation of adenine-requiring amino acids by methylated poly U , G. It appears probable that an occasional miscoding, of the order of 1% of the 7-methylguanine, as postulated, would not be detectable by this method. There remains the question, why methylation was the most mutagenic of all alkylations in RNA (@), while the ethylating agents were generally found to be the most effective mutagens in double-stranded DNA. It has been suggested that the 7-ethyl group in deosygunnosine is more effective than the methyl group on the basis of greater stability, in that the deoxy‘See Addendum, p.26.

ROLE

OF CONFORMATION I N CHEMICAL

MUTAGENESIS

21

riLosyl bond at N-9 is vcry lubile w l i ~ i :iL incthyl group quaternizes the N-7 positioti, but :tccording to ittost :iuthors it is less cffected by an ethyl 101). Thc f x t that l h s l e r el al.(91) found diniethyl substituent (100, sulfate to be a good mutagen for transforming DNA may be due to either the low pH used for reaction (pH S.2), or to more rapid testing of the alkylated samples. As a consequence of the protecting action of double-strandedness, side reactions play a much greater role in DNA and may account for a great part of the inactivating and mutagenic events obtained with reagents affecting the amino groups. One of these is the cross-linking caused by which niay lead to extensive depurination nitrous acid in DNA (92, 93), (81) and excisions of genetic material ( l o g )and , thus to mutations almost unrelated to the known chemistry of deamination. The cross-linking action of bifunctional alkylating agents (e.g., mustard gas) leads to similar consequences, unrelated to alkylation per se, which are favored in double stranded as compared to single-stranded DNA (4,103). Single-stranded DNA is more readily inactivated by bifunctional alkylating agents (104). In line with the above working hypothesis (Fig. 3),we suggest that alkylation of adenine may contribute inactivating events in addition to depurination (103). Nitrosoguanidine also seems to be a more effective mutagen on transforming DNA (96) than on single-stranded TMV-RNA suggests that native (48) although the use of elevated temperatures (60’) DNA is not mutated by this reagent. However, the difference in the reaction conditions employed with DNA and RNA do not justify analogies as regards the mechanism of mutagenesis, since nitrosoguanidine is quite unstable even at 25".

V. The Reactivity and Mutability of Protein-Encased Nucleic Acids We consider here the three classes of viruses that have been more or less intensively studied in terms of their reactivity and mutability, namely (a) TMV, (b) 'I'-even phages, and (c) small single-stranded DNA phages. Then we briefly discuss the mutagenic reactions to which resting bacteria have been subjected. We must realize, however, that it is always difficult to establish with certainty that the action of any chemical on a particle as complex as a bacteriophage, and particularly a microorganism, represents a true in vitro effect, as contrasted with conditions in which the reagent can be retained by, or bound in, the particle so as to produce its effects concurrently with subsequent metabolic activity. (a) The RNA encapsulated in the TMV particle is in general less reactive than free TMV-RNA, so that reactions usually must be performed under more rigorous conditions or for much longer time periods to obtain

22


coinparable levels of inactivation. However, there is also at least one definite qualitative difference that has iiiteresting consequences, the nonreactivity of the guanine residues in the TMV particle toward HN02. Thus Schuster and Wilhelm (9) found the guanine content of TMV to remain unchanged at a time when about 20% of the adenine and over 30% of the cytosine had been deaminated (144 hours, pH 4.2), as contrasted to the deaminations in isolated RNA of 28,24, and 17% for G, A, and C, respectively, after 29.5 hours a t pH 4.2. Apart from the dramatic difference in the reactivity of guanine, i t is evident that the adenine is also less reactive, as compared to the cytosine in the virus than in the RNA. Since the action of HN02 on guanine is probably always inactivating, and the other two deaminations are mutagenic (Fig. l),one would expect the action of HNO2 on intact TMV to show a markedly higher ratio of mutagenic to inactivating events than results from the deamination of free RNA. This has been demonstrated by us in recent experiments, in which interference by the deaminated virus protein was ruled out by isolating the RNA from the treated virus and reconstituting it, prior to testing its residual infectivity and its level of mutagenesis, as compared to that of RNA directly (see Table I). deaminated arid reconstituted (3.2) The nature of the protecting action of the TMV structure on the guanine, and, to a lesser extent, on the adenine, residues is not known. Since only purine-containing polynucleotides seem to reconstitute readily one may surmise that these are the ones that with TMV protein (lob), interact with protein groups and become firmly bonded. It also appears logical that the protein groove holding the RNA should fit most tightly around the biggest nucleotide residues, and thus prevent access of reagents to it. This seeming nonreactivity of the guanine residues in the TMV particle may supply an explanation for the recent findings that nitrosoguanidine causes many mutations and little inactivation when acting on intact TMV, However, earlier studies quite in contrast to its action on TMV-RNA (48). (IOG’),recently confirmed by us, had indicated that the 7 position of guanine in the TMV particle, in contrast to its amino group, is available for alkylation. Since it was shown with TMV-RNA that this principal action of nitrosoguanidine, in contrast to typical alkylations, is dependent 49), it appears probable that the on the base-stacked conformation (48, alkylation of guanine in the TMV particle is inhibited by the absence of base stacking, rather than by the inaccessibility of its N-7 group to this particular reagent. As a consequence of this diminished reactivity of guanine, and possibly of adenine, to alkylation by nitrosoguanidine, it seems that a slower and highly mutagenic reaction is able to become predominant. The nature of this reaction has not yet been identified.

BOLE O F CONFORMATION

IN CIlEMICAL MUTAGENESIS

23

Comparatively much 3-methyl cytidirie is formed in TMV, as it is formed if RNA (or poly C) is treated with nitrosoguanidine in dimethyl formamide, and the possibility that this is the mutagenic event has not yet been ruled out. On the other hand, the possibility that deamination of adenine is responsible for the mutagenicity of nitrosoguanidine is suggested by the results of Chandra etal.(47’). Hydroxylamine reacts so slowly with intact TMV, in contrast to that it is usually quoted as not inactivating several spherical viruses (lor), TMV at all. This reagent causes no mutants when acting on TMV (32). It is therefore suggested that the observed slow inactivation results from the decomposition products of NHzOH arising during several days at 37 C. We can offer no explanation for the failure of the cytosine residues in TMV to react with NHzOH (at pH 6), while being quite reactive to HNOs (at pH 4.5)and probably also to HC'HO (at neutrality). (b) In view of the strong protecting action of the TMV protein on its RNA, it is surprising that the T-even phages are quite susceptible to many mutagenic agents. This is also in distinct contrast to the unreactivity of double-stranded DNA or polynucleotides. Freese and Strack (94) have offered an interpretation for this phenomenon, namely, that the multiple folds necessary to accommodate the T-even DNA in a shell 1/600its length render small segments of the molecule effectively single-stranded. The guanine residues of T2 appear to react much more with HN02 than do the cytosine residues, and the adenine residues seem least reactive (IS). This is the same result obtained under similar conditions with transforming DNA (go), and thus indicates that the bulk of the DNA in T2 shows a pattern of variably low reactivities similar to that of double-stranded DNA. This is also indicated by the fact that the T-even phages are not noticeably affected by formaldehyde (83). Hydroxylamine is 1000 times inorc effective as a mutagen on T4 than on native B.subtilis transforming DNA (94) and even more effective than HN02. This can be explained on the basis that NHzOH acts atpH 6 only on the hydroxymethykytosine residues of the phage, and almost only according to reaction c (Fig. 2), and that therefore almost every chemical event is mutagenic, whereas with HNOz the concurrent deaminations of A, C, and G give comparable numbers of mutagenic and inactivating events. (c) The number of papers dealing experimentally with the chemical results of modification of double-stranded DNA's, including the T-even phages, is regrettably small, but this number is high compared to that dealing with the chemical modification of single-stranded DNA and/or the phages containing such DNA's. Mundry (108) arid Tessman (128) compared tjhe effect of H N 0 2 arid NI1,OH on intact $,X174and its DNA,

24

B. SINGE11 AND

H. FRAENKEL-CONItAT

and found no difference resulting from the presence of the protein coat. Otherwise we seem to have only deductions concerning the chemical events caused by agents used for reverse mutation, based on supposed primary mutagenic events. These deductions, often derived from in vivostudies, are frequently equated with chemical data, notwithstanding the fact that some of the conclusions based on genetic evidence are difficult to reconcile with known chemical facts. We do not review these papers, since we are not convinced ofthe validity of such deductions. We only advocate that more research effort be devoted to anaIytica1 studies of the results of mutagen treatment of single-stranded DNA, double-stranded DNA, and phages or viruses containing these two t-ypesof riucleic acid. We believe that the availability of such data would be of great benefit for our understanding of mutagenesis. (d) The invitro studies on mutagenesis in bacteria cannot readily be correlated with those on simple or more complex nucleic acids. Most typical mutagens have been successfully used with microorganisms with the seeming and surprising exception of NHzOH and its analogs (fO9). Although quantitative comparisons have not been made, it appears that the DNA in bacteria, like that in the phages, is more susceptible to mutagenic agents than isolated native transforming DNA (109). If this is true, it would suggest that DNA in its natural habitat is, in part, less “native” On the other hand, it is also possible that enough reagent than in vitro. remains in the cell to produce effects during the later growth and/or replicating phases, instead of acting strictly on noninetabolising bacteria. One clear example of the great difference in sensitivity of bacteria as compared to pure nucleic acids is nitrosoguanidine, which even at high concentrations is a poor mutagen for RNA (48). Under such conditions, it is apparently quite a good mutagen for transforming DNA (3), but is highly mutagenic for bacteria a t very much lower concentrations (51) (Table 11).

Vl. Conclusions I n Figs. 1-3 we have summarized the chemistry of the three most extensively studied types of mutagenic reactions to illustrate the working hypothesis proposed by Watson and Crick (11U) that mutagenesis requires a tautomeric shift in the N-1, C-6 region of the purines or the N-3,C-4 region of the pyrirnidine~.~ We have for the purposes of this argument listed our interpretation of the hydroxylamine and alkylation data, although we recognize that this is an arbitmry choice, and that future research may prove us wrong. We w i s h to stress ngairi in this context that the efficiency of a mutagen is dependrnt, not ordy on the extent of tautomeric shift it produces, but on the ratio of the frequencies of the mutagenic to the

ROLE

O F CONFORMATION

IN CIlEMICAL MUTAGENESIS

25

inactivating events occurring simultaneously, and such quantitative evaluations are not possible for most experiments. We have attempted to summarize in Tables I and I1 most of the mutagenesis, with particular reference to literature dealing with invitro nucleic acid conformation and milieu. In Table 11, we have attempted quantitative evaluations of the various mutagens acting on the various systems, but any such attempt leads to arbitrary choices, which should not be taken too seriously. We have also indicated the relative reactivity of the bases, without indicating the reaction mechanisms (which has been done in , when available, on the chemical data of the authors, and Figs. 1 4 ) relying, giving our judgment of the modified bases in parentheses when such data were not available. It must be stressed again that mutagenesis is usually observed at an early stage of the reaction, before the base changes reach the level of analytical detectability for most reactions. It will be noted that in Table I1 there is only one column for transforming DNA, and that this is headed “Denatured Transforming DNA.” The absence of a column for native transforming DNA is due to the lack of pertinent data. Double-stranded DNA cannot be treated with nitrous acid, which requires a pH below 5, without incipient denaturation, and its nonreactivity at pH 5 (IS)indicates that double-strandedness is critical in this respect. The same is true for NH20H, which has therefore generally been used under incipient denaturing conditions. Also nitrosoguanidine was used at elevated temperatures (96) presumably because it did not produce mutants at lower temperatures. The only reaction one would expect to be effectiveon genuinely double-stranded DNA, alkylation, has very recently been shown to be quite effective as a mutagen for transforming DNA (91). The last column contains results obtained with synthetic polynucleotides, acting as templates for RNA polymerase, or as messenger in the cell-free amino acid incorporating system. We have there used the terms “positive” and “inactivated” to denote when a polymer acquires a new function (a mutagenic event), and when it only loses some of its activity as template or messenger. From a survey of this table, it is evident that much more remains to be done. It is also evident that no extrapolation from one system to another (mononucleotides, polymers, or single-stranded nucleic acids, doublestranded polymers or nucleic acids, intraviral nucleic acid) is justified, since in many instances the differences in the behavior of nucleic acids in different milieus is not only quantitative but qualitative. We have seen some extraordinariIy sweeping and definitive statements about the mode of action of mutagens in our survey of the literature, many nil effort to avoid this attitude, and to of them erroneous. We havc nintle emphasize instead how iniich and what remains to be done, rather than what we, or others, believe to be known.

26

B. SINGER AND

H. FRAENKEL-CONRAT

We summarize the established facts pertaining to reactivity and mutability of various classes of nucleic acids as follows: Single-stranded polynucleotides or deoxypolynucleotides: generally less reactive than monomeric form, with the exception of methylation of guanine and possibly adenine by nitrosoguanidine, which requires basestacking; readily mutated by nitrous acid, hydroxylamine and methoxyamine, bromine, and to a lesser extent by many other agents. Double-stranded polynucleotides and deoxypolynucleolides: the hydrogenbonded regions (N-1, C-6 of purines; also C-2 of guanine; N-3,C-4 of pyrimidines) generally less reactive than in single-stranded polynucleotides; the N-7of guanine and the N-3of adenine equally and more reactive, respectively; the 5,6 double bond of pyrimidines less reactive than in single-stranded polymers; generally resistant to mutagenesis, except by alkylation under denaturing conditions. Protein-encapsulated single-stranded RNA ( T M V ):the amino groups of guanine and to a much lesser extent of adenine unreactive; the N-7 of guanine reactive to typical alkylating agents, but less to nitrosoguanidine; cytosine unreactive only toward hydroxylamine; readily mutated only by nitrous acid and nitrosoguanidine. Protein-encapsulated double-stranded D N A (Tb,T4):generally similarly resistant as free double-stranded DNA t o modification in the hydrogenbonded regions of the bases; reactive to typical alkylating agents; readily mutated by all highly mutagenic agents, including ethylation, probably because of the existence of single-stranded segments.

ADDENDUM Recent studies (32) of the alkylation of poly A using W-labeled dimethyl sulfate have shown that about 5 and 10% of the total alkylation is on the 3 and 7 positions, respectively. I n poly (A,U) the latter dkylations remain unchanged, only the 1position becoming quite unreactive. I n RNA the proportion of 3, and particularly, 7 substitution as compared to 1-methyl A, is much greater. Similar studies with [14C]nitrosoguanidinehave shown that this reagent acts similarly on poly A and the adenine residues of RNA, yielding allthree possible derivatives. A recent report on the template activity of poly C containing 6.3% of 3-methylcytosine showed incorporation of uridylic acid in the polyguanylic acid. The mechanism of this mutagenic event, which made cytosine code like adenine, is not understood [D. B. Ludlum and R. C. Wilhelm, J .B i d .Chem.243, 2750(196S)l.

ACKNOWLEDGMENTS We wish to thank Dr. L. Hirth of the University of Strasbourg, France, for his hospitality and interest during the gestation period of this review, during which we were partly supported by research grants GB 3107 and GB 6209X from the National Science Foundation and by National Aeronautics Space Adrninist,ration grant, NsG 479.

ROLE

O F CONFORMATION

I N CHEMICAL MIJTAGENESIS

27

We are also iiidebted to Doctors P. Brooltes, P. L). Lawley, T. H. Jukes, A. M. Michelson, and D. Shugar for reading the manuscript and making helpful suggestions.

REFERENCES 1. D. R. Krieg, This series 2, 125 (1963). 2.L. E. Orgel, Aduan.Enzymol. 27,289(1965). 3. E.Freese and E. B. Freese, Radiation Res.Suppl. 6, 97 (1966). 4.P. D. Lawley, This series 6,89 (1966). 6.U. Winkler, Fortschr. Botun. 28, 172(1966). 6. J. H. Phillips and D. M. Brown, This series 7,349 (1967). 7. H. Fraenkel-Conrat, in “The Molecular Basis of Virology” (H. Fraenkel-Conrat, ed.). Reinhold, New York, 1968. 8. H. Schuster and G. Schramm, Z. Nuturforsch. 13b, 697 (1958). 9.H. Schuster and R. C. Wilhelm, Biochim. Biophys. Acta68, 554(1963). 10. R. Shapiro, J. Am. Chem.SOC.86, 2948(1964). 11.R. Shapiro and S. H. Pohl, Biochemistry 6, 448 (1968). 12. A. Gierer and K. W. Mundry, Nature 182, 1457(1958). 13. H. Schuster and W. Vielmetter, J .Chim.Phys.68, 1005(1961). 14.A. M. Michelson and C. Monny, Biochim. Biophys. Actu129, 460(1906). f5. M. J. Bessman, I. R. Lehman, J. Adler, S. B. Zimmerman, E. S. Simms, and A. Kornberg, Proc. Natl. Acad.Sci. U.S. 44,633 (1958). 16.C. Basilio, A. J. Wahba, P. Lengyel, J. F. Speyer, and S. Ochoa, Proc. Natl. Acad. Sci.U. S. 48, 613(1962). 17. D. W. Verwoerd, H. Kohlhage, and W. Zillig, Nature192, 1038(1961). 18. D. M. Brown and P. Schell, J. Mol.Biol. 3,709 (1961). 19. D. M. Brown and M. J. E. Hewlins, J .Chem.SOC.( C )p. 1922(1968). 20.D. M. Brown and J. H. Phillips, J .Mol.Biol. 11, 663(1965). 21.J. H. Phillips, D. M. Brown, R. Adman, and L. Grossman, J. MoZ. Biol. 12, 816 (1965). 22.R. G. Wilson and M. J. Caicuts, J. Biol. Chem.241, 1725(1966). 23. P. D. Lawley, J. Mol.B i d .24, 75 (1967). 24.N. K. Kochetkov, E. 1. Budovsky, E. D. Sverdlow, R. P. Shibaeva, V. N. Shibaev, and G. S. Monastirskaya, Tetrahedron Letters p. 3253(1967). 26.D. M. Brown, M. J. E. Hewlins, and P. Shell, J .Chem.Soc.( C )p.1925(1968). 26. C. Janion and D. Shugar, ActaBiochim. Polon. 16,107 (1968). 27. C. Janion and D. Shugar, ActaBiochim. Polon.12, 337 (1965). 28. E. Freese, E. B.Freese, and E.Bautz, J. MoZ. Biol. 3,133(1961). 99. J. D. Phillips, D. M. Brown, and L. Grossman, J. MoZ. Biol. 21, 405 (1966). 30. G. D. Small and M. P. Gordon, d .Mol.Biol. 34, 281(1968). 31. H. Schuster, J .Mol.Biol. 3, 447 (1961). 32.B. Singer and H. Fraenkel-Conrat, In preparation. 33.N. K. Kochetkov, E. I. Budowsky, and R. P. Shibaeva, Biochim.Biophys. Acta68, 493(1963). 34. E. B. Freese and E. Freese, Proc. Natl. Acad.Sci.U.S.62, 1289(1964). 36. E. Freese, E. B. Freese and S. Graham, Biochim. Biophys. Acta123,17 (1966). 36. J.H. Van de Pol and G. A. Van Arkel, Mutation Res.2,466(1965). 37. A. K. Kriek and P. Emmelot, Biochim. Biophys. Acta91, 59 (1964). 58. A. K. Kriek and P.Emmelot, Biochemistry 2, 733(1963). 59. D.B. Ludlum, R. C. Warner, and A. J. Wahba, Science 146, 397 (1964).

28

B. SINGER AND

H. FRAENKEL-CONRAT

40. R. C. Wilhelm and D. B. Ludum, Science 163, 1403 (1966). 41. A. M. Michelson and M. Grunherg-Manago, Biochim. BiophTys. Acta91, 92 (1964). 42. J. H. Chen and F.F. Davis, Biochem. Biophys. Res.Commun. 20, 124 (1965).

43. H. Fraenkel-Conrat, Biochim. Biophys. Acta49, 169 (1961). 44. L. T. Thiry, ViroZugy 19, 225 (1963). 4.6. A. Loveless, Proc. Roy.SOC.B160, 497 (1959). 46.E. Bautz and E.Freese, Proc. Natl. Acad.Sci. U .S. 46, 1585 (1960).

47. P. Chandra, A. Wacker, R. Siissmuth, and F. Lingens, 2. Naturforsch. 22b, 512 (1967). 48.B. Singer and H. Fraenkel-Conrat, Proc. Natl. Acad.Sci.U.S. 68,234 (1967). 49. B. Singer, H. Fraenkel-Conrat, J. Greenberg, and A.M. Michelson, Science 160,1235 (1968). 50.J. B. Mandell and J. Greenberg, Biochem. Biophys. Res.Commun. 3, 575 (1960). 61. E. A. Adelberg, M. Mandel, and G. Chen, Biochem. Biophys. Res.Commun. 18,788 (1965). 62. F. Lingens and 0. Oltmanns, 2. Nuturforsch. 21b, 660 (1966). 65. L. S. Lerman, J .Mol.Biol. 3, 18 (1961). 54. D. Freifelder, P. F. Davidson, and E. P. Geiduschek, Biophys. J. 1, 389 (1961). 65. M. I. Simon and H. Van Vunakis, J. Mol.Biol. 4, 488 (1962). 66. A. Wacker, G. Tiirck, and A. Gerstenberger, Ndumissenschaften 60, 377 (1963). 67. B. Singer and H. Fraenkel-Conrat, Biochemistry 6,2446 (1965). 68. K. S. Shastry and M. P. Gordon, Biochim. Biophys. Acta129, 32 (1966). 69. K. S. Shastry and M. P.Gordon, Biochim. Biophys. Acta129, 42 (1966). 60.L. A. Waskell, K. S. Shastry, and M. P.Gordon, Biochim. Biophys. Acta129, 49 (1966). 61. M. I. Simon, L. Grossman, and H. Van Vunakis, J .Mol.Biol. 12, 50 (1965). 68.P. Chandra and A. Wacker, 2.Naturforsch. 2lb, 663 (1966). 65. B. Singer and H. Fraenkel-Conrat, Biochemistrp 4, 226 (1965). 04.J. K. Setlow, Current Topics Radiation Res.2,197 (1966). 65.R. L. Sinsheimer and R. Hastings, Science 110, 525 (1949). 66. K. L. Wierzchowski and D. Shugar, ActaBiochim. Polon. 8,219 (1961). 67.R. L.Sinsheimer, Radiation Res.6, 121 (1957). 68.H. E. Johns, J. C. LeBlanc, and K. B. Freeman, J .Mol.B i d .13,849 (1965). 69.H. E. Johns, S. A. Rapaport, and M. Delbruck, J .Mol.B i d .4,104 (1962). 70. K. B. Freeman, P.V. Hariharan, and H. E. Johns, J.Mol.B i d .13, 833 (1965). 71. L. Grossman, Proc. Natl. Acad.Sci.U .S. 48, 1609 (1962). 72. L. Grossman, Proc. Null. Acad.Sci.U.S. 60,657 (1963). 75. J. Ono, R. G. Wilson, and L. Grossman, J. Mol.Biol. 11, 600 (1965). 74.M. Daniels and A. Grimison, Biochem. Biophys. Res.Commun. 16, 428 (1964). 75. H. Schuster and H. G. Wittman, ViroZogy 19,421 (1963). 76.R. M. Litman and H. Ephrussi-Taylor, C m p t .Rend.Acad.Soi. 249, 838 (1959). 77. E. Cabrera-Juarez and R. M. Herriott, J .Bacterial. 86,671 (1963). 78. A. Tsugita and H. Fraenkel-Conrat, J .Mol.Riot. 4, 73 (1962). 79. K. W. Brammer, Biochim. Biophys. Acta72, 217 (1963). 80. T. A. Trautner, M. N. Swartz, and A. Kornberg, Proc. Natl. Acad.Sci. U.S.48,449 (1962). 81. R. Kotaka and R. L. Baldwin, J .Mol.Biol. 9, 323 (1964). 82. F. Pochon and A, M. Micheleon, Biochim. Biophys. Acta149, 99 (1967). 83. H. Fraenkel-Conrat, Biochim. Biophys. Acta16, 308 (1954). 84.M. Staehelin, Biochim. Biophys. Acta29, 410 (1958).

ROLE O F CONFORMATION I N CHEMICAL MUTAGENESIS

29

S. Zamenhof, H. E. Alexander, and G. Leidy, J .Exptl. Med.98, 373 (1953). R. Haselkorn and P. Doty, J .Bid.Ciient. 236, 2738 (1961). D. Freifelder and P. F. Davison, Biopfiys. J .2, 249 (1962). E. E. Horn and R. M. Herriott, Proc.Natl. Acad.Sci. L .S.48, 1409 (1962). S. H. Goodgal and E. H. Postel, Science 148, 1095 (1965). 90. R. M. Litman, J .Chim.Phys.68,997 (1961). 91. S. E.Bresler, V. L.Kalinin and D. A. Perumov, Mutation Res.6, 1 (1968). 92. E. P. Geiduschek, Proc. Xatl. Acad.Sci. U .S.47, 950 (1961). 93. E. F. Becker, Jr., B. K. Zimmerman, and E. P. Geiduschek, J .Mol.Bid. 8, 377

85. 86. 87. 88. 89.

(1964). 94. E. Freese and H. B.Strack, Proc.Null. h a d .Sci.U.S.48, 1796 (1962). 95. E. Bautz-Freese, J. Gerson, H. Taber, H.-J. Rhaese and E. Freese, Mutation Res.4,

517 (1967). 96.E. Freese and E. B. Freese, Biochemistry 4, 2419 (1965). 97.K.L. Wierzchowski and D. Shugar, Photochem. Photobiol. 1, 21 (1962). 98. R. B. Setlow, Proc. Xatl. h a d .Sci. U.S.63, 1111 (1965). 99. D. B. Ludlum, Biochim. Biophys. Acln96,674 (1965). 100. P. D. Lawley and P. Brookes, Biochem. J .89, 127 (1063). 101. P. Alexander, J. T. Lett, and G.Parkina, Biochim. Biophys. Acta48, 423 (1961). 102. I.Tessman, J.Mol.Biol. 6,442 (1962). 103. P. D. Lawley, J. H. Lethbridge, P. A. Edwards, and K. V. Shooter, Biochem. J. 106, 52P (1967). 104. N. Yamamoto, T. Saito and M. B. Shimkin, Can,cer Res.26, 2301 (1966). 105.H. Fraenkel-Conrat and B. Singer, Virology 23, 354 (1964). 106. P. Brookes and P. D. Lawley, Biochem. J .77, 478 (1060). 107. It. M. Franklin and E. Wecker, Nutzcre184, 343 (1959). 108.K. W. Mundry, impublished observations. 109.W. Hayes, “The Genetics of Bacteria and Their Viruses.” Wilcy, New York,1964. 110. J. D. Watson and F. H. C. Crick, Nature171, 964 (1953). 111. K. W. Mundry, 2. Vererbztngdehre 91,87 (1960). 112.W. Vielmetter and C. M. Wieder, 2. N n t i ~ ~ f o r14b, s c h312 . (1959). 113. I. Tessman, Virology 9, 375 (1959). 114. V. F. Chubukov and S. Tatarinova, Zliur. Mikrobiol. Epidemiol. Immunobiol. 42,80 (1965). 115. D. M. Green and D. R. Krieg, Proc.Null. Acad.Sci.U .S .47, 64 (1961). 116. D. A. Ritehie, Genetics Res.Cambridge 6, 168 (1964). 117.J. W. Drake and J. McGuire, .I.Virology 1, 260 (1967). 118. B. D. Howard and I. Tessmm, J. Mol.Hiol.9, 372 (1964). 119. J. W. Drake, J .Bacteriol. 92, 144 (1966). 120.F. Kaudewitz, Nature83,1829 (1959). 121.B. S. Strauss, J .Bacteriol. 83,241 (1962). 123.H. E. Kubitshek, Proc. Natl. Acad.Sci. Cr.S.62, 1374 (1964). 123.B. R. Webb and H. E.Kubitschek, Biochem. Biopfys. Ites. Commun.13, 90 (1963). 12.4. A. Zampieri and J.Greenberg, Mutation Res.2, 552 (1965). 125. I. Tessman, H. Ishiwa, and S. Kumar, Science 148, 507 (1065). 126.I. Tessman, R.. I1[ml = 13/(nP 2)1(Mo/l00)bl

(3)

228

JEN TSI YANG

AND TATSUYA SAMEJIMA

where n is the refractive index of the solvent, which is also wavelength is widely used for ORD of proteins and dependent. The quantity [m ] polypeptides, but theoreticians still disagree on the validity of this Lorentz correction for polymers in solution. It is rarely used for the ORD of nucleic acids. The dimension for [MI,in?],and [?a is] degree X cm2/decimole. or m,and others use R and Some workers prefer to use the symbol @ for ill R instead of m and m . The CD can be expressed simply as the difference in molar absorptivity EL - eR,which equals (AL - AR)/mZ. Here the subscripts L and R refer to the left and right circularly polarized components, the A sare the absorbance, m is the molar concentration, and 1 is the light path in centimeters. The dimension of EL - ER is cm2/mole. As in the case of optical rotation, CD can also be expressed in terms of its ellipticity. Thus, we have the specific ellipticity, [*I, molar ellipticity, [el,and mean residue ellipticity, [el, simply by replacing a. M , and m in Eq. (3)by \k, 8, 0. Their dimensions are identical with the corresponding quantities for optical rotation. It can be shown (80) that EL - eB is related to its ellipticity by

[*I

=

3 3 0 ( A-~ ApJ/ZC

(4)

and

Here again the light path, Z, is in centimeters, and the concentrations, C and m, are in grams per milliliter and moles per liter. The A sfor [el, of course, refer t o the absorptivity based on mean residue rather than the entire polymer molecule.

C. Correlation between ORD and CD ORD, a dispersion property, and CD, an absorption property, share a common origin, the optical activity, and are thus closely related. Theorems of very general validity niake it possible to calculate the dispersion characteristics of a molecular system from its absorption properties orvice versa. Indeed, Moscowitz, using the Kronig-Kramers transform, has given explicit formulas that correlate ORD and CD (18,2% :)

imd(~>l =

(z/T)

hm

[ei(x‘)1[h’/(x2 - x’z)] ~JX’

(6a)

[m;(A )][X 2/(X2 - X’7] dX

(6b)

and [&(A)]

=

- (2/rX)

Equation (6a, b) can easily be solved with a computer program. For a

ORD AND

220

CD OF NUCLEIC ACIDS

Gaussian CD, Eq. (62)would give an ORD profile similar to that shown in Fig. 1. An important theoretical quaiitity of optical activity is the rotational strength, Ri, which is related to the experimental ellipticitv, I P j ,by (18):

or

Here h is Planck's constant, c is tthe speed of light in vacuum, N I is the number of molecules per milliliter, and \Eiis in radians. The dimension of Ri is erg X cm3 X radian. The rotational strength determines the sign and magnitude of the partial CD and the corresponding ORD.

0. Drude Equation Drude, in 1896, first introduced a general expression for rotations in the region farfrom any of the optically active absorption bands:

where X i is the wavelength of the ith electronic transition and k; is a constant proportional to the rotational strength, Ri. Equation (8) was It later derived correctly from quantum mechanical considerations (23). can also be shown that for an idealized Cotton effect Eq. (6a) is reduced to one Drude term in a wavelength range distant from the absorption band (18). Equation (8) can be approximated as [m]A =

k / ( x2 A:)

(9)

if - purine . pyrimidine > pyrimidine pyrimidine. Second, charges of the same sign on both bases cause unstacking, but a charge on one base does not. Exceptions to the above generalization are that at pH 7, UpG and CpU stack, at pH 11.5, CpU- stacks and ApG- does not, and a t pH 1, G+pU, UpG+, C+pG+, and G+pC+ stack. (Because of lack of titration data on these compounds, there is uncertainty about the last two compounds, which may actually be charged singly). Bush and Tinoco (74)also calculated the ORD ofthese dinucleosidephosphates by considering exciton interactions of the electronic transition moments above 220 mp as well as the directions and magnitudes

-

ORD

AND

CD OF NUCLEIC

THE

a

241

ACIDS

CLASSIFICATION

TABLE 111 DINUCLEOSIDE PHOSPHATES

OF

(61)

Borderline.

of these moments from the absorption spectra and found the position and relative magnitude of the first peak and trough (on the long wavelength side) in good agreement with experiment. That base-stacking interactions give rise to the observed multiple Cotton effects is also confirmed by the ORD study of GMP gel (76). In concentrated solution (10 mg/ml) and at low temperature (27, the monomers of this compound are stacked to form a left-handed helical structure (76). Accordingly, the Cotton effects of the gel are multiple instead of a single one around the 260-mp absorption band for the monomer. However, the ORD profile is inverted from that shown in Fig. 3 and resembles that of the helical poly I (see Section V, B), that is, it displays two troughs and one peak near the 260-mp absorption band. Raising the temperature of the solution causes a reversible transition from the helical aggregates to the monomer with a T,,, around 15". Brahms et al.(66) studied the CD and also absorption spectrum of n variety of 3 ’ 35' and 2'- 5' dinucleoside phosphates in a range of temperature between -20 and 80". Their results were classified into two categories. One is represented by essentially two adjacent positive and negative CD bands of almost equal rotational strength in magnitude witsorption mnximuin. Since the sum ofthe rotnt8ionnIstrengths in the spectrnl region untlcr investigation is approximately zero, the Cn is therefore designated "con-

242

J E N TSI YANG AND TATSUYA SAMEJIMA

servative,” for instance, ApA. The second category is composed of one or two positive CD bands, characteristic of the guanine and cytosine derivatives (Fig. 4). Since the sum of rotational strengths in the range of wavelengths studies is not zero in this case, the CD is termed “nonconservative” (for instance, CpC). The conservative type is in accord with the that is, the interactions among the prediction of the exciton theory (73), chromophores in a dimer give rise to a splitting of the monomer into two bands. The nonconservative type is attributed to contributions other than the exciton splitting, that is, t,o those arising from the interactions between near and far ult,raviolet transitions (77; see Section VI, C). The difference 10

8 6 4% 2 ;

0 -2

-4

220

240

260

200

300 220 X (mp)

240

260

280

300

FIG.4. CD and absorption per base of four dinucleoside phosphates at neutral pH in 4.7 M KF plus 0.01 M Tris and at -18 to -20°C (66). The absorption spectra are: left, UpG ( - - - ) and GpU (- . -); right. CpA ( - - - ) and ApC (- -). The dots for CD are the average of the monomer constituents.

.

between these two kinds of CD cannot be readily seen from their corresponding ORD. On the other hand, the results in Fig. 4 also reveal the sequence dependence of the CD of dimers just as ORD does (see Fig. 3).

EFFECT OF TEMPERATURE The rotation of the stacked dinucleoside phosphates decreases in magnitude with increasing temperature. This can be interpreted as the unstacking of the bases at high temperature, although the possibility that increasing torsional fluct,uation rather than base unst,acking causes the decrease in rotation cannot be completely ruled out. Similarly, the intensity of the CD of dimers decreases markedly with increasing temperature. With the linear van? Hoff plots, Brahms et nl.(56) estimated the thermodynamic parameters, AH , AS”, and AF , of the stack-unstack processes and could find no fundamental diflerenre among the dinucleoside phosphates studied warranting a division into L‘stxked”and “unstacked” groups. This is contrary to the widespread acceptance of such a division, as shown in

ORD AND CD O F NUCLEIC ACIDS

243

Table 111. However, it is not too difficult to reconcile this viewpoint with that deduced from optical properties. For instance, if the two stacked bases rotate relative to each other, the Cotton effects are markedly reduced, but the thermodynamic quantities may not be affected significantly. Note that these thermodynamic quantities are determined on the basis of a two-state model, which assunies that a dinucleoside phosphate molecule can have only two conformations, one stacked and the other unstacked. The stacked form is favored at low temperatures, and the equilibrium between the two states is shifted toward the unstacked form with increasing temperature. This model could be oversimplified, since the dinucleoside phosphate molecule is expected to have a continuous spectrum of the stacked conformations and it is difficult to visualize a sharp division on the molecular level between the two states. Davis and Tinoco (78)further pointed out that the calculated thermodynamic quantities of, for instance, ApA are not self-consistent, since optical rotation and hypochromicity methods gave very different results. Davis and Tinoco (78)and Glaubiger et aE.(79) thus proposed as an alternative a torsional oscillator model in which the parallel bases oscillate with respect to each other. This model predicts that the ORD of dinucleoside phosphates should show an exponential decrease at higher temperatures (as contrasted with the van't Hoff equation) and that the hypochromism should be almost independent of temperature. While the model fits the ORD data as well as the two-state model, it still does not predict the correct temperature dependence of the hypochroniism. David and Tinoco (78)concluded that both the two-state model and the oscillating dimer model fit some of the experimental data, but neither is completely satisfactory. Needless to say, we still cannot draw any definite conclusion about a specific conformation or conformations of dinucleoside phosphates in solution in spite of many physical studies of these and related compounds (free bases, nucleosides, and polynucleotides). True, the bases tend to stack at low temperatures in aqueous solutions and unstack at high temperatures and in denaturing solvents. But we cannot speculate much beyond this statement. Brahms et al.(56) also reported the CD of 2' --f 5' dinucleoside phosphates such as CpC, ApC, and ApA. The general profile is vcry similar to that of the 3’ 5' isomers, but the intensity in this case is relatively small and also the band position is slightly displaced. The changes in intensity of the 2 ’ - 5' dimers were some 104070 from 80" to -20 ,whereas the intensity of the 3 ’ 4 5 ’dimers can change by a factor of three. These findings suggest that the 2’ + 5’ dinucleoside phosphates also have a dissymmetric structure with weakly interacting bases and that their predominant conformation is probably close to the unstacked form in the region of temperature used. Brahms et al.(56) postulated that the 2’--f

244

J E N TSI YANC AND TATSUYA SAMEJIMA

hydroxyl group of ribose might form an intramolecular hydrogen bond, probably with an oxygen atom, which in turn favors the base stacking. This, they conclude, is supported by the study of deoxyribonucleoside phosphates such as dGpdG, which lacks the 2’-hyclroxyl group and, therefore, yields an exceedingly weak CD. We note that Ts’o et at. (80) aIso measured the ORD of dApdA, which shows Cotton effects similar to those of ApA except that their magnitude appears to be smaller than that of the ribosyl dimer.

6. Trinucleoside Diphosphaies and Other Oligomers Cantor and Tinoco (81) measured the ORD and hypochromism at pH 1, 7, and 11.5 of seven trinucleoside diphosphates: A-A-A, A-A-C, A-A-U, G-A-U, A-G-U, G-G-U, and G-G-C (Fig. 5). Again, by designating those with hypochromism less than 3% as “umtacked,” greater than 6% as “stacked,” and from 3 and 6% as “partially stacked,” these authors found that the first five compounds are stacked at pH 7,and A-A-C and A-A-U also stacked at pH 11.5. G-G-C is the only compound to show much stacking at pH 1. More recently, Cantor and Tinoco (82) reported the ORD of four additional trimers, A-C-U, G-C-C, A-G-Cp, and G-A-Cp at pH 7. The last two compounds contain 3’-terminal phosphates, which

220

260

300

340

220

260 300 X (rnp)

340

220

260

300

340

FIG.5. Molar rot.ation per base of seventrinucleoside diphosphates at pH 7 and ionic st,rength 0.1 (0.01 M ph0sphat.ebuffer plus 0.08 M KCIO,) (81).Solid line, experirnent,al; broken line, calculated from Eq. (14).

ORD

AND

CD OF NUCLEIC

245

ACIDS

seem not to affect greatly the optical properties of the trimers. This is also in accord with the finding of Vournakis etal.(83) that the ORD profiles of A-A-Cp and A-A-C are in excellent agreement. Inoue et al.(8%) also reported the ORD and hypochromism at pH 1, 7, and 11 of seven trinucleotides all ending with Gp : A-A-Gp, A-C-Gp, C-A-Gp, C-C-Gp, U-A-Gp, A-U-Gp, and U-U-Gp. The first five compounds are stacked at pH 7. Trimers having one or no protolytic base (uridylyl or guanylyl residue) are still stacked at pH 11, whereas those having two or three protolytic bases are unstacked at t,his pH. C-C-Gp appears to be the only compound to exhibit appreciable stacking at, pH 1. Inoue et al.(83a), however, believe that the S’-terminal phosphate is a significant factor in the formation of ordered conformations in oligonucleotides, and consequently to their ORD and hypochromicity, although its importance may diminish with increasing chain length as the ratio of base to phosphate approaches unity in polynucleotides. Like dinucleoside phosphates, sequential isomers of trinucleoside diphosphates show very different ORD profiles, for example, G-A-U and A-G-U at pH 7 (Fig. 5 ) .Ideally, all 64 trimers should be prepared and their ORD measured, but this would be a formidithle task for any single laboratory. Enthusiasm for studying higher oligomers becomes even less when one realizes that there are 4N possible oligomers of chain length N . Thus, Cantor and Tinoco (82) have developed a simple semiempirical approach for calculating and predicting the optical properties of these 64 trimers from those of their constituent dinucleoside phosphates. This is based on the assumption that only nearest-neighbor interactions between nucleoside bases contribute to the optical properties (cf. Felsenfeld and Hirschman, 8%).

NEAREST-NEIGHBOR INTERACTIONS The molar rotation, [MI,at any wavelength of a dinucleoside phosphate, NpN’, can be written as [MNN’] = [ M N ]

+

f [MN ] INN!

(11)

where I N N is the contribution of the nearest-neighbor interactioIi to the rotation. Since the molar rotation of a nucleoside and its corresponding nucleotide are usually very close and small compared to the rotation of a dinucleoside phosphate, one can take the average of the molar rotation of pN’ and N for [n/!,,], for instance. Likewise, for a trinucleoside diphosphate, we have [ M N N N J t ]= [ M N I

f

[MNt]

+

[2vN’t1

+

JNN~

f JN’N”

+

JNS

,

(12)

Here again, J , and J N f N , ,are the terms for nearest-neighbor interactions,

246

JEN TSI YANG

AND

TATSWA

SAMEJIMA

but J ,,= 0, since the effect of the next-nearest-neighbor interaction is ignored in this treatment. If the bases in the dimer and trimer have the same relative conformation, J N N ~ = I ,and J N 1 = I N t N . Combining we have Eqs. (11)and (12), [hf

N ]

= [.$fNN ]f LiTfNtNtC]-

[nrNr]

(13)

or, in terms of mean residue rotation, [m], [ ? I t N N N ~= l

(a[m,iY~]

+

2[rnN,N,tI

- [,nxrI l/3

(14)

Ignoring the end effects, Eq. (14)can be extended to higher oligomers and polymers :

where X N N ~ is the mole fraction of NpN’ and XN of nucleoside N. If the sequence of a polynucleotide is random, we can further assume that XNN’ = X N . X N ~ ,and Eq. (15) becomes A

A

For a homopolymer, Eq. (16)is greatly simplified as [mpolgrner] = a[mNN]

-

[mX1.

(17)

The calculated ORD, using Eq. (14)for the trinucleoside diphosphates, agreed very well with the experimental values (Fig. 5 , broken lines), thus supporting the theory of the nearest-neighbor interactions between bases. It also gives a strong argument for similar conformation of bases in dimers and trimers and makes it possible to predict the ORD of oligomers from their constituent dinucleoside phosphates. While this practical approach promises to become a new tool in determining the sequence of oligonucleotides, its usefulness may be limited to trimers and perhaps tetramers because differences among the ORD of longer sequence isomers will probably become very small or vanish. It should also be pointed out that the work of Inoue et al.(8%)on seven trinucleotides shows a rather poor agreement between experimental ORD and that based on Eq. (14), assuming a negligible effect of the 3’-terminal phosphate. These workers cannot draw any definite conclusion as to whether the disagreement is greater in trinucleotides than in trinucleoside diphosphates (cf. Fig. 5). They further suggest that the dinucleotide rather than dinucleoside phosphate should be used as a unit structure in the nearest-neighbor calculations, ifthe effect of the 3’-terminal phosphate on the ORD is not small in


247

oligonucleotides. Equations similar to Eq. (14)can easily be obtained for the mean residue rotation of a trinucleotide and a trinucleoside diphosphate. The importance of this terminal phosphate effect must be clarified by future investigations. In the case of polymers, the use of Eqs. (15), (IS), and (17) requires that the rotatory contributions of the nearest-neighbor bases, -NpN'-, can be approximated by those of the corresponding dinucleoside phosphates, NpN'. This again must await more experimentation. measured the ORD of a series of oligoadenylic Michelson et al.(84) acids with a degree of polymerization varying from N = 2 to 12 and also one polymer with N = 200. At pH 6.75 the amplitude between the first peak (at 277 mp) and trough (at 255 mp) increased gradually with chain length and more than doubled for N = 200 over N = 2. Note that Eq. (17) actually predicts that the mean residue rotation of a homopolymer is about twice that of its dimer. At pH 4.86, the increase in the amplitude was also gradual between N = 2 and 12, but a drastic difference in the amplitude was observed for the polymer, which was about &fold that for N = 2. This is undoubtedly due to a transition of the protonated double-stranded, helical form of poly A, known to exist below pH 5 (see Section V, B). The ORD of di-, tetra-, and hexamers of adenylic acid, PA)^, PA)^, and (pA)e, at neutral pH over the temperature range of 5 to 85" were also reported by Poland et al.(85). Their results can be accounted for by a theory of cooperative stacking of bases for the single-stranded oligomers, which shows that the stacking in this case is only slightly cooperative. 86) corroborated well the The CD studies of oligoadenylic acids (57, ORD results of Michelson etal.(84). The general spectrum is the same for all the oligomers snd high polymers, as illustrated by the trimer, heptamer, and polymer at neutral pH in Fig. Ga. The intensity of the CD bands are an order of magnitude higher than, and entirely different in profile from, that of the monomer AMP. As the degree of polymerization increases, the positive band becomes broader arid increases in intensity more rapidly than does the negative band. There is also a general shift toward lower wavelengths with increasing degree of polymerization. As expected, the intensity reduces at elevated temperatures as a result of unstacking of the bases. The mean residue rotational strength of the positive band is also gradually enhanced with increasing number of residues (Fig. 6b). In contrast, the positive rotational strength in acid solution (pH 4.5) shows a sudden increase a t about the level of the heptamer and the sharp increase approaches a constant plateau at about 15 residues. These results support the conclusion that oligoadenylic acids and poly A at neutral pH adopt single-stranded conformations stabilized by base stacking, whereas in acid solution a double-stranded, helical conformation can be assumed only by oligomers larger than the heptamer. Brahms etal.(57) also determined the

248

J E N TSI YANG

60 c UI ?lo

* 40

-- 2 o

x

E E

+ 0 z LT

TATSUYA

SAMEJIMA

-

5

+

AND

7.4

20

O2

’ 1, ’ 6

I

---

I lb I ; 1

N (b)

FIG.6. (a) CD per base of ApApA, A ~ ( A P ) ~and A , poly A at p H 7.4in 0.1 M NaCl plus 0.01 M Tris and at various temperatures (57). Left, curves 1-6‘: -2,0.5, 4.5, 18,25, and 47 C.Center, curves i4 : -2,0.5,8, 18.5,32, and 40 C.Right, curves 1-6: - 2 t o 6, - 17,34,42,57, and 74 C;curve 7,quaternary ammonium salt of p l yA in 98% ethanol at 0 C;and curve 8, AMP at 0°C. (b) Rotational strength of the positive band of oligo- and poly A as a function of the Open circles, p H 4.5; filled circles, pH 7.4. Temperature: degree of polymerization, N (67). about 0 C.

thermodynamic parameters of these compounds and concluded that thermal ‘Ldenaturation”of these single-stranded structures is largely a noncooperative process, whereas that of the double-stranded ones in acid solution is cooperative.

ORD

AND

CD OF NUCLEIC

ACIDS

249

Adler et al,(8’7) studied iu detail the ORD and absorption of both oligomers and polymers of CMP and dCMP at neutral pH over a wide temperature range. They concluded that ribosyl oligo- and polymers display greater rotatory power, hypochromicity, and heat stability than do the comparable deoxyribosyl compounds. Comparison of the two oligomer series further showed that asymmetric macromolecular structure was attained more readily, as a function of chain length, in the oligoribonucleotides. Detailed CD studies of oligocytidylic acids and poly C have also ) . these compounds at neutral pH possess a singlebeen reported ( 8 7 ~Again stranded, stacked-base conformation, but in acid solution (pH 4.0) the double-stranded, helical structures begin with the heptamer. In contrast with the above natural 3’-+ 5’ oligocytidylic acids, a series of 2' 4 5 ’ oligocytidylic acids at neutral pH showed no marked temperature dependence for the CD spectra, suggesting the lack of significant stacking between the bases in this case ( 8 7 ~ ) . Simpkins and Richards (87b) reported the spectrophotometric titration, hypochromism, and ORD ofoligouridylic acids (up to the undecamer) and also of poly U and concluded that these compounds are devoid of temperature-dependent base-stacking interactions at 20 Cin 0.1 M NaCl. The Cotton effects of the oligomers (from dimer up) and polymer are closely similar; their shape and the magnitude of the mean residue rotation show little dependence on chain length.

V. Synthetic Polynucleotides A. Poly A, Poly U, and Their Complexes Studying synthetic polynucleotides will help us understand the more complicated nucleic acids which contain a multiplicity of bases and base sequences. (For reviews on polynucleotides, see refs. 88-95.) Like the oligomers, the general ORD profile of all polynucleotides (except poly I) consists of two peaks and one trough centered around the 260-mp absorption band, and their CD has a positive band (on long wavelength side) and a negative one above 22&230 m p . Additional Cotton effects are also observed below 220-230mp. and Lamborg etal.(32) first reported the ORD's Sarkar and Yang (94) of poly A and poly U above 200 mp, and, more recently, Ts'o et al.(80) have extended the measurements to about 190mp. Holcomb and Tinoco (95) also presented the ORD of poly A in neutral and acidic solutions at various temperatures. Figures 7 and 8a illustrate the ORD spectra of these polymers and their complexes ; Table IV lists the pertinent numerical values of the ORD of various polynucleotides. The multiple Cotton effects

250

JEN TSI YANG

AND

TATSUYA

SAMEJIMA

40

4oc

,IT

30 20 10

G ;x

y 10 u 20 30 40

50

200

300

250

350

X (mp) (b)

FIG.7.ORD of (a) poly A and (h) poly U at, pH 7.5in 0.15M KF. Insets: Variation ofrotat,ion a t t,he first, peak with temperature. From Sarkar and Yang (94).

I

30

E

I

I

I

I

I

4

\

-

I

27 OC

A

-20 -30

-40

200

250

300

350

1 (mp) (0)

FIG. 8. (a) ORD of poly A . poly U a t p H 7.5 in 0.15 M KF. Inset: Variation of rotation and optical density with temperature. From Sarkar and Yang (94). (b) CD of poly A . poly U and poly (A,U)a t pH 7.4in 0.005 M NaCl plus 0.01 M Trisand at 14-15°C (106). Curves: 1, poly A poly U, measured separately; 2, poly A . poly U; 3,poly (A,U)using 0.1 M NaCl instead of 0.005 M ; and 4,same as curve f except at 2°C.

+

0

3.

z

TABLE I V THE COTTOX EFFECTS OF POLYNUCLEOTIDES Peak 1 Polymer and solvent

Poly A (0.15 M KF, pH 7.5) Poly U (0.15 M KF, pH 7.5) Poly A . poly U (0.15 M KF, pH 7.5) Poly C (0.1 M NaCl plus 0.01 M cacodylate, pH 7.0) Poly Go (0.1 M NaC104 0.001 M cacodylate, pH 7.0) Poly G . poly C (0.1 M Tris, pH 7.5) poiy ~ ( A - T )(0.15 n l NaCl 0.015 M sodium citrate)

+

+

(1

- 101 - 76

+5 +35 +17

257 2-57 257 257 260 260 250 257 268 268

271

$15 5

249

-29

276 294 283 287

+33 +9 +7 5 +2 7

245 270 253 263

-37 -26 - 17 -11

1-2 27 80 1-2 27 80 27 80 27 80

283 283 283 284 284 284 286 284 293 293

25

27 -85 95 27 85

+:33 +26 +6 +25

$10 +6 +21

Jq-ith another small C o t t o r i effect near 290 mp

Peak 2

Trough 1

Temperature (“c) X (mp) [m]X 10-” A (mp) [ m ]X 1 0 P

( [ ? ~ i ] :(peak) ~ ~ =

-21

-46 - 18

U 0

Troiigh 2

4

X (mp)

238 838 238 230 220

- 15

m

- 36

23’2 235

- 17 -45 - 19

U

c1

[m] X l O F A (mp) [nil X

-15 -15 -11 -0 -

-2 +3

212 212 212 213

-90

(94 1

-66 -16 -15

(:/4)

-

-

-

-

218 212

-21

-

-

-

-

-

-

-

-

-

220-30 240-45 236 235

$1

n

-

-

-1 3

-

-

+165 +8 3

-

-

-

-

-23

+9 X lo3 and [ m ] z s s(trough)

=

7,

2 F

E *z z

(24)

-5

224

1:eferenc.e

(96) -

(96a)

(96)

(97)

+8 X lo3). f.3

E

252

JEN TSI YANG

AND

TATSUYA

SAMEJIMA

all have a large peak at 282-286mp and trough at 252-260mp, followed by a small peak near 230-240mp. Their magnitude is reduced by the disruption of the secondary structure ofthe polymers at elevated temperature. Poly A at neutral pH is known to be single-stranded; small-angle X-ray scattering study indicates a molecule consisting of rodlike segments with a linear dimension of 3.4A per nucleotide residue (98). Thus, the melting curve in this case is rather broad (Fig. 7a, inset). The ORD profile of poly A in acid solution is similar to that at neutral pH, but the magnitude of the Cotton effects is larger at pH 4.55than at pH 7.5and a sharp transition occurs in acidic buffer (for instance, 0.1 M sodium acetate plus 0.1 M NaC1) with a T, of about 60"(94, see also 96). This agrees with the X-ray diffraction study of oriented fibers (99) and the small-angle X-ray scattering study (98), which show that poly A possesses a double-stranded helical conformation in acidic solution. The Cotton effects of poly U have a much smaller magnitude (except at 1-2’)than those of poly A. There is no significant change in rotation with heating at temperatures above 20",but a sharp increase in the magnitude of the Cotton effects occurs below 20"(Fig. 7b,inset). These results are in accord with conclusions drawn from optical, hydrodynamic and titration studies; poly U lacks any regular, cooperative structure at room temperature and thereby exhibits little stacking of bases (100). Such helical conformation at low temperature, however, is rather unstable, as witnessed by its low T,. The conclusion of Richards etal.(100) has been challenged by Michelson whose various optical studies of single-stranded poly U and Monny (IOOa), indicate a significant amount of helical structure arising from interplanar interactions. Simpkins and Richards (87’b), however, point out that the existence of a Cotton effect per se cannot be taken as evidence of stacking interaction as implied by Michelson and Monny (IOOa),since UMP also shows a Cotton effect. Simpkins and Richards (87’b, 100b)also attempt to reconcile this disagreement by suggesting that base-stacking in poly U is favored only in high salt solutions, e.g., above 0.5 M NaCI. This is also consistent with the CD evidence of stacking interaction of UpU in 4.7M KF even at 20 C(56). Poly A and poly U are known t.0 form two complexes, polyA poly U and poly A 2 poly U,when appropriate proportions of poly A and poly U respectively (IOI-lO4). are mixed at an A:U molar ratio of 1:1 and 1:2, The Cotton effects of poly A . poly U (Fig. 8a) and poly A . 2 poly U (not shown here) have a magnitude (per base) between t.hat of poly A and Both complexes show a sharp melting curve in agreepoly U (Table 111). ment with their double- and triple-stranded helical conformation, and the variation of the first peak (on the long wavelength side), parallels the

ORD

AND

CD OF NUCLEIC

253

ACIDS

hyperchromicity of t.liese complexes at, eicvated temperatures. The first trough also undergoes a shift t,oward the red with increasing temperat>ure. Comparison of the experimental ORD of poly A . poly IJ with that calpoly U indicates that the latter is located at longer culated from poly A wavelengths than the experimental curve (94).It implies that base pairing in a double-stranded helical conformation causes a shift toward the blue of the ORD of ribosyl polymers (see Section VI, B). Brahms (106, 106)and, more recently, Hashizume and Imahori (107) reported the CD of poly A, poly U,arid their complexes, some of which are also found forpoly A shown in Fig. 8b. Hashizume and Imahori (107) another large positive band at 220mp with a shoulder around 235mp and a negative one near 208mp. They also reported that the melting curves of poly A, poly U, and poly A .poly U were very close to the ORD results found that at pH 4.9, where poly A shown in Figs. 7 and 8.Brahms (105) is double-stranded, the positive band is much enhanced and the negative one reduced; this is accompanied by a blue shift of the CD spectrum. Poly U at neutral pH shows a CD profile very similar to that of poly A, except that its intensity is much less than that of poly A and its variation with temperature is also small (106). The CD of the complex of homopolymers, poly A poly U (Fig. Sb, curve .2), and the random copolymer, poly (A,U)(curve S), are very similar; the slight difference between these two curves may indicate a less ordered structure for the copolymer due to some random distribution of Poly (A,U)also shows a broader melting bases according to Brahms (106). curve (based on hyperchromicity) than poly A . poly U. The CD spectra of these two complexes have the same features, that is, a large positive band at about 262-265mp and a very reduced negative one at about 240-244 mp, whereas the CD bands of their components, poly A poly U, are located on the longer wavelength side (curves i and 4). This again indicates that the formation of the double-stranded helical conformation is accompanied by a blue shift of the spectrum. The CD of the alternating copolymer, poly (A-U),appears to be quite different from that of poly A . poly U (K. Imahori, private communication). One interesting feature in Fig. 8b is that the CD of poly A poly U and poly (A,U)is markedly reduced in intensity and the positive band is poly U (curves 1 and 4).[The broadened as compared with poly A and 4 (at 2’)occurs because poly U difference between curves 1 (at 14-15’) adopts an ordered structure at 2" and its CD is more intense than at Brahms (106) suggested that the antiparallel temperatures above lo0.] arrangement in the double-stranded poly A - poly U and poly (A,U) could account for their low CD intensities. Since the only difference between a parallel and an antiparallel double-stranded helix in this case is

+

-

+

-

+

254

J E N TSI YANG A N D TATSUTA SAMEJIMA

that the orientation of the ribose residucs in the two strands is in the same direction in one case and opposite iii the other, it is hard to believe that this can be the cause of the observed reduction in intensities of the CD spectra. It seems more likely that interactions among bases in adjacent strands in addition to those among stacked bases of the same strands could be responsible for tfhemarked change in optical properties of these polymers.

B. Poly C, Poly G, Poly G Poly C, Poly I, Poly I Poly C, and Poly A 2 Poly 1 Fasman etal.(31) found that the ORD of poly C has a single Cotton Cantor etal. effect between 255and 350 mp (see also Ulbricht etal.(108);

FIG. 9. (a)ORD of poly C at pH 8.4 in 0.05 M sodium phosphate. From Ts o etul.(80). (bj CD and absorption of poly C at pH 4.0 (in 0.1 M NaCl plus 0.05 M acetate) and 7.5(in 0.1 M K F plus 0.01 Af Trisj (Vaj. Solid lines, CD at about 0°C; broken lines, absorption a t 25 C.

(108a)J; Sarkar and Yang (96) extended the measurements to 225 mp and observed another shoulder around 250 mp, thus indicating that poly C also has muItipIe Cotton effects with two peaks a t 293 and 250 mp and one trough at 267mp; more recently, Ts’o et at. (80) reexamined the ORD of poly C to about 200 m p (Fig. 9a). The magnitude of the first peak and trough (on the long wavelength side) for poly C is much larger than that of other polynucleotides, although the melting curve a t neutral p H was very broad and indicative of a structure for the single-stranded polynucleotide not highly ordered. In acid solution, e.g., p H 4.0-4.4, where poly C is believed to be a double-stranded helix (88, 109, 110); the magnitude of the first peak dropped and that of the first trough was enhanced. This is

ORD

AND

225

CD O F NUCLEIC ACIDS

quite different from the p H effect on poly A (Section V, A), where the magnitude of the peak and trough was larger a t p H 4.85 than a t neutral Since protonation of AMP does not alter its absorption spectrum pH (94). very much, although the spectrum of CMP is significantly changed in acid solution, protonation as well as conformational change (to a doublestranded helix) must be responsible for the difference. Sarkar and Yang (111)concluded that protonation of the cytosine base was a t least partially responsible for the reduction in magnitude of ORD for poly C in acid solution, since the same phenomenon has also been observed for DNA's and RNA's even in the absence of any conformational change. The Cottoil effects of poly C are shifted to the longer wavelength upon acidification instead of the blue shift characteristic of the formation of double-stranded helices of other polynucleotides such as poly A in acid solution. This must be attributed t o the drastic change in optical properties when cytosine is protonated. The C D and absorption spectrum of poly C at p H 7.5and 4.0 (Fig. 9b) fully substantiate this conclusion. At neutral pH, poly C has only one main positive CD band a t 276 mp, but in acid solution it has one positive band a t 287 mp and a negative one at 266 mp (86, 87a). Poly G was until recently very difficult to prepare; it aggregates easily in aqueous solution. Ulbricht elal.(108) reported that a t neutral pH and a total ionic strength of 0.15, poly G has an ORD with two peaks a t 266 and 222 mp and one trough at about 243 mp (the amplitude of the first Cotton effect on the long wavelength side was +39,400). The ORD changed little as the pH of the solution was lowered to 1,suggesting that the conformation of the polymer survived complete protonation. On the other hand, the ORD at pH 12.2 showed only a single, negative Cotton effect typical of the purine p-mononucleotides, indicating a complete loss of secondary strucobserved similar ORD for poly G a t p H 7.0(in ture. S. K. Arya (96a) 0.1 M NaC1O4 0.001 M cacodylate), except that the two peaks and trough were located at 271,224, and 249 mp (see Table IV), which were The several millimicrons longer than those reported by Ulbricht elal.(108). corresponding C D showed a large positive and a small negative band with extremes a t 260 and 237 mp and another extremely small positive one near 288 mp (9th). Fig'ure 10 shows the ORD of poly C poly C (prepared by synthesizing poly G on a poly C template), which has two peaks a t 276 and 22&230 mp and one trough at 245 mp (96). This polymer complex is believed to be a double-stranded helix and therefore has a very sharp, but irreversible, melting curve with a T, near 90"as contrasted with about 60" for poly A poly U (Fig. Sa). At elevated temperatures, both the peak and trough of the ORD reveal a large red shift together with a reduction in their magnitude. No CD of poly G . poly C is as yet reported. Ihe260 mp absorption band (96). melting of poly I . poly C occurs near 60" and is accompanied by a red shift. Poly A . 2 poly I melts around 40"with a blue shift; furthermore, unlike other polyribonucleotides it has n serond peak larger than its first one at room temperature.

C. Poly d(A-TI, Poly d,A Poly dT, Poly dC, Poly dA, and Poly dT Figure 11 (97) shows the ORD of poly d(A-T), a copolymer of alternating sequence that forms a double-stranded helix, which may be represented as poly d(A-T) . poly d(A-T), through the base-pairing between dA and dT in the opposing strands. Note that the second peak is about twice as large as the first one. This appears t o be true for all double-stranded polydeoxyribonucleotides as well as DNA's (see Section VI,A), whereas the polyribonucleotides always have a second peak close to zero rotat,ion. Poly d(A-T) has a reversible transition with a T, at about 60". Recently, Ts'o etal.(80) examined the ORD of poly d(A-T) from two sources: one synthetic as that used in Fig. 11 and one natural, isolated from Cancerantennarius. The Cotton effects for the two poly d(A-T)'s are very close. Those ofthe synthetic one are essentially the same as those shown in Fig. 11, except for some small differences in magnitude of the peaks and

258

JEN T S I YANG

AND

TATSUYA

SAMEJIMA

FIG.11.ORD of poly d(A-T) at pH 7.4in0.15M KF.Curves: 1,27-60 %’C; J 65 C; and ,!IJ81i"C. From Samejima and Yang (97).

troughs and in the fine splitting of the peaks. These workers also made measurements to about 200 mp and observed another trough and peak below 225 m p . The subtle difference between the natural and synthetic copolymers was the effect of temperature on the magnitude of the first,peak just before the onset of the melting. The synthetic copolymer had a smaller peak a t 282mp than the natural one, but its magnitude increased with t,emperature (between 20’and 50") up to the level of the natural one, whereas the natura1 one was not so sensitive to heating in this temperature range. This phenomenon is probably due to some difference in the secondary struct.ure of these two polymers at room temperature. The natural d(A-T)7L has been shown to be a straight-chain, double helix without branching or hairpin-like structures (118); it also contains about 3% guanine plus cytosine in its base composition, which is unlikely to have a serious effect


259

on conformation and ORD. The synthetic d(A-T),, however, has been known to have branches and hairpin-like structures through intrachain 120). Ts'o et al.(80) attributed the difference in tembase-pairing (119, perature response of the peak at 282inp prior to melting to this dissimilarity in structure of the two copolymers. The ORD of poly dA . poly d T (see Fig. 13,Section V, D), which like its counterpart poly rA . poly rU forms a double-stranded helix ( l d l ) , shows more complicated Cotton effects than that of poly d(A-T) (Fig. 11). At room temperature, the ORD curves of the two kinds of polymers are distinctly different, but above the T , they show much similarity. The T, of poly d(A-T) is also 5" lower than that of poly dA . poly dT (80). Recent X-ray study on fibers indicated that the structure of poly d(A-T), but not 123). Thus, poly dA - poly dT, is similar to that of the B-form of DNA (122, 1 I I I I I I

I I l l I I I

la)

1

20

L "

230

190

40 30

-

-20

-

('I

310

270

u

dA

-40

-

;

-

270

230

190

'.

310

* ,

x

E

u

-

-

-30-

F

350

1 1 1 1 1 1 1 i 1 1 1 1 1 1 1 .

--__

350

-

-4

*.

*

-8 -12

-16

I

b

190

230

270

X hpL)

310

350

FIG.12.ORD of polydeoxyribonucleotides. (a) Poly dC at pH 8.4 in 0.05 $1 sodium phosphate; (b) poly dA at pH 7.3.5 in0.13M XaClOa; ( c )poly dT at p H 7.0 in 0.05 M KaClO,. From Ts'o et al. (80).

260

JEN TSI YANG

AND TATSUYA

SAMEJIMA

the difference in ORD may be due to this difference in helical structure. An alternate interpretation is that the sequential difference in the constituent polynucleotide chains can give rise to a dissimilarity of the exciton splitting of the absorption bands and thereby the difference in ORD. The ORD profile of poly d(G-C) is reported to be more complicated than that of poly d(A-T) (97). The copolymer used, however, was suspected to contain more dG than dC; a reinvestigation of it seems warranted. Ts'o etal.(80) measured the ORD of poly dC, poly dA, and poly dT, and Adler ef al.(87) the ORD of poly dC. The shape of the curve for poly dC (Fig. 12) is quite similar to that of poly C (see Fig. 9),but the magnitude of its Cotton effects is only about 3040% that of the latter. This implies much less stacking interaction for poly dC than for poly C, a conclusion also supported by the finding of less than 1% hyperchromicity when the solution of poly dC is heated from 23 to 90".In acid solution, both the shape and magnitude of the ORD curves for poly dC and poly C are very similar, except that the second peak is positive for poly dC and negative for poly C. The pH of the single-to-double-strand transition at room temperature is 7.2 for poly dC and 5.7for poly C in 0.05 M sodium ions. Thus, the absence of the 2'-hydroxyl group seems to stabilize the helical structure of poly dC in arid solution. Ts'o et al.(80) suggest that the intramolecular hydrogen bonding of the 2'-hydroxyl group to the 2-carbonyl group greatly reduces rotational freedom around the glycosyl bond, thus enhancing the stacking of bases of poly C. The lower stability of the double-stranded helix ofpoly C in acid solution can be explained as follows : (a) the intramolecular hydrogen bonds greatly hinder participation of the 2-carbonyl group in interchain hydrogen bonding; and (b) the dissociation constant of the ribosyl cytosine group is lowered by intramolecular hydrogen bonding. Both effects will tend to lower the transition pH ofpoly C as compared with poly dC. The difference in ORD between poly dA and poly A at neutral pH is very striking (cf. Fig. 7a). The prominent first peak and trough for poly A between 250and 320mp are much reduced in the case ofpoly dA. But in acid solution their ORD curves are very similar, except that the magnitude of the Cotton effects is smaller for poly dA and its first peak shows a blue shift of about 10 mp. The transition pH for poly dA was about 1.5units lower than that required by poly A, suggesting that the poly dA helix in acid solution is less stable than that of poly A (80). This may be ascribed to the intramolecular hydrogen bonding of the 2'-hydroxyl group to the base, which enhances the base stacking of poly A as compared with poly dA. The ORD's of poly dT and poly U are much the same at room teniperature and in the absence of Mg*+ (cf. Fig. 7b). But unlike poly U, the ORD ofpol9dT in solutions containing Mg2+ was found to be insensitive

ORD

AND

CD OF NUCLEIC

26 1

ACIDS

to temperatux siid to lack :my tr:nwition at low tempertbture, thus indicating little stacking interaction arid secondary structure (80).

D. Hybrids of Polyribonucleotides and Polydeoxyribonucleotides The observation that the ORD of double-stranded polydeoxyribonucleotides always has a much larger second peak than the first one and the has led Ts’o el aE.(80) t o opposite is true for polyribonucleotides (10) examine the hybrids of polyribonucleotides and polydeoxyribonucleotides. Figure 13 includes the ORD of all four permutative pairs of 1:1 mixtures of poly r h. poly dT, poIy dA ’ poly dU, poly rA poly rU and poly dA poly dT (the last two are included for the sake of comparison). The positions of the peaks and troughs of these curves are roughly the same, but their magnitude is quite different (poly dA . poly d T also has more than one peak in the 260-290mp region). The two hybrids aIso have sharp 1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

I

I

1

I.,

20

.

I

15

10

5

0

5

-5

x

T

E u

-10

-15

-20

- 25 1

1

1

1

1

1

I

A (mp)

FIG.13. ORD of ( 1 )polyrA . polyrU,(2)polydA . polyrU,(31polyrA . polydT, (4)poly dA * poly dT (enzymatic) in0.05 M NaCI04at 20°C. FromTs’o etul. (SO).

and

262

JEN TSI YANG

AND

TATSUYA

SAMEJIMA

melting curves with T,’sof 41.5" for poly dA . poly rU and 59"for poly rA . poly d T in 0.05 M NaC104 (pH 7.0), as compared with 51"for poly rA poly rU and 61.5" for poly dA . poly dT under the same conditions. We have already mentioned that the ORD of double-stranded deoxyribosyl complexes has a second peak much larger than the first one. But the formation of the hybrids brings an increase in the first peak as compared to the original deoxyribosyl complex and a decrease in the second peak as compared to the original ribosyl complex, resulting in an ORD profile approaching that of the double-stranded rihosyl complex. This phenomenon cannot be attributed to the rotatory cont,ributions of ribose and deoxyribose and these differences must reflect different types of helical structure (see Section VI, C for further details).

-

VI. DNA and RNA A. Similarities and Differences between DNA and RNA Accurate ORD results of nucleic acids have been accumulating since 124-127). DNA and RNA from vari1963 (see, for example, 10,32,59,97, ous sources have similar ORD profiles of two peaks and one trough centered around the 260-mp absorption band, as typified by the examples shown in Fig. 14.Table V lists the pertinent numerical values of several DNA's and RNA's. ForDNA, the first peak on the long wavelength side occurs at 290mp, the first trough near the absorption maximum, and the second peak around 230 mp. These positions may vary a few millimicrons among the DNA's studied, but the general shape has been predicted by the theory of Tinoco (see Section VI, C). When measurements are extended below 220mp, a second trough is observed near 215mp and a third peak, much larger than the first one, can be located near 200mp. The ORD of RNA also has three peaks and two troughs between 190 and 300 mp, but its first peak is centered near 280 mp, about 10 mp less than that of DNA. The first trough again can be found near the 260-mp absorption maximum. The most striking difference between DNA and RNA is the relative magnitude of the first two peaks; invariably, DNA has a second peak that is about twice as large as the first one (on the long wavelength side), the reverse being true for RNA, whose second peak is always very small and has a rotation very near zero. An explanation for this dissimilarity between DNA and RNA is given in Section VI, C. Brahms and his co-workers (11, 14,86,106,106) reported in detail the CD of nucleic acids and polynucleotides. Their measurements were, however, limited to wavelengths above 220-230mp. They found a positive band on the long wavelength side and a negative one immediately following

OltD AND

CD OF NUCLEIC ACIDS

263

it on the short side with a crossover near the absorption maximum. This is true for a wide variety of nucleic acids, irrespective of their origin. However, DNA has two almost equally large (in magnitude) CD bands with a maximum a t about 272 mp and a minimum near 245mp. In contrast, RNA has a very large positive band centered at 265 mp, several millimicrons shorter than that for DNA, and an extremely small negative one around 252mp, a few millimicrons longer than that for DNA. It should be noted also that the intensit,y of the first positive band is stronger in RNA than in DNA. When measurements are extended below 220 mp, both DNA and RNA ?how additional negative arid positive CD hands (Fig. 14), which are undoubtedly related to the absorption h:Lnd near 200mp.

TABLE V THE COTTON EFFECTS OF NUCLEICACIDS".* T,

Substance or source DNA 1. Aerobacter aerogenes 2. Bacillus megaterium 3. Esckichia coli 4. M~cobacterium tuberculosis

5. Serratia marceseens

6. T2 phage 7. Calfthymus 8. Salmon sperm

9. Poly d(A-T) 10. Poly dG * poly dC

Peak 1 Temperature ("C) X (mp) [a]

27 96 27 87 27 90 27 96 27 96 27 90 27 96 27 96 27 85 27 90

290 292 290 292 290 292 290 292 290 292 290 292 290 292 290 292 283 287 310 320

+2310 t-1200 +1820 +790 +2200 1080 4-2610 +I260 4-2410 +1170 +1380 +770 +1910

27 90 27 90 27 90

280 292 283 290 280 290

+3140

+

+850

+1930

+850 +2450 +900 +250 -130

Trough 1

Peak 2

h (m#)

Iul

X (mp)

[ul

257 260 258 260 257 260 256 260 258 260 258 262 257 260 256 262 255 263 285 295

-2920 -2770 -860 -1180 -2230 -2590 -3690 -1380 -2540 -1420 -2410 -2120 -1920 -1980 -2000 -1900 -5780 -3730 -830 -600

225 225 225 225 225 228 225 228 228 228 235 230 228 230 228 228 236 235 264 270

+4650 +615 +4480 +2070 +4200 +1640 +4360 +4130 +1450 +3900 +lo60 +3730 +660 +3920 +950 +5480 +2770 +2520 +I610

251 258 253 258 252 260

-2770 -2040 -3220 1790 -3500 -2580

227 227 225 225 228 228

-160 -750 +240 -120 +120 -750

+870

Trough 2 X

(mr) 217

[PI

Peak 3 A (mp)

[a]

From 290 Literature mp peak value ("C) ("C)

+2980

-

-

215

f3.550

-

-

215

+3090

91

93.5

82

85

-

-

86

90.5

215 215 217

+3390 -1160 +2680

92

97

90

93.5

80

83

84

87

85

87.5

63.

65

-

-

218

+3150

216

f2830

-

-

-

-

215

+2940

-

-

-

-

247 242

-4590 -2190

217 215 215 210 217 210

-1360 -1030 -1430 -1380 -1150 -3100

-

RNB 11. Yeast tRNA 12. Tetrahynena rRNA 13. Rat liver

+800 +3060 +730 +3400 +980

-

Data taken from ref. (87). M K F (pH 7.4), except substance 1, 0.01 M NaC1; substance 3, 0.13 M KF; substance 9, 0.15 M NaCl 10, 0.02 M NaCl 0.01 M phosphate buffer (pH 7.1); substance 12, 0.15 M KF 0.01 M phosphate buffer (pH 7.0). c From 283 m# peak. a

6 Solvents used: 0.15

+

+

+ 0.015 M sodium citrate; substance

ORD AND

265

CD O F N U C L E I C ACIDS

A new negative CD band around 300 mp that had escaped detect>ion by previous workers because of its extremely low intensity (128, 129)has recently been reported. The minimum of this band for DNA is located near 310 mp and its magnitude is about 1/500 of the first maximum; the minimum for RN A is found between 295 and 298 mp, and its magnitude is 1/10 to 1/30 of the first maximum. None of the synthetic polynucleotides or their complexes, however, shows such a negative C D band near 300 mp, with the exception of poly I, which seems to have a very small one. Since the intensity of the first positive band is much stronger than that for nucleic acids, it could be that an adjacent small negative band is submerged and escapes detection. K. Imahori (private communication) has informed us that the CD of poly (A-U) and poly (G,C), but not poly G poly C, did show a small negative band between 290 and 300 mp.

-

1. TEMPERATURE DEPENDENCE

The Cotton effects of the nucleic acids are conformation dependent (Fig. 15). The first and second peaks of the ORD for DNA are obviously temperature-dependent; so is the first peak of RNA (the first trough in both cases seems not very sensitive to temperature). Since the denaturation of DNA is known to be a cooperative phenomenon, the Cotton effects of DNA remain little changed below its T ,and thereafter fall drastically with further increase in temperature. A plot of the rotation at 290 mp, for instance, versus temperature would show a sharp helix-coil transition, which parallels closely the hyperchromicity of the 260-mp absorption band ; indeed, the T ,as determined from both methods agrees very well. RNA, on the other hand, is known to have a broad melting curve; accordingly, its Cotton effects also fall gradually with increasing temperature. It should be noted also that a t elevated temperatures the peak and trough of RNA undergo a red shift of about 2-12 mp. Since the base chromophores in nucleic acids give rise to the observed Cotton effects, the interactions among various bases are expected t o contribute different rotations. Empirically, a simple linear relationship has been found between the rotation at the 290-mp peak and the base composition of DNA in terms of the content of guanine plus cytosine (Fig. 16) (97) : [a]290 =

26.5 X mole percent (G

+ C) + 550

(18)

15.4 X mole percent (G

+ C) + 220

(19)

for native DNA’s and [a1290=

for heat-denatured DNA’s. No comparable C D data on DNA’s are available, hut sudi linear relntionships shoultl cxist . Two notahlc exreptioris in arid poly (l(A-T). The formrr contains hylroxyFig. 10 arcthe T2 phtigc

I

I

I

I

Salmon sperm DNA

I

I

200

250

,

A

I

300

1

Yeast iRNA

,

I

I

350

200

250

I

300 X (mp)

(a) I

I

I

I

I

1

I

I

I

I

I

I

I

42-

o -2

\

-

DNA

- -4I

I

I

2

I I

J

I

I

I

I

6I

4-

2-

0

I

I

I

I

I A (mp)

(b) F1c:. 15a a11db.

I

A

I

350

(

ORD AKD CD O F NUCLEIC

ACIDS

267

mcthylcgtosine which may not necessarily contribute the same rotation as that of cytosine. Poly d(A-T) has a, much larger rotation than that read from the straight line; unlike thc irregular sequence of bases in DNA, the regvlarity of the alternate sequence in poly d(A-T) may have enhanced the Cotton effects and thus the rotation at 290 mp (cf. Fig. 11). Also, poly d(A-T) is an exception in the melting curve reported by Marniur and Doty (130). Equation (18) provides a simple means for determining the base composition of DNA having standard bases, adenine, thymine, guanine, and cytosine. It is complementary to other analytical methods such as chromatography (131), CsCl density gradient (232, I$$), absorbance ratio of 260 t o 280 mp a t pH 3 (134), bromination (135), and melting temperature (130). It has the advantage over the T,-technique in that only a single rotation a t room temperature is measured, thus avoiding the difficulties associated with heating (e.g., turbidity) that often make measurement difficult. OF PROTONATION 2. EFFECT

Although the Cotton effects of nucleic acids as well as polynucleotides have been demonstrated to be conformation dependent, protonation of the nucleoside bases can also change the magnitude of these Cotton effects without disruptbig the secondary structure of the polymers (111). For instance, the rotation at the 290-mp peak for calf thymus and sperm whale DNA's is reduced about 10% when the p H of the solution is lowered from 7 to 4,whereas the second peak near 230 mp and the trough near 260 m p are almost identical a t the two pH's. Further lowering of the p H of the solution t o 3,however, did reduce both the peaks and the trough; the rotation at the 290-mp peak a t p H 3 is about half that a t pH 7.In addition, another small Cotton effect appears near 270 mp, its peak and trough being levorotatory; the rotation of the peak is close to zero. The effect of p H on the multiple Cotton effects is more drastic for RNA than for DNA. For instance, the rotation a t the 280-282 mp peak for several RNA's may decrease about one-half from p H 7 to 4; this is accompanied by a red shift of this peak by several millimicrons. Unlike DNA, however, RNA does not show a new Cotton effect between 265 and 275 mp, FIG.15. (a) ORD ofsalmon sperm D N A and yeast tRNA at pH 7.4in 0.15 AT KF and a t various temperatures. Left, curves 1-4: 27",80", 85", and 96°C. Right, curves 1-4: 27",55",go", and 27"after slow cooling from 90°C. From Samejima and Yang (97). (b) CD ofcalf thymus DNA and tRNA a t p H 7.4and a t various temperatures (14). Upper, DNA in 0.01 M NaCI, 0.01 M Tris, and 0.001 M EDTA: curves 1 ,20 C;2, 45 C;3,heated to boiling temperature for 10-15min and cooled rapidly; and 4,80 C. Lower, RNA in 0.15 M NaCl, 0.01 M Tris, and 0.001 M EDTA: curves 1-4,22",45", 70", and 80°C.

268

JEN ‘PSI YANG

AND

TATSUYA

SAMEJIMA

FIG.16.The relationship between therotation atthe290-mppeakandthe (guanine cytosine) content ofDNA. Symbols: 1, Mycobacterium tuberculo sis; 2, Serratia mrcescens; 3,Aerobacter aerogenes; 4,Escherichia coli; 6,salmon sperm; 6, calfthymus;7, Bacillus ,o megaterium; 8,T2 phage;and 9,poly d(A-T).From SamejimaandYang (97).

+

G + C (mol%)

even at a pH as low as 2.5(32). Difference absorption spectra ofnucleic acids at pH 4 with a pH 7 solution as reference show almost no change at the 260-mp absorption maximum, but a hyperchromic effect occurs around 280-290mp. A similar study of poly d(A-T) shows no such pH effect,but the Cotton effects of poly I . poly C are markedly changed at low pH (111). In Section 111,A we have already mentioned that the Cotton effects ofthe adenine derivatives show little changes even at pH 2,but marked displacement (red shift) of the Cotton effects are observed for the cytosine derivatives upon protonation. It is thus stipulated that protonation of the base cytosine, not adenine, is responsible for the observed changes in ORD. Since DNA is known to be stable at pH 4 (in the presence of salt), this effect of protonation cannot be associated with any gross conformational change of the polymer. The same may be true for RNA’s such as yeast tRNA (111). Similar conclusions have now been reached by Zimmer et al.(136), who

269


studied the protonation of Xtreptonzyces clwysomallus DNA and found that the new Cotton effect for this (G C)-rich DNA began to appear at pH below 4, when the cytosine base is 3040% protonated; the peak at 260262 mp has a positive rotation, and the two troughs are located at 276-279 and 245-247 mp.

+

B.

Single- versus Double-Stranded Structure

The differences in the ORD arid CD of DNA and RNA are an intriguing question, and their origin is still not fully elucidated. Two possibilities may be considered. First, the major difference in chemical composition of the nucleic acids is the constituent sugar, deoxyribose in DNA and ribose in RNA. This, we believe, plays an important role in determining the geometric arrangement of the polynucleotide chains (see Section VI, C). Second, most DNA molecules form double-stranded helices, whereas the RNA molecules are usually single-stranded and they may adopt hairpinlike or loop-like models. Thus, there is a difference in the conformation of nucleic acids that could account for the observed differences in the ORTI and CD profiles. This question can be clarified by studying RNA’s having rice the double-stranded helical conformation such as reovirus R,NA (137), dwarf virus RNA (RDV-RNA) (138, 139), cytoplasmic polyhedral virus RNA ( I @ ) , and the replicative form of MS2 bacteriophage RNA (141). Figure 17illustrates the ORD and CD of RDV-RNA. Evidently, this RNA possesses the same profiles as a single-stranded RNA; the second peak of the ORD is much smaller than the first one (on the long wavelength side) and the negative CD band is extremely small, unlike the ORD and CD of DNA. Thus, we can reasonably rule out the second possibility that singleversus double-stranded conformation of the nucleic acids is responsible for the described differences in the ORD and CD profiles. The intensity of the positive CD band for RDV-RNA in Fig. 17 is much stronger than that of any ot,her nucleic acids studied. This undoubtedly can be attributed to the high degree of base stacking in this polymer molecule. Raising the temperature of the RDV-RNA solution also causes a sharp, irreversible helix-coil transition just as does the “melting” of the double-helical DNA (139). One interesting finding in Fig. 17 is that the extremes of the positive and negative bands are located at 261 and 235 mp, several millimicrons shorter than the corresponding ones for a single-stranded RNA. I t strongly suggests that the formation of a doublestranded helix of RNA results in a blue shift, albeit small, of the CD bands and the corresponding ORD profile. In Section V, A,we showed that the formation of poly A . poly U from the two homopolymer components also results in a blue shift of the CD bands (Fig, Pb). The ORD in this case

270

JEN TSI YANG

I I

I

I

200

250

300

350 200 X (mp)

AND

TATSUYA

I

I

250

300

SAMEJIMA

FIG.17.ORD and CD ofricedwarfvirus RNA in0.01 M SSC solutions at various temperatures (139).Curves: I ,24"; 2,93"; 3,’24 C after slowcooling from93 C.

(Fig. Sa), however, was less clear-cut; the first trough of poly A . poly U was located at a shorter wavelength than either poly A or poly U, but the first peak did not show a blue shift. On the other hand, the ORD of the double-helical poly G . poly C is shifted toward the long wavelength side when the complex is disrupted upon "melting" (see Fig. 10). These results together with the recent theoretical calculations of the ORD of RNA (see Section VI,D) have led Hashizume and Imahori (107) to suggest that the location of the CD band at 261 mp reflects the presence of a perfect double-stranded helical conformation of the RNA molecule. (Note, however, that the crossovers of both poly A . poly U and poly G . poly C are located near 265mp. It remains to be seen whether the exact position of the crossover is an accurate and sensitive indicator for detecting such helical conformation.) These workers further stipulated that the separation of paired bases through the breaking of hydrogen bonds causes a red shift of the CD bands, whereas the unstacking of the bases markedly reduces the int,ensity of the CD bands. Similar conclusions were also reached by Adams et al.( l 4 d who ) , found that the crossover of the ORD of yeast tRNALeuwas shifted from 260.5to 263.3mg and the CD maximum from 260.3to 262.3 m p on going from the biologically active to the inactive form. [The biologically inactive form is considered to have nearly the same amount, of

ORD

AND

CD O F NUCLEIC

ACIDS

271

secondary structure as the active form; for further details, see Fresco et al.(143), Muench (144), Sueoka et al.(145).] ddams et al.(14% at) tributed the observed small shifts to the disruption of several base pairs in the RNA molecule. These shifts are also accompanied by a very small decrease in the rotational strength of the Cotton effects, indicating some degree of unstacking during the conversion of an active molecule to its inactive form. It is, however, not possible at present to decide whether the unpaired bases entered into single-stranded stacking or unstacking conformations. Neither can we speculate how the conformation of other stacked bases is affected by the breaking of a few paired bases. We simply do not have the detailed knowledge about the effect of base composition and sequence of RNA on the position, shape, and magnitude of its CD and ORD. But it is clear that both techniques are potentially very powerful tools for studying the fine details of the RNA conformation in solution.

C. Base

Tilting

The Cotton effects of the derivatives of each purine or pyrimidine are very similar, whether the constituent sugar is ribose or deoxyribose (Section 111,A). But data on polynucleotides as well as nucleic acids clearly demonstrate the pronounced effect of the 2’-hydroxyl group of the pentose on the ORD and CD of these polymers. We are led to believe that differences in conformation of the polyribonucleotides and polydeoxyribonucleotides rather than the configuration of the 2’-carbon atom of the pentose give rise to the striking differences in the Cotton effects of ribosyl and deoxyribosyl polymers. Since we can eliminate the explanation based on singleversus double-stranded structure, the alternate possibility is then the difference in geometry of the stacked bases between DNA and RNA. It may be instructive to consider the known structures of the double-stranded nucleic acids. Figure 18 illustrates the helical models of the A- and B-form of DNA, RNA, Table VI lists the pertinent parameters and the hybrid DNA.RNA (146). for all four models. Clearly, the mode of stacking is quite different for the A- and B-form of DNA. Furthermore, the sugar rings in the A-form are incorporated into the main helical chain, but those in the B-form are situated radially from the helical axis. Another salient feature is that the pIanes of the base pairs are almost perpendicular to the helical axis for the B-form, but tilted by about 20 degrees for the A-form. The distance between the helical axis and the paired bases also differ in the two forms (see Table VI).Of particular interest is the finding that the structure of double-stranded RNA or of DNA.RNA hybrids is very similar to that of the A-form of DNA. The replacment of thymine in DNA by uracil in RNA has no influence on the molecular structure, since the DNA from PBS

272

JEN TSI YANG

AND

TATSUYA

SAMEJIMA

. ,

I

I IA)

(8)

(Cl

IDl

FIG. 18.Doublestranded helical modelof nucleic acids. (A) A-form of DNA; (B)B-form of DNA; (C) RNA; (D)hybridof DNA and RNA. Symbols:0 , oxygen; 0 , phosphorus; 0 , nitrogen; 0, basepair. From Tsuboiand Higuchi(146).

2 bacteriophage, which contains uracil has the same structure as normal DNA’s (151). Indeed, model building of the RNA molecule shows the interference of the Z'-hydroxyl group of the ribose resulting in formation of a double helix similar to the B-form of DNA. The successive bases in this case are tilted or twisted (that is, the base pairs are not coplanar) to accommodate such steric hindrance. We therefore suggest that the mode of base stackmgs, the orientation of the sugar rings, and the orientation of the base pairs with respect to the helical axis are responsible for the observed differences in ORD and CD between DNA and RNA. [Atpresent the

ORD AND

CD OF NUCLEIC

273

ACIDS

TABLE VI THEHELICALSTRUCTUREOF DOUBLE-STRAKDED NUCLEIC ACIDS* DNA Measurement Pitch ofhelix Number of residues per turn Inclination of base pair from horizontal line Distance between axis of helix and base pair Orientation of PO;: (a) Angle between axis ofhelix and 0-- - -0 line (b) Angle between lhe helical axis and the bisector of angle L OPO

A-forms 28.2A 11 ’LO"

4 i

55"

B-ford

3 3 . 7 A 30.5A 10 or 11 10 2"

15"

0

4-5 K

.55"

3 65"

3 65"

li S&

DNAdlKA hybridsd 28 A 11

20"

4A

70"

6606(in DSA

40"

chain, 65"; in RNA chain, 70") 55"e(in DNA chain, 70"; in RS.4 chain, 40")

____

*From Tsuboi and Higuchi (146). a Fuller etat. (147); Sutherland and Tsuboi (14Ta). Langridge etal.(148, 1.68~). Sat0 etaf (158); Fuller P t al.(148); Arnott etal.( 1 4 9 ~ ) . Milman etal.(150); Higuchi eta/.( 1 6 0 ~ ) . e These values are the effective orientation by superimposing the orientation ofboth the DNA and RNA chains of the hybrids.

exact dimension of the double-stranded RNA remains somewhat uncertain, that is, whether it, has 10 or 11 base pairs per turn. The RNA helix in solution is even Iikely to have 12 base pairs per turn and, therefore, less tilting, probably 10 degrees, than that listed in Table VI (P. 0. P. Ts'o, private communication). This will not alter our working hypotheses (see below) .I We further speculate that the same rules apply to single-stranded as well as double-stranded polymers. Our working hypotheses for nucleic acids in aqueous solution can be summarized as follows (poly I is excluded in this discussion since it has a n inverse profiIe of the Cotton effects; see Section V, B). 1. The relative magnitudes of the two peaks of ORD on the long wavelength side and of the corresponding positive and negative CD bands of polynucleotides as well as nucleic acids are primarily determined by the geometry of the stacked bases, which in turn is influenced by the presence or absence of the 2'-hydroxyl group on the pentose. 2.T he stacking of bases perpendicular to the helical axis would lead to a higher second peak (near 225-230nip) of O R D and a large negative CD h n d as in DNA. Tilting of the Imes woultl reduce the second peak nntl

274

JEN TSI YANG AND TATSUYA SAMEJIMA

increase the first one, and decrease the negative and increase the positive CD band as in RNA. 3.The deoxyribosyl polymers can have stacked bases perpendicular to the helical axis or tilted, whereas the stacked bases of the ribosyl polymers or DNA.RNA hybrids are always tilted. 4. For any polynucleotide or nucleic acid, the magnitude of the Cotton effects decreases with unstacking of the bases (e.g., at elevated temperature). A large change in the magnitude indicates a stacking-unstacking process, but a small change does not necessarily preclude the presence of stacked bases. 5 . Protonation or deprotoriation of the bascs can change the magnitude of the Cotton effects, even though the stacking of bases may remain unchanged. 6. For RNA and ribosyl polymers, the formation of base-pairs leads to a significant blue shift of the ORD and CD. In contrast, only a very small blue shift could be detected for the formation of a double helix of DNA (see Table V). Brahms and Mommnerts (14 reported ) an intermediate form of DNA in 80% ethanol at low salt concentration and between 40 and 55 Cwhich differs from both native and completely denatured DNA. The CD in this case resembles that of RNA; the positive rotational strength of this intermediate form was about three times that of native DNA and the negative CD band almost completely disappeared. It is highly tempting to suggest that this intermediate form has tilted bases instead of bases perpendicular to the helical axis. Similarly, we found that poly dT and poly dC can have an ORD similar to that of a ribosyl polymer, but no ribosyl polymer has been found to have an ORD profile of the B-form of DNA. Ts’o et al.(80) have also discussed in detail the large influence of the 2’-hydroxyl group of ribose on the conformation and helix-coil transition of polydeoxyribonucleotides and polyribonucleotides. They explain the differences in ORD between these two classes of polymers on the hypothesis that the 2 hydroxyl group forms hydrogen bonds with the 2 keto group of cytosine and uracil and with the N-3of the adenine in the polymer, even though the predominance of such hydrogen-bonded structure of ribosyl polymers in solution has not yet been demonstrated experimentally. Ts’o et al.(80) further stipulated that the stack of the bases of the ribosyl helix is morc oblique and that of the deoxyribosyl helix more parallel. Ultimately, our understanding of the dissimilaritiesof the Cotton effects of nucleic acids must depend on advances in theoretical calculations. But a qualitative description here may suffice for our purpose. The exciton t,heory of optical activity (15-17, 73)prellicts that the m r * transitions in a helical molecule such as DNA produces many overlapping 0hands arid the

ORD AND

CD O F NUCLEIC

275

ACIDS

resultant spectrum shows two nearly equal CD bands of opposite signs with :I crossover at a wavelength of maximum absorption. The opposite rotat,ionalstrengths sum to zero and the spectrum is therefore conservative. The corresponding ORD as calculated from the Kronig-Kramers relationship [Eq. (Sa)] shows two peaks and one trough (or two troughs and one peak) with the trough (or peak) located near the wavelength of maximum absorption. Qualitatively, this is in good agreement, with the experimental CD and ORD ofDNA (Fig. 14). In contrast, the CD of RNA does not appear in pairs of nearly equal positive and negative bands and the spectrum is t,herefore nonconservative over the range of wavelength studied (above 220 mp). Bush and Brahms (77)examined Tinoco’s general theory of and concluded that. interactions of the CD bands in optical activity (152) the region of 200 and 300mp with far-ultraviolet transitions can give rise to a CD of t,he nonconservative type such as found in RNA. They further suggested that base tilting cannot be expected to give large nonconservative CD bands, which of course is contrary to our speculations mentioned above. Bush and Brahms considered the striking difference between the CD of DNA and RNA to be due t,o some geometrical factors and they hypothesized that formation of a double-stranded structure would be reflected in changes of spectra and in t,heappearance of a conservative type of CD such as in DNA. This explmation, however, cannot, be applied to the CD of double-helical RNA, for instance. Thus, the matter is still an open question and must await more detailed theoretical calculations. has now refined his exciton theory by taking into conTinoco (253) sideration both the conservative and nonconservative types of spectra. The theory can be qualitatively described as follows. For a polymer array of N identical monomer units the singlc electronic transition of each monomer will be combined into N polymer transitions, that is, N absorption bands at N frequencies for each band in the monomer. If the bands in the polymer are not too shifted relative to the monomer band, the optical properties of the polymer can be represented as t~ weighted sum of the monomer absorpt,ion band plus its first derivative, second derivative, ete. For simplicity the second derivat,ive and higher derivatives will be ignored. Thus, the CD of the polymer at any given frequency, can be written as CL

-

+ bz(d~i\l/du).

CR = ( X ~ C ~ I

(20)

Here EM represents the molar absorptivity of a single electronic transition of the monomer at frequency u. The coefficient al characterizes the interactions among different absorption bands of the monomers (nonconservative type) and the coefficient b2of the same band in all identical monomers of the polymer molecule (conservative type). The corresponding molar rotation at a.ny wavelength can he obtained from Eq. (20) by using the

270

JEN TSI YANG

AND TATSU’I-A SAMEJIMA

Kronig-Kramers transform. Figure 19 illustrates the CD 011 the left-hand side and ORD on the right for a modified Gaussian band (curves 1 and l a ) and its negative derivative (curves 2 and Sa). The Cotton effects of the polymer can have the shape of these characteristic curves with either sign In or any combination of these curves (curve 1 f 2 ; curve l af.%’a>. general, both the conservative and nonconservative types will contribute to the CD and ORD and their contributions are very dependent on the geometry of the molecule. Our speculation is simply that the nonconservative contributions are small for a helix having bases perpendicular t o the helical axis such as the B-form of DNA. The above general idea can be applied to real polymers; heteropolynucleotides can be repreeented by an average effective monomer, and double-stranded polymers can be treated as a single-stranded polymer of monomeric units having two transitions (for a paired base). Native DNA's are represented mainly by curves d and 2u,and the RNA's characterized mostly by curve 1 2 and curves l a 2u.

+

+

t 0 c

0 - aa l -

U

> L

0 C

c

+

+ -e

.-

0 5

- a 0

+ 0

-

1(mpl

Fro.19. Theoretical CD (left) and ORD (right) cwves (155). The top two curves are the characteristic curves for a modified Gaussian band and its first derivative which combine to give all others. Each curve can be positive or negative.

ORD

AND

CD O F NUCLEIC

277

ACIDS

The exciton theory deals with the T-T* transitions; the existence of an n-r* transition in nucleic acids remains unsettled. We merely wish to suggest that the small negative CD band found near 300 mp (Fig. 14) might be attributed to this n-r* transition.

D. Calculation of the ORD of RNA Cantor e2 al.(10812) recently extended the nearest-neighbor formalism (Section IV,B) to take into account the double-stranded conformation, using an equat,ion analogous to Eq. (15): (21)

The summatioris are carried over the base sequence of oxily one of the two strands, and the choice of the strand is irrelevant. Here [m&J is the molar rotation per base of the double-stranded dinier containing the sequence NpN' and its antiparallel complement and [m:] the molar rotation per base of the double-stranded monomer. With the approximations of random nearest-neighbor frequencies, identical base composition of both strands, and separable effects of the single- arid double-stranded interactions, Eq. (21)can instead be written as (cf. 836): [WLRNA]

+

= [?n1]/2 f [ w . L ] / ~

+

X ~ ~ ~ A T ~ Z - A UX&’~A?~-GC

+

XAUXGC2Am--Au/GC

(22)

Here [ml] arid [ m ~are ] the mean residue rotations of the two single strands; the 2Am's are the additional contributions of two sequential A U pairs and G C pairs, and the average interactions of all the remaining possible sequential base-pairing combinations; the X A U and XGC are the mole fractions ofthe A . U and G . C pairs. In other words, the three 2Am terms are the increments in rotation caused by the interstrand interactions, due to the G . C and A U hydrogen bondings, with respect to the curves calculated from the single-stranded base-stacking model. They can be roughly estimated from the data on double-helical polynucleotides (94,96'), that is,

-

-

2 0-AU~ = ~ [mvOiy A . poiy u] -

and 2Arn-cc =

[mpoiy G

-

poty

cl -

([?))poiy A]

-b

[mPoiy u])/2

GI + [ m p o lc~I ) / ~

([mpoiy

(23)

(24)

Cantor etal.(10812)calculated the [ m p o l yaccording ~] to Eq. (17), since no experimental data were available for single-stranded poly G. The term Am-AUlGC is assumed to be the average of AW~-AU and A m - G C , since no experimental estimate is :tvailshle. This of course is a very rough estimate and is the best we can do at present.

278

JEN TSI YANG

AND

TATSUYA

SAMEJIMA

Using the known sequence of yeast alanine tRNA (154), Cantor etal. (208a) constructed a model of this polymer with a maximum number of antiparallel A . U and G . C pairs but without unstacking any bases in the single-stranded polynucleotide chain. They were able to calculate and almost reproduce the peak, trough, and crossover of the experimental ORD, but the magnitude of the calculated rotations was much larger than the observed ORD, due undoubtedly to the approximations and assumptions used in such computation. Vournakis and Scheraga (127) also used the ) calculated the ORD for the possible method of Cantor et al.( 1 0 8 ~ and conformation of both alanine tRNA and tyrosine tRNA previously proand Madison et al.(155). Their results were posed by Holley etal.(254) . two slightly different very similar to those of Cantor ef al.( 1 0 8 ~ )That models of alanine tRNA gave equally satisfactory agreement between experimental and calculated curves suggests a degree of uncertainty in predicting the correct model for the RNA molecule. It seems therefore premature to conclude that the crude calculations support the conformation also ) of any particular chosen model for the tRNA. Cantor el al.( 2 0 8 ~ found that the calculated ORD of the base-pairing model for alanine tRNA is shifted to the shorter wavelength side when compared with that based on a single-stranded RNA without base pairings, a finding in agreement with experimental observations (see Section VI, B). McMullen et al.(156) have also applied the method of matrix rank analysis to a large body of experimental ORD data to determine and identify the minimum number of independent components contained within it. They found the ORD of tobacco mosaic virus RNA to be a superposition of only two basic spectra of the single- and double-stranded helical forms of the molecule. That leads to a direct calculation of the percent composition of the double strands at any of the conditions considered, if a structural model in terms of an equilibrium between the two forms is postulated. This method of analysis seems to be powerful and of wide applicability, since it is independent of the source of data. But it has the disadvantage of yielding only the shape of the spectral components and variations in amplitude are specifically ignored in the reduction process. It is therefore necessary to find the molar rotation at any given set of conditions for the two forms. The method of caIcuIation described in this section should be equally applicable to the CD of RNA, but no comparable detailed experimental data on the 16 dinucleoside phosphates and poly G . poly C exist at present. Theoretical calculations of the ORD and CD of DNA have also not yet been tried. Comparison of the ORD results in Sections V and VI shows that the magnitude of the Cotton effects of synthetic polyribonucleotides is much larger than that of RNA. This cannot be attributed to the imperfect base

ORD AKD

CD O F NUCLEIC ACIDS

279

pairing in RNA as compared with poly A . poly U, for instance, since a t elevated temperature where the secondary structure of the polymers is disrupted the first peak of the ORD of polynucleotides is still higher than have suggested that the two that of RNA. Brahms and Mommaerts (14) intertwined double-stranded helical structure of poIynucleotides such as poly A . poly U and poly A in acid solution may be parallel as contrasted with the antiparallel base pairings in RNA. Accordingly, the rotational strengths of the base pairs are assumed to he additive in the former case and subtractive in the case of RNA. This suggestion, however, fails to account for the findings that poly U, for instance, is known to have little ordered structure above 10” and it still has a higher first peak of its ORD than do most RNA’s. Rather, we suspect that the difference arises from the regularities in base sequences. It is quite possible that the interactions among an array of identical bases or bases of alternating sequence differ significantly from those of mixed bases of irregular sequence. This hypothesis seems also to apply to polydeoxyribonucleotides; for instance, poly d(A-T) has larger Cotton effects than those expected for DNA (see Figs. 11 and 15a). It is contended that the regularity of the base sequence would enhance the Cotton effects as contrasted wit,h the irregular sequence found in nucleic acids. I n view of this difference in rotation, estimation of the amount of secondary structure in RNA using synthetic polynucleotides as model compounds seems very uncertain. Vournakis and Scheraga (I%?),however, suggested that the amount of G . C hydrogen bondings in a RNA molecule can be made by comparing the mean residue rotation at 276mp with that of the 276mp peak of poly G . poly C, neglecting the small contributions due to the A . U base-pairings. They recognized that the lack of adequate model compound data and calculation techniques made possible only crude ) a different approach to the estimates at best. Cotter etal.( 1 5 6 ~suggest calculation of ORD for RNA. These workers question the correctness of Eqs. 23 and 24,because they are based implicitly on the single-stranded states of homopolymers, which are unlike the RNA molecule with its “random” sequence of bases. Instead, Cotter etal.derive the composition of the base-paired part from thermal difference absorption spectra. The rotations for the unpaired part are then calculated from Eq. (15) using shndard dinucleoside phosphate data and assuming a random sequence of the appropriate base composition of RNA, as applied by Cantor et al. (108a). However, the rotations for the double-stranded part are still obtained from the data of Sarkar and Yang for poly A . poly U and poly G poly C (94,96),which differ from the double-stranded RNA of a “random” sequence, thus making such calculations somewhat uncertain. Suffice it to say, any quantitative interpretation of the ORD of RNA should presently

280

JEN TSI YANG

AND

TATSUYA

SAMEJIMA

be viewed with reservations. In this respect),the use of a double-helical RNA is perhaps a more realistic as a model [see Hashizume and Imahori (lor)] approach and worth further investigation. Even here we have to bear in mind that structural possibilities with many base-pairing arrangements other than those proposed by Watson and Crick cannot be overlooked;4 furthermore, structures with more than two strands are also possible.* These are the challenges that must be met if ORD can be used for such calculations. The very nature of any empirical analysis cautions us against overenthusiasm toward its quantitative interpretations, at. least at this stage of development,.

VII. Complexes of Nucleic Acids A. Ribosomes, Viruses, and Deoxyribonucleoproteins Nucleic acids are known to form complexes with a variety of substances, from small molecules, such as metallic ions, to macromolecules, such aa proteins. The study of the optical properties of these complexes might enable us to elucidate the interactions and the conformational changes, if any, that occur when these constituents are incorporated into the complexes. Blake and Peacocke (157)initiated the ORD study (above 230mp) of rabbit reticulocyte ribosomes, and their isolated constituents, rRNA, and soluble proteins. Examination of the Cotton effects and also the rotation of RNA at a given wavelength led to the conclusion that the secondary structure of RNA in mammalian ribosomes is the same as the one that the isolated RNA molecule possesses in solution. McPhie and see also Gratzer, 1,5959) studied the ORD of ribosomes from Gratzer (158; coli as well as rabbit reticulocyte. Quantitatively, the yeast and Escherichia results of the three species were very similar. Dissociation of the yeast ribosomes and their disorganization by high concentrations of chelating agent essentially had no effect on the ORD results. Sarkar and Yang ( l a gsee ; also Sarkar et al., 160) investigated both the ORD and CD of Eseherichia coliribosomes (70S ) ,their two basic subunits (the 30 S and 50 S particles), and the corresponding 16 S and 23 S RNA's and found no notable difference in the mixtures of 70 S, 50 S, and 30 S particles and also no difference in mixtures of 50 S and 30 S particles. This implies the absence of any conformational change when the 30 S and 50 S particles associate to form the active 70 S ribosomes. Figure 20 summarizes the results for the subunits of Escherichia coliand their corresponding RNA moieties. Since the ribosomal proteins have no CD bands above 250mp, the portion of the spectrum in this wavelength range approximates that of 4

See article by Pullman arid Pullman in this volume.

ORD

AND

CD OF NUCLEIC

281

ACIDS

A (rnpL1

(rnFLJ 250

300

I

I

200

250

300

I

I

3

1 P)

+ '0

3 2

-25 4

4

2 P)

+ 'Q

0 2

-9

2

X (mp)

A (mp)

FIG.20. CD (upperpart)and ORD (lower part) of 30 S arid 50 S Escherichia coli ribosomal subunittr and their corresponding RNA moieties (139).Allsolvents are made up of 0.005 M Tris, 0.0005 M MgZC,and 0.1 M KC1.

the corresponding RNA's, which is about 63% of the ribosomes. Significant difference in the spectrum between ribosomal subunits and the RNA moieties, however, occurred below 250mp, where the contributions of the peptide chromophores become prominent. While the 23S RNA, for instance, displays a small positive CD band at about 230mp, the ribosomal subunit actually shows a shoulder of a negative CD band and also a negative shoulder in ORD near 230 and 235mp. It is now well recognized that the a-helical (right-hand) polypeptides show three CD bands between 185and 250mp, one positive with a maximum at 191 mp and two negative with a and the corresponding ORD double minimum at 210and 222mp (161,162), has a trough at 233mp and a peak at 198m p (16.2, 163). Thus, the results in Fig. 20 strongly suggest the presence of helical segment,sin the ribosomal

282

JEN TSI YANG

AND TATSUYA

SAMEJIMA

protein moieties. Analyses ofthe difference ORD and CD spectra between ribosomal subunits and their RNA moieties indicated that the ribosomal proteins, at least in thecase of 50 S particles, are partially a-helical (about 20-25%).The portion of the isolated ribosomal proteins of Escherichia coli that could be solubiIized was also found to have about the same amount of helical content. On the other hand, the protein moiety of the yeast ribosomal proteins contained about 30% a-helix, but once isolated from the RNA moiety the sdubfe portion of the proteins had no detectable a-helical content and was presumably denatured (158). THE COTTON Peak 1

TABLE VII EFFECTSOF VIRUSES(166) -

Trough 1

Peak 2

Trough 2

Class 10

T2 T2(gt) T4 T6 T6(gt)

x,

290 290 290 290 290 290

270 270 27 1 272 270 270

-3960 -3000 -4050 -4650 -3650 -3620

240

240 240

2280 2060 1680 2350 2430 2240

620 771 627 825 640

255 265 260 260 258

-1820 -2360 -2580 -2240 -2200

230 240 237.5 225 240

3200 630 238 1320 180

520 4840 4030 3450

260 252 255 257

-3660 -7860 -8000 -5800

245 -

-1180 -200 -1500 -1700 -1370 -480

237.5 240 241

Class I1

T5 Tli B3 X(C)

MK)

290 290 295 290 290

Class 111 292.5 +X1?4 290 MS2 285 f2 285 R17 ViralDNA and RNA 290 a T2 DNA 290 X(C) 290 DNAb 290 DNAb ax174 290 DNA T7DNAb 290 R17RNA 285 0

-2350 235 - 235 - 232 235

2780 258 1280 260 2230 (2140) 258

-1910 236 -2100 230 -1920 228

2730 4480 4380

2210 (2140) 258

-1540

228

4290

1920

-2180

230

2870

260

4050 -2690 225 2090 (20%) 258 4070 252.5 -4100 227.5 -1070 217

-4290 -8860 -11800 -10100

- 1780

These viruses have no peak at 290 mp. yt = nonglucosylated. The values in parentheses we the rotations at 290 m,u computed from Eq. (18).

ORD

AND

CD OF NUCLEIC

283

ACIDS

Maestre and Tinoco (164, 165)attempted to elucidate the internal structure of the nucleic acids and their relation to the protein coats in Each virus has viruses; they measured the ORD of 1 G viruses (Table VII). a characteristic ORD curve, by which it can be easily distinguished even in the cases of very closely related phages. The general profiles (Fig. 21)can be classified into three types: (I) t,he T-even class has levorotations above 250 mp with a trough at about 270 mp, although its ORD retains the two-peak-and-one-trough shape of nucleic acids; (11)the T-odd class has an ORD very similar to that of nucleic acids (Section VI) with a peak near 290 mp and a trough near 260mp; (111) the third class shows a characteristic deep trough near 235 m p in ORD caused by a high percentage of ;i3 5

2

c 0

0

1

41 0 E -1

-$ - 2 T)

no- - 3 2-4 U

Y

1

'

"

'

MS2

-

\

-

-

l

220

i

l

240

,

l

L

[

280

260

,

I

300

,

I

,

320

XCmP) CC)

FIG.21.ORD of (a) T-evenphages, (13) T-odd phages, and (c)viruses containing a large contribution from theprotein coat(166).

284

JEN TSI YANG

AND

TATSUYA

SAMEJIMA

protein. The rotations of the nucleic acid and protein moieties were found to be nonadditive, that is, the ORD of the intact phage cannot be explained as the simple sum of the ORD of isolated protein coat (virus ghost) and nucleic acid. On the other hand, the ORD of osmotically shocked T2 phage can be identical, within experimental errors, with that of the sum of the purified components. Since osmotic shock releases the nucleic acid from the head of the phage, but leaves the protein coat essentially intact, the difference in ORD between intact and osmotically shocked phage must be the result of the packing of the DNA molecule inside the head of the phage. + relemedDNA, Furthermore, this difference in rotations, [aIphage- [aIgho8t shows qualitative similarities among all the DNA phages tested; all displayed a negative Cotton effecttwith a trough between 280 and 290 mp (the low wavelength regions, however, were obscured by the rotation of the protein). This implies that the packing of the DNA molecule inside the suggested, as virus has a common characteristic. Maestre and Tinoco (166) did Pollard (166) and Tikchonenko et al.(167), that the DNA molecule inside a virus is in a dehydrated state that altered the stacking structure of the bases with the resultant change in optical properties. In support of this contention, lowering the activity of water by using a high LiCl concentration does alter the ORD of the T2 and T7 phages, for instance, and the difference ORD between normal DNA and that in high salt concentration is very similar t o that between intact and osmotically shocked phages. We note, however, that the ORD of T2 phage in 24.4% LiCl (pH 7.4) (165) still retains a larger second peak than the first one characteristic of all normal DNA’s. If our hypothesis about the base tilting versus base perpendicular to the helical axis (Section VI, C) is valid, then the DNA molecule inside the viruses may still have a structure similar to the B-form. It could be that other types of geometrical change that have escaped our detection are involved in the condensation of the DNA molecule into the compact package of internal DNA. Maestre and Tinoco (164) also found a linear relationship between the specific rotation at the 290-mp peak (based on the concentration of DNA) and the percentage by weight of DNA in each phage after corrections were made for the rotations due to the glucosylation of the 5-hydroxymethyl cytosine of the T-even phage family (Fig. 22). The deviation A[LY]~~,, for T-even phages from the straight line (Fig. 22) was corrected by assuming that T2, T4, and T6 were 70, 100, and 147% glucosylated (168). The correlation shown in Fig. 22 is also consistent with the hypothesis that the main influence on the ORD of a polymer is the iriteractioii among the polymer units, and the differences in rotations for the different phages mirror differences in conformation of the DNA molecule inside the phage (164). Thus, a high DNA concentration in the phage should lead to a

285

ORD AND CD O F NUCLEIC ACIDS I

I T 7

1

T-6

I

30

I

I

* I

40 50 60 Weight % DNA in bacteriophage

I

I

70

a0

FIG.22.The relationship between the rotation at the 2 9 0 - upeak (based on the concentration ofthe DNA moiety) and the weight percent of DNA in the phages (164).

conformation of DNA different from that in solution. However, an entirely satisfactory explanation for these results is still lacking, because extrapolation of the straight line in Fig. 22 to zero DNA content (presumably approximating free DNA in solut,ion) leads to an [ c Y ] ~ ~higher o than any measured in solution or expected from Eq. (18) (ef. Fig. 16). Whatever interpretation for these results is finally established, the ORD is apparently very sensitive to the state of DNA inside the phage and will be very useful in the study of bacteriophages. Maestre and Tinoco (165) have also analyzed the ORD of the T2 ghost (protein coat) using both the bo and methods see refs. 2 and 20) and 233-mp trough methods for proteins (for concluded that the helical content of the phage ghost is quite small (5205Z0). They also found the influence of the protein coat on the rotations above 233 mp (away from the Cotton effects of the protein) due to the whole phage was small compared to that of the nucleic acids. Oriel (169) has studied the ORD of calf thymus deoxyribonucleo-

286

JEN TSI YANG

AND

TATSUYA

SAMEJIMA

protein (DNP). Both the particulate fraction and the soluble DNP showed the same ORD [the particulate fraction is the gellike fraction and amounted to about 15% of the final preparation (170)l. It had a trough at 235 mp in addition to one near 260 mp, typical of the DNA molecule; this second trough can be readily recognized as the characteristic 233-mp trough foran a-helix of proteins. Using the 233-mp trough method, Oriel estimated that particulate DNP, soluble DNP, and the corresponding histones (separated from the DNA molecule in 2 M NaC1) contained about 20% a-helix. This result agreed well with the estimated helical content from the bomethod for acid-extracted histones in 1 M NaCl (171, 272). Thermal denaturation studies indicated that histones associated with the DNA molecule are more stable than those dissociated from DNA, suggesting a strong interaction between the proteins and DNA in DNP (169).

B. Complexes with Small Molecules The binding of dyes to nueleic acids has been studied for two decades with respect to the staining properties of the dyes and their mutagenic properties (88, 90,173-176). This interaction is known to cause a shift in the wavelength ofthe absorption maximum of the bound dyes (177). The binding of acridine orange to DNA (178-182) and of proflavine to DNA and RNA (179,183, 184)induces Cotton effects in the absorption band of studied the ORD and absorption the dyes. Blake and Peacocke (279) spectra of acridine orange bound to native DNA (Fig. 23a). At low DNA

400

500 X (mp)

600

460

500

540

X (mp)

( al (bl FIG. 23.(a)ORD andabsorption spectra ofacridine orange boundto native DNA at, pH 7.0in 0.009 M NaCl plus0.001 M sodiumphosphate at20 C. DNA (phosphorus)/ dye ratios: low ( - - - 1, 28.48 p M to15.68 high(-), 85.44 p M to15.68 pM. Theuppercurveineachpairistheabsorption spectrum. FromBlakeandPeacocke (179). (b)CD and absorption spectra ofacridine orangeboundtonative DNA atpH 6.6. DNA (phosphorus)/dye ratios: (-), 3 and ( - - -), 9 in 0.001 M buffer, and ( . . . ), 15 at0.1ionic strength. The uppercurves areabsorption spectra; (- - -) absorption spectrum of acridine orangein ethanol. Dye concentration: 3 x 10-6If. From Gardnerand Mason (182).

a;

ORD AND

CD OF NUCLEIC

ACIDS

287

(phosphorus) :dye ratios (about 1.8), the intensity of the 292-mp absorption maximum became smaller as compared with the free dye but that of the shoulder near 470 mp was enhanced. The ORD of the complex showed a small negative Cotton effect with a trough near 475 mp and a peak at however, about 440 mp. At high DNA (phosphorus) :dye ratios (about 5.4), the intensity of the absorption maximum near 492 mp was enhanced; this was accompanied by a gradual red shift of the band. Two Cotton effects were induced in this case, a relatively large positive one centered at the band on the long wavelength side and a negative one superimposed on it. The magnitude of the positive Cotton effect first increased gradually with increasing DNA concentrations, reached a limiting level, and thereafter decreased with further increase in the DNA:dye ratios. A similar study has been reported by Yamaoka and Resiiik (180), but comparison of the results from different laboratories under various experimental conditions is difficult, since the Cotton effects of the DNA-dye complexes are very sensitive to the solvent composition (ionic strength and pH) and also to temperature. Figure 23b shows the CD and absorption spectra of the The CD consisted of three DNA-acridine orange complexes (181, 182). bands, one positive at 505 mp and two negative at 488 and 465 mp. At DNA:dye ratios less than 3 and at low ionic strength, only the 505- and 465-mp bands could be observed; they were, however, preferentially suppressed with increasing DNA :dye ratios. In all cases, lowering the ionic strength of the solution increased the intensity of these two bands but reduced slightly that of the 488-mp band. Gardner and Mason (182) attributed the 488- and 465-mp negative CD bands t o the interactions between bound dye monomers and between bound dye dimers, whereas the 505-mp positive band is largely due to the dye dimers. On the other hand, Yamaoka and Resnik (180) analyzed their ORD data of the DNAacridine orange complexes, using the Kronig-Kramers transform (see Eqs. 6a, b), and concluded that at least 4 CD bands were required to fit the experimental ORD curves. Several laboratories have also attempted theoretical calculations to explain the observed ORD results. The problem is a complicated one; for instance, we are still unable to differentiate between the fractions of monomers and poIymers of the total dye molecules bound to DNA, or between the degrees of aggregation in the polymeric dyes. However, it is the current belief t!hat the monomeric dye molecules are intercalated between neighboring base pairs and that the dye aggregates may be bound to the phosphate groups of the DNA molecule. Blake and Peacocke (179, 183)found that the binding of proflavine to DNA produced a positive Cotton effect centered at the 443-mp absorption band of the dye. Its magnitude depended on the ionic strength, tempernratio. Lowering thr ionic strength of the solution from ture, and DNA :(lye

288

JEN m I YANG

AND TATSUYA

SAMEJIMA

0.1 to 0.001, for instance, doubled the magnitude of the Cotton effect. A t a constant concentration of proflavine, the magnitude of the Cotton effect increased linearly with the DNA concentration until the molar ratio of the DNA (phosphorus) to proflavine was about 4 or 5, and then decreased with further increase in the DNA concentration. These findings suggest that the bound dye molecules contribute less to the rotation when they are sparsely distributed over the DNA molecule than when they are near-neighbors. In other words, a group of such ligands bound to neighboring sites is required for induced opticaI activity. Yamaoka and Resnik (184) restudied the binding of DNA with proflavine and found the first Cotton effect to have a peak and trough at 482 and 457 mp. They also observed another broad peak near 410 mp; the rotations approached zero between 420and 400 mp, but became increasingly negative below 400mp. These authors were able to fit their ORD data with two Gaussian CD bands, one positive and the other negative. More recently, Yamaoka and Ziffer (185)reported the ORD and CD of DNA-actinomycin D complex (see also Permogorov and Lazurkin, 186). Free actinomycin D shows a major absorption band near 440 mp, which is weakly optically active (187).However, the absorption maximum shifted to about 460 mp with the appearance of a strong negative CD band when actinomycin D was mixed with DNA, thus indicating the participation of the actinomycin chromophore in the complex formation. Mahler et al.(188) studied the interactions at low ionic strength between DNA and steroidal diamines such as irehdiamine A (pregn-5-ene3P,2Oa-diamine) and malouetine [5a-pregnan-3~,20a-ylenebis(trimethyliodide)] by means of various physical methods. They found that the positive CD band of DNA and the corresponding 290-mp peak of its ORD is first enhanced by the addition of steroids, reaches a maximum effect at a steroid :DNA(phosphorus) ratio of about 0.2 to 0.3, and then decreases at still higher steroid :DNA(phosphorus) ratios. These results suggest the formation of two types of complexes, depending on the molar ratios of steroids to DNA. The first complex is characterized by enhanced thermal stability as compared with native DNA. The second complex is less stable thermally, and at room temperature its optical parameters are those characteristic ofpartially disoriented DNA. The interactions between nucleic acids and metallic ions have been studied with a variety of physicochemical methods, but only recently did Cheng (189) report the ORD of caIf thymus DNA in the presence of divalent ions such as Ca2f, 2n2+,Mg2+,Mn2+, Cu2+,and Hg2+ (Fig. 24). In all cases the ORD spectra were altered. While manganese, magnesium, calcium, and zinc ions did not significantly shift the positions of the peaks and troughs, cupric ion showed a characteristic reduction of the magnitude ofall cxtmmes and eliiniimtion of tJhrseconil peak. Mercuric ion produced

ORD

289

AND CD O F NUCLEIC ACIDS

X (mp)

FIG. 24.ORD of calf thymus DNA in the presence of divalent cations at26 C.A , Ca2+;C,ZnZ+; D,Mg2+;E,Mn2+;F ,none; G, C U ~ +and ; H, Hgz+.Concentrations: DNA, 1.0 X W 4M (phosphorus); divalent ions, 2 0 x 10-4M ; buffer, 5 X 10-3M NaCIOl M Tris-HC1 (pH 7.5). From Cheng (189). plus 5 X

an even more remarkable change in the spectrum; the Cotton effect turned negative on the long wavelength side. These changes were attributed to the specific action of the divalent ions on the DNA molecule. The less sensitive absorption spectrum could not, detect such interactions between DNA and these metallic ions.

VIII. Visible Rotatory Dispersion Prior to 1963,almost all the ORD studies of nucleic acids, polynucleotides, and their constituents were confined to the visible and near ultraviolet regions (109,110, 190-200). Although we have shown that CD and ORD in the ultraviolet region can provide a wealth of information about the conformation of these polymers, it seems appropriate to conclude this review by mentioning briefly their rotatory dispersion in visible light. The nucleic acids and their constituents all show featureless and monotonous ORD in the visible region. With the exception of TMP and UMP (28), the ORD usually obeys a one-term Drude equation and the constants of k and A, in Eq. (9) are conformation dependent. The range of wavelength over which the Drude equation applies varies widely with the compounds studied. Table VIII summarizes the pertinent numerical values for some of the nucleic acids and polynucleotides. The lists are not intended to be complete, but they serve to illustrate the lack of any genera1 trends. The

290

JEN TSI YANG

AND

TABLE VIII THEONE-TERMD R U D E EQUATION OF NUCLEIC ACIDS AND

Substance

+

0 15 M NaCl 0.015 M Nn citrate, pH 7-7.5 Salmon sperm DNA 0.15 M KF, pH 7.4 (a) 27" (b) 80" (c)goo 0.1 M NaCl 0.1 M Na TMV RNh citrate 0.001 21.1 MgC12, pH 5.50 0.1 M KF, pH 7.4 Yeast tRNA (a) 27" (b) 55" (c)90" 0.02 M phosphate buffer 0.18 M NaC1, pH 7, 27" 0.02 M phosphate buffer Yeast rRNA 0.18 &f NaCl, pH 7 (a) 27" (b) 80" Turnip yellow mosaic 0.02 M phosphate buffer 0 18 M NaCI, p H 7 virus RNA (a) 27" (b) 80" 0.02 M phosphate buffer R17 viral RNA 0.18 M NaC1, p H 7 (a) 27" (b) soo 0.1 M Na acetate 0.1 M PolyA NaCI, pH 4.85 (a) 20" (b) 27" 0.05 M Tris + 0.1 M NaC1, pH 7.8 ( 4 6" (b) 27" (c) 75" 0.1 M N a acetate 0.1 M Poly NaCl, pH 4.85 (a) 20" (b) 80" 0.05 M Tris 0.1 M NaCl, pH 7.8

+ +

38.0

230 220

36.0 38.5 4.7 46.5

227 228 286 256

34.5 18 .O 1.2

245 267 316

28.0

248

45.0 3.8

245 296

50.0 8.5

255 292

55.0 8.0

239 334

+

+

+

+

+

+

u

+

100 94

50

37 1.0

4.2 1.0

SAMEJIMA

POLYNUCLEOTIDEd

k X lo8, deg cm' decagram-1 Xe (mp)

Solvent

Calf thymus DNA

TATSUYA

279 278

275 276 314

302 307

Reference

ORD AND

CD OF NUCLEIC

291

ACIDS

TABLE VIII (Continiied) 6

Substance ~

Poly

c

Solvent ~

~

x

106,

degcm* decagram-’A. (mp) Reference ~

(a)6-7" (b)27" (e) 75" 0.1 M Na acetate 0.1i l l NaCl, pH 4.85 (a) 20" (b) 80"

+

9 .,5 5.0

20.5

290 298 316 (109)

4.2

1.6

302 307

only generalization for the data on these polymers is that X, increases and k decreases when their secondary structure is disrupted. Unlike the measurements of the Cotton effects in the ultraviolet region, study of the visible rotatory dispersion usually requires a large

quantity of samples to ensure precise measurements. Since not much information is derived from these studies as compared with those on Cotton effects, the ORD in the visible region is not usually used for studies on nucleic acids and their constituents. On the other hand, in solvents that absorb strongly in the ultraviolet region, e.g., many organic compounds, chloride ions, we must use measurements in the visible region 200). (see, for example, refs. fgr,

IX. ConcludingRemarks Accurate measurements of the Cotton effects of nucleic acids, polynucleotides, and their constituents began to accumulate only five years ago. The purine and pyrimidine base chromophores themselves are optically inactive, but once attached to the optically active sugars they are induced to produce Cotton effects. These are conformation dependent and provide us with a new means for probing the molecular structure of these biopolymers in solution and for following their conformational changes when exposed to different environments. Significant advances have also been made in theoretical treatments that help us to understand and interpret the experimental observations, although quantitative calculations are still a formidable task at present. The mononucleosides and mononucleotides are in a class by themselves as far as optical activity is concerned. A11 display a single Cotton effect around the 260-mgabsorption band. For natural compounds having the 0-D-furanese f i g , the purine derivatives have negative signs and tha

292

JEN TSI YANG AND

TATSUYA

SAMEJIU

pyrimidine ones have positives ones. Replacing the ,&glycosyl bond by an a-linkage reverses the sign. The purine and pyrimidine base rings are considered to favor the anti rather than the syn form with respect to the sugar ring as they do in the nucleic acids. Furthermore, since the a-D-and p-L-anomers have the same configuration at C-l', the Cotton effects of these purine or pyrimidine derivatives are of the same sign. The ORD and CD of dinucleoside phosphates and trinucleoside diphosphates are sequence-dependent ; the positions of the extremes and their magnitudes vary among the isomers, even though the general spectra are very similar. Thus, optical activity can become a new means for determining the base sequence in oligonucleotides. There will probably be applications for the study of the primary structure of nucleic acids when they are degraded and separated into various fractions of oligomers. The very simplicity and the small quantity of samples required for the optical method make it very attractive. But it remains to be seen whether this approach has definite advantages over other physicochemical methods such as chromatography. The oligonucleotides and polynucleotides as well as nucleic acids display multiple Cotton effects around the 260-mp absorption band. The general ORD profile shows two peaks and one trough between 230 and 300 mp, with the trough centered near the wavelength of absorption maximum (poly I and GMP gel are the exceptions, their Cotton effects having two troughs and one peak over the same wavelength region). The corresponding CD has a positive band (or two bands as in the case of some dinucleoside phosphates) on the long wavelengt,h side and a negative band following it. These results can be interpreted in terms of the stacking interactions among the bases, which cause a splitting of the mr*transitions and thereby the appearance of the positive and negative bands around the 260-mp absorption band. The most important contributions to the optical activity of a polynucleotide chain are the nearest-neighbor interactions, which are present from dimers to polymers. These base interactions are largely responsible for the stability of helical polynucleotides, be it singleor double-stranded. Both DNA and RNA also show a small negative CD band near 300 mp, which is probably attributable to an n?r* transition. Our concept of the molecular forces that stabilize the helical conformation of polynucleotides and nucleic acids has modified remarkably in the past several years. Originally, the hydrogen bonds between the base pairs in the Watson-Crick model were thought to be the major factor holding the DNA double helix together in aqueous solution as well as in solid state. The linear variation of the T, of DNA’s with the G C content had been attributed to the formation of three hydrogen bonds in the G . C pair and only two in the A . T pair. We now believe that the hydrophobic interac-

+

293

ORD AND CD O F NTJCLEIC ACIDS

tions giving rise to base stacking best explain the stability of the helical polynucleotides. The importance of such hydrophobic interactions is fully supported by recent theoretical treatments of helical polynucleotides. I n aqueous solutions, the base planes tend to stack on top of each other rather than to be exposed to the water molecules. The fact that organic solvents break up the helical structure can be interpreted in terms of the weakening of the hydrophobic interactions among the stacked bases in the presence of nonaqueous solvents. We have already mentioned that oligo- and polynucleotides can exist in single-stranded helical conformations stabilized by base stackings without the benefit of any hydrogen bondings. Unstacking of the bases would disrupt the helical conformation. This is manifested in the reduction of their Cotton effects and is accompanied by a hyperchromic effect, just as in the denaturation of double-helical DNA. Study of the dinucleoside phosphates clearly indicates that, of the four RNA bases, uracil stacks the least with neighboring bases, be it adenine, guanine, cytosine, or another uracil. Since thymine is very similar to uracil, DNA T content would therefore be expected to be the most with the lowest A stable. Thus, the dependence of the T ,of DNA on the G C content can be explained in terms of stacking interactions without resort to hydrogen bondings. The same is true for the linear correlation between the G C content and the [m]290 of DNA [see Eq. (18)]. Of course, hydrogen bondings among the paired bases would enhance the stability of the helical conformation of polynucleotides, as reflected by the sharp T, of doublestranded helical polynucleotides. Although DNA and RNA show the same two-peak-and-one-trough ORD profile around the 260-mp absorption band, the relative magnitude of the two peaks is in marked contrast. Invariably, the first peak (on the long wavelength side) is smaller than the second for DNA, whereas the second peak is close to zero rotation for RNA. This rule applies irrespective of whether the nucleic acid is single- or double-stranded. Undoubtedly, the 2’-hydroxyl group plays an important role in the geometric arrangement of the polynucleotide chains. Madel building indicates that a double-stranded polyribonucleotide can only have paired bases tilted to the helical axis, unlike the B-form of DNA that has the bases nearly perpendicular to the helical axis. It is therefore suggested that bases perpendicular to the helical axis lead to a second peak larger than the fist, whereas tilted bases drastically reduce the magnitude of the second peak. It is further speculated that the same rule applies even to single-stranded ribosyl and deoxyribosyl po1ynucleotides, and that the double-stranded hybrids will resemble the ribosyl polymers. This difference in the mode of stacking between ribosyl and deoxyribosyl polymers is believed t o affect the interactions between near and far ultraviolet interactions of the bases, thus accounting for the

+

+

+

294

JEN TSI YANG

AND TATSUYA

SAMEXMA

observed “conservative” and “noncoriservative” OED and CD spectra. Ultimately, our hypothesis must be proved or disproved by future experimental results as well as theoretical calculations. The theory of nearest-neighbor interactions explains clearly the enhancement of the Cotton effects with base stacking in a single-stranded polynucleotide chain. For double-stranded helical conformation, however, we must also consider additional interactions among the paired bases and diagonal interactions among the nearest-neighbor bases in the two separate strands. We have shown that base pairing between ribosyl polymers, and to a lesser extent between deoxyribosyl polymers, causes a blue shift of the Cotton effects. This blue shift is also substantiated by empirical calculations of the ORD of RNA based on results of synthetic polyribonucleotides. Thus, in principle we can estimate, albeit very crudely, the extent of base pairing in a RNA molecule from the position of the peak and trough of its ORD. We have also mentioned that the magnitude of the Cotton effects of homopoIynucleotidesor polynucleotides of regular sequence is much larger than that found in DNA and RNA. It is for this reason that any quantitative calculations of the extent of base stacking in a natural polynucleotide based on model synthetic polynucleotides should be viewed with reservation; much depends on future modifications and refinements. The ORD and CD studies of complexes of nucleic acids with other compounds, as in ribosomes, viruses, and nucleohistones, would enable us to detect any change in conformation when the constituents are incorporated to form the complexes. With the newly developed technique of difference ORD, it will be possible to measure small changes in rotations with more confidence. Introduction of chromophores into nucleic acids could induce new Cotton effects as in the case of the binding of dyes, which in turn would provide additional information concerning the conformation of the nucleic acids. Even the binding of small ions such as Hg2+could drastically change the optical properties and thus indicate an alteration of the conformation of the nucleic acids. In this review we have briefly described only a few examples. The vast field of interactions between nucleic acids and many other compounds remains to be explored, as far as ORD and CD are concerned. There is every reason to believe that such investigations will be accelerated in the next few years. In this review we have not listed any numerical values of the CD of nucleic acids, partly because this technique has not yet been utilized as extensively rn ORD and partly because the numerical values reported in the literature might be subject to further refinement due to the lack of some universally accepted standard for the calibration of the CD instrument (see Section 11, E). We are confident, however, that both CD and

ORD

AND

CD OF NUCLEJC

ACIDS

295

ORD will play an even more important role than they do now in advancing our knowledge of the structures ofnucleic acids. ACKNOWLEDGMENTS We thank Professor Masamichi Tsuboi formany valuable discussions and for calling to our attention the models of double-stranded nucleic acids (Fig. 18),J. T. Y. is indebted to Professor I. Tinom, Jr., for several stimulating discussions. Doctors H. Drucker, W. B. Gratzer, I(.Imahori, P. I

G-C A. c G.T C*T A .G

> < < <

C-T

-6.5

-10.2

>

A .G

-7.5

c.c T *T T *T G-G

-7.0

-19.2

strength of the different types of observed associations in solution have confirmed, as indicated in Table XI, the different detailed predictions of the theory. 3.As concerns the appaxent absence of any interaction between bases unsubstituted at their glycosyl nitrogen, this is accounted for, at least in part, in Table XII. In this case, it is obvious that one has to consider a complementary mode of self- or cross-association of the bases, which would involve the glycosyl nitrogens themselves, as exemplified in structures VI and VII forthe self-association of adenine and the cross-association of adenine with thymine. The modification which this new situation introduces with respect to the previom results, as summed up in Table X, is indicated in Table XII. The essential modification concerns the maximum value of EXPERIMENTAL RESULTSON Result

A .U

-

>A.A

or IT.U

-

G C > G G or C . C G - C > A Tor A . U

THE

TABLE XI RELATIVE STRENGTHOF HYDROGENBONDTNO Solvent

CDClS CDCla CDC1, CDC13 Me2S0

CHC18 G*G>C.C U-U>A*A

+ CHCls

Method

IR IR IR IR NMR

IR

MezSO

NMR

CHCb CHCla

IR IR

Refereu ces

ELECTRONIC

STRUCTURE

O F NUCLEIC

365

ACIDS

TABLE XI1 INTERACTION ENERGIES BETWEEN UNSUBSTITUTED BASES (KCAL/MOLE) A-T

-8.13

-- 7 . 0

-5.2

G.G

c.c

-14.5 - 13

. .4

-8 . 1 3

A

c .c G.G T-T

-19.2 7.8

- 13 -14.5

G.T

-7 . 4

C.T

-- 6 . 5

A.G

-7 . 5

-5.2

T.T

- 13 -5.2

A .A G.G

-14.5

C.C

c.c

-8.13

A-C G-T C.T A.G

< < <

>

V

u

I

1

2

'

3

4

5

t

I

I

L

1

1

0.1 0.2 0.3 0.4 0.5 0.6 0.7

-

-

NH20H

FIG. 9.Dependence of the ratio of the reaction products of cytidine with (A)hydroxylrtmine (pH 6.0) and (B)0-methylhydroxylamine (pH 4.9) on the reciprocal of the concentration ofthe modifying reagent (180).

reaction. After direct determination of CvI/G'vm and RBI the values of K 4 and K4/K6 could be readily calculated from Eq.(1). The pH of the reaction mixture considerably affects the rate of the reaction of cytosine with hydroxylamine. The dependence of the rate of the modification on pH is described by curves with maxima at pH 6 for hydroxylamine and at pH 5 for 0-methylhydroxylamine. The data suggest that the protonated cytosine residue reacts with a neutral hydroxyIamine molecule. Although the above experimental pH optima do not coincide with the calculated values (pH 5.1and 4.5, respectively), the discrepancy finds a reasonable explanation. First, the rates of the conversions V -+ VI and V + VII must depend on pH in an andogous manner, whereas the rates of reaction VII+ V must increase con-

431

THE CHEMICAL MODIFICATION O F NUCLEIC ACIDS

siderably with decreasing pH (cf. 74,187). Second, the optimum pH of reaction VII + VIII must be higher than that of reaction V + VI as the pK of the compound VII is higher than that of cytidine (cf. 185, 184). Hence, the optimum pH of the overall reaction, measured by the decrease of cytidine concentration, must be somewhat higher than that calculated from the known pK values of cytidine and hydroxylamine. The conclusion is in accord with the experimental data. An important prediction following II by increasing pH, from the data is the increase of the ratio C ~ I / C ~ Icaused also confirmed by experiment (Fig. 10).

0

4.5

5.5

6.5

PH FIG. 10. The dependence on pH of the reaction product ratio of cytidme with hydroxylamine (curve A, 30 ,1M NH20H)and with O-methylhydroxylamine (curve B, 37",2.5M NHpOCs) (180).

The rates of the stages ofthe reaction depend also on the ionic strength of the reaction mixture. Change of the ionic strength from 0.1 to 4.5M results in a change of the CVI/CVIHratio for the reaction with O-methylhydroxylamine by a factor of 1.5. A considerable isotopic effect is also the casesubstitution of D20for HzO results in increase of the CVI/CVIIIratio from 0.55 to 1.05. The values and ratio ofthe rates of reactions at C-4 and C-5, C-6, and, respectively, the CVI/CVIII ratio depend essentially on the nature and position of the cytosine substituents. For example, Janion and Shugar demonstrated that the presence of a methyl group a t C-5 or C-6 and also the presence of a hydroxymethyl group at C-5 ofthe cytosine inhibits the reaction V + VII, so that the reaction proceeds as a simple conversion ofV into VI (186, 186).

432

N. K. KOCHETKOV AND E. I. BUDOWSKY

The rate of this reaction is rather low. An analogous scheme may be applied to explain the reactivity of 4-akoxy- and 4-thio-2-ketopyrimidine with hydroxylamine. I n this case, the reaction at C-4 is much faster than that at C-5, C-6,and faster than that at C-4 of cytosine and of 5-or &methylcytosine (186). On the other hand, the substitution of methyl groups for hydrogen atoms of the exocyclic amino group inhibits the reaction at C-4 and increases the CVI/CVIII ratio (187). The effect of the substituents upon the rates of reactions at C-4 and (3-5, C-6 correlates with the electron localization energies a t these centers, calculated by Hiickel’s MO method (188). (See article by Pullman and Pullman in this volume.) The obtained parameters of the reaction of cytosines with hydroxylamine and 0-methylhydroxylamine enables a choice of conditions to obtain a given ratio of modified cytidine residues of the types V I and VIII over a wide range. For example, it appeared possible to convert the cytidines in poly C quantitatively to units of the type VIII orVI (189) (cf. 252). An inverse problem may be also solved-knowing the conditions of reaction and the structure of the starting pyrimidine nucleus, one may predict the extent of modification and the amount and ratio of the various types of modified units. At the same time, it is to be remembered that the action of hydroxylamine upon DNA involves also a number of side reactions; the most important of these are the following: (i) Modification of the adenines to N-6-hydroxyadenine. The reaction consists of the substitution on the amino group of a hydroxylamino (or 0-methylhydroxylamino) residue. The rate of the reaction is about the same as that of the substitution of the amino group in cytidine (190). (5)At low concentrations of hydroxylamine, the latter is decomposed to afford peroxides. The peroxides may modify not only cytidine, but also thymidine and adenosine (191, 192). The formation of peroxides is inhibited by mercapto compounds, pyrophosphate, Versene, etc. (178,193), together with the corresponding side reactions. Allthe side reactions mentioned are slower than those modifying the cytosine nucleus. However, they must be taken into account, or, if possible, eliminated in functional studies.

B. Application of the Reaction of Hydroxylamine with Cytosine Residues to Functional Stud,ies

The above data are of essential importance for the correct interpretation of evidence on functional alterations in nucleic acids caused by the action of hydroxylamine or its derivatives.

THE CHEMICAL MODIFICATION O F NUCLEIC ACIDS

433

Hydroxylamiiie and 0-methylhydroxylamine are widely applied to genetic studies because of their extremely high mutagenicity and the small extent of inactivation of the genetic material. Both the genetic studies (19.4) and the studies of the effect of modification by hydroxylamine and 0-methylhydroxylamine on the template activity of poly C in the RNA polymerase system (195-197) revealed that the action of the reagent results in transitions of the C + U (C -+ T) type along with inactivation. As only two types of stable modified units are formed during the modification of cytosine nuclei, it seemed reasonable to expect that one of them is responsible for the transition, and the other for the inactivation. Most probably, the t#ransitionsare due to units of type VI. The following evidence is in favor of this conclusion. The transitions are observed with T-even phages (198, 199), containing 5-hydroxymetliylcytosine instead of cytosine. The former may only form units of type VI, as demonstrated by Yanion and Shugar (185, 186). The inactivating effect of hydroxylamine upon transforming DNA is higher than that upon DNA of T-even phages by a factor of 104 (dOO), in accord with the fact that units of types VI and VIII may be formed in transforming DNA, whereas only units of type VI are formed in DNA of the T-even phages. Analysis of the data of Phillips et al. (195) leads to the conclusion that the units leading to transitions are formed by a single-stage process, whereas the inactivating ones are formed in a two-stage reaction. Taking into account the instability of units of type VII, only units of types VI and VIII need be considered; of them, units VI are formed in a single-stage reaction, and units VIII in a two-stage reaction. Finally, the increase of the inactivating action with increasing pH (194, 201) is in accord with the decrease of the CvI/CvIII ratio at acidic pH values. Allthe above evidence is in favor of the general mechanism involving the CVIICVIII ratio, which can be deduced from the reaction conditions and the nature of the nucleus subject to modification. The availability of the data on side reactions also makes it possible to attempt a rational treatment of the mechanisms of template synthesis, particularly of the mechanism of chemical mutagenesis caused by hydroxylamine.

REFERENCES 1.C. T.Yu and P. Zamecnik, Bzochirn. Biophys. Acta46, 148 (19ti0). 2. H. Weit and P. Gilham, J .Am. Chem.Soc. 89, 5473 (1967). 3. B. Singer and H. Fraenkel-Conrat, Biochim. Bzophp. Acta72,534 (1963). 4. A. Steinschneider and H. Fraenkel-Conrat, Biochemzstry 6, 2735 (1966). 6.E. Wagner and V. M. Ingram, Biochmistry 6,3019(1966). 6. A. Annqtrnng, IT. Tlntlopinn, V. hr. Ttlgritm, :indE. W:igner, 13roc hrmzslrr/ 6, 3027 (100ti).

434

N . K. KOCHETKOV

AND E. I. BUDOWSKY

7 .G. Brownlee, F. Sanger, and B.Barrel, J. Mol.Biol. 34, 379 (1968). 8.R. W. Holley, J. Apgar, G. A. Everett, J.T. Madison, M. Marquisee, S. H. Merrill, J.R. Penswick, and A. Zamir, Science 147, 1462 (1965). 9.H. G. Zachau, D. Dutting, and H. Feldman, Angew.Chem.78, 392 (1966).

10. A. A.Bayev, T. V. Venkstern, A. D.Mirsabekov, A. I. Krutilina, L. Li, andV. D. Axelrod, Mol.Biol. 1, 7541 (1967). 11.J. Madison, G. Everett, and H. Kung, Science 163, 531 (1966). 12.V. RajBhandary, S. Chang, A. Stuart, R. Faulkner, R. M. Hoskinson, and H. G. Khorana, Proc. Natl. Acad.Sci.U.S. 67, 751 (1967). 18.H. M. Goodman, J. Abelson, A. Landy, S. Brenner, and J. D. Smith, Nature217, 1019 (1968).

14.R.Rudner, H. Shapiro, and E. Chargaff, Biochim. Biophys. Acta129, 85 (1966). 15.H. S. Shapiro and E. Chargaff, Biochemistry 6, 3012 (1966). 16.V. Habermann, Collection Czech. Chem.Commun. 26, 3147 (1961); 28, 510 (1963). 17.G. B. Petersen and J. Reeves, Biochim. Biophys. Acta129,438 (1966). 18.A. Masin and B. Vanjushin, Biokhimiya 33, 377 (1967). 19.N. Kochetkov, E. Budowsky, M. Turchinsky, and V. Demushkin, Dokl.Acad. Nauk SSSR162, 1005 (1963). 20.W. Seifert and W. Zillig, 2.Physiol. Chem.348, 1017 (1967). 21.R.Naylor, W. Nancy, and P. Gilham, J. Am. Chem.SOC. 87,4209 (1965). 22.0. Ivanova, D. Knorre, and E.Malygin, MoZ. B i d .1, 335 (1967). 2.9. R. Chambers, Biochemwtry 4, 219 (1965). 24.P. R.Witfeld and H. Witzel, Biochim. Biophys. Acta72, 338 (1963). 26. N. Kochetkov, E. Budowsky, N. Broude, and L. Klebanova, Biochim. Biophys.

Acta134,492 (1967).

W.M. Beer and E. N. Moudrianakis, PTOC. Natl. Acad.Sci.U . S. 48,409 (1967). 27.M. Beer and C. Zobel, J. MoZ. Biol. 3,717 (1961). 28.B.Ulanov and A. Malyshev, Biophysics (USSR) 12, 325 (1967). 29. B. Ulanov, A. Serebrjany, and R. Kostjanovsky, Dokl. Acad.Nauk SSSR176,474 (1967). SO. P. J.Highton and M. Beer, J. Mol.B i d .7 ,70 (1963). S1. M. Yoshida and T. Ukita, Biochim. Biophys. Acta167, 466 (1968). 32.T. Morosova and R. Salganik, Biokhimiya 29, 17 (1964). SS. N. Kochetkov, E. Budowsky, and

V. Demushkin, Dokl.Akad.Nauk SSSR 168,

102 (1966).

S4. G. Augusti-Tocco and G. L.Brown, Nature206,683 (1965). 36.V. Drewitch, D. Knorre, E. Malygin, and R. Salganik, Mol.Biol. 1, 249 (1967). 36. H. Seidel and F. Cramer, Biochim. Biophys. Acta108, 367 (1965). 37.A. Stuart and H. G. Khorana, J .Biol. Chem.239, 3885 (1964). 38.D. Knorre, N. Pustoshilova, N. Teplova, and G. Shamovsky, Biokhimiya 30, 1218 (1965).

39. D. Knorre, A. Malysheva, N. Pustoshilova, A. Sevastyanov, and G. Shamovsky, Biokhimiya 31, 1181 (1966). 40.D. Knorre and G. Shamovsky, Biochim. Biophys. Acta142, 555 (1967). 41.D.Knorre and G. Shamovsky, Mol.B i d .2,33 (1968). 42.D. Knorre, N. Pustoshilova, and A. Sevastyanov, Biokhimiya 33, 56 (1968). 4s.P. Whitfeld, Biochem. J .68,390 (1954). 44.D. M. Brown, M. Fried, and A. Todd, J. Chem.SOC.p. 2206 (1955). 45.H. Ney and L. Heppel, J. Hiol. Chem.239, 1927 (1964). 46.M. Ogur a n d J. Small, J. Biol. C h m . 236, P. C .ti0 (19fiO).

THE CHEMICAL

MODIFICATION

OF NUCLEIC

ACIDS

435

47.J.X. Khym, Biochemistry 2,344 (1963). 48.D. Livingston, Biochim. Biophys. Acta87, 538 (1964). 49.M. Tomass, Y. Sanno, and It. Chambers, Biochemistry 4, 1710(1965). 60. G. Moss, C. B. Reese, K.Sehofield, R. Shapiro, and A.Todd, J. Chem.Soc.p.1149 (1963). 61. K. Pfitsner and J. Moffat, J .Am. Chem.Soc.87, 5661(1965). 52.U. RajBhandary, R. Young, and H. G. Khorana, J .B i d .Chem.239, 3875(1964). 63. R. Ralph, R. Young, and H. G. Khorana, J. Am. Chem.SOC. 86,2002(1963). 64.J. Cox and 0. Ramsay, Chem.Rev.64, 317 (1964). 55.K.-H. Scheit and A. Holy, Biochim. Biophys. Acta149, 344 (1967). 56.A. Holy and K.-H. Scheit, Biochim. Biophys. Acta138, 230 (1967). 67.R. Markham and J. D. Smith, Biochem. J .49, 401(1951). 68. H. Fritz and B. Rottger, Z.Naturforsch. 188, 124(1963). 69.E. Budowsky and L. Klebanova, Vopr.Med.Khim.13,299 (1967). 60. B. Lane and G .Butler, Biochim. Biophys. Acta33,281 (1959). 61. B. Lane and G .Butler, Can.J .Biochem. PhysioZ. 37, 1329(1959). 6R. K. Dimroth and H. Witzel, Ann.Chem.620, 109(1959). 63.W. Farkas, Biochim. Biophys. Acta166,401 (1968). 64.C.Tamm, M. Hodes, and E. Chargaff, J. Biol. Chem.196,49 (1952). 65. C. T a m and E. Chargaff, J .Biol. Chem.203, 689 (1953). 66.A. Jones and D. Letham, J .Chem.SOC.p. 2573(1956). 67.A. Jones, D. Letham, and M. Stacey, J .Chem.SOC.pp. 2579,2584(1956). 68.K. Burton and G. B. Petersen, Biochem. J .76, 17 (1960). 69.K. Burton, Biochem. J .77, 547 (1960). 70.F.Micheel and A. Heesing, Chem.Ber.94, 1814(1961). 71.H. Venner, Z.Physiol. Chem.339, 14 (1964). 72.T. V. Venkstern, Usp.Biol. X h i m .6,3 (1964). 73.D. Shugar, in“The Nucleic Acids” (E. Chargaff and J. Davidson, eds.), Vol. 111, p.39.Academic Press, New York, 1960. 74.J. McLaren and D. Shugar, “Photochemistry of Proteins and Nucleic Acids,” Macmillan (Pergamon), New York, 1964. 7 5. A. Wacker, This series 1, 369 (1963). 76.P. D. Lawley, This series 6,89 (1966). 77.G.Wheeler, CancerRes.22, 651 (1962). 78.K. Lohman, Biochem. Z.264, 381 (1932). 79.H. Schuster, Z.Naturforsch. 16b, 298 (1960). 80.R.Shapiro, J .Am. Chem.Soc. 86,2948(1964). 81.I.Carbon, Biochim. Biophys. Actu96,550 (1965). 82.T.Kotaka and R. L. Baldwin, J. Mol.Biol. 9, 323 (1964). 83. T.Ushida and F. Egami, J .Bioclrem. (Tokyo) 67, 742(1965). 84.J. Nelson, S.Ristov, and R. Holley, Biochim. Biophys. Acta149, 590(1967). 85.H. G.Wittman, Nnturwissensrhaften 48, 729(1961). 86.T. I. Tikhonenko, E. N. Dobrov, T. Velikodvorskaya, and N. Kisseleva, J .Mol. Biol. 18, 58 (1966). 87.S.Zamenhof, H. Alexander, and G. Leidy, J .Exptl. Med.98, 373(1953). 88.D. E. Hoard, Biochim. Biophys. Acta40,62 (1960). 89.M. Feldman, Bi0chi.m. Biophys. Acta149, 20 (1967). 90.S.Lewin, J .Chem.SOC.p. 792 (1964). 91. S. Lewin, Arch.Biochem. Biophys. 113,584 (1966). 92.S.Lewin and M. Barnes, J .Chem.SOC.(B), p. 478 (1966).

436

N. K. KOCHETKOV

AND

E. I. BUDOWSKY

93.L. Grossman, S. Lewin, and W. Allison, J .Mol.Biol. 3, 47 (1961).

!/4.J. Penniston and P. Doty, Biopolymers 1, 145(1963). 95. E. N. Dobrov, T. Tikhonenko, and S. Klimenko, Biophysics USSR 12, 957 (1967). 96. E. Triphonov, Yu. Lazurkin, and M. Frank-Kamenetsky, MoZ.Biol. 1, 164(1967). 97. M. Staehelin, Biochim. Biophys. Acta31, 448 (1959). 98. R. Shapiro and J.Huchmann, Biochemistry 6,2799(1966). 99. N. Broude, E. Budowsky, and N. Kochetkov, Mol.Biol. 1, 214(1967). 100. N. Broude and E. Budowsky, in preparation. 101. P.Witfeld and H. Witzel, Biochim. Biophys. Acta72, 338 (1963). 102. R. Shapiro and S. C. Agarwal, J .Am. Chem.SOC.90,474(1968). 103. P. T.Gilham, J. Am. Chem.SOC.84, 687 (1962). 104. S. Brostoff and V. Ingram, Science 168,666 (1967). 106. N. W. Ho and P.Gilham, Biochemistry 6, 3632(1967). 106. A. Girshovich, M. Grachev, and L. Obuchova, MoZ. Biol. 2,351(1968). 107. D. Knorre, E. Malygin, G. Mushinskaya, and V. Favorov, Biokhimiya 31, 334 (1966). 108. R. Naylor and P. Gilham, Biochemistry 6,2722(1965). 109. D. Knorre and G. Mushinskaya, Izv. Sibirsk. Otd.Akad.Nauk SSSR7, 132(1967). 110. R. Salganik, V. Dashkevich, aiid G. Dynuhits, Biochim. Biophys. Acta149, 603 (1967). 111. V. Drevitch, R. Salganik, D. Knorre, and E. Malygin, Biochim. Biophy.?. Acta 123, 207 (1966). 118. M. Yoshida and T. Ukita, J. Biochem. (Tokyo) 67,812(1965). 113. H. Priess and W. Zillig, 2. Physiol. Chem.342, 73 (1965). 114. N. Kochetkov, E. Budowsky, V. Shibaev, and G. Eliseeva, in press. 115. F. Cramer, K. Randerath, and E. Schiifer, Biochim. Biophys. Acta72, 150(1963). 116. F. Cramer and H. Seidel, Biochim. Biophys. Acta72, 157 (1963). 117. H. Seidel and F. Cramer, Biochim. Biophys. Acta108, 387 (1965). 118. F. Cramer and G. Schlingloff, Tetrahedron Letters 43, 3201(1964). 119. H. Seidel, Biochim. Biophys. Acta138, 98 (1967). 120. R. Bergquist and A. Deutch, ActaChem.Seand. 8,1880(1954). 12f. R. Caputto, L. F. Leloir, C. E. Cardini, and A. C. Paladini, J. Biol. Chem.184, 333 (1950). 192. T. Suzuki and E. Ito, J. Biochem. 46, 403(1958). 123. C. T.Yu and P. C. Zamecnik, Bioehim. Biophys. Acta76, 209(1963). 124. T. Venkstern, A. Mirsahekov, B. Gorshkova, and A. Bayev, Biokhimiya 28,712 (1963). 125. R. Shapiro and S. C. Agarwal, Biochim. Biophys. Res.Commun.24, 401 (1966). 126. A. Jones, J. Tithemor, and R. Walker, J .Chem.Soe. (C), p.1635(1966). 127. K. Brammer, Biochim. Biophys. Acta72, 217 (1963). 1% R. Holmes and R. Rolins, J. Am. Chem.SOC.86, 1242(1964). 129. F. Ascoli and I?. Kahan, J .Biol. Chem.241, 428 (1966). 130. W. Seer and D. Shugar, ActaBiochim. Polon. 8, 363 (1961). 131. M. Bessman, I. R. Lehman, J. Adler, A.S. Zimmerman, E. S. Simms, and A. Kornberg, Proc. Natl. Acad.Sci. U.S.44, 633 (1958). 132. A. M. Michelson, J.Dondon, and M. Grunberg-Manago, Biochim. Biophys. Acta 66, 529 (1962). 133. P. Cerutti, H. Todd Miles, and J. Frazier, Biochem. Biophys. Res.C o m m m . 22, 5 (1966).

T H E CHEMICAL MODIFICATION OF NUCLEIC ACIDS

437

134.P. Cerutti and N. Milles, J .Mol.Biol. 26, 55 (1967). 236.G. Belle, P.Cerutt,i, and B. Witkop, J .Am. Chem.SOC.88, 3946 (1966). 136. F. Baron and D. Brown, J. Chem.SOC. p. 2855 (1955). 137.S. Takemura, Biochim. Hiophys. Acta29, 447 (1958); Bull. SOC.Chem.Japan32, 920 and 926 (1959). 138. D. Hayes, F. Hayes-Baron, J .Chem.Soc.( C )p. , 1528 (1967). 139. A. Temperli, H. Turler, P. Rusl,, A. Danon, and E. Chargaff, Biochim. Biophys. Actu91, 462 (1964). 240.D. M. Brown, in“Methods in Enzymology,” (L. Grossman and I(. Moldave, eds.), Vol. 12, Part A, p. 31,Academic Press, New York, 1967. 141.B. Ellery and R. Symons, Nature210, 1159 (1966). 142,D. Brown, A. McNaught, and P.Shell, Biochim. Hiophys. Res.Comnzun. 24, 967 (1966). 143.E. Budowsky, J. A. Haines, arid N . Kochetkov, Dokl.A k d . h uukSSSR168, 379 (1964). 144. F. Lingens and H. Schneider-Bernlohr, Biochim. Biophys. Acta123, 611 (1966). 146.F. Lingens and H. Schneider-Bernlohr, Ann.Chem.686, 134 (1965). 146. R. Shapiro and R. Klein, Biochemistry 6, 235 (1966); 6, 3576 (1967). 247.A. B. Patel and H. D. Brown, Nature214, 402 (1967). 148. K. Kikugawa, A. Muto, H. Hayatsu, K. Miura, and T. Ukita, Biochim. Biophys. Acta114, 85 (1966). 149. H. Hayatsu and T. Ukita, Biochim. Biophys. Acta123, 458 (1966). 160. M. Yoshida and T. Ukita, Biochim. Biophys. Acta 123, 214 (1966). 151. L. Gal-Or, J. Mellema, E. N. Moudrianakis, and M. Beer, Biochcmistry 6, 1909 (1967). 166. C. Janion and D. Shugar, ActaBiochim. Polon. 16, 107 (1968). 153.H. Shuster, J .Mol.Biol. 3, 447 (1961). 154. D. Brown and P. Shell, J.Mol.Biot. 3, 709 (1961). 166. E. Freese, E. Bautz-Freese, and E. Bautz, Proc. Natl. Acad.Sci. U.S .47,845 (1961). 156. D. Verwoerd, H. Kohlhage, arid W. Zillig, Nature 192, 1038 (1961). 167. N. Kochetkov, E. Budowsky, and N. Simukova, Biokhimiya 27, 520 (1962). 268. V. Demushkin and E. Budowsky, Unpublished. 159. N. Kochetkov, E. Budowsky, V. Demushkin, M. Turchinsky, N. Simukova, and

E. D. Sverdlov, Biochim. Biophys. Acta142, 35 (1967). 160. D. Brown and P. Shell, J .Chcm.Soc.1966, 268.

161, N. Kochetkov, E. Budowsky, M. Turchinsky, and V. Demushkin, Dokl.Akad. Nauk SSSR 162, 1005 (1963). f62. S. Takemura, J .Biochim. (Tokyo) 46, 1285 (1959). 163. E. Rosenberg and S. Zamenhof, J .Uiol. Chem.236, 2845 (1961). 164.N. Kochetkov, E. Budowsky, and N. Simukova, Dokl.Akad.Nauk SSSR 163, 597 (1963). 265.E. Budowsky, N. Simukova, R. Slubaeva, and N. Kochetkov, Biokhimiya 30, 902 (1965). 166.E. Budowsky, N. Simukova, and L. Gus’kova, Biochim. Biophys. Acta166, 755 (1968). 167. M. Turchinsky, L. Gus’kova, arid E. Budovsky, Mol. BioZ.1, 792 (1967). 168. N. Kochetkov, E. Budowsky, M. Turchinsky, and N. Simukova, Biochem. Biophys. Res.Commzin.19, 49 (1965). 169. E.Budowsky, M. Turchinsky, aid L. Gus’kova, in prepsrstion. 170.N. Kochetkov, E. 3udowsky, and V. Demushkin, Mol.Bid.1, 583 (1967). 171. M. Turchinsky, E. Sverdlov, and E, Budowsky, Mol.Biol. 1, 623 (1967).

438

N. K. KOCHETKOV AND E. I. BUDOWSKY

17.2. N. Kochetkov, E. Budowsky, and R. Shibaeva, Biochim. Biophys. Acta88,493 (1963). 173.N. Kochetkov, E. Budowsky, and V. Domkin, Mot.Bio2. 1, 558 (1967). 174.N. Kochetkov, E. Budowsky, and V. Domkin, Khim.Prorodnych Soedin. p. 230 (1968). 176.J.Phillips and D. M. Brown, This series 7,349 (1968). 176.L.Frolova and L.L. Kiselev, Dokl. Acad.Nauk SSSR167, 1466(1964). lY7.S. Belman, T. Huong, E. Levinc, and W. Troll, Biochem. Biophys. Res.Commun. 14, 463(1964). 178.E.Budowsky, R. Shibaeva, E. Sverdlov, and G. Monastyrskaya, MoZ. Biol. 2,321 (1968). 179,D. Brown and J.Phillips, J. Mol.BioZ.11, 663(1965). 180.E.Budowsky, E.Sverdlov, R. Shibaeva, and G. Monastyrskaya, MoZ. Biol. 2,329 (1968). 181.P. Lawley, J. Mol.Biol. 24,75 (1967). 182.H. Johns, J. LeBlanc, and K. Freeman, J .Mol.Biol. 13, 861;883(1965). 18s.C. Janion and D. Shugar, ActuBiochim. Polon. 7 ,309 (1960). 184.I.Tessman, R. Poddar, and S. Kumar, J .Mol.Biol. 9,352 (1964). 186.C. Janion and D. Shugar, Biochem. Biophys. Res.Commun. 18, 617 (1965). 286. C. Janion and D. Shugar, ActuBiochim. Polon. 14, 293(1967). 187.R. Shibaeva, E.Budowsky, and N. Kochetkov, in press. 188.N. Kochetkov, E. Budowsky, E. Sverdlov, R. Shibaeva, V. Shibaev, and G. Monastyrskaya, Tetrahedrvn Letters 34, 3253(1967). 189.E. Budowsky, E.Sverdlov, and G. Monastyrskaya, in press. 190.E. Budowsky, E. Sverdlov, G. Monastyrskaya, in press. 191.F. Freese, E. Bautz-Freese, and S. Graham, Biochim. Biophys. Actu123, 17 (1966). 192.H.-J. Rhaese, E. Freese, and H. 8. Melzer, Biochim. Biophys. Acta166,491(1968). 19s. E.Freese and E. B. Freese, Biochemistry 4,2419(1965). 194.E. Bautz-Freese and E. Freese, Proc. Nutl.Acad.Sci.U.S.62, 1289(1964). 196.J.Phillips, D. Brown, R. Adman, and L. Grossman, J. MoZ. Biol. 12,816 (1965). 196.R. 0. Wilson and M. J.Caicuts, J .BioE.Chem.241, 1721(1966). 197.J. Phillips, D. Brown, and L. Grossman, J .Mol.Biol. 21, 405 (1966). 198.E. Freese, E. Bautz, and E. Bauta-Freese, Proc.Nutl. Acid.Sci.U.S. 47,845 (1961). 199.V. Chubukov, J .Mierobiol. Epidemiol. Immunobiol. (USSR), p. 129(1965). ,900.E.Freese and H. Strack, Proc. Nutl. Acad.Sci. U . S .48,1796(1962). 201.J. H. Van de Pol and G. Van Arkel, Mutat. Res.2 ,466(1965).

Author Index Numbers in parentheses are reference numbers and indicate that an author’s work is referred to, although his name is not cited in the text. Numbers in italics show the page on which the complete reference is listed.

A Abdulnur, S., 341(110, l l l ) , 373,(110, 111), 397

Abelson, J., 80(60), 113, 405(13), 434 Abrams, R., 160(227), 219 Achey, P. M., 118(64, 72, 73), 120(64, 72, 73), 139, 142054, 160), 151(64, 72, 73, 154), 152, 153(72, 160), 154(72), 213 (64,72, 73, 154, 160), 216, 217 Adam, R., 184(296), 221 Adams, A,, 270,271(143), 299 Adman, R., 6(21), 7(21), 14(21), 27, 145 (182), 181(182), 218, 433(195), 438 Adelberg, E. A., 9(51), 15(51), 24(51), 28 Adkins, B. J., 231(27), 296 Adler, A,, 249,260,297 Adler, J., 5(15), f l , 415(131), 4% Afanaseva, T. P., 96(120), 116 Agarwal, S. C., 413(102), 415(125), 4.90 AjdaEiE, Z., 186(306), 221 Alexander, Ch., 375(261), 401 Alexander, H. E., 18(85), 29, 412(87), 435 Alexander, M., 76(12), 112 Alexander, P., 21(101), 29, 118(1, 78, 80, 105), 120(78, 80, 1051, 128(78, 80), 130(78, 801, 131(1), 133(137, 147), 156 (78, 80), 213(78, 80), 214,2lG,217, 393(319), 402 Alfert, M., 15812221, 211(222), 213(222), 219 Allison, W., 412(93), 436 Alpers, D. H., 79(36), 112 Anderegg, J. W., 156(210), 164(210), $19 Anderlovb, A., 68(111), 73 Anden, M., 80(51), 94(113), 101(113), 113,114, 170(260), 820 Anderson, E. C., 187(308), 221 Anderson, J. S., 98(127), 116

Anderson, W. F., 118(117), 126(117), 116 Anthony, D. D., 105(153), 110(153), 116 Antoni, F., 194(324), 198(324), i?11 Aoki, A., 45(60), 72 Aoyagi, S., 245(83a), 246(83a), 197 Apelgot, S., 118(19), 151(19), 211(19), 212 (19), 213(19), 214 Apgar, J., 278(154), 299, 405(8), 434 Appel, K., 330(18), 341(154), 396, 399 Appleman, A. W. M., 155(200), 164(200), 218

Applequist, J., 240(64), ,997 Arbogast, A., 118(27), 120(27), 213(27), 214

Armstrong, A., 405 (61,433 Armstrong, A. T., 339(53), 396 Arnott, S., 273, ,999 Arya, S. K., 251(96a), 255, 256, 297 Asano, K., 107(159), 116 Ascione, R., 271(143), 299 Ascoli, F., 374(242), 401,415(129), 430 Attardi, G., 76(14), 77(16), 112 Augenstein, L., 350(177, 178,179), 399 August, J. T., 76(6), 84(6), 116 Augusti-Tocco, G., 406(34), 434 Axelrod, V.D.. 405(10), 4.94

B Baba, H., 332(38), 396 Babinet, C., 80(55), 113 Bach, D., 32(5), 45(80), 48(80, 82, 83), 70, 78

Bacq, Z. M., 118(1), 131(1), 214 Bar, H., 45(74), 46(74), 48(74), 64(74), 68(74), 72

Baird, S. L., Jr., 374(232, 2471, 400, 401 Balabuha, V. S., 133(142), 217 Baldwin, R. L., 14(81), 17, 19(81), 21(81),

439

28, 85(77), 114, 259(119, 122), 198, 304(11), 310(11), 326,412(82), 435

440

AUTHOR

INDEX

Belts, R. E., 164(236), $19 Ball, M. A., 341(85), 3997 Bennet, E.L., 160(228), 219 Ballard, D.,76(3), 112 Bertinek, J., 45(62), 7’2 Baltimore, D., 88(93), 114 Berenbom, M.,165,220 Bangerter, B. W , 240(67), 297 Berends, W.,377(264, 265, 266, 267, 268, Banghart, F. W., 162(233), 213 269),382,401 Baptist, J. E., 118(65), 120(65), 151(65), 213(65), 216 Berg, H., 45(73, 741, 46(74), 48(73, 74, 84, 85),57(85), 64(74), 68(74), 72 Barker, G.C., 38(26), 5O(26), 71 Berg, P.,80(50), 85(77, 781,86,90(98), 92 Barnabei, O.,198,222 (1051, 94(50), 99(98, 1301, 100(98), Barner, H.D.,118(31), 120(31), 213(31), lOl(135, 1381, 102(50, 98), 105(98, 214 130), 108(105), 110(98), 113,11/, 116, Barnes, M., 412(92), 436 170(259), 10,304(11,15), 310(11,15), Barnett, L.,286(174), 300, 385(293), 402 326 Baron, F., 415(136), 437 Barone, T.F., 118(90), 173, 174(90), 211 Berg, T. L., 189,205(317), 221 Bergmann, E. D., 341(65), 349(65), 366 (90),226 Barrel, B., 405(7), 434 (209),396,.boo Bergmann, F., 351(If@), 399 Basilio, C.,5(16), 14(16), 2’7 Bergquist, R., 415(120), 436 Baudet, J., 341(143), 398 Bergson, G ., 341(721,696 Bauer, H.,37(14), 71 Baugnet-Mahieu, L.,164(238), 187(238), Bernardi, G.,57(96), 73 Berns, K.,118(117), 126(117), 217 205(238), 213(238), 219 Berry, R.,118(42), 120(42), 155(42), 216 Bauts, E., 417(155), 433(198), 437 Bauts, E. K. F., 116,307, 308, 310(28), Bersohn, R.,374(232), 400 Bertani, G.,118(48), 120(48), 121(48), 135 3% (481,213(48), 2f6 Bauts, F. A.,308,326 Bautz-Freese, E., 1(3), 6(28), 7(34, 351, Berthier, G.,328(2), 341(143), 396,396 23, 24, 26, 271, 338, 8(34), 12(28),13(3), 14(28, 34,46,95. Berthod, H., 330(22, 341(22,23,24,26,27,63,98, 163), 346 961, 15(95), 16(3), 19(35, 95, 96), 21 (22,26), 347(26), 349(22), 350(22, 23, (95), 24(3), 25(95), 27,28, 118(94), 24), 354(22, 23, 24), 365(98), 382(22, 204(94), $is, 383(285), 402,417(155), 23), 383(22, 24), 385(26), 392(63), 432(191, 193), 433(194, 198, 437,4% 396,396,397,399 Bayev, A. A.,405(10), 415(124), 434,43C Bessman, M.J., 5(15), 67, 143(165), 218, Beaven, G. H.,350(172), 399 415(131), 436 BebareviE, A., 118(44), lZO(441, 165, 166 (249), 190(318), 191(318), 193(318, Betel, I.,155,164(201), 818 321), 197(318), 199, 200(334), 201 Beukers, R.,377(264, 265, 266, 267, 269), 382, 401 (3351, 202(335), 203(337), 213(318), Beychok, S., 224(5), 296 a 4 , 220, ,@i, 9.@ Biczo, G.,341(155), 399 Becker, E. D., 357(194), 364(194), 399 Billen, D.,118(33, 66, 66a, 71, 72), 120 Becker, E. F., Jr., 19(93), 21(93), d9 (33, 66, 66a,71, 721, 124(66a), 142, Beckwith, J. R.,111 (174), if 6 151(66, 66a, 71, 72), 153, 154, 173 Bedford, J. S., 130(124), 156(208), 217, (270), 213(33,66,%a, 71,72, 154, 160, 219 269), $14,916,9i7 Beer, M., 406(26, 27, 30), 416(151), 434, Birnstiel, M., 311(39), 326 437 Beers, R. F., 249(88), 254(88), 286(88), Bishop, C.W., 163,219 Blake, A., 280,286(179, 183). 287,299,300 297 Blather, F. R., 103(150), 116 Belle, G.,415(135), 437 Blazsek, V.,68(106), 73 Belman, S., 428(177), 438

AUTHOR

441

INDEX

Blears, A.J., 374(235), 400 Blohina, V. D., 118(4), 21.4 Blout, E. R., 224, 225(8), 281(163), 296, 299

Boag, J.W., 382(275), 401 Boedtker, H., 289(193, 194), 300 BorBni, E.,194(323), 198(323), 221 Bogdanova, S.L., 96(120), 116 BohitEek, J., 45(71), 61(103), 72, 73 Bolle, A.,78(21, 24), 79(46), I06(21), 112, 113

Bollum, F. J., 143(167, 1741, 151(177), 156(210), 163(235), 164(210), 218,819, 244(80), 249(80), 254(80), 257(80), 259(80), 260(80), 261(80), 274(80), 297, 305, 306,526 Bolton, E. T., 310(37), 317(44), 326 Bonner, J.,168,170,22'0 Botrk, C., 374(242), 401 Boublik, M., 57(98),73 Boudnitskaya, E. V., 185, 205(303), 221 Bourgeois, S., 93(108), 110(108), 114 Boxer, G. E., 89(94), 109(94), 114 Boyce, R. P., 118(13, 91, 97, 113), 119 (13, 91), 120(13), 123(13, 91), 125 (113), 151(13), 154,210(13), 213(13),

(174, 175), 300, 385(293), 402,405 (13),4S4 Brent, T. P.,118(79), 120(79), 128(79), 130(79), 151(79), 155(79), 156(79), 157(79), 158,213(79), 216 Bresler, S. E., 14(91), 15(91), 18(91), 19 (91),20,21,25(91), 29 Breyer, B., 37(13, 14), 71 Bkzina, M., 32(7), 36(7), 68(7), 70 Britten, R. J., 302,317,318,319(5), 321, 324

Brody, E., 94(112), 114 Brody, E.N., 96(119), 116 Brookes, P., 20,21(100), 22(106), 29, 374 (249, 2501, 385(290, 291, 292), 392 (309,310, 311, 312, 313, 314), 393 (318), 401, 402 Broom, A. D., 238(49), 296, 369(213), 370 (2161,400 Brostoff, S.,413(104), 4%’ Broude, N.,406(25), 413(99, IOO), 434, 436

Brown, D., 415(136), 416(142), 417(154), 419(160), 427(160), 428(160), 429,433 (195,1971,437, 438 Brown, D. D., 322(52), 326 Brown, D. M., 1(6), 6(18, 19,20,21,25, 214,816 291, 7(6, 21,291, 14(21, 291, 17(20), Boy de la Tour, E., 78(21), 106(211, 113 19(18), 27, 391(302, 304, 3051, 402, Boyland, E., 374(238,239,240), 400 409(44), 415(140), 42811751,434,437, Bradbury, E.M., 286(172), 300 Bradley, D. F., 225(15, 16), 274115, 161, 438 286(173, 1781,295, 300, 305(17), 326, Brown, G. B.,39(32), 71 330(28), 341(28, 89, 157), 355(28), Brown, G. L.,406(34), 434 Brown, H. D.,416(147), 437 356(89), 395, 397, 399 Brahms, J.,57(99), 73, 225, 238(56, 57), Brown, J. C., 79(41), 89(41), 113 241,242(56, 771,243,247(57,86), 248 Brown, R. A.,202 (571,249(87a), 250(106), 252661,253, Brownlee, G.,405(7), 434 254(87a), 255(86, 87a),261, 267(14), Bruner, R.,99(133), 104(151), 105(133, El), 106(133), 116 274,275,279,296, 297, 698 Brunfaut, M.,185(303), u)5(303), 221 Brammer, K. W., 12,28, 41511271,436 Buc, M. H., 183(294), 221 Brdar, B., 174(265), ,920 Buchanan, J.M., 78(22), 112 BrdiEka, R., 32,YO Bremer, H.,79(38), 82.(67), 83(67), 87 Bucher, N.L., 187(313), 221 (86),88(38), 89, 90(86), 92(86, 101, Budilova, E. V., 164(240), 187(240), 205 (240),219 1021, 93(101), 98, 99(133), 104(38, 1511, 105(86, 133,1511,106(38, 1331, Budnikov, G., 37(20), 71 107(86), llO(67, 1661, 113, 114, 116, Budowsky, E. I., 6(24), 7(24, 331,B', 405 (19, 33),406(25), 410(59), 413(99, 116 loo), 414(114), 417(157, lSS), 418 Brenner, S., 78(29), 79(46, 471, 110(29), (158, 159), 419(159), 420(159), 421 112,113, 207(348), 210(349), 222, 286

442

AUTHOR

INDEX

(161), 423(164, 165, 166, 167, 168, Carrier, W. L., 118(12), 119(12), 120(12), 123(12), 151(12), 154, 210(12), 213 169), 424(168), 425(33, 159), 426(33, 1701, W(164, 165, 170, 171, 172, 1731, (121,214 428(164, 165, 172, 178), 429(178), 430 Carroll, D. G., 339(53), 396 Caskey, C.T., 320(48), 326 (1801,431(180,187),432(178,187,188, Cassani, G.,305,306,326 189, 1901, 434,434 436,437,438 Cassim, J. Y., 230,296 Bugarski, M., 186(306),221 Cavalieri, L. F., 40,71 Bukaresti, L.,68(106), 73 Cecil, R., 70,73 Bulant, V.,45(61), 72 Burgess, R.R., 80(591,83(59), 84(59, 651, Cernf, R.,45(72), 72 Cerutti, P., 145(181), 218,391, 402,415 113,116 (133,134, 135),@6,437 Burki, H. J., 118(122), 120(122), 130(122), Chamberlin, M. J., 80(50), 85(77, 781,86, 2f7 87(85), 94(50), 101(138), 102(50,139), Burton, A.,87(89), 114 113, 114,116,259(121), 273(150), 298, Burton, K.,102(142), 116,411(68, 691, 299, 304(11, 12, 13, 14, 151, 310(11, 437 15), 3.96 Bush, C. A., 240, 242(77), 262(59), 275, Chambers, R., 405(23), 409(49), 414(23), 297,341(88), 397 Butler, G.,410(60,61), 436 434,436 Butler, J. A.V., 118(79), 120(79), 128(79), Chambon, P., 76(5), 105(154), llO(154, 165), 112,116,118(47), 120(47), 816 130(79), 133(134), 151(79), 155(79), 156(79), 157(79), 158(79), 213(79), Chambron, J., 374(231), 4UU Champe, S.P., 78(20), 11% 216,817 Chan, S. I., 240(65, 66, 67, 701, 297,341 C (141), 369(211, 212), 398, 400 Cabrera-Juarez, E., 12(77), 15(77), 19 Chandra, P.,9, 11(62), 15(47,62,127), 23, 28,29 (77),28 Caicuts, M. J., 6(22), 7, 14(22), 27,433 Chang, L. O., 162(233), 219 Chang, S., 405(12), 434 (I%), 438 Caillet, J., 341(92, 93, 94, 95, 96, 97, 98, Chantrenne, H.,173(271), 213(271), 220 104, 105), 359(92), 361(94, 96, 203), Chapman, J. D., 139, 217 365(98), 367(96), 369(93,97),370(93), Chargaff, E.,102(141), 116,405(14, 151, 410(64, 65), 411(14, 15), 415(139), 373(!33), 374(105), 388(297), 397, 400, 402

Cairns, J., 142(164), 218 Caldwell, I., 118(80), 120(80), 128(80), 130(80), 156(80), 213(80), 216 Calendi, E., 48(81), 72 Callis, P.R., 351(183),399 Calvin, M.,374(229), 400 Cammarano, P.,199,822 Cannelakis, E.S., 143(168), 218 Cantor, C. R., 244,245,254,277,278,279, 297,2998

Cantor, S. M., 39(28), 71 Caputto, R.,415(121), 436 Carbon, I.,412(81), 436 Cardini, C. E., 415(121), 436 Carlson, J. G., 158(224), 211(224), 213 (224),219

434,435,437

Charlier, M., 382(277), 4 O i Chase, M.,122 Chen, G., 9(51), 15(51), 24(51), 2s Chen, J. H., 9(42), 28 Cheng, Ping-Yao, 288, 289(189), SO0 Chevalley, R.,78(21), 106(21), 112 Chinali, G.,199(333), 922 Chipchase, M.,321 (49), 386 Cho-Chung, Y. S., 204(339), 205(339), 213(339), 222 Chubukov, V. F., 14(114), 29, 433(199), 438 Clark, B. F. C., 98(127),116 Clark, J. B.,131(128), 217 Clark, L. B., 234(46), %96,350(175), 351 (175), 399

AUTHOR

443

INDEX

Clarke, G. A., 339(54), 396 Claverie, P., 341(92, 93, 94, 95, 96, 100, 101, 103, 104, 108, 109), 357(103), 359(92), 361(94,96), 367(96), 369(93), 3701931, 373(93), 374(104), 397 Cleaver, J. E., 155(194), 156(194), 157 (1941, 218 Clementi, E., 331(35, 36), 339, 396’ Clementi, H., 331(36), 396 Cohen, J. A., 174(276), 175(276), 220 Cohen, S. S., 118(27, 31), 120(27, 31), 213 (27, 31), ,914 Cohen, S. N., 95(117), 96(124), 116 Cole, L. J., 133(136), 917 Cole, T., 375(262), 401 Colvill, A. J. E., 83(70), 94(111), 95(111), 96(122), 108(122), 113, 114, 116 Commings, D. E., 210(353, 3541, 222 Contesse, G., 79(43), 113 Cotter, R. I., 279,299 Coulson, C. A., 118(6), 214 Cox, J., 436 Cox, R. A., 133(148), 217,231(29), 232 ( B )233(29), , 296, 350(174), 399 Crab%, P., 225 (19),296 Cramer, F., 406(36), 414(115, 116, 117, 1181, 434, 436 Crane-Robinson, C., 286(172), 500 Crathorn, A. R., 118(79), 120(79), 128 (79), 130(79), 151(79), 155(79), 156 (79), 157(79), 158(79), 213(79), 216 Crawford, E. M., 83(71, 74), 99(74), 100 (741, 102(74), 113 Crawford, L. V., 83(71, 74), 88(91), 99 (741, 100(74), 102(74), 113,114 Creasey, W. A., 155(195), 164(195), 187 (310), 218, 221 Crick, F. H. C., 24,29, 207(346), 222,286 (174), SOU, 253(99), 298, 320(47), 321 (50), 326, 326, 359(196), 385(293), 399, 402

Crothers, D. M., 306, 309, 325 Curtis, H. J., 206(343, 3441, ,922 Cusachs, L. C., 339(52), 340(52), 39G Cushley, R. J., 238(54), 296 Cusin, F., 210(349, 3521, 222

D Daljanski, F., 194(326), f?22 Damjanovich, S, 158(225), 213(225), 2f9

Damle, V., 240(64), 297 Dangluk, S. S.,374(235), 4UO Daniels, M., 12(74), 28 Danilov, V. I., 341(69, 1351,396,398 Danon, A., 415(139), 437 Darnell, J. E., 85(172), 116, 195, 221 Das, H. K., 79(34), 112 Das, N. K., 158(222), 211(222), 213(222), 219

Dashkevich, V., 413(110), 436 D a m e , M., 374(231), 4OO Davidson, J. N., 143(166), 163, 218, 219 Davidson, N., 258(118), 298, 304(8, 101, 306, 309(30), 316, 317(8), 326,351 (181, 1821, 399 Davidson, P. F., 10(54), 28, 133(145), 135 (145), 217 Davie, E. W., 80(61), 90(61), 92(61), 94 (611, 96(61), 110(61), 113 Davies, D. R., 9(42), 28,241(76), 252(99, 101, 1021, 257(117), 259(122), 297, 298, 374(236), 400 Davis, D. R., 331(35, 361,396 Davis, R. C., 243,297, 341(124), 374(124), 398

Davison, P. F., 18(87), 29 Dawid, I,. B., 322(52), 326 Dean, C. J., 118(18, 80, 1051, 119(18), 120(18, 80, 105), 124(18), 128(80), 130(80), 151(18), 154, 156(80), 210 (18), 213(18, 80), 214,216 Delahay, P., 43(52), 45(52), 72 Delbriick, M., 11(69), 28, 154(192), 218 De Lerme, B., 374(242), 401 Dellweg, H., 15(127), 29, 377(2701, 401 Del Re, G., 330, 337, 341(158), 396, 399 De Mars, R. I., 109(163), 116 Dembitzer, H. M., 194(327), 222 Demerec, M., 118(25), 120(25), 213(25), $14

Demushkin, V., 405(19, 331, 417(158), 418 (158, 1591, 419(159), 420(1591, 421 (161), 425(33, 1591, 426(33, 1701, 427 (1701, 434, 437 Denliardt, D. T., 118(101a), 149(101a), 216 Denhardt, G, H., 78(21), 106(21), 11.9 Denia, A., 330(25), 331(321, 341 (25, 32), 350(25), 354(25), 382(25), 3g5 Depireux, J., 341(129), 398

444

AUTHOR

INDEX

DuBois, K. B., 118(38), 120(38), 160(38), Deutch, A., 415(120), 436 916 de Vellis, J., 206(342), 213(342), 2.92 De Voe, H., 240(72), 297, 329(10), 341 DuBois, R. P., 205(341), 213(341), 2Z.8 (10, 831, 346(10), 350(173), 355, 366 Duchesne, J., 341(127), 349(166), 398, 399 Diitting, D., 405(9), 434 (101, 372,396, 397 Dunn, J. J., 116 Devreux, S., 173(271), 213(271), 220 Dupuy-Mamelle, N., 341(611, 391(611, Dewey, D. L., 133(151), 136(151), 217 396 Dewey, K. F., 79(41), 89(41), 113 Dymshits, G., 413(110), 456 Dewey, W. C.,156(209), 157, 219 di Bitonto, G., 198(332), 222 E Dickstein, S., 351(1801,399 Dicckmann, M., 92(105), 99(130), 105 Earle, J. D., 118(110), 124(110), 125(110), 126(110), 127(110), 216 (1301, 108(105), 114,116 Ebal, J. P., 225(13), ,996 Di Marco, A,,48(81), 7.8 Echols, H., 78(25), 119 Dimroth, K., 410(62), 436 Eckstein, H., 118(86), 120(86), 126, 151 Dirksen, M., 78(22), 112 (86), 155(86), 158(86), 165(86), 205 Diner, 8., 341(158), 399 (861, 213(86), 216 Ditmars, W. E., 374(230), 400 DjordjeviE, B., 118(74), 120(74), 127, 151 Edelman, A., 174(276), 177(276), 920 Edgar, R. S., 78(21), 106(21), 111 (741, 156(74), 158, 213(74), 816 Edmonds, M., 181(284), ,920 Djordjevi6, N., 118(106), 124(106), 216 Dobrov, E. N., 57(97), 73, 284(163), 300, Edwards, P. A., 21 (103), 29 Egami, F., 412(83), 436 412(86, 951, 456,436 Eguchi, S., 46(57), 72 Doermann, A. H.,12.2 Ehrenberg, A., 375(255), 401 Doida, Y., 187(307), 221 Doly, J., 76(5), 105(154), 110(165), 112, Ehrenberg, L., 375(255), 401 Eigner, J., 31(1), 70 116 Eisenstadt, J. M., 173(273), 213(273), 220 Domkin, V., 427(173, 174), 438 Eisinger, J., 375(257, 2791, 401 Dondon, J., 415(132), 436 Doty, P., 18(86), 29, 79(41), 89(41), 113, Ekert, B., 118(19, 201,151(19), 211(19), 212(19, 201, 213(19, 20),,914 118(117), 126(117), 184(296), ,917, 221, 224(2, 8),225(8), 229(24), 231(2, 291, Eliseeva, G., 414(114), 4% 232(29), 233(29), 267(133), 280(180), Elkind, M. M., 158, 211(216, 217, 2181, 213(216, 217, 218), 219 281(161), 285(2), 286(170), 289(193), 296, 298,,999, 300, 301, 303, 304(7), Ellery, B., 415(141), 437 316, 317(42), 324,326,350(174), 373 Ellis, F., 156(203), 218 Ellis, M. E., 133(136),617 (220), 399, 400, 412(94), 436 Elson, D., 76(2), 112 Douzou, P., 382(276), 401 Dove, W. F., 78(23), 11.2,304(8), 317(8), Elving, P. J., 38, 39(27, 34, 351, 40(35), 41(27, 45), 42(34, 41, 451, 43(34, 45), 3.26 71, 349(167, 168, 169, 170), 399 Downing, M., 370(218), 400 Drake, J. W., 15(117, 119), 29, 387(296), Emerson, T. R., 233(38, 39, 40, 41), 234 (40, 41, 45), 235, 237, 238, 247(84), 390(289), 40.2 .89G, 697, 354(184), 399 DmkuliE, M., 118(59), 120(59), 138(59), 151(59), 173(59), 174(265), 175(59), Emery, A. J., Jr., 80(63),82(63), 83(63), 113 213(59), 216,220 fl Drewitch, V., 406(35), 412(35, I l l ) , 414 Emmelot, P., 8(37, 38), Emrich, J., 79(40), 89(40), 113 (35,I l l ) , 434,436 Englesberg, E., 77(19), 112 Drobnik, J., 350(177, 178, 179), 399 Dubinin, N. P., 118(93), 120, 204(93), ,916 Ennis, H. L., 93(106), 114

AUTHOR

445

INDEX

Ephrussi-Taylor, H., 12(76), 15(76), 19 (76), 28 Epstein, R. H., 78(21, 22, 241, 80(61), W (611, 92(61), 94(61), 96(61), 106(21, 1571, 110(61), 112, 113,116 Ermolaeva, N. V.,133(142), 217 Errera, M., 118(2,30,32), 120(30,32), 133 (32, 1351, 151(32), 173(32), 174(32), 185(301, 303), 205(301, 305), 213(30, 32), 214,221 Evans, A., 94(113), 101(113), 114, 170 (2601, 220 Evans, H. G., 130(123), 217 Everett, G. A., 278(154, 1 5 3 , 299, 405f8, ll),494 Eyring, H., 231(30, 33), 235(30), 238(30), 296

F Fancher, H., 99(130), 105(130), 115 Falk, M., 373(223), 400 Farkd, J., 234(47), 296 Farkas, W., 410(63), 436 Fasman, G. D., 231,249(87), 254,260(87), 262(124), 296, 297, 2D8 Faulkner, R., 405( 121,434 Fausto, N., 118(83), 120(83), 160(83), 162, 163(83), 164(237), 187(316), 189(316, 316a), lW(83, 316, 316a), 205(237, 316), 213(237), 216, 221 Favorov, V., 413(107), 436 Feldman, H., 405(9), 434 Feldman, M., 412 (89), 436 Feldschreiber, P., 118(18), 119(18), 120 (18), 124(18), 151(18), 154(18), 210 (is), 213(18), 214 Felsenfeld, G., 31(2), 57(101), 70, 73, 240 (63), 245, 249(93), 252(102, 103, 1041, 277(83b), 296, 297, 298, 341(86), 397 Fernandes-Alonso, J. I., 329(12), 341(12), 395

Fernindez-Morin, H., 83(70), 113 Fiala, S., 44(59), 72 Ficq, A , 185(301), 205(301), 221 Filipovich, I. V., 118(4), 214 Filipowicz, B., 44(58), 72 Fisher, H., 109(163), 116 Fitch, F. W., 118(38), 120(38), 160(38), 215

Flamm, W. G., 309, 323(32), 325 Flessel, C. P., 252(100), 298 Fluke, D. J., 150(188), 218 Fontaine, F., 267(134), 298 Forejt, J., 37(17), 71 Forssberg, A,,118(2, 35, 36), 120(35, 361, 160(35, 36), $14 Fowler, G. N., 341(871,397 Fox, C. F., 93(lG9), 99(129), 100(129), iio(iog), 114, 116 Fox, E., 118(49), 120(49), 121(49), 135 (49), 213(49), 216 Fox, J. J., 234(44), 238(54), 296 Fradkin, G. E., 118(100), 121(100), 213 (1001, 216 Fraenkel-Conrat, H., 3(7), 7(32), 9(32, 43, 48, 49), lO(32, 57), Il(32, 57, 631, 12(32, 78), 13(32), 14(32, 431, 15(32, 48, 57), 17(48, 49), 18(83), 19(32), 20 (43), 21(48), 22(32, 48, 49, 1051, 23 (32, 83), 24(48), 26(32), %7, 28, 433 (3, 4), 433 Frampton, E. W., 118(71), 120(71), 142 (159), 151(71), 153(159), 173(268), 178, 179, 198(268), 213(71, 159), 216, 217, 220 Frank-Kamenetsky, M., 412(96), 436 Franklin, R. M., 23(107), 29 Frary, B. D., 45(76), 46(76), 50(76), 60 (76), 61, 68(76), 72 Frascicatti, M., 374(242), 401 Frazier, J., 145(181), 218, 415(133), 436 Fredericq, E., 267(134), 298 Freeman, E. J., 106(156), 116 Freeman, K. B., ll(68, 701,12(68), 15 (68), 28, 391(303), 402, 438 Freese, E., 1(3), 6(28), 7(34, 35), 8(34), 9(46), 12(48), 13(3), 14(28, 32, 46, 94, 95, 96), 15(95), 16(3), 19(35, 94, 95, 961, 21(95), 23, 24(3), 25(95), f l , 28, 29, 118(94), 204(94), 216, 383(283, 284,285), 387(283,284), 393(284), 401, 402, 417(155), 432(191, 192, 1931, 433 (194, 198, 200), 437,438 Freifelder, D., 10(54), 18(87), 28, 29, 118 (51, 52), 120(51, 52), 121(51, 521, 133 (51, 52, 1451, 13561, 52, 129, 1 4 8 , 136(51, 52, 1291, 138, 150(51, 52, 129), 213(51, 52, 1291, 215,217

446

AUTHOR

Freifelder, D. R., 118(52), 120(52), 121 (52), 135(52), 136(52), 150(52), 213 (52), 216 Frenkel, E. P., llP(82), 120(82), 160(82), 816

Fresco, J. R., 224, 225, 252(100), 256, 270 (142), 271(142), 289(193, 194, 195, 198), 290(198), ?296,298, 899, 300, 310 (351, 326 Freund, A. M., 57196),73 FriE, I., 234, 296 FriE, J., 268( 136), 299 Fried, M., 409(44), 434 Fritz, H., 410(58), 436 Frolova, L., 428(176), 438 Fromageot, P., 106(158), 109(162), 116 Fuchs, E , 80(53), 83(69), 90(97), 93 (97), 104(152), llO(97, 152), 113, 114, 116 Fujii, I., 269(139, 140), 270(139), 299 Fujiki, H., 143(175), ,918 Fujioka, M., 156(211), 186(211), 187(312), 219,2?,91 Fujita, H., 330(21), 341(76, 134), 354(76), 3996,397, 398 Fuke, M., 269 (140), 899 Fukuda, N., 339(54), 341(101), 396, 397 Fukui, X., 341(151), 385(151), 398 FulIer, W., 273(149a), 89g Furth, J. J , 76(4, 12),80(51), 94(113), 101(113), 112, 1f3, 114, 170(260), E20 Futai, M., 238(55), 296

G

INDEX

Gellert, M., 241(76), 297, 309(33), 326 Georgiev, G. P., 168, ,980 Gersch, N. F., 341(106), 374, 3.97 Gerson, J., 14(95), 15(95), 19(95), 21(95), 25(95), 29 Gerstenberger, A., 10(56), 28 Gierer, A., 4, 14(12), f l Giessner-Prettre, C., 330(22, 23, 241, 331 (331, 341(22, 23, 24, 33, 63, 1401, 346 (22), 349(22), 350(22, 23, 241, 354(22, 23, 241, 382(22, 23, 241, 383(22, 241, 392(63), 396, 396, 398 Gilbert, M., 331(32), 341(32, 108, 1091, 374,396,397 Gilbert, W., 76(11), 77(17, 181, 111(18), 112 Gilham, P. T.,405(2), 413(21, 103, 105, 1081, 43% 434, 436 Gill, S. J., 370(218, 219a), 400 Gillespie, D., 85, 116, 301(3), 307, 323(3), 324,326 Ginosa, W., 150(184), 218 Giovanella, B. C., 374(241), 401 Girshovich, A., 413(106), 436 Gladstone, L., 76(1), 112 Glaubiger, D., 243, 997 Goel, N. S., 341(101), 397 Gold, M., 101(137), 116 Goldberg, I. H., 109(160), 126 Goldstein, A., 79(34), 112 Goldthwait, D. A., 105(153), 110(153), 116 Gollmick, F. A., 45(73, 741, 46(74), 48 (73, 74, 84), 64(74), 68(74), 72 Gomatos, P. J., 269(137), 299, 310(36), 3% Goodgal, S. H., l4(89), 18(89), 29 Goodman, H. M., 76(10), 112, 405(13),

Gage, 1,. P., 78(28), 111 Gaines, K., 79(38), 88(38), 98(38), 104 (381,106(38), 113 Gal-Or, L.,416(151), 437 Ganesan, A. T., 210(351), 222 434 Gordon, J. A., 373(224), 400 Gardner, A. W., 38(26), 50(26), 71 Gardner, B. J., 286(182), 287(182), 300, Gordon, M. P., lO(58, 59, 60),12,27 Gordy, W., 341(147, 148, 1491, 375(147, 374(233), 400 148, 149, 253, 254, 258, 259, 260,2611, Gartland, W. J., 271(145), 299 376,377,398,401 Gayevska, E., 118(117), 126(117), 917 Geiduschek, E. P., 10(54), 19(92, 931, 21 Gorlenko, Zh. M., 96(lm), 116 (92, 9 3 , 28, 29, 78(24, 28), 84, 85 Gorn, R., 224(8), 296 415(124), 436 (75), 94(111), 95011, 115, 118), 96 Gorshkova, B., (119, 121, 1221, 97, 108(122), 118, 113, Goutarel, R., 288(188), 300 Goutier, R., 164(238), 187(238), 189, 205 11.4, 116 (238, 3171, 213(238), 219, 281 Gelbard, A. S., 185(304), 281

AUTHOR

447

INDEX

Grachev, M., 413(106), 436 Grady, L. J., 139,141,217 Graham, S.,7(35), 19(35), 27,432(191), 438

Granick, S., 286(177), 300 Grasemann, J. M., 118(49), 120149), 121 (491,135(49), 213(49), 215 Gratzer, W. B., 231(29), 232(29), 233(29), 350 279(15&.),280,281(158),296,299, (1741,599 Graziosi, F.,172,220 Green, B., 374(238, 239,240,248),400,4Ol Green, D. M., 14(115), 29 Green, D.W., 249(89), 2.97 Green, G., 288(188), 300 Greenberg, J., 9(49, 50), 15(50, 1241,17 (491,22(49), 28,29 Greer, S., 118(109), 124(109), 216 Grimison, A.,12(74), 28 Gros, F.,76(11), 77(16), 78(27), 79(43, 48), 92(104), 93(108), 96(123), 110 (108), 112, 113,114,116 Grosjean, M.,224(4), 225(4), 230(4), 296 Grossman, L., 6(21,29), 7(21, 29), lI(61, 73), 12(73), 14(21, 291, 15(61, 71,72, 73), H,28, 145(182), 171(262), 181 (182), 218, 220, 231(31), 249(87), 254 (311,260(87), 296,297,391(301, 305, 306,307),402,412(93), 433(195, 1971, 436,438

Grunberg-Manago, M., 9(41), 14(41), 19 (411,28,89(95), 114, 415(132), 436 Giinther, H. L., 379(273), 401 Guild, W. R.,118(8), 214 Gulyas, S.,118(75), 120(75), 128(75), 130 (75), 156(75), 213(75), 215 Gumport, R. I., 99(129), 100(129), 115 Gurskii, G . V.,374(234), 400 Guschlbauer, W., 63(104), 64(104, 1051, 73,249(92), 29Y Gus kova., I,., 423(166,167,169), 437 Gut, J., 43(50), Y2,341(165), 399 Guttes, S.,158(223), 211(223), 213(223), 219

H Habermann, V.,45(72), Y2, 405(16), 416

(IF), 434 Harhagen, J. M., 267(135), 298 Hadopian, H., 405 (6),433

Hadeib, Lj., 118(44), 120(44), 816 Hagen, U.,118(88, %a), 133(150), 144 (881, 145(88), 148(88), 167(88, 88a), 169(88, 88a), 172,213(88, %a), 216, 2lY

Hsger, G., 216 Haines, J. A.,393(316), 402,43Y Haines, R. B.,118(6), a14 Hall, B. D.,90(99), 114, 116, 289(193), 300,301(21,324 Hall, C. E., 83(73), 100(73), 113 Hall, E. J., 130(124), 156(208), 217,219 Hall, J. B., 102(143), 115 Hamer, D., 39(30), 40,42(30), 68(30), Yl Hamilton, 11.D., 273(147), 299 Hamlin, R. M., Jr., 36412041,400 Han, A,,158(219, 2201,211(219,220), 213 (219,220), 219 Hanawalt, P. C.,118(11), 119(11), 120 (Il), 123(11), 151(11), 153,174(275), 210(11), 213,214,220 Hanlon, S., 370(215), 400 Hanson, K.P., 155(199), 164(199), 218 Harbem, E., 184,185(300), 221 Hariharan, P. V.,11 (701,28 Harm, W., 118(98, 991, 121(98, 991,213 (98,991,816 Harrington, H., 118(37, 84,85), 120(37), 144,145(84, 851, 146,147,160(37), 167 (84,85), 169(84,851, 213(84, 8 5 ) , 215, 216

Harris, F. E,, 341(119, 120,121,1221,3.98 Hartman, K.A,,Jr., 373(223), 400 Hartman, Ph. E., 143(163), 207(163), 218 Hartmann, G., 110(167), 111(170), 116 Haschemeyer, A. E. V.,238(52), 296,357 (188,189),374(251, 2521,399, 4 O l Haselkorn, R., 18(86), 29, 257(115), 289 (193,194), 2.98, 300 Hashizumc, H., 253, 269(139), 270(139), 280,698,299

Hastings, R.,11(65),25 Hatch, F. T., 118(87), 213(87), 616 Haug, A.,382(276), 401 Hayashi, M., 87(88), 94(110), 114,175, 260

Hayashi, M. N., 94(110), 114 Hnyatsu, H., 416(148, 149), 43Y Haves, D., 415(138), 43Y HRyes, F.N., 82(68), 113, lXlf286), 221

448 Hayes, W., 24(109), 29, 383(287), 40B Hayes-Baron, F., 415(138), MY Haynes, R. H., 118(15, 65, 92, 1161, 119 (15, 92), 120(15, 651, 123(15, 92), 151 (15, 65), 154(15),210(15), 213(15, 65), 214, 216, 216, 917 Heath, J. C., 39, 40(29), 41(29), 42(29), ri Heesing, A., 411(70), 436 Heidelberger, Ch., 184(300), 185(300), 221, 374(241), 401 Helene, C., 382(277), 4Ol Heller, H. C.,375(262),401 Helmkamp, G. K., 240(65), 254(109, 110), 289(109, 110, 197, 200), 290(109>,291 (109, 197, 2001, 297, 298, 300, 341 (lal), 369(211), 398, 400 Henley, D., 271 (1431,299 Hennig, S. B., 143(176),218 Henry, J., 80(52), 113 Heppel, L., 305(17), 325, 409(45), 434 Herak, J. N.,341(147, 148),374(147, 1481, 375(260), 398, 401 Herriott, R. M., 12(77), 14(88), 15(77), 18(88), 19(77), 28, 29, 383(288), 402 Hevesy, G., 118(120), 159, 160, 217, 219 Hewitt, R., 118(66, 66a, 721, 120(66, 66a, 72), 124(66a), 151(66, 66a,721, 153 (72), 154(72), 213(66, 66a, 72), 216 Hewlins, M. J. E., 6(19, 251, 7, 27 Heyrovsk?, J., 32, 36(9), 37(16, 17), 38 (2'2, 241, 39(9), 71 Hiatt, H., 76(11), 118 Hidvbgi, E. J., 194(323, 324), 198(324), 981 Hietbrink, B. E., 205(341), 213(341), 222 Higasi, K., 332(381, 396 Highton, P. J., 406(300),434 Higuchi, S., 269(138), 271(146), 272(146), 273,999 Hillova, J., 118(107), 124(107), 916 Hilz, H., 118(86), 120(86), 125(86), 151 (861, 155(86), 158(86), 165(86), 205 (861, 213(86), 216 Hirschfelder, J. O., 355(185), 6999 Hirschman, S. Z., 245, 277(83b), 297, 341 (86), 597 Hishizowa, T., 186(305), 197(305), 991 Hnilirn, I,., 286(171), 300

AUTHOR

INDEX

Ho, N. W., 413(105), 436 Ho, P., 76(4), 118 Hoard, D. E., 412(88), 136 Hodes, M., 410(64), 436 Hofelich, F., 341 (1021,997 Hoffer, M., 234(43, 44), 296 Hoffman, T. A., 341(153), 598 Hoffmann, R., 338,396 Hofschneider, P. H., 83(69), 119 Holand, J. J., 182(289), 921 Holcomb, D. N.,249(91), 252(95), 297 Holiday, E. R., 350(172), 399 Holland, J., 194(323, 324), 198(323, 3241, 221

Holiey, R. W., 278(154), 299, 405(8), 412 (841, 415(84, 4% 435 Hollis, D. P.,238(49), 896, 341(142), 369 (1421, 374(142), 598 Holmes, D. E., 375(263), 401 Holmes, R., 415(128), 436 Holoway, B. W., 133(145), 136(14!i), 217 Holy, A., 410(55, 56), 436 Holewarth, G. M., 229, 230(25), 281(161), 996, 999 Honig, B., 330(28), 3411281, 355(28), 396 Honikel, K. O., 110(167), 116 Hoogsteen, K., 357, 699 Hooper, C. W., 273(148, 148a), 999 Horn, E. E., 14(88), 18(88), 99 Hory, K., 143(175), 218 Hoskinson, R. M.,405(12), 434 Hotz, G., 118(102, 102a), 121(102), 123, 149(102a),916 Howard, A., 118(39), 120(39), 156(202), 160(39), 216,918 Howard, B, D., 15(118), 29, 390(299), 409 Howard-Flanders, P., 118(13, 91, 971, 119 113, 911, 120(13), 123(13, 91),151(13), 154, 910(13), 213(13), g14, 216 Howsden, F. L., 118(110, l l l ) , 124(110, lll), 125(110, 1111, 126(110), 127 (110), 216 Hoyer, B. H., 317,325 Hradecna, Z., 95(116), 103(116), 115 Huang, P. C., 76(14), 119 Huang, R. C.,168, $20 Huchmann, J., 413(98), 436 Hudnik-Plevnik, T., 118(106), 124(106), 133(138), 142(158), 151(161), 174

AUTHOR

449

INDEX

(138), 175(138, 278, 279), 176, 177, Janik, B., 38, 39(27, 35), 40(35, 42, 43), 41(27, 42), 42(43, 46,47,481,43(46), 180(279), 183,195(330), 196(330), 197, 66(46), 71 198(278, 330), 213(278), 216,217,220, Janion, C., 6(27), f l , 416(152), 429(186), 222 430(186), 431(183, 185,1861,432(152, Hudson, C. S., 234(42), 296 1861,433,437,438 Humlovh, A.,43(51), 45(51), 72 JankoviE, V.,165(249), 166(249), 220 Humphrey, R.M.,156(209), 157,219 Jaskunas, S. R., 254(108a), 277(108a), 278 Huong, T., 428(1771,@8 (108a,156), 279(108a), 298,299, 341 Hurwitz, J.,76(6, 121,79(39), 80(51, 58), (1241,374( 1241,398 81(58), 82,83(58), 84(6), 88(39, 931, 89,92(39), 94(113), 95(117), 96(124), Jehle, H., 341(90, 91), 397 97(39), 98, 101(113,137), I04(39), 105 Jencks, W. P.,373(224), 400 38,71 (39,155), 109(155), 112,113,114, 116, Jenkins, I. L., Jennings, J. P., 233(35), 296 116,170(260), 220 Jensen, R.,258(118), 298,309(30), 325 Huston, D. C., 138(152), 217 Hutchinson, P., I18(8), 214,2730491, Jensen, L.H.,238(53), 296 Jirgensons, B., 286(171), 300 299 Johns, H.E., ll(68, 69, 70), 12(68), 15 (68),28, 391(303), 402, 438 I Johnson, E. A.,35011721,399 Ibuki, F., 45(60), 72 Jones, A.,411(66, 671,415(126), 496,4% Ijlstra, J., 377(264, 265, 2661,401 Jones, D. S., 102(140), 116 Imahori, K.,253,269(139), 270(139), 280, Jones, 0. W., 90(98), 92(105), 99(98, 1341, 298,299 100(98), 102(98), 105(98), 106(134, Imamoto, F., 78(44, 45),113 156), 108, 110(98), 114, 116 Imamura, A.,330(17, 21), 341(17, 76, 133, Jones, R. Norman, 364(208), 400 134,151), 354(76), 374(245, 2461,385 Jordan, D.O., 341(IM), 374,997 (1511,596,597, 598,401 Jordan, F.,331(31), 341(31), 374(31), 383 Ingalh, R. B.,375(263), 401 (282),396,401 Ingram, V. M., 405(5, 6),413(103), 433, Jorgenson, G., ll8(66), 151(66), 213(66), 456

Inman,

R.B., 143(172, 173), 918, 258(119,

1201, 298

Inoue, Y., 245(83a), 246,297 Inouye, M.,79(40), 89(40), 115 Isaars, L.N., 78(25), 112 Isenberg, I.,374(232,247), 400, 401 Ishihama, A.,80(57), 113,170(261), 210 Ishimoto, M.,15(127), 29 Ishiwa, H., 14(125), 29 Isupova, L.S.,133(142), 217 Ito, E., 415(122), 436 Ivanova, O., 405(22), 413(22), 434 Twai, I., 231(34), 237(34), 238(34), 296

216 Josse, J., 31(1), 70

Jovih, D.,158(219), 211(219), 213(219), 219 Jovicki, G., 165(249), 166(249), 220 Joyner, A.,78(25), 112

K

Kabat, S., 76(14), 112 KaFanski, K.,118(44), 120(44), 616 Iiafiani, K. A.,194,212 Kaga, M., 187(312), 121 Kagi, J., 231(32), 249(32), 262(32), 268 (32),29G J Kahan, F., 415(129), 436 Jacob, F., 77(15, 161,112, 210(352), 22.2 Kaiser, A. D., 306(22), 310(22), 3% Kakefuda, T., 210(254), 2% Jacobson, B., 157(214), 819 KalLb, D., 45(64, 65, 66,67, 68,69,70), Jaffk, H.H., 332(39), 396 72 James, T.W., 289(191, 192), 300

450

AUTHOR

INDEX

Kalinin, V. L., 14(91), 15(91), 18(91), 19 Kikugawa, K., 416(148), 437 Kim, J. H., 185(304), 221 (91), 20(91), 21(91), 25(91), 29 Kim, S. XI., 185(304), $21 Kallenbach, N.R., 306, 309(34), 326 Kimball, R.F., 118(119), 126(119), 217 Kalvoda, R.,37(20, 21), 38(22, 23), 71 Kameyama, T.,80(57), 113, 167(254), Kimura, M., 319, 320(46), 326 170(261), 173(273),205(254),213(254, Kin-Ichiro Miura, 183(295), 221 Kirk, J. M., l09(161), 116 273), 220 Kiselev, L.L., 428(176), @8 Kamiya, T., 82(66), loo(%), 113 Kanazir, D., 118(30,32, 106, 115), 120(30, Kiseleva, N.P., 284(167), 300 32), 124(106), 126(115), 1331321, 151 Kishimoto, S., 157(212), 18612121, 187 (309),219,221 (32), 173(32), 174(32), 199(334), 200 (334), 201(335), 202(335), 203(337), Kita, M., 238(58),996 Kjeldgaard, N. O.,76(8), 78(8), 79(49), 213(30, 321,$14,216,217,222 112,113 Kanmir, D. T., 165(249), 166(249), 190 (318), 191(318), 195(330), 196(330), Klamerth, O., 173(267), 175, 178, 213 (2671,,220 197(318), 198(330), 213(318), 220,221, Klebanova, L., 406(25), 410(59), 434,436 228 KIee, W. A,,235,296 Kanner, L. C., 94(lll), 95(111), 124 Klein, G., 118(35, 361, 120(35, 36), 160 Kano-Sueoka, T.,271(145), M9 (35,36), 214 Kaplan, H.S., 118(17, 54, 55, 56, 63, 68, 110, 111, 112), 119(17), ZO(17, 54, 55, Klein, R., 416(146), 437 56, 63, 681, 123(17), 124(110, 111, Kleinschmidt, A. K., 87(89), 114 112), 125(17, 110, 111,1121,126(110, Kleinwachter, V.,350(179), 399 112), 127, 133(17), 137, 138, 140, 151 Klimenko, S.,412(95), 436 (17,54, 55, 56, 63, 681,207W81, 210 Kline, B,,239(62), ,997 (17), 213(17, 54, 55, 56, 63, 68),214, Klouwen, H.M., 155(200), 164(200), 218 Klyne, W.,233(35), 296 215,216,222 Knijnenburg, C.M., 88(92), 114 Kasinski, H. E., 44(59),72 Knorre, D.,405(22, 35),409(38,39,40, 41, Katz, L.,357(190), 399 42), 413(22, 35, 107, 109, lll), 414 Katz, Z.,364(207), 400 (35,Ill), 434,436 Kaudewitz, F.,14(120), 29 Kaufman, B. N., 118(34), 120(34), 160 Kniisel, F., 110(167,168), 116 Kochanski, E.,332(32), 341(32, 791, 354 (341,214 397 (791,396, Kawase, S.,269(140), 299 Kochetkov, N. K., 6(24), 7(33), R,405 Kay, C.M., 262(126), 298 (19, 33), 406(25), 413(99), 414(114), Keck, K.,118(88), 144(88), 145(88), 148 417(157), 418(159), 419(159), 420 (881, 167(88), 169(88), 213(88), 226 (159), 421(161), 423(164, 165, 1681, Keir, H. M., 143(166), 218 424(168), 425(33, 159), 426(33, 1701, Kellenberger, E.,78(21), 106(21), 119 427(1&4, 165, 170, 172, 173, 1741, 428 Kelly, G.W., 194(327), 222 (164, 165, 172), 431(187), 432(187, Kelly, L. S., 118(41), 120(41), 160(41, la), -434, &6,437,4% 228), 216,919 Kelner, A.,118(29), 120(29), 213(29), 214 Kodama, M., 330(17), 341(17), 374(245, 246), 330,401 Kestner, N.R., 373(221), 400 Kodoya, M.,99(128), 116 Khesin, R.B., 96(120), 116 Khorana. H.G., 102(140), 126,305(16), Koga, M.,156(211), 186(211), 219 6(17), 6(17), R,417(156), 307(24), 526,405(12), 409(37, 52, 53), Kohlhage, H., 418(156), 437 434,@6 Kohne, D. E., 302, 317, 318, 319(5), 321, Khuong-Huu, Q., 288(188), 300 Khym, J. X.,409(47), 436 324

AUTHOR

451

INDEX

Kollin, V., 79(37), 113 Kondo, N. S., 370(217), 400 Konrad, M. W., 79(38), 87(86), 88(38), 89, 90(86), 92(86), 98(38), 99(133), 104(38), 105(86, 133), 106(38, 133), 107(86), 110(166), 113, 114, 116,116 Konstantinova, V. V., 133(140), 617 Kopama, M., 341(133), 398 Koranda, J. J , 118(871, 213(87), 216 Kornberg, A., 5(15), 16(80), 27, 28, 95 (114), 114,143(165, 169, 170, 171, 172, 173), 218, 259(120), 298,309(31), 325, 415(131), 436 Kornberg, R. D., 99(130), 105(130), 115 Kornhauser, A., 15(127), 29 Kos,E., 174(265), 220 Koscheenko, N. N., 118(4), 21.4 Kostjanovsky, R., 406(29), 434 Kotaka, R., 14(81), 17, 19(81), 21(81), 28

Kotaka, T., 412(82), 435 KrajinFaniE, B., 118(106), 124(106), 216 Krakow, J. S., 93(107), il4 Kraut, J., 238(50,53), 296 Kreuckel, B., 160(228), 219 Krieg, D. R., 1(1), 13(1), 14(115), 16(1), 27,29, 383(286), 402 Kriek, A. K., N37, 38),27 Kritskii, G. A., 118(43), 120(43), 133 (1391, 165, 225,217 KrSger, H., 118(88, %a), 144(88, %a), 145(88), 148(88), 167(88, 88a), 169 (88, 88a), 172(88a), 213(88, 88a), 816 Kroes, H. H., 174(277), 220 Kruglyak, Yu A., 341(69), 396 KrupiFka, J., 43(50), 45(62), 7 2 Krutilina, A. I., 405(10), 434 Kubinski, H., 102(144, 145, 146), 103(145, 146), 115 Kubitshek, H. E., 15(122, 123), 29 KuFan, Z., 54(93), 73, 118(59, 60, 61, 701, 120(59, 60, 61, 701, 138(59, 60,61, 70), 151(59, 60, 61, 70), 173(59, So), 175 (59, 60, 611, 182(291), 213(59, 60, 61, 70), 115, 221 Icumar, S., 14(125, 126), 29,431(184), 438 Kung, H., 278(155), 299, 405(11), 434 Kuprievich, V. A., 330(20), 341(20), 596 Kurland, C. G., 78(30), 110(30), l l d Kfita, J., 36(9), 39(9), 7 1

Kuzin,A. M., 118(3, 45, 121), 120(45, 1211, 128(121), 130(121), 133(144), 135(3), 138, 155(197), 1&4(197), $14, 217,218 Kwiatkowski, J. S., 341(74, 751, 350(74, 75), 354(74, 75), 397 Kyogoku, Y., 269(138), 299, 357(195), 364(195, 2051, 399, 400

1 Labana, L. L., 361(201), 400 Labaw, L. W., 118(28), 120(28), 150(189), 213(28), 214, 218 Lacroix, M., 341(129), 598 Ladik, J., 329(13), 330(18), 341(13, 153, 154, 155, 156), 396,398,399 Laipis, R., 88(90), l i d Laird, C., 118(49), 120(49), 121(49), 135 (49), 213(49), 816 Lajtha, L. G., 118(42, 46), 120(42, 46), 155(42), 156(203), 216,118 Lakshminarayanan, A. V.,238(51), 296 Lamb, B., 39(31), 42(31), 71 Lamborg, M. R., 231, 249, 262(32), 268 (32), 296,298 Lamola, A. A,,382, 4ffl Landy, A., 405(13), 434 Lane, B., 410(60, 61), 436 I,ane, D., 118(117), 126(117), 817, 317 (41),325 Langridge, R., 86(83), 103(149), 114, 115, 259(123), 269(137), 272(151), 273 (149a, 150), 2998,299 Lapthisophon, T., 118(72), 120(72), 151 (72), 153(72), 154(72), 213(72), 216 Larkiewicz, Z., 118(108), 124(108), 216 Laser, H., 118(14), 119(14), 120(14), 124 (14), 151(14), 210(14), 213(14), 814 I,atarjet, R., 118(19, 25, 261, 120(25, 261, 151(19), 211(19), 212(19), 213(19, 25, 261,214 Lavik, P., 118(37), 120(37), 160(37), 215 Lawley, P. D., l(41, 61231, 8, 13(4), 16 (4), 20(4), 21(4, 100, 1031, 22(106), 27,29,374(249), 385(290, 291, 292), 392(309, 310, 311, 312, 313, 314, 3151, 393(318), 4ff1, 402, 412(76), 455,429, 438 I,awrence, M., 86, il4 Lazar, J., 240(68), 297

452

AUTHOR

INDEX

Limperos, G., 133(143), 217 Lazurkin, Yu.,412(96), 436 Lin, C. Y., 231, 238, 296 Lazurkin, Y. S., 286(178a), 288, 300 Lindahl, T., 270(142), 271(142, 1431, 299 Lea, D. E., 118(5, 61, 214 Leach, W. M., 158(224), 211(224), 213 Lindblow, C., 231(31), 254(31), 262(124), 296, 298 (224), 219 Le Blanc, J. C., 11(68), 12(68), 15(68), Lingens, F., 9(47, 52), 15(47, 52), 23(47), 28, 416(144, 1451, 4.37 28,391 (3031,402,438 Lipsett, M. N., 241(76), 297,289(196), Lebowits, J.,88(90), 114 SOO, 305, 326 Lederberg, J., 210(351), 2.92 Liquori, A. M., 374(242), 401 Ledoux, L., 165(245), 213(245), 219 Litaka, Y ., 269 (138), 273(150a), 299 Lee, T., 157(214), 219 Legrand, M., 224(4), Z25(4), 230(4), 296 Litman, R. M., 12(76), 14(90), 15(76), 18 (901, 19(76), 23(90), 28, 29 Lehman, I. R., 5(15), 27, 143(165, 1701, Little, J. W., 309(33), 3.96 218, 415(131), 436 Livingston, D., 409(48), 436 Leidy, G., 18(85), 89,4120371, 436 Lodeman, E., 15(127), 89 Leive, L., 79(37), 118 Lowdin, P. O., 341(115, 116, 117, Leloir, L. F., 415(121), 4.36’ 388(116, 117, 118), 398 Leng, M., 240(63), .W7 Logan, R., 185, 205(301), 3.91 Lengyel, P., 5(16), 14(16), 97 Lofroth, G., 375(255), 4Ol Lemieux, R. V., 234(43), 296 Lerman, L. S., 10(53), 28, 109(164), 116, Lohman, K., 412(78), 436 286(176), 300,374(226, 227,228), 400 Looney, W. B., 162(!233), $19 Loveless, A., 9(45), 14(455),28 Lesk, A. M., 224(8), 225(8), 896 Lowney, L. I., 79(34), 112 Leslie, R. B., 374(232), 400 Lowy, B. A., 40,71 Letham, D., 411(66, 671, ,436 Lloyd, D. A., 243(79), 297 Lethbridge, J. H., 21(103), 29 Lett, J. T., 21(101), 29, 118(18, 78, 80, London, E. S., 289(190), 300 105), 119(18), 120(18, 78, 80, 105), Longworth, J. W., 382(2'78), 401 124(18), 128(78, 801,130(78, 801, 133 Lord, R. C., 357(195), 364095, 204,205), 373(223), 399,400 (147), 151(18), 154(18), 156(78, 801, 210(18), 213(18, 78, 801,214, 2f6,817, Lord Todd, 393(316), 402 Lowry, T. M., 224(1), 225(1), 296 393(319), 402 Lubin, M., 83(72), 93(106), 113, 114 Levedahl, B. H., 289(191, 192), SO0 Luck, G.,268(136), ,999 Levene, P. A., 289(190), 300 Ludlum, D. B., 9(39, 40), 14(39, 40), 19 Levin, S., 373(225), /to0 (99),20(99), R,g8, 89 Levine, E., 428(177), 438 Lukhovd, E., 54(91, 921, 55(92), 72 Levine, L., 373(224), 400 Lunell, S., 341(123), 398 Levinthal, C., 109(163), 116 Lunt, M. R., 102(142), 116 Lewin, S., 412(90, 91, 92, 93), 436,436 Luria, S. E., 78(22), 109(163), 112,116 Leyko, W., 44(56, 58), 72 Luthy, N. G., 39(31), 42(31), 71 Lezius, A. G.,143(176), 218 Luzzati, V., 57(100), 73,252(98), 298 Li, L., 405(10), 434 Li, T. K., %1(32), 235, 249(32), 262(32), Lykos, P. G., 341(58), 396’ 268(32), 296 M Lichstein, H. C., 173(270), 213(270), 220 Lieberman, I., 156(211), 157(212), 186 Maal@e,O., 76(8), 78(8,30), 110(30), 112 (212), 187(309,312,314,315), 219, 2.91 McArdle, H. H., 143(166), 818 McCaffery, A. J., 286(181), 287(181), 500 Lielausis, A., 78(21), 106(21), 112 McCallum, M., 323(32,53), 326 Lifschits, Ch., 341(65), 349(65), 396 Lifsan, S., 330(28), 341(28), 355(28), 3% McCarter, J, A., 374(248), 401

AUTHOR

453

INDEX

McCarthy, B. J., 194(322), 201(338), 221, 222, 307, 308, 310(37), 311, 317(44),

S26 McConaughy, B. L., 307,308,326 McDonald, C. C., 240(68), 297 McFlya, A. B., 156(210), 164(210), 219 McGinn, F. A., 39(32), 71 McGlynn, S. P., 339(53), 3% McGrath, R. A., 118(16, 691, 119(16), 120 (16, 69), 123(16), 134(16), 137, 138, 139, 151(16, 69), 154, 158(221, 2241, 210(16), 221(221, 2241, 213(16, 691, 213(221, 224), 214, 216, 217, 219 MeGuire, J., 15(117), 29 Maehmer, P., 349(166), 399 McKinney, L. E., 374(241), 401 McLachlm, A. D., 341(85), 597 McLaren, A., 302(4), 309(32), 317, 322, 324, 325, S26 McLaren, J., 412(74), 431(74), 435 McMullen, D. W., 278, 299 McNaught, A., 416(142), 437 McPhie, P., 279(156a), 280, 282(158), 299 McQuillen, K., 79(33), 112 Madison, J. T., 278(154), 299, 405(811),

434 Maestre, M. F., 283, 284, 285(164), 999 Magasanik, B., 167(253), 205(253), 213 (253), 220, 267(131), 298 Mahler, H. R., 181(285), 220, 239, 288, 297, so0 Maidlovh, E., 45(72), 72 Main, R. K., 144(179), 145(179), 163 (179), 218 ‘Maisin, J. R., 165(245), 213(245), 219 Maitra, U., 79(39), 80(58), 81(58), 82, 83 (58), 88(39, 931, 89, 92(39), 96(124), 97(39), 98, 104(39), 105(39, 155), 109 (155), 113, 114, 116, 116 Mak, S., 156(205), 213(205), 219 Malamy, M., 76(12), 112 Malcolm, D., 323(54), 926 Maling, B., 304(13), S26 Malrieu, J. P., 341(77), 354(77), S97 Malygin, E., 405(22, 35), 413(22, 35, 107, 1111, 414135, I l l ) , 434, 436 Malyshev, A., 406(28), 434 Malysheva, A., 409(39), 434 ManEiC, D., 118(44), 120(44), 216

Manclel, M., 9(51), 15(51), 24(51), 28 Mnndel, P., 76(5), 110(165), 112, 116, 118 (471, 120(47), 615 Mandell, J. B., 9(50), 15(50), 28 Mangiorotti, G., 79(35), 112 Mann, D. E., 339, S96 Maooukk, O., 42(49), 72 Mantione, M. J.. 3411114, 131, 132, 145, 1461, 374(114), 375(146), 377(131, 1321, 382, 398 Mantsavinos, R., 143(168),218 Marcker, K. A., 98(127), 116 Marinova, Z., 145(183),918 Markham, R., 410(57), 411(57), 436 Marmur, J., 118(117), 126(117), 217, 267 (133), 272(151), 298, 299, 301, 303, 304(7a, 7b), 316, 317(41), 324, 326, 373(220), 400 Marquisse, M., 278(154), 299, 405181, 434 Marsh, R., 359(198), 400 Marshall, R. E., 320(48), S26 Martinez, A. M., 82(68), 113 Marvin, D. A., 273(148, 148a), 299 Masin, A., 405(18), 416(18), 4 4 Mason, S. F., 286(181, 182), 287(181, 182), 300, 350(171), 351(171), 374 (2331, ~99,400 Masson, F., 57(100), 73, 252(98), ,998 Massoulie, J., 63(104), 64(104), 73, 249 (92), 297 Mathews, F. S., 357(191, 1921, Y99 Mathis, A,, 57(100), 73, 252(98), 298 Mataudaira, H., 186, 197,211 Matsushita, S., 45(60), 72 Matthews, L., 118(117), 126(117),217 Mauger, A. B., 288(187), 300 Maunzot, J. C., 238(56), 241(56), 242(56), 243(56), 249(87a), 252(56), 254(87a), 255(87a), $96 Mautner, H. G., 341(72), 396 Maxwell, Ch. R., 202,292 Mayneord, W. V., 206(345), %1 Mehrotra, B. D., 239(62), 297 Meites, L., 36(10), 71 Meljnikova, H. A., 194(328), 222 Mellema, J., 416(151), 437 Melvin, I. S., 240(69), 297 Melzer, H. S., 432(192), 438 Mennigmann, H. D., 118(114), 126(114),

216

454 Menael, C., 143(176),818 Merrill, S. H., 278(154), 899, 405(8), 494 Meselson, M., 267(132), 298 Metlas, R., 201, 202,288 Meta, E., 143(176), 818 Meyers, D. K., 155(193), 156(193), 157 (1931, 158(193), 162, 165, 186, 918 Michaelis, L., 286(177), 300 Micheel, F., 411(70), 436 Michelson, A. M., 4(14), 9(41, 491, 14 (411, 17(49), 19(41), 22(49), %7', 28, 63(104), M(104), 73, 238(56, 67), 241 (56), 242(56), 243(56), 247(57), 248 (571, 249(87a, 90, 921, 252661, 254 (87a,108, 255(87a, 108), %6(108), 286(90), 296, 897, 898, 305, S26, 354 (184), 399, 415(132), 436 Micka, K., 38(24), 71 Miles, D. W., 231, 996 Miles, H. T., 31(2), 70, 145(181), 818, 249 (93),897,357(195), 364(194), 399,361 (200),400, 415(133), 436 Miletii., B., 118(59, 60, 611, 120(59, 60, 61), 138, 151(59, 60, 611, 158(219), 173(59, 601, 175(59, 60, 611, 182(290), 211(219), 213(59, 60, 611, 213(2191, 816, 219, 881 Miller, I. R., 32(4, 61, 45(80), 48(80), 48 (82,83), 66(86),70,78 MiIIer, J.H., 359(197, 2061, 399, 400 Milles, N., 415(134), 437 Millette, R. L., 80(53), 90(97), 92(100), 93(97), 110(97), 113,114 Mihan, G., 86(83), 114, 273, 899 Milosavljevih, A., 186(306), 221 Milogevib, M., 199(334), 200(334), 928 Mirsabekov, A. D., 405(10), 415(124), 434, @6 Mitsui, H.,%(I%), 116 Mitsui, Y.,269(138), 899 Mittal, J. P., 382, 401 Miura, K., 269(138, 139, 140), 270(139), 299, 416(148), 437 Miauno, S., 111(171), 116 Moffat, S., 409(51), 436 Moffitt, W., 227, 228(22), ,996 Moldave, K , 92(103), 114 Mommaerts, W. H. F. M., 57(99), 73, 225(12, 13, 14), 262(14), 267(14), 274, 279, 296

AUTHOR

INDEX

Monastirskaya, G., 6(24), "(241, 27, 428 (178), 429(178), 430( 180), 431(1&0), 432(178, 188, 189, 1901, 438 Monny, C., 4(14), 27, 252, 898, 305, 325 Monod, J., 77(15), 112 Moohr, J., 95(118), 116 Moore, J. L., 118(87), 213(87), 216 Moore, T. A., 341(1601,399 Mori, J. F., 185, 205(302), 821 Morita, T., 185, 205(302), 8'21 Morosova, T., 406(32), 434 Morris, D. W., 79(49), 113 Moscowita, A., 225(18), 228(18, 22), 229

(It?), 996 Moseley, B. E. B., 118114, 104), 119(14), 120(14), 124(14), 151(14), 210(14), 213(14), $14, 216 Mosher, W. H.,133(143), 817 Mosley, V. M., 118(28), 120(28), 150 (189), 213(28), 814,918 Moss, G., 409(50), 9 5 Moudrianakis, E. N., 406(26), 416(151),

434, .4sr

Mudd, 8. H., 235, $96 Mueller, G. C., 182(288),221 Miiller-Hill, B., 77(17, lS), 111(18), 11% Muench, K., nl, 899 Muller, R. L., 341(58), 396 Mundry, K. W., 4, 14(12), 15(111), 23,

R Mushinskaya G., 413007, log), 436 Muto, A., 416(148), 437 Myers, L. S., Jr., 375(263), 401

N Nadkami, G. B., 173(272),213(272), 220 Nagakura, S., 330(19), 341(73), 350(73), 354(73), 396, S996 Nagata, Ch., 330(17, 21), 341(17, 76, 133. 134, 151), 354(76, 245, 246), 385(151), 396, 397,sgs,40t Naito, T., 21 (1041, 89 Nakada, D., 167(253), 205(253), 213 (2531, 820 Nakajima, T., 329(6, 7, S ) ,341(6, 71, 392 (61,396 Nakamoto, T., 84, 85(75), 113 Nakamura, Ch., 186(305), 197(305), 221 Nakanishi, K., 245(83a), 246(83a), .897

AUTHOR

INDEX

455

Okada, S., 118(122), 120(122), 130(122), NaBata, Y., 105(155), 109(155), 116 144(180), 145(180), 163(180), 187 Nancy, W., 405(21), 413(21), ,434 (3071, 217,218,221 Nandi, U. S., 258(118), 298,309(30), 326 Naono, S., 77(16), 78(27), 79(43, 481, 96 Okada, Y., 79(40), 89(40), 113 Okun, L., 118(49), 120(49), 121(491, 135 (123), 112,113,116 (491, 213(49), 216 Nash, H. A., 341(89, 1571, 356(891, 394, Oliver, R., 118(42), 120(42), 155(42), 216 397,399 Olivera, B. M., 258(118), 298, 309(30), Naylor, R., 405(21), 443(21, 108),434,436 326 Neidhardt, F. C., 76(7), 112 Olner, R., 156(203), 218 Neifan, A. A., 194(328, 329), 22.2 Olson, A. C.,240(69), #7 Nelson, J., 4121841, 415(84), 436 Oltmanns, O., 9(52), 15(52), 28 Nesbet, R. K., 330(16), 3411161, 596 Negkovih, B., 128, 156, 157(207), 186 Ono, J., 11, 12, 15(73), 28,171, 220,391 (301, 306), 402 (306), 219,221 Neville, D. M., 286(178), 300, 374(236), Opara-Kubjnska, Z., 102(144), 103(145), 116, 118(10), 119(10), 120(10), 124 400 (lo), 125(10), 151(101, 210(10), 213 Newton, J., 79(40), 89(40), 113 (lo), 214 Ney, H., 409(45), 434 Ord, M. G., 118(40), 120(40), 155(40), Nikolik, S., 186,221 160(40), 163, 165(247, 247a), 166 Nirenberg, M., 320 (481, 526 (247a), 216, 220 Nishimura, S., 102(140), 116,183(292), Ordy, J. M., 206(343), 222 221 Nishimura, T., 231(34), 237, 238(34, 551, Orgel, A,,286(174, 1751, 300,385(293), 296 409 Orgel, L. E., l(21, 13(2), 16W, 27, 118 Nitta, K., 111(171), 116 (%I), 212(24), 212(24), 214,383(289), Niyogi, S. K., 307, 308, 310(27), 326 40.2 Norman, A., 150(184), 218 Novak, D., 118(61), 120(61), 138(61), 151 Oriel, P. J., 269(141), 285, 286(169), 299, 300 (61), 175(61), 213(61), 216 Novelli, G. D., 1670541, 173(273), 205 Ormerod, M. G., 118(78), 120(78), 128 (781, 130(78), 156(78), 213(78), 216 (254), 213(254,273), 260 Osawa, S., 99(128), 116 Novogrodsky, A., 88(93), 114 Noyes, W. D., 118(42), 120(42), 155(42), Oshinsky, C. K., 309(33), 326 Otaka, E., 99(128), 116 216 Nuesch, J., llO(167, 168, 1691, 111(169), Oth, A,,267(134), 29.8 116 P Nutwell, D., 202, 222 Paduch, V., 1 1 8 ( 8 6 ) , 120(86), 125(86), 151 Nygaard, A. P., 90(99), 114,301(2), 324 (86), 155(86), 158(86), 165(86), 205 Nygaard, 0. F., 118(8l>, 120(81), 158 (86), 213(86), 216 (223), l60(81), 161, 162, 163(230), Paigen, K., 85(79), 114,118(34), 120(34), 211(223), 213(223), 216,219 160(34), 914 Painter, R. B., 118(76, 771, 120(76, 77), 0 128(76, 771, 130(76, 771, 155(76), 156 (76, 77), 157(76), 158, 184(76), 211 Obuchova, L., 413(106), 436 (215), 213(76, 77, 215), 616 Ochoa, S., 5(16), 14(16), 2’7 Paladini, A. C., 415(121), 436 Ogur,M., 409(46), 434 PnleFek, E., 38(18, 19), 39(33), 40(19, 33, Ohno, K., 332(43), 396 39, 42, 43), 41(33, 39, 42, 441, 42(33, Ohtsuka, E., 305(16), 326 39, 43, 46, 471, 43(33, 44, 46), 45(33, Oikawa, K., 262(126), 998

456 75, 76, 77, 78, 79), 46(18, 19, 75, 76, 77, 78), 48(75, 771, 49(75), MF(18, 75, 761, 51(19), 52(19, 75, 77), 53(19, 44, 87, 88, 89, 901,54(44, 79, 901,55(79), 56(18, 75, 78, 90, 94, 951, 57(75, 77, 781, 58(102), 59(75, 951, 60(18, 19, 76, 79), 61(103), 63(19), 64(19), 66 (19, 33, 461, 67(19), 68(19, 76, 87, 88, 901, 71,72,7 3 Palm, P., 104(152), 110(152), 116 PmteliE, M., 186(306), 881 Panusz, H., 44(56), 72 Pardee, A. B., 167(252, 2551, 205(252, 255), 213(252, 255), 8.20 Pardue, M. L., 162(233), 819 PGizkov6, H., 45(61), 72 Parke, W. C.,341(90,91), 397 Parkins, G. M., 21(101), 29,118(78), 120 (78), 128(78), 130(78), 156(78), 213 (781, 216,393(319), 402 Parr, R. G., 332(41), 396 Patel, A. B., 416(147), 437 Patrick, M. H.,118(65), 120(65), 151(65), 213(65), 216 Patten, R. A., 375(254), 401 Patterson, D. L., 304(12), 3% Peacocke, A. R., 133(149), 817,280,286 (179, 1831, 287, 299,300, 317 (431, 326 Pesevski, I., 182(290, 291), 881 Pelc, S. R., 156(202), 818 Penistan, 0. P., 39(28), 71 Penman, S., 364(207), 400 Penniston, J., 412(94), 436 Penswick, J. R.,278(154), 899,405, 434 Perevertajlo, G. A.,57(97), 73 Permogorov, V. I., 286(178a), 288, 300 Perrault, A. M., 341(159), 399 Perry, R. P., 182(287), 281 Perumov, D. A., 14(91), 15(91), 18(91), 19(91), 20(91), 21(91), 25(91), 89 Peschel, G. G., 350(176), 399 Peter, H. H., 240(67), 897 Peters, E., 165,280 Petemen, D. F., 118(38), 120(38), 160(38), 187(308), 216,221 Petersen, E., 118(88a), 144(88a), 167(88a), 169(88a), 172(88a), 213(88a), 216 Petersen, G. B., 102(142), 115,405(17), 411 (681,434,436

AUTHOR

INDEX

Petrovib, D., 158(219, 220), 211(219, BO), 213(219, 220), 919 PetroviE, J., 192(321), 193(321), 281 PetroviE, 5. P., 165(249), 166(249), 190 (3181, 191, 192, 193(320, 321), 197, 213(318), 880,221 Petters, V. B., 194(327), 288 Pettijohn, D., 82(66), 100(66), 113, 118 ( l l ) , 119(11), 120(11), 123(11), 151 ( l l ) , 153, 210(11), 213(11), 214 Pfitzner, K., 409(51), 436 Phil, A., 153, 158(225), 213(225), 218, 219 Phillips, D. M. P., 286(172), 300 Phillips, J.,428(175), 429, 433(195, 197), 438

Phillips, J. H., 1(6), 6(20, 21, 29), 7(6, 291, 14(21,29), 27,391(302,304,305), 402 Phillips, W. D., 240(68), 897 Piekala, A., 341(164), 399 Pitha, J., 341(164, l a ) , 364(208), 399,400 Pithova, P., 341(164), 364(208), 399,400 Pitot, H. C., 204(339), 205(339, 3401, 213 (339,3401,322 Pivec, L., 57(98), 73 Pleticha, R., 45(63),72 Pochon, F., 17, 88,256,298 Podder, R. K., 14(126), 89,431(184), 438 Pohl, S. H., 4(11), 18(11), 27 Poland, D., 247, 297 Pollak, M., 341(99,100,101), 397 Pollard, E. C.,118(8, 62, 64,73, 89, 90), 120(62, 64), 138(152), 139, 141, 142 (1541, 150(188), 151(62, 64,1541, 152, 173(269), 174(90), 175(62), 210(355), 211(90), 213(62, 64, 73, 154, 2691, 814,215,816,817,8.0, 284, 899 Pons, S., 199(333), 822 Pople, J. A., 340(55, 56, 57), 396 Postel, E. H., 14(89), 18(89), 29 Potter, R. L., 118(81,82), 120(81,82), 160 (21, 82), 161, 162, 163(230), 216 Potter, V. R., 156(210), 163(M5), 164 (2101, 219 Pouwels, P. H., 88(92), l l 4 Prestidgp, L. S., 167(252, 255), 205(252, 255), 213(253, 255), 820 Preston, B. N., 133(149), 817 Preuss, A., 83(69), I13

AUTHOR

457

INDEX

Priess, H., 82(64), 83(64), 113,414(113), 436 Printz, M. P.,32, 57(3), 70, 101(136), 116 Pruden, B., 375(258, 2591,401 P ~ S O R ,w. H., 379(273),401 Pullman, A.,328(l, 231,329(1, 4,5,6,9, ll), 330(22, 23, 24, 251, 331(32, 33, 341, 332(1, 40,44), 338,341(1, 6,22, 23, 24,25, 26,27, 32, 33, 34, 63,64, 67,68,71, 78, 79, 113,125,130, 144, 150, 161, 162, 163), 346(22, 26), 347 (26,341,3491221,350(22, 23,24,25), 354(22, 23, 24,25,78, 791,361(202), 370(202), 375(125), 382(22, 23,24,25, 78), 383(22, 24,78, 150), 385(1, 2,6, 2951,387(67), 390(300), 392(1, 6,631, 393(150, 300,321), 396,396,397,398, 399, 400, 402 Pullman, B., 328(1, 23), 3290,4,5,7,8, 9), 331(31), 332(1), 341(1, 31,59,60, 61,62, 66,66, 67,70, 92,93,94,95, 96,97,98,104,105,113,125,126,130, 131, 132, 136, 137, 138, 140,143,145, 152, 158, 159, 161,1621, 349(62, 65, 661,359(92), 361(94,96,202),365(98), 367(96), 369(93,97), 370(93,202),373 (93), 374(31, 104,105), 375(125, 126, 146), 377(131,132), 382,383(136,282), 385(1, 295), 387(67), 388(297), 390 (300), 391(61), 392(1), 393(300, 320, 322), 396, 396,397,39S,399, 400,401,

Rapaport, S. A.,11(69), 28, 244(80), 249 (801, 254(80), 257(80), 259(80), 260 (SO),261(80), 274(80), 297 Rash, E., 187(311), 291 Rasmussen, R. E., 118(77), 120(77), 128 (77), 130(77), 156(77), 158,211(215), 213(77,215), 216 Ratliff, R. L., 80(56), 82(68), 83(56), 113, 181(286), 221 Rebhum, L.,187(311), 221 Reese, C.B., 393(316), 402,409(50), 436 Reeves, J.,405(17), 434 Reggiani, M.,48181),72 Reich, E.,109(160), 116 Rein, R.,339(54), 341(99, 119, 120,121, 122), 396, 397,398 Reiter, H., 118(103), 123(103), $26 Rembaum, A.,332(38), 396 Resnik, R.A,,286(180), 287,28S,300 Reuschl, H., 118(102), 121(102), 216 Revel, M., 92(104), il4 Reybeyrotte, N.,ll8(19), 151(19), 211 (191,212(19), 213(19), 214 Reynolds, J. W., 339(52), 340(52), 396 Rhaese, H.J.,14(95), 15(95), 19(95), 21 (95),25(95), 29, 432(192), 437 Rhodes, W., 341(81, 821,397 Rich, A,, 76(10), 85(81), 11.3,114,249 (89), 252(99, 101,102,103), 257(114, 116,117), 297, 298,357(190, 191,192, 193, 195), 359(199), 364(204, 205), 374(252), 399,400,401 Richards, E. G., 249,252(100), 297,698 409 Pustoshilova, N., 409(38, 39,421,434 Richardson, C .C., 950141,11.6,143(173, 1731,218 R Richardson, J.P., 80(54, 621,81(54), 82 (54, 62), 83(74), 87(62, 871, 91(54, Rxckus, J. A,,194(328), 29% 131), 92(62), 96(62), 99(62, 74,1311, Radloff, R., 88(90), 114 lOO(74, 1311, lOl(62, 131), 103(62), Rahn, R. O.,382(278), 401 104(62, 1311, 105(87, 1311, 109(87, Raimondi, D .I,., 339,396 131), llO(62, 1311,l f 3 116 , Rajalakshni, K.V., 341(128), 398 Richer, M.,341(128),398 RajBhandary, U.,409(52), 4% Riley, M., 167(252), 205(252), 213(252), RajBhandary, V., 405(12), 434 620,304(13), 335 Ralph, R., 409(53), 436 Ristov, S.,412(84), 415(84), 436 Ramachandran, G.N . ,238(51), 696 Rltchie, D. A.,15(116), 29 Ramsny, O., 436 Ramuz. M.. 76(5), 105(1!54), 110(1 54, Roberts, R . B., 76(13), 112 I&), 112,116' Robertson, F. W., 321(40),326’ Robev, S., 145(183), 218 Randerath, K., 414(515), 436 '

458

AUTHOR

Robins, R. K., 231(33), 29G, 393(317), 402 Rorsch, A., 174(276, 2771, 177(256), 220 Rolfe, R., 267(132), 298 Rolins, R., 415(128), 436 Romancev, E. F., 118(4), $14 Rosa, E. J., 351(183), 399 Rosenbaum, M., 202 Rosenberg, E., 437 Rosenbluth, J. R., 374(232), 4UO Rosenfeld, F. M., 133(133), 217 Rosenfeld, V. L., 229(23), 295 Rossetti, G.P., 370(219), 400 Rossi, M., 341(64), 396 Roth, S. S., 164(239), 187(239), 205(239), 213(239), 219 Rottman, F., 391,402 Rottger, B., 410(58), 455 Rouviere, J., 77(16), 79(48), 112, 115 Rownd, R., 303(7b), 304(7b), 325 Rudner, R., 405(14), 411(14), 454 Ruger, W., 307, 310(28), 325 Rust, P., 415(139), f l Ruet, A,,160(158), 116 Rumano, B., 198(332), 222 Rushizky, G. W., 167(252), 205(252), 213 (252), 2ZU, 245 (83),297 Ruttkay-Nedeckjr, G., 68(107, 108, 109, 110, 1111, 73 Ryter, A., 210(352), 222

S Sadron, C., 225(12), 295, 374(231), 400 Sakaki, T., 269(140), 299 Salas, M., 79(42), 89(42), 113 Salem, L., 332(42), 396 Salganik, R., 406(32, 351, 413(35, 110, 1111, 414(35, 1111, 434, 436 Salovcy, R., 375(256), 4 O l Salser, W., 78(24), 112 Salto, H., 341(151), 385(151), 598 Salyers, A,,341(90, 911,397 Samejima, T., 225(10), 231(9, 28), 232 238(58), 240(28), 251 (28), 233(28), (971, 253(94), 257(97), 258(97), 260 (97), 261(10), 262(10, 971, 264(97), 267(97), 268(97), 269(139), 270(139), 289(28), 290(97), 295, 296, 298, 299 Samorayski, T., 206(343), 29.2

INDEX

Sander, C.,254(109), 289(109, ZOO), 290 (1091, 291(109, 200), 298, 300 Sanger, F., 405(7), 434 Sanner, T., 153, 158(225), 213(225), 218, 219

Sanno, Y., 409(49), 436 Sarabhai, A. S., 79(46), 113 Sarkar, P. K., 231(27), 232(28), 233(28), 240(28), 241(75), 249, 250(94), 251 (94, 961, 252(94), 254, 255(94, 96), 256(96), 257(96), 263(128), 265(128, 129), 267(111), 268(111), 277(94, 961, 279(94, 961, 280, 281(129), 289(28), 290(94, I l l ) , 296, 297, 298 Sarnat, M. T., 94(111), 95(111, 115), 96 (122), 108(122), 114,116 Sasisekharan, V.,238(51), 206 Sato, T., 269(13!3), 299 Sauerbier, W., 150(187), 818 SaviE, D., 118(106), 124(106), 180, 210, 220 Sawada, F., 238(58), $96 Scaife, J. F., 184(298, 299), 8.91 Scarpinato, B., 48(81), 78 Schaechtler, M., 79(33), 11.2 Schafer, E., 414(115), 436 Scheit, K.-H., 410(55, 561,436 Schell, P., 6(18), 19(18), 87 Schepmm, A. M., 174(277), $20 Scheraga, H. A., 245(83), 247(85), 262 ( I n ) , 278,279, $97, 898 Scherrer, K., 85(172), 116, 195, ,222 Schildkraut, C. L.,95(114), 114, 143(171, 172), 218, 259(120), 298, 301, 303(7b), 324, S%5 Schlessinger, D., 79(35), 111 Schlingloff, G., 414(118), 436 Schmidt, H., 37,71 Schmier, I., 281(163), 299 Schneider-Bernlohr, H., 416(144, 145), 437 Schofield, K., 409(50), 435 Scholes, G., 118(21), 133(130, 131, 132), 135(21), 212(21), 213(21), 914, 217 Schramm, G., 4, ?87 Srhuster, H., 4(9), 7(31), 12(13, 751, 14 (13, 751, 18, 22, 23(13), 25(13), 97, 412(78). 455 Schweizer, M. P., 238, 240(65, 66), $96,

AUTHOR

459

INDEX

297,341(141, 142), 369(142, 211,212, Shigeura, J. T., 89(94), 109(84), 114 Shimkin, M.B.,21(104), 29 213), 370(216), 374(142), SDS, 400

Scope, Y.M., 233(35), 296 Scott, J. F., 183(294), 221 Seaman, E., 262(124), 298 Searashi, T.,118(103), 123(103), 216 Seeds, W. E., 273(148), 299 Segal, G.A.,340(55, 56,571,396 Seidel, R.,406(36), 414(116, 117, 119), 434, 436 Seifert, W.,405(20), 434 Sein, K.T., 203,222 Sekiguchi, T., 186(305), 197(305), 221 Selzer, R., 15(127), 29 Semal, M., 164(238), 187(238), 205(2381, 213(238), 219 Sentenae, A.,106(158), 109(162), 116 Serebrjany, A.,406(29), 434 Sereni, F., 198(332), 822 Setlow, J. K., 11, 12(64), 15(64), 19(98), 28,99, 151,218 Setlow, R. B., 118(8, 12,96), 119(12), 120 (12),123(12), 143,151(12, 177, 1901, 154,167(251), 170,174(274, 2751,205 (251), 210(12), 213(12, 2.51, 274), 214,216, $18, 290 Sevastyanov, A.,409(39, 42), 4.94 Shaffer, C .R., 139,217 Shamovsky, G.,409(38, 39, 40, 411,434 Shapiro, H.S.,405(14, 15), 411(14, 15), 434

Shapiro, R., 4110,ll), 18(11). 27,409(50), 412(80), 413(98, 102), 415(125), 416 (146),436,436, 437 Shastry, K. S., lO(58, 59,60), 28 Sheats, G.F., 370(218),400 Sheldrick, P.,102(146, 147), 103(146, 1471, 116 Shell, P.,6(25), 27,416(142), 417(154), 419(160), 427(160), 4281154,160), 437 Shemyakin, M.F.,96(120>,116 Sheppard, D., 77(19), 112 Sherrer, K.W., 194(322), 221 Shibaev, V. N., 6(24), 7(24), 27, 414 (114), 432(188), 436,438 Shibaeva, R. P., 6(24), 7(24, 331,27,423 (165), 427(165, 172), 428(165, 172, 1781, 429(178), 430(180), 431(180, 187), 432(178, 187, 1881, 487,438 Shields, H., 375(253), 401

Shimizu, B.,231(34), 237(34), 238(34, 551, 286

Shin, D. H., 92(103), 114 Shooter, K. V., 21(103), 29 Shoup, R. R., 357(194), 364(194), 399 Shmurak, S.X., 286(178a), 300 Shramko, 0. V.,341(691,996 Shugar, D., 6(27), 11(66), 19(97), 27,28, 29, 382(274, 275), 401,412(73, 741, 415(130), 416(152), 429(186), 430 (186), 431(74, 183, 185, 186), 432 (152,1861,433,436,436,437,438 Shulman, R. G., 375(256, 257, 279), 401 Shuster, H., 417(153), 428,437 Siebke, J. C., 102(142), 116 Siekevitz, P., 802 SimiE, M. M., 195,196,198(330), 222 Simmons, N.S., 224,281(163), 296, 299 Simms, E. S., 5(15), 27, 143(165, 170), 218, 415(131), 436 Simon, M. I.,10(55), 11(61), 15(61), 28 Simpkins, H., 249,252,297,298 Simpson, E., 118(91), 119(91), 123(91), 216

Simpson, W. T., 351(183), 899 Simukova, N., 417(157), 418(159), 419 (159), 420(159), 4’23(164, 165, 166, 168), 424(168), 425(159), 427(164, 165), 428(164, 165), 437 Sinanoglu, O.,341(110, 111, 1121, 373 (110, 111, 112, 221,2221,397, 400 Singer, B., 7(32), 9(32,48,491,lO(32, 57), ll(32, 57,63), 12(32), 13(32), 14(32), 15(32, 48,571,17(48, 49), 19(32), 21 (48), 22(32, 48,49, 105), 23(32), 24 (48), 26(32), Y7,28, 405(3), 433 Sinsheimer, R.I,., ll(65, 67), 28,84(76), 86,87(89), 102(143), 114, 116, 118 (lola), 149(101a), 21G Sippel, A.,111(170), 116 Skalka, A.,78(27), 112 Skoda, J.,45(62), 78 Skov, K.,155(193), 156(193), 157(193), 158(193), 162,165,1%), 818 Slayter, H. S., 83(73,74), 99(74), lOO(73, 74), 113 Sly, W. S.,78(25), 11.2 Small, G.D., 12,Q

460

AUTHOR

INDEX

Steinberg, C. M., 78(21), 106(21), 11% Small, J., 409(46), 434 Smekal, J., 234(47), 296 Steiner, R. F., 24(3(88), 254(88), 286(88), Smellie, R. M. S., 143(166), 181(283), 818, 297 220 Steinschncider, A., 405(4), 43s Smith, D. A., 80(56), S2(68), 83(56), 113, Stent, G. S., 78(29), 79(38), 82(67), 83 181(286), 921 (671, 85(80),88(38), 92, 98(38), 104 (38), llO(29, 67), 112,113,U 4 , 150 Smith, D. E., 37(15), 71 (185), 118, 284(168), 300 Smith, D. L., 39(34), 40, 41(45), 42(34, 41, 45), 43(34, 45), 71,349(167, 1681, Sternberger, N., 80(63), 82(63), 83(63), 99(132), 100(132), 106(132), 110(132), $99 113,116 Smith, J. D., 405(13), 410(57), 411(57), Stevens, A., SO(52, 63), 82(63), 83, 99 434, 435 (132), 100(132), 106(132), 110(132), Smith, K. C., 118(55, 561, 120(55, 56), 151 113, 116 (55, 56), 213(55, 56), 216 Smoot, A. O., 118(83), 120(83), 160(83), Stewart, R. F., 3511181, 182), 399 162(83), 163(83), 189(83), 190(83), Stocken, L. A., 118(40), 120(40), 142, 151 (161), 155(40, 195), 160(40), 163, 164 216 (195), 165(247, 247a), 166(247a), 168, Srnrt, J., 45(62), 7 2 215, 217, 218, 820 Sniper, W., 375(258, 2591, 401 Stoesser, P. R., 370(219a), 400 Snyder, L., 96(122), 97, 108(122), 116 So, A. G., 80, 90, 92, 94(61), 96(61), 110 Storrer, J., 130(125), 131(125), 217 Strack, H. B., 14(94), 19(94), 23, ,99, 433 (61), 113 Sobell, H. M., 357(188, 189, 1931, 359 (200), 438 (1971, 361(201, 2061, 374(251), 399, Strauss, B. S., 14(121), 29, 118(103), 123 (103), 816 400,401 Streisinger, G., 79(40), 89(40), 113 Sober, H. A., 245(83), 297 Streitwieser, A,,Jr., 332(37), 339(51), 396 Song, P. S., 341(1601,399 Stretton, A. 0. W.. 79(46, 471, 115 Sorm, F., 45(62), 78,341(164), 399 Struchkov, V. A., 133(144), l65(246), 217, SormovB,Z., 57(98), 73 ,919 Spahr, P. F., 76(11), 89(96), 112,114 Struck, W. A., 349(169), 399 Sparrow, A. H., 130(123), 133(133), 217 Stuart, A,,405(12), 409(37), 434 Spencer, J. H., 102(141), 116 Studier, F. W., 289(200), 291(200), 300 Spencer, M., 273(149), 299 Stuy, J. H., 118(57, 58), 12M.57, 581, 138, Sperber, G., 341(123), 398 151(57, 58), 175(57, 581, 213(57, 58), Speyer, J. F., 5(16), 14(16), ,97 216 Spiegelman, S., 76(9), 85, 94(110), 112, 116, 116, 175(281), lSO(2811, 220, 301 Subirana, J. A., 317(42), 395 Sueoka, N., 267(133), 271, 2.98, 299 (31, 307, 323(3), 324,326 Sudararajan, T. A,,79(41), 89(41), 113 Sponar, J., 57(98), 73 Sussmuth, R., 9(47), 15(47), 23(47), 28 Sreton, A. 0. W., 207(348), ,922 Sugino, Y., 118(82), 120(82), 160(82), 816 Stacey, K. A., 133(137), 144, ,917 Suit, J. C., 118(66a), 124(66a), 151(66a), Stacey, M., 411(67), 485 213(66a), 216 Staehelin, M., 18(84), ,98, 110(168), 116, Summers, W. C., 103(148), 116,118(101), 413(97), 436 216 Stahl, F. W., 118(49), 120(49), 121(49), 818 Suskind, S. R., 143(183), 207(163), 135(49), 213(49), ,916 Stanley, W. M., Jr., 79(42), 89(42), 113 Susman, M., 78(21), 106(21), 118 Stannem, C. P., 118(75), 120(75), 128(75), Sutherland, G. B. B. M., 293, 299 Sutton, H., 158(216), 211(216), 2131216), 130(75), 156(75), 213(75), 816 219 Stead, N. W., 99(134), 106(134), 116

AUTHOR

INDEX

461

Taylor, E. W., 157(213), 219 Susuki, H., 99(128), 116,175, 990 Taylor, J. H., 143(162), 818 Suzuki, T., 415(122), 436 Sverdlov, E. D., 6(24), 7(24), Z7, 418 Taylor, K., 95(116), 103(116), 116 (159), 419( 159), 420(159), 425(159), Temperli, A., 415(139), 437 427(171), 428(178), 429(178), 430 Teplova, N., 409(38),434 (1801, 431(180), 432(178, 188, 189, Terasima, T., 157(206), 157, 819 Terzaghi, E., 79(40), 89(40), 113 I N ) , m, 438 Tessman, E. S.,150(185), 818 Swafficld, M. N., 187(314, 315), 2.91 Swan, R. J., 233(38, 39, 40, 411, 234(40, Tcssman, I., 14(113, 125, 1261, 15(118), 19 (128), 21(102), 23, 89, 150(185, 1861, 411, 235(39, 41), 237(41), 238(39), 247 218,390(299), 402,431(184), 438 (84), 254(108), 255(108), 256(108), Thach, R., 79(41), 89(41), 113 296, 297, 298, 354(184), 399 Theriot, L., 118(91), 119(91), 123(91), 216 Swartz, M. N., 16(80), 98, 309(31), 326 Swartzendruber, C. S., 118(69), 120(69), Thikry, J., 230, 296 Thiry, L.T., 9, 88 139(69), 151(69), 213(69), 216 Thomas, C. A., Jr., 80(60), 103(150), 113, Sweet, R. M., 359(198), 400 116,302(6), 306, 307, 308, 310(27), Swenson, P. A., 164(251), 174(274), 183 324,325 (292), 205(251), 213(251, 2741, 220, Thomas, R., 304(9), 386 221 Thompson, L. R., 204(338), 289 Swift, M., 187(311), 221 Thrower, K. J., 317(43), 3.96 Symons, R., 415(141), 437 Tikhonenko, T. I., 57(97), 73, 284, $00, Ssabo, L. D., 194(324), 198(324), ad1 412(86, 95), 436,436 Szer, W., 415(130), 436 Szybalski, W., 95(116), 102(144, 145, 146, Till, J. E., 118(75), 120(75), 128(75), 130 147), 103(116, 145, 146, 147, 148), 116, (751, 156(75, 204, 2051, 21305, 204, 118(10, 67, 74, 101, 108, 114), 119(10), 205), 216, 219 120(10, 67, 74), 124(10, log), 125(10), Timofeeva, M., 194(328,329), 922 126(114), 127, 133(146), 134(129), 151 Timofieff-Ressowsky, N. W., 118(7f, 214 (10, 67,74), 156(74), 158,210(10), 213 Tinoco, T., Jr., 225, 234(46), 238(59), 239 (10, 67, 74), 214,816,916 (W),240(60, 61, 71), 241, 242(73), Sztumpf-Kulikowska, E., 382(274, 2751, 243(79), 244, 245, 249(91), 252(95), 401 254(108a), 262(59), 274(15, 16, 17, 73), 275(152), !276(153), 277(108a), T 278(108a, 156, 1631, 279(108a), 183, 284, 285(164), 296,996,297,298,$99, Taber, H., 14(95), 15(95), 19(95), 21(95), 329(10), 341(10, 80, 83, 84, 88, 1%), 25(95), 29 346(10), 350(173, 175, 176), 351(175), Tagashira, Y., 330(17), 341(17, 1331, 374 355, 366(10), 372, 374(124), 396, $97, (245,246), 396,598,401 898,899 Takagi, Y., 99(128), 116,143(175), ,918 TissiBres, A,,80(61), 90(61), 92(61), 93 Takemura, S., 415(137), 437 (1081, 94(61), 96(61), 110(61, log), Tarnaoki, T., 182(288), 221 118,114 Tamm, C., 410(64,65), 436 Tittensor, J., 415(126), 4-36 Tamm, I., 310(36), 326 Tanaka, M., 330(19), 341(73), 350(73), Tobey, R. A., 187(308), 221 Tocchini-Valentini, G. P., 94(111), 95 354(73), 396,396 (111, i m , 114 Tanooka, H., 118(50), 120(50), 121(50), A., 409(44, 501, 434,436 Todd, 135(50), 213(50), 816 Tokarskaya, V. I., 118(45, 531, 120(45), Tashiro, Y., 809 166, 816,,920 Tatarinova, S.,14(114), 99

462 Tolmach, L, J., 156(206), 157, ,919 Tomasi, V., 198(332), 222 Tomasz, M., 409(49), 436 Tomin, R., 186(306), 221 Tomita, K., 357(190, 193), 399 Tomkins, G. M., 79(36), 112 Tomlin, P., 118(54, 55, 561, 120(54, 55, 56), 151(54, 55, 561, 213(54, 55, 56), 216 Toropova, G. P., 133(141), 155(198), 164 (198, 2421, 217,218 Townsend, L. B., 393(317), 402 Trager, L., 15(127), 89 TrajkoviE, D., 201(335), 202(335), 22,9 Trautner, K. W., 16,898 Trautner, T. A., 309(31), 325 Travers, A,, 98,115 Travers, A. A., 116 TrgovEeviF, Z., 118(70), 120(70), 138(70), 151(70), 213 (70),216 Trincher, K. S., 155(197), 164(197), 2198 Triphonov, E., 412(96), 436 Troll, W., 428(177), 438 Trujillo, T. T., 80(56), 83(56), 113,181 (286), 221 Ts’o, P. 0. P., 238(49), 240(65,66,69, 701, 244, 249, 254(109, 1101, 257, 259, 260, 26l(SO), 274, 289(109, 110, 197, 199, 200), 290(109), 291(109, 197, 200), 296,297,,998, $00, 341(141, 1421, 369, 370(216), 374(142), 398,400 Tsuboi, M., 269(138), 271(146), 272(146), 273( 150a), 299 Tsugita, A., 12(78), 88, 79(40), 89(40), flJ Tubbs, R. K., 374(230), 400 Tiirck, G., 10(56), 15(127), 298,%9 Turchinsky, M., 405(19), 418(159), 419 (159), 420(159), 421(161), 423(167, 168, 169), 424(168), 425(159), 427 (171), 434,437 Turler, H., 415(139), 437

AUTHOR

INDEX

Ulbricht, T. L. V., 233(35, 36, 37, 38, 39, 40, 41), 234(41, 45), 235(39, 41), 237 (41), 238(39), 247(84), 254, 255, 256, 296,297 , 298,354(184), 399 Umezawa, H., 111(171), 116 Urks, M., 45(61), 72 Urnes, P. J., 224(2), 231(2), 285(2), 296 U r n , D. W., 231(30), 235(30), 238(30), 296 Usida, T., 412(83), 456

V Valdemoro, C., 341(159), 399 Valentini, L., 48(81), 78 Vallee, B. L.,231(32), 249(32), 262(32), 268(32), 296 Van Arkel, G. A., 7(36), 14(36), F7, 433 (2011, 438 Van Bekum, D. W., 155(196), 164(196, 2411, $18,219 van Bruggen, E. F. J., 83(70), 113 Van de Pol, J. H., 7(36), 14(36), 27,433 (201), 438 Van de Vorst, A., 341(128, 1291, 3998 Van Duuren, B. L., 374(243, 2441, &Of Van Holde, K. E., 238(57), 247(57), 248 (57), 296, 370(219), 400 Vanjushin, B., 405(18), 416(18), 434 Van Lancker, J. L., 118(83), 120(83), 160 (831, 162(83), 163(83, 2311, 164(237), 187(316), 189(83, 316, 316a), 190(83, 316, 316a,), 205(237, 316), 207, 209, 213(237), 216,819,221,622 van Rotterdam, J., 88(92), 114 Van Vunakis, H., 10(55), 11(61), 15(61), 28

Van Winekle, Q., 374(230), 4OO VBrt&esz, V.,194(323, 3241, 198(323), 221 Velikodvorskaya, G. A., 284(167), 300 Velikodvorskaya, T., 412(86), 4.96 Veillard, A., 330, 341(137, 138, 1391, 396, 398 U Velluz, L., 224(4), 225(4), 230, 295 Uchiyama, T., 164(237), 187, 189(316, Venkataraman, P. R.,l81(285), 220 316a), 190(316, 316a), 205(237, 3161, Venkstern, T. V., 405(10), 411(72), 415 (124), 434,435,436 213(237), 219,221 Ukita, T., 406(31), 414(112), 416(148, Venner, H., 268(136), 299,411(71), 436 Verwoerd, D. W., 6(17), 7(17), $7,417 149,NO), 434, @6,437 (1561, 418(156), 43 7 Ulanov, B., 406(28,29), 434

AUTHOR

INDEX

Vetter, V., 40(36, 37), 43(36, 53), 44(36, 37, 54, 55), 45(77), 46(77), 48(77), 52(77), 57(77), 71,72 Vielmetter, W., 4, 12(13), 14(13, 1121, 18, 23(13), 25(13), 27, 29 Vinograd, J., 88(90), 114 Voet, D., 231(29), 232(29), 233, 296, 350 (174), 399 Vogler, C., 118(62), 120(62), 151(62), 175 (62), 213(62), 216 Voiculetz, N., 165(245), 213(245), 219 Volkin, E., 118(33), 120(33), 213(33), 214 von Hippel, P. H., 32, 57(3, 1011, 70,73, 101(136), 115 von Stackelberg, M., 37, 71 Vos, O., 155(196), 164(196), 218 Vournakis, J. N., 245, 247(85), 262(127), 278, 279, 297,298

W Wacker, A., 9(47), 10(56), 11(62), 15(47, 62, 127), 23(47), 28,29,118(22), 120, 212(22), 213(22), 214, 377(270, 271, 2 7 3 , 401,412(75), 436 Wagle, M. M., 173(272), 213(272), 220 Wagner, E., 405(5, 6), 433 Wahba, A. J., 5(16), 9(39), 14(16, 39), 27,79(42), 89(42), 113 Wainson, A. A., 118(121), 120(121), 128 (121), 130(121), 217 Wakes, J. R., 181(283), 220 Waldron, D. M., 39(30), 40(30), 42(30), 68(30), 72 Walker, P. M. B., 302(4), 309(32), 317, 319(45), 321(45), 322, 323(32, 53), 324,325,326 Walker, R., 415(126), 436 Wallace, H., 311(39), 325 Walsh, W. M., 375(256), 401 Walter, G., 90(97), 93(97), 104, 110(97, 152), 11.4, 115 Walwick, E. R., 144(179), 145(179), 163 (1791, 218 Wang, J. C., 258(118), 298, 306, 309(30), 325 Wang, S. Y., 267(135), 298 Waring, M. J., 88(91), 114

Warner, R. C., 9(39), 14(39), 27 Warshaw, M. M., 238, 239(60), 240(60, 61), 241, 261(59), 296, 297 Waskell, L. A.,10(60), 28 Watanabe, K. A., 238(54), 296 Watson, D. G., 359(198),@0 Watson, J. D., 24, 29, 76(11), 112,207 (347), 222,252(99), 29s Watson, R., 88(90),114 Webb, B. R., 15(123), 29 Weckcr, E., 23(107), 29 Wchrli, W., 110(168), 116 Weil, G., 374(229), 400 Weil-Malherbe, H., 374(237), 400 Weill, J. H., 225(13), 2996 Weinblum, D., 377(270), 401 Weiss, J. J., 45(63), 72,118(23), 120, 133 (130, 131, 132), 135(23), 167(256), 168 (256), 170(258), 173, 204(23), 212(23), 213(23), 214,817,220 Weiss, S. B., 76(l), 84(75), 85(75), 93 (log), 95(118), 99(129), 100(129), 110 (log), 112,113,114,116 Weit, H., 405(2), 433 Weitzman, P. D. J., 70, 7 3 Weller, P. K., 210(355), 22!? Wells, B., 263(128), 265(129), 298 Wells, R. D., 305, 326 Wempen, I., 234(44), 296 Wetmur, J. G., 304(10), 316, 317, 326 Wheeler, C. M., 144(180), 145(180), 163 (180), 167(256), 168(256), 170(25.8), 173, 218, 220 Wheeler, G., 412(77), 43656 Wheland, G. W., 339, 385(294), 396, 402 Whitfeld, P., 409124, 43), 413(101), 434, 43s Whitfield, J. W., 164, 219 Whitmore, G. F., 118(75), 120(75), 128 (75), 130(75), 213(75), 815 Wiberg, J. S., 78(22), 111 Widholm, J., 309(30), 315 Wieder, C. M., 14(112), 89 Wierschowski, K. L., 11(66), 19(97), 88, 29

Wilder, J., 269(141), 299 Wilhelm, R. C., 4(9), 9(40), 14(40), 20,

n,u, 28

Wilholm, J., 258(118), 298

464

AUTHOR

Wilkins, M. H. F., 273(147, 148, 149, 149~4,299 Wilkinson, A. E., 131(126, 1271, 6f7 Williams, D. L., 82(68), 113,181(286), 661 Williams, R. W., 118(16, 691, 119(16), 120(16,69), 123(16), 134(16), 137,138, 139(69), 151(16,69), 154,210(16), 213 (16, 69), 614,916 Williams-Ashman, H. G., 76(3), 11.8 Wills, E. D., 131(126, 1271, 9f7 Wilson, D. L., 96(119), 116 Wilson, H. R., 273(147, 148, 148a), 699 Wilson, R. G.,6(22), 7, 11(73), 12(73), 14(22), 15(73), 2'7, 68, 171(262), 660, 391(301, 3M), 402, 433(196), 438 Win, H., 339(54), 396 Winkler, U., 1(5), 13(5), 16(5), dY Witkin, E. M., 118(95, 118), 120, 126 (118), 204(95), 216,217 Witkop, B., 415(135), 437 Wittman, H. G., 12(75), 14(75), 28, 412 (89,436

INDEX

233, 240(28), 241(75), 249, 250(94), 251(94, 96, 971, 252(941, 253(94), 254, 255(94, 96), 256(96), 257(97), 258 (97), 260(97), 261(10), 262(10, 971, 263(128), 264(97), 265(128, 1291, 267 (97, 110, 268(91, 110, 277(94, 961, 279(94, 961, 280(160), 281 (129, 162), 285(20), 289(28), 290(94, 97, 1111, 696, 296,297,998 Yanofsky, C., 79(44, 451,115 Yanofsky, S.A., 76(9), 116 Yegian, C., 110(166), 116 Yoshida, M., 406(31), 414(112), 416(150), 4349 @61 437 Youdale, T., 164, 219 Young, R., 409(52,53), 435 Yu, C. T., 405(1), 409(1), 415(123), 433, 436

Yuan, D., 92(102),114 Yung, N. C., 234(44), 996

Z

Zachau, H. G., 183(293), 221,405(9), 434 Witz, J., 57(100), 73, 252(98), Z98 Witzel, H., 4O6(24), 410(62), 413( 101), Za.jec, Lj.,118(59, 601, 120159, 60), 138 (59, 81, 151(59, 601,17369, 60),175 @4, 436,@6 916 (59, 601, 21369, a), Wolf, M. K., 286(173), 300 Wood, W. B., 101(135), 115,170(259), Zamecnik, P. C., 231(32), 249(32), 262 (32, 125), 268(32), 296, 298, 405(1), 220 409(1), 415(123), 433,436 Woodard, L., 187(311), $21 Woodhouse, D. L., 39(30), 40(30), 42 Zamenhof, S., 18(85), 29, 412(87), 436, 437 (30),68(30), 71 Zamir, A., 278(154), 699, 405(8), 434 Woodson, B., 172(264), 173(264), 260 Zampieri, A., 15(124), g9 Woody, R. W., 225(15, 16), 274(15, 161, Zavarine, R., 118(63), 120(63), 126, 151 296 (63), 213(63), 616 Wu, R., 306(22), 310(22), 326 Zemm, W., 206(343), 266 Wyckoff, R. R., 118(28), 120(28), 150 Zeszotek, E., 105(153), 110(153), 116 (189, 914 Ziffer, H., 288(187), SO0 Zillig, W., 6(17), 7(17), 87, 80(53), 82 Y (641,S(64, 69), 90(97), 93(97), 104 (152), llO(97, 1521, 113,114,405(20), Yamane, T., 382,401 414(113), 417(156), 418(156), 434, 436, Yamaoka, K., 286(180, 184), 287, 288 @Y (187), 300 Zirnm, B. H., 309(34), 325 Yamamoto, N., 21(104), 69 Zimmer, C., 268,299 Yamamoto, O., 184, 221 Zimmer, K. G., 118(7), $14 Yamazaki, H., 111(171), 116 Yang, J.T., 225(10, 20), 227(20, 21), 228 Zimmer, K. Z., 118(9),81.4 (% 229(24), I), 230, 231(27), 232(28), Zimmerman, A. S., 415(131), 436’

AUTHOR

IXDEX

Zimmerman, B. X., 19(93), 21(93), 29 Zimmerman, S. B., 5(15), 27, 309, 3215 Zimmermann, F., 118(88), 144, 145(88), 169(88),213(88), 9f6 148, 167(88),

465 Zobel, C.,406(27), 434 Zubay, G.,85(82), 11.4,286(170), 300 Zuman, P.,32(7), 36(7, ll), 42(49), 68 (71,70,71,72

Subject Index A

replication, 142-143 biochemistry, 143 Adenine, polarography of, 39-40 radiation effects, Alkylating agents, chemistry of action, in witro, 143-149 8-9 in vivo, 149-166 ribonucleic acid polymerase binding to, B 99-101 Bacteriophage, deoxyribonucleic acid, role in ribonucleic acid synthesis, 84-88 lethal changes produced by radiaas target for lethal radiation effects in tion, 121-123 living systems, 120-131 Brominating agents, chemistry of action, 12-16 transcription, 1&167 radiation effects, genetic effects in v i m , 171-204 C priming activity, 167-171 Circular dichroism, see Optical Rotatory Dispersion Deoxyribonucleoproteins, optical rotatory Cytosine, dispersion, 281-286 modification hydroxylamine, 427- Dinucleoside phosphaw, optical rotatow 433 dispersion and circular dichroism, 23&244 polarography of, 40-41 Drude equation, optical rotatory disperD sion and, 229 Deoxyribonucleie acid, families in higher organisms, 318-322 E optical rotatory dispersion, Electronic structure, base tilting and, 271-277 interbase interactions, comparison to ribonucleic acid, 262forces involved, 354-357 269 related problems, 373-374 single- veixus double-stranded, 269van der Waals-London interactions, 271 357-369 polarography, 45-16 vertical interactions, 369373 estimation of denaturation, 60-61 methods of calculation, 332-333 native and denatured, 46-54 extended Hiickel theory, 338-339 premelting temperatures and, 55-60 Huckel approximation, 336337 single-stranded breaks and, 54-55 iterative extended Hiickel theory, radiation-induced damage, 339-340 biological consequences, 204-207 representation of o-bonds, 337-338 breakdown, 138-142 self-consistent field method, 333-336 physical and chemical nature, 132self-consistent field procedure for all138 valence electrons, 340 working hypothesis of, 207-213 problems in radioand photobiology, repetition rate, spin densities in free radicals, 375deoxyribonucleic acid-deoxyribonu377 cleic acid interaction, 322 thymine photodimerization, 377-383 deoxyribonucleic acid-ribonucleic problems investigated, 340 acid interaction, 322-324 purine and pyrimidine bases, 34&354 466

467

SUBJECT INDEX

F

cleavage of N-glycosyl bonds, 410-411 complexes, optical rotatory dispersion, Free radicals, nucleic acid bases, spin 280-289 densities in, 375477 components, polarography of, 38-45 G electronic structure, interbase interactions, 354-374 Guanine, polarography of, 41-42 methods of calculation, 332-340 H mutagenesis and, 383-393 problems investigated, 340 Hybrids, purine and pyrimidine bases, 340-354 rates of reassociation, radio- and photobiology and, 375deoxyribonucleic families and, 318383 322 types of calculation, 329-332 factors affecting,316318 heterocyclic bases, reaction at, 411-417 stability, hybrid stability and, 306-308 artificial polymers and, 304-306 phosphate grouping, chemical modifidiscrimination and sequence homolcation of, 409-410 ogy and, 310-316 protein-encased, reactivity and mutanoncomplementary regions and, 308bility of, 21-24 310 visible rotatory dispersion, 289-291 nucleic acids, 306-308 Nucleosides, Hybridization, specificity of, 302-303 optical rotatory dispersion and circular Hydroxylamine, di ch roism, chemistry of action, 5-8 purine and pyrimidine derivatives, cytosine and, 427-432 231-233 functional studies, 432-433 a- versus /?-linkages of sugars, 233uracil nucleus and, 417-427 238 M Nucleotidea, optical rotatory dispersion and circular Mammalian cells, death, damage to dedichroism, oxyribonucleic acid and, 127-131 purine and pyrimidine derivatives, Mercury electrode, nucleic acid com231-233 ponents and, 4-4 a- versus /?-linkage of sugar, 233-238 Microorganisms, inactivation, radiationinduced damage to deoxyribonucleic acid and, 123-127 0 Mutagens, chemistry of action, 4-16 Mutagenesis, electronic factors in, 383- Oligonucleotides, optical rotatory dispersion and circular dichroiam, 238-249 393 Optical activity, origin of,225-227 N Optical rotatory dispersion, calibration of instruments, 229-230 Nitrosoguanidine, chemistry of action, correlation with circular dichroism, 9-10 228-229 Nitrous acid, chemistry of action, 4-5 difference ORD, 230-231 Nucleic acids, Drude equation, 229 carbohydrate moiety, chemical modifiexpressions for, 227-228 cation of, 408-409 nucleic acids, 269-280 chemical modification, nucleic acid complexes and, 280-289 functional studies, 406408 nucleosides and nucleotidea, 231-238 reactions used, 408-417 structural studies, 404406 oligonucleotides, 238-249

SUBJECT INDEX

origin of optical activity, 225-227 synthetic polynucleotides, 249

secondary structure, hydroxylaminolysis and, 425-422 synthctic, optical rotatory dispersion P and circular dichroism of, 249-262 Photobiology, electronic structure and, Polyribonucleotides, polarography of, 61376-383 68 Photochemistry, mutagens and, 10-12 Polyuridylate, optical rotatory dispersion Polarography, of, 249-254 analytical applications, 44-45 Purine (s) , claesical, 3S36 derivatives, optical rotatory dispersion deoxyribonucleic acids, 45-61 and circular dichroism of, 231-233 modern techniques, 36-38 electronic properties, nucleic acid components, 38-45 distribution and dipole moments, polyribonucleo tides, 34m47 natural, 68 molecular orbitals, 347349 synthetic, 61transitions, 34S354 principles of,32-38 polaromaphy of, 4 2 4 3 Polyadenylate optical rotatory dispersion Purine nucleotides, preferential chain of, 24!3-254 initiation and, 97-98 Polyadenylate -2polyinosinate, optical ro- Pyrimidines, tatory dispersion of, 254-257 derivatives, optical rotatory dispersion Polycytidylate, optical rotatory disperand circular dichroism of,231-233 sion of, 264257 clectronic properties, Polydeoxyadenylate, optical rotatory disdistribution and dipole moments, persion of,257-261 340-347 Polydeoxyadenylate poly deoxy thymidylmolecular orbitals, 347-349 ate, optical rotatory dispersion of, transitions, 349-354 257-261 polarography of, 4 2 4 3 Polydeoxy (adenylate-thymidylate) , opR tical rotatory dispersion of, 257-261 Polydeoxycytidylate, optical rotatory Radiation, biological consequences of, 204-207 dispersion of,257-261 deoxyribonucleic acid as target for Polydeoxythymidylate, optical rotatory lethal effects, 120-131 dispersion of, 257-261 deoxyribonucleic acid replication and, Polyguanylate, optical rotatory dispcr142-166 sion of, 254-257 physical and chemical nature of damPolyguanylate - polycytidylate, optical rotatory dispersion of, 254-257 age, 132-138 ribonucleic acid synthesis and, 166-204 Polyinosinate, optical rotatory dispersion Rndiobiology, electronic structure and, of,254-257 Polyinosinate-polycytidylate,optical ro375-383 Ribonucleic acid, tatory dispersion of, 254-257 deuridylic, preparation and properties, Polymers, artificial, hybrid stability and, 421425 304-306 interaction with deoxyribonucleic acid, Polynucleotides, 322-324 doubledranded, reactivity and mutamechanism of chain initiation, 99-107 bility of,17-21 optical rotatory dispersion, hybrid, optical rotatory dispersion of, base tilting and, 271-277 261-262 calculation of, 277-280 reactivity and mutability of,16-17

-

SUBJECT INDEX

comparison to deoxyribonucleic acid, 262-269 strandedness and, 269-271 regulation of synthesis, general concepts, 76-79 selective transcription, asymmetric, 94-96 further restrictions on, 96-97 preferential chain initiation with purine nucleotides, 97-98 transcription mechanism, de mvo synthesis and direction of growth, 88-89 growth rate of chain, 92-93 release of molecules, 89-92 role of deoxyribonucleic acid, 84-88 Ribonucleic acid polymerase, attachment site, nature of, 101-103 binding to deoxyribonucleic acid, 99101 deoxyribonucleic acid priming, radiation effects, 167-171 inhibition of, initiation, 109-11 1 polymerization, 109 initiation reaction of, 104-106 initiation sites, 1061W preparations,

469 physical properties, 81-84 purity, 80-81 termination signals, 107-109 Ribosomes, optical rotatory dispersion, 281-286

T Thymine, photodimerization, mechanism of, 377-383 Transcription, restrictions on, 96-97 selective, 94-98 Trinucleoside diphosphates, optical rotatory dispersion and circular dichroism, 244-249

U Uracil, modification with hydroxylamine, 417-427 Uridine, hydroxylamine and, 417-421

V Van der Waals-London interactions, electronic structure and, 357-369 Viruses, optical rotatory dispersion, 281-286 Visible rotatory dispersion, 289-291


No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

No title

Recommend Documents