THE MOLECULAR BASIS OF EVOLUTION Th e MOLECULAR BASIS NEW YORK - JOHN WILEY 6 SONS, INC. London o Chapman 6 Hall, Limited OF EVOLUTION CHRISTIAN B. $+NFINSEN National Heart Institute National institutes of Health Bethesda, Maryland TO MY MOTHER AND TO THE MEMORY OF MY FATHER Copyright @ 1959 by John Wiley & Sons, Inc. All rights reserved. This book or any part thereof must not be reproduced in any form without the written permission of the publisher. Library of Congrorr Catalog Card Number: 59-11794 Printed in the United Statbs of America PREFACE T 1 he writing of this book has been stimulated by the excitement and promise of contemporary protein chem- istry and genetics and by the possibilities of integration of these fields toward a greater understanding of the fundamental forces underlying the evolutionary process. It has become, inevitably, a highly personal volume expressing the experimental and phil- osophical outlook which has resulted from a process of self-education in an unfamiliar area of science. As with many biochemists, pure biology, including genetics, has not been a major exposure in my education, and the process of learning something about these subjects has been both a revelation and a struggle. Various kind, and frequently amused, friends, versed in the complexities of modem genetics, have sifted through these pages, and I hope that most of the misinterpretations and frank mistakes have been eliminated. It has been highly interesting of late to observe how many scientists, working either in protein chemistry or in genetics, or for that matter in relatively unrelated fields, have arrived at long-range research plans that are similar to. my own, down to almost the last detail of experi- mental planning. This book, therefore, will undoubtedly represent the point of view of numerous other biologically oriented individuals. On the other hand, some of the ideas to be discussed are so new and controversial that for every well-informed reader of this book who, vii in general, approves them there may be another who considers them nonsense. The recent advances in the development of techniques for the study of protein structure have made it possible to elucidate the complete amino acid sequences of a number of rather large polypeptides pos- sessing hormonal activity, and nearly complete sequences should soon be available for several with enzymatic activity as well, Concomitantly, there have been developed methods for the analysis of the finer, more subtle, structural aspects of proteins, concerned with folding, intramolecular bonds of various types, and intermolecular interactions. These advances now begin to enable us to discern the vague outlines of macromolecules, in the three-dimensional sense, and to ascribe their physical behavior and biological activity to specific covalent and noncovalent structural features. We like to believe that Nature has been extremely wise and effi- cient in the design of the chemical compounds, however large and complicated, which make up the structure and machinery of living things. Thus, although chemical differences are found among the representatives of a given protein as isolated from a variety of species, we tend to suppose that such variations, rather than being fortuitous and unimportant irrelevancies, are part of a complicated and highly integrated set of variations in all the functionally and structurally important elements of the cell, the summation of which accounts for the unique morphology and phenotypic character of the individual organism. In the last few years, a number of studies have shown that various biologically active molecules may be subjected to considerable degra- dation without loss of functional competency. It becomes necessary, therefore, to consider that the "macromoleculariness" of proteins and other large molecules may, in many cases, be concemed not only with a specific biological property but with other, more subtle, phenomena of cellular activity and engineering not yet apparent, such as adsorp- tion to surfaces or substrates. We must, perhaps, expect a multiplicity of variables in the "natural selection" of variants in molecules, all of which may, together, determine the biological suitability of a particu- lar molecular species. An understanding of the underlying principles governing the species specific variations in molecular structure and of the effect of such variations on species characteristics must involve a clarification of the process of translating information present in the genetic ma- terial of the cell into the chemical language of enzymes, regulators, and the like. Such considerations are only now becoming possible as Viii PREFACE the result of the dramatic strides taken in the last few years in genetic theory and methodology, and a portion of the discussion in the fol- lowing pages will have to do with the experimental background of the analysis of genetic fine structure and with the possible significance of such analysis to the question of protein biosynthesis. It is abundantly clear that the metabolic organization of all living cells, whether plant or animal, shows a remarkable uniformity. Even a cursory examination of the literature of comparative biochemistry and physiology indicates that such biochemical functions as glycolysis, proteolysis, and fatty acid degradation, as well as more integrated processes such as electromotor activity and active transport through membranes, are ubiquitous in nature. Discounting the likelihood of completely parallel evolution in the plant and animal kingdoms, and in their major branches, we are led to conclude that, long before significant specialization, there existed in the waters of the earth vari- ous primeval forms of life which were endowed with representatives of most of, if not all, the important biological processes characterizing living things as we know them today. Although it is unlikely that we shall ever have more than opinions regarding the origin of life, it does seem possible to approach, experimentally, the nature of the speciation which began when such primeval cells had become estab- lished. This approach must involve a backward extrapolation of the information we can obtain on the chemical and genetic factors in organisms chosen from our modem environment. Before examining for the reader the aspects of the mechanism of evolution that have been particularly illuminated by recent advances in biology and chemistry, it has been necessary to outline, in a broad sort of way, some of the basic fundamentals of evolution and the specific sciences, particularly genetics, that have contributed so essen- tially to its understanding. In the opening chapter, therefore, I have collected and rephrased some gleanings from the massive literature of morphological evolution to serve as a background for what follows. In several subsequent chapters is presented further preparatory ma- terial dealing with classical and contemporary genetics and with the basic facts of protein structure. Finally, after some discussion of the rapidly expanding body of knowledge relating structure to function in biological systems, I have considered a few aspects of natural selec- tion in evolution which suggest themselves as a result of contemporary research, as well as some experimental approaches at the molecular level. This book was written for pleasure, with the desire for self-en- lightenment as the major stimulus. Since I cannot help but feel that PREFAC: IX everyone in science must be interested in the evolutionary process as the central theme of biology, I have listed a number of the original articles and books which contributed to the subject matter of this book. I am greatly indebted to many of my friends and colleagues, includ- ing Dr. W. F. Harrington, Dr. Daniel Steinberg, Dr. W. Il. Carroll, Dr. E. D. Kom, and Dr. W. D. Dreyer, who have read and helped improve various chapters of this book. I should also like to express my gratefulness to Dr. Bruce Ames for his patient help in connection with some of the discussions of genetic subjects. My special thanks are due Professor John T. Edsall of Harvard University and Dr. Michael Sela of the Weizmann Institute of Science, Rehovoth, Israel, who have read the entire manuscript and whose suggestions have been invalu- able in the avoidance of error and in the improvement of style and organization. Finally, I should like to thank my wife, Florence Anfin- sen, for the cheerful and understanding support she gave to a fre- quently rather morose husband. Bethesda, Maryland May 1959 CHRISTIAN B. ANFINSEN GROUND RULES FOR THE READER A prospectus of this book was circulated to a num- ber of experts by the publisher before the actual job of writing was begun. The opinions received have been very helpful in establishing what I hope is the proper slant. One opinion, in particular, expressed a point of view so similar to my own that I have asked its author, Dr. William Stein, of the Rockefeller Institute for Medical Research, for permission to reproduce it here. It has always seemed to me, and I may be wrong in this, that when an expert communicates with the relatively non-expert, he has a responsibility to stay pretty close to the facts. without scruple. An expert can speculate to other experts They have the equipment to meet him on his own grounds, evaluate the evidence and accept or reject the speculation as they choose. The non-expert has no such basis for evaluation. He has to accept rela- tively uncritically what the expert tells him, and hypothesis and fact soon become confused in his mind. A plausible speculation-and the speculations of the true expert are always plausible-can soon masquerade successfully as gospel. In the present state of our ignorance, I would regard this as unfortunate. It would seem to me that a book such as this one should aim to stimulate thought and experiment among practicing scientists, and should not lull the uninitiated into thinking that we understand more than we do. xi x PREFACE We shall deal, in this book, with many contemporary hypotheses, some of which are far from general acceptance. Consequently, I would like to emphasize two points of caution to the reader. First, simple examples have purposely been chosen to illustrate the presen- tation of subjects which in fact are sometimes complicated by excep- tions and inconsistencies. Second, it will be apparent that the author, like anyone else, has occasionally taken sides. CONTENTS 1 . . . . The Time Scale and Some Evolutionary Principles 1 2.. . . Genes as Determinants of Heredity 15 3.. . . The Chemical Nature of Genetic Material 39 4.. . . The Substructure of Genes 67 5.. . . Protein Structure 98 6.. . . The Biological Activity of Proteins in Relation to Structure 126 7.. . . Species Variation in Protein Structure 142 8.. . . Genes as Determinants of Protein Structure 164 9.. . . On the Accuracy of Protein Synthesis 185 lo.... The Biosynthesis of Proteins 195 11 . . . . Genes, Proteins, and Evolution 211 xii GROUND RULES FOR THE READER xiii chapter 1 THE TIME SCALE AND SOME EVOLUTIONARY PRINCIPLES T o most of us, paleontology is the name of a sort of genteel outdoor science concerned with the collection and gross de- scription of old bones and hardened mud blocks containing preserved animal tracks. To the paleontologist and, for that matter, to any novice who has had the good fortune to pass through what might be called the "Darwin-to-Simpson reading stage," no definition could be further from the truth. Just as history, to the historian, is alive and a part of the continuing pageant of human experience, so is the study of the life of the past a living science to its devotees. The study of fossils cannot tell us a great deal about the natural forces that shape the evolutionary process, but it does furnish us with guidelines for the consideration of information derived from other sciences. As G. S. Carter' has put it, "The part of paleontology iu the study of evolutionary theory resembles that of natural selection in the process of evolution; it serves to remove the inefficient but cannot itself initiate." It is clear that we can, and should, present only the most superficial survey of the fossil record and its interpre- .Y P z \ \ \ \ \ / \ \ \ \ /I \ \ \ \ \ // \ \ \ 1. \ / \ \ I ' I `. ! ! I 1' 2 THE MOLECULAR BASIS OF EVOLUTION tation in the present volume. For our purposes here we need only arrive at some general appreciation of the arbitrary divisions of geo- logical time and outline the phylogenetic relationships that exist be- tween the various living and extinct forms of life. Measurements of the extent of decay of long-lived radioactive ele- ments in the rock strata of the earth's crust enable us to make reason- able estimates of the ages of various strata. Utilizing such data as check points, but relying mostly on time estimates arrived at by classical geological methods, the paleontologist can arrange the fossilized remnants of life in a consecutive order with reasonable accuracy. He can also, in many cases, make certain deductions con- cerning the relation of specific upheavals and rearrangements of the earth's surface to the changing patterns in the nature and distribution of life as it was in the past. For the purposes of those interested in the earth sciences, time may be expressed perfectly well on a linear scale, as shown on the left of Figure 1. Such a scale serves to emphasize the relatively small fraction of global time during which life has existed on the earth. The biologist is, however, more naturally preoccupied with "protoplasmic" time and must magnify the portion of the time scale that has to do with living things. The right half of Figure 1 is more useful to the biologist and lists some of the landmarks in evolution, assigned to their proper paleontological time period. The earliest fossils that occur in any abundance may be assigned to the Cambrian and Ordovician periods and include a large propor- tion of the basic types of aquatic animals and the possible beginnings of the vertebrates. The record for the Pre-Cambrian period is ex- tremely sparse and is represented mostly by the relatively primitive plants, the algae. At the end of the Pre-Cambrian, most of the in- vertebrate phyla were relatively well differentiated, although the ab- sence in most instances of structural elements that could survive as fossils makes the reconstruction of their phylogenetic tree somewhat controversial. One scheme is presented in Figure 2. This arrange- ment of the phyla, which includes the higher vertebrate forms for comparison is, according to its author, L. H. Hyman, not to be taken literally but is only suggestive. It is based on an arrangement of animals in order of structural complexity, without separation of the allied phyla. The bacteria, yeasts, etc., are not shown, for they branched off at some early point in time when the momentous biologi- cal accident occurred which led to the establishment of plant and animal kingdoms. Another way of looking at the phyla is shown in Figure 3, taken THE TIME SCALE AND SOME EVOLUTIONARY PRINCIPLES 3 Figure 2. Relationships of the phyla of the animal kingdom. The arrangement here is based on the scheme given by L. H. Hyman in The Inoertehrates, volume 1, McGraw-Hill Book Company, p. 38, 1940. Figure 3. A schematic diagram of the history of life. The various phyla of animals are rcprescnted by paths, the widths of which are proportional to the known variety of each phylum during the various biological periods. Redrawn from G. G. Simpson, The Meanfng of EuoZutfon, 1950, by permission of Yale University Press. 4 THE MOLECULAR BASIS OF EVOLUTION THE TIME SCALE AND SOME EVOLUTIONARY PRINCIPLES 5 from George Gaylord Simpson's fascinating book, The Meaning of Evolution. Here we see the major phyla, as they have existed through most of biological time, in terms of their relative abundances. We can observe here some of the correlations between geology and biology which the paleontologist is able to make. For example, the distinct contractions in the abundances of almost all the phyla in the Permian and Triassic periods and the actual extinction of the Graptolithina correlate well with the geological evidence for great mountain building and climatic rigor during these times. A final illustration for this phylogenetic orientation is given in Figure 4, in which the vertebrates are arranged in their proper as- tendency (to use an "anthropophilic" expression). In our discussions of the relations between the biochemistry and genetics of various or- ganisms we shall refer from time to time to the contents of these figures. We shall be interested, for example, in the structure of pro- teins as they occur in various species and in the possibilities of mak- ing some crude estimates from such data of the rates at which specific genes have become modified. The basic characteristics of the evolutionary process vary consid- erably, depending on the level of evolution which is being considered. Evolutionary change, measured broadly in terms of the origin of new systems of animal organization, is an expression of avemge change. ,jj\ Arthropoda Tllarslc - Permian - Pennsylvanian Mismsippian Devonian lative variety in losril record Ordovic!an Duration and diversity of the prmcipal groups of ammals (baud mainly on counts 01 genera and higher groups) Figure 4. A schematic diagram of the history of the vertebrates. The widths of the pattern for each vertebrate class is proportional to the known varieties of the class in each of the geological periods. Redrawn from G. G. Simpson, The Mean- ing of Euolution, 1950, by permission of Yale University Press. As Simpson has put it, "It is populations, not individuals, that evolve." As we approach the level of immediate cause and effect, however, certain aspects of evolution become more highly significant, and when we consider a small experimental population of the fruit fly, Drosophila, we must become more concerned with individual mutations and their contribution to the survival or death of these specific flies than with theoretical, infinitely large populations. This example is obviously not evolution in the grand sense. It emphasizes "the survival of the fittest," a phrase which, in the light of modem ideas, we know must be replaced with "the survival of the branch of a population which is adapted well enough to its environment to live to procreate." Nevertheless, all evolutionists will agree that the basic cause of change must be gene mutation (although some authors will hold out for the additional involvement of something more ethereal in the way of causation, variously termed "aristogenesis," "dun u&al," "entelechy," among other names-we shall return briefly to these terms later in this chapter). As our store of information concerning species variations in bio- chemical properties, and specifically in protein structure, increases, we will do well to have before us, as a constant frame of reference, a clear picture of the phylogenetic relationships between various forms of life and, particularly, of the time, in terms of numbers of genera- tions, required to accomplish these variations. 6 THE MOLECULAR BASIS OF EVOLUTION To develop some appreciation of the magnitude of time involved in the differentiation of a species in relation to that required for a more sweeping phylogenetic change, let us briefly examine those divisions of the process called micro-, macro-, and megaevolution. To quote the capsule summary given by Carter,* "There is, first, the origin of the smallest evolutionary differences as seen in continuous series of strata; secondly, there is the differentiation of the members of a group in adaptive radiation; and thirdly, the evolution of a new type of animal organization from its predecessors." Microevolution In certain favorable instances, when geological processes have re- sulted in the formation of a continuous local succession of strata, paleontologists have been able to reconstruct the morphological pro- gression of a species as it took place over many hundreds of thousands of years. An elegant example of such a reconstruction is the work of Trueman and his collaborators on the evolution of the coiled lamellibranch, Gryphaea, a mollusk derived from oysters of the genus Ostrea which is frequently found in the strata of the Mesozoic era. Mollusks of the genus Gryphuea arose frequently and independently from flat-shelled predecessors, presumably in response to the neces- sity for raising the mouth of the shell above its muddy environment. Four stages in the progressive development of a line of Gryphueu are shown in Figure 5. During the evolution from Ostrea irregulurb to Gryphueu incurvu a number of morphological characters were modi- fied, and each of these was changed at different rates. Any one of these characters may be used as a measure of rate of change; in Fig- ure 6 is shown a plot of the variations in one of these, the number of whorls in the shell, as a function of the vertical location of the sample studied within the superimposed strata. The populations ex- amined by Trueman from any given stratum gave a unimodal distribu- tion curve, strongly suggesting that the population was single and was not a mixture of independent populations. In a case such as this there is little question that microevolution has occurred without any large and sudden changes (saltations). The general characteristics of the evolution typified by the Gryphaeu, with its succession of imperceptible gradations and with its uni- formity around a mean, led Trueman to suggest that "such an evolv- ing stock must be regarded as a `plexus' or `bundle of anastomosing lineages.' " The example has been presented here mainly to illustrate THE TIME SCALE AND SOME EVOLUTIONARY PRINCIPLES 7 About 6x lo6 years Ostrea I$ irregularis Gryphaea dumortieri Gryphaea aff obliquuta Gryphaea aff incurva Figure 5. Four stages in the evolution of Gryphaea from its oyster-like ancestor. Redrawn from A. E. Trueman, Biol. Revs. Biol. Proc., Cambridge Phil. Sot., 5, 296 ( 1930). THE MOLECULAR BASIS OF EVOLUTION About 6 x lo6 years Lower angulata zone Number of whorls Figure 6. Distribution curves showing the variation in the number of whorls of the shells of successive populations of evolving Gryphaea. Redrawn from A. E. Trueman, Biol. Revs. Biol. Proc. Cambridge Phil. Sot., 5, 296 (1930). that microevolution is a population phenomenon and that the sep- arate development of radiating lines becomes almost impossible in a restricted population since continual interbreeding prevents the SUC- cessful rise of deviant groups. Macro- and Megaevolution When a mutation confers some benefit on an organism within the framework of the environmental restrictions on the population to which it belongs, the characteristic controlled by the mutant gene may ultimately become firmly entrenched in the heredity of the en- tire group. However, a limited horizon, such as that available to the Gryphueu, permits only a limited phenotypic change. Thus, even though a few "advanced" Gryphnen might have appeared which were: THE TIME SCALE AND SOME EVOLUTIONARY PRINCIPLES 9 B endowed with some unique and specially favorable character, they would not be likely to be perpetuated as a unique strain because of their random interbreeding with the standard average organism. The major factor responsible for the larger changes in evolution that lead to distinct new specializations, or to new systems of animal or plant organization, is adaptive radiation. Adaptive radiation is the term used by evolutionists to describe the separation of populations into smaller groups having different natural histories. The more mobile the group and the more demanding the environmental changes to which adaptation must be made, the greater the diversity of form (and the number of unsuccessful "experiments"! ) that results. This diversity and mobility, together with the concomitant high rate of evolutionary change, make the fossil record scattered and incomplete as opposed to the situation for the sedentary Gryphaea. Neverthe- less, paleontologists have been able to reconstruct the phylogeny of numerous lines with great success, and certain distinct parameters of macroevolution are well delineated. The macroevolution of a particular population of organisms leads to great complexity of form, most of the examples of which are false starts and become extinct after a relatively short time (paleontologi- tally speaking). For an evolutionary development to be successful, all the various morphological parts must change in a correlated way to insure survival. The evolutionists can express such correlations in relative growth and development of parts by means of double logarithmic plots, as shown in Figure 7. Here are represented the relations between the heights of the paracones (a cusp of the molar teeth) and the lengths of the ectolophs (the ridge on the outer border of the crown of the same tooth) of the teeth of horses during their progression from the primitive Eohippus to the modem animal. Characters that may be related by such straight-line plots (of the general form Y = bXk) are said to be undergoing allometric change, and the slopes of the lines (k) g ive a measure of the relative rates at which two specific bodily characters are changing. A sudden modification in the slope of the plot relating two allo- metric changes indicates a sudden shift in evolutionary direction. For example, such an indication is given several times during the evolution of the horse. As horses underwent adaptive radiation they became exposed to new types of environmental opportunities in- volving both new kinds of food and new terrain. The changes in the position of the eye and in the structure of the foot and of other physical characteristics have been described in a fascinating way by Simpson in his book Horses. The modification of the molars 10 THE MOLECULAR BASIS OF EVOLUTION Figure 7. Changes in the structure of the molars during the evolution of the horses. After G. G. Simpson, Tempo and Mode in Evolution, 1944, by per- mission of Columbia University Press. 4-, , , I III1 8910 15 20 2530 Length of ectoloph during equine evolution is particularly instructive in connection with our present consideration of sharp changes in evolutionary direction. AS ecological conditions made browsing more favorable than grazing, the whole plan of the molar was modified by natural selection in a direction compatible with the abrasive action of hard grasses. Thus the crown of the molar became thicker and, together with the de- velopment of cement, permitted the animals to enjoy a fertile life span in spite of the erosive nature of their natural food supply. A schematic representation of the adaptive radiation of horses is shown in Figure 8. This figu re shows the eating habits of the various suc- cessive members on the main evolutionary line. The correlative plot of two allometric structural features of the molars, the size of the tooth and its height, shows that there occurred an abrupt increase in the relative height of the tooth in the horses that converted to grazing, whereas in another evolutionary offshoot, Hyohippus, which continued to browse on soft, easily chewed plants, such a change did not occur. Most authorities seem to agree that the evolution of a particular line of organisms, like the horses, can be explained without compli- cation on the basis of the selection of mutants that confer a survival value on the individual and on the population to which he belongs. The occurrence of a particularly advantageous mutation has fre- quently led to an almost explosive change in structure or habit, and Simpson has proposed the name "quantum evolution" for such major jumps. The view is frequently expressed, however, that the process of natural selection might still be an adequate explanation for these rapid shifts. Their suddenness is perhaps overemphasized because THE TIME SCALE AND SOME EVOLUTIONARY PRINCIPLES 11 Figure 8. The evolution of the horses. The diagram shows the geographic dis- tribution of the various forms and indicates their manner of securing food by browsing or by grazing. Redrawn from G. G. Simpson, Horses, 1951, by per- mission of Oxford University Press. 12 THE MOLECULAR BASIS OF EVOLUTION of gaps in the fossil record that resulted from the rapidity of the changes and the limited geographical region in which they occurred. In discussions of those portions of evolution in which whole new systems of biological organization arose, the terminology and interpre- tations of experts becomes varied and, sometimes, delightfully mysti- cal, at least to window-shoppers such as myself. We have already mentioned the terms entelechy, e'lan vital, and aristogenesis. Such terms have been coined to explain (explain away, perhaps) the fre- quent, puzzling phenomena in which new structures and physiologies have arisen in the absence of obvious adaptive value or selective in- fluence. During the evolution of reptiles, for example, there occurred a simplification of the jaw structure which made superfluous the quadrate and articular bones of the reptilian jaw. Ultimately, mil- lions of years later, these "liberated" units became involved in a major change in the structure of the middle ear and made possible the chain of small bones which is characteristic of this organ in mammals. This "aristogenic" change, leading to an entirely new anatomical or- ganization at a much later time, is not easy to explain on the basis of selection and adaptation alone. The phenomenon has implied to some that the evolutionary process has, built into it, some knowledge of the future and that temporarily useless structures may be stored away for later use according to some master plan. From the standpoint of maintaining a more unified picture of the evolutionary process, `aristogenesis" and the "preadaptation" of an organism for some subsequent evolutionary event do not appear to be necessary concepts. Simpson has pointed out that, in small pop- ulations, a mutation which confers no adaptive value (or, indeed, which may be detrimental) can become established, although "al- most always this would lead to extinction." In those rare cases when the word "almost" applies, a change in the natural history of the or- ganism might then cause an enormously rapid and major evolutionary modification owing to the sudden usefulness of this otherwise dis- advantageous gene, fortuitously harbored in the heredity of the strain. From this point of view we may explain the whole of evolution, from the localized, sedentary sort of microevolution to the dramatic ap- pearance of new phyla, on the basis of mutation and selection alone. As we shall discuss in a later chapter, certain structural parts of biologically active proteins appear to be superfluous from the stand- point of function. A tendency to assume that such parts are non- essential might simply reflect the fact that we have not yet developed sufficiently sensitive methods for the detection of subtle, second-order relationships between structure and function. On the other hand, THE TIME SCALE AND SOME EVOLUTIONARY PRINCIPLES 13 certain structural configurations may now actually be unessential and may have been preserved as chemical vestiges of earlier molecules, much as the bones of the mammalian ear were retained from the re- arranging components of the reptilian jaw. The information available to us on proteins and other chemical components of protoplasm is, of course, insufficient to permit a rational choice between these alternatives at the present time. We can only hope, in analogy to the paleontologist and his "fossil record," that as the "protein record" relating the proteins of various species to one another becomes more complete, some basic ground plan for phylogenesis and speciation may begin to emerge at a molecular level of understanding. REFERENCES 1. G. S. Carter, Animal Evolution; a Study of Recent Views of Its Causes, Sidgwick & Jackson, Ltd., London, 1951. 2. A. E. Trueman, Geol. Mag., 61,360 (1924). SUGGESTIONS FOR FURTHER READING Colbert, E. H., Evolution of the Vertebrates, John Wiley & Sons, New York, 1955. Huxley, J. S., Evolution, the Modern Synthesis, Allen 81 Unwin, London, 1942. Oparin, A. I., The Origin of Life on the Earth, translated from the Russian by Ann Synge, Academic Press, New York, third edition, 1957. Simpson, G. G., The Meaning of Eoolutfon, Yale University Press, New Haven, third printing, 1950. Simpson, G. G., Horses, Oxford University Press, New York, 1951. Simpson, G. G., Life of the F'ust, Yale University Press, New Haven, 1953. Smith, H. W., From Fish to Philosopher, Little, Brown & Company, Boston, 1953. THE MOLECULAR BASIS OF EVOLUTION chapter 7 L GENES AS DETERMINANTS OF HEREDITY D arwin thought of evolution as a process of adap- tation to environment by means of the natural selection of favorable "variations." Within the context of the knowledge of his day he could not, of course, replace the word "variations,, with "mutations,,, since the science of genetics had not yet been invented. However, being a man with a strong urge to tie up loose ends, Darwin sug- gested that "variations,,, * m&ding those that he felt might be ac- quired in response to environmental pressures during the lifetime of the organism, were inherited by a mechanism in which all the somatic (body) cells contributed information to the germ cells. We know now that acquired characteristics are not inherited and, with the emergence of genetics, it became possible to speak of the inherited characteristics of an organism (his phenotype) as the expression of the sum of his chromosomal genes (his genotype).* We may now * It should be stressed that environmental conditions, during development, can exert a profound influence on the phenotypic expression of the genes. A classical example of this is the effect of temperature on the number of eye facets in Drosophila whose chromosomes bear the mutations "low-bar" and "ultra-bar."' Two organisms with identical developmental potentialities may look or act quite differently, although their respective offspring will be back to the .old standard 15 describe evolution in terms of the natural selection of favorable gene mutations in a population and the perpetuation of these through re- production. Since this book is directed at biochemists, many of whom may have had as little formal training in genetics as I have, it is necessary to present, as a starting point for further reading, an abbreviated sur- vey of the gene concept and of some of its experimental consequences. We shall restrict ourselves to the Mendelian genetics of normal bi- sexual reproduction as it occurs in the higher plants and animals. The mechanisms involved, although by no means universal, can serve as a qualitative basis for considering the reproduction of even such specialized genetic systems as the bacterial viruses, if we are willing to cut some comers. Parent generation 0 RR \ / Rxr Nearly a hundred years ago, Gregor Mendel made the observa- tions that established the fundamental laws of genetics. Mendel crossed strains of garden peas which differed in one contrasting char- acter (e.g., purple or white flowers) and observed that the progeny (the so-called F, generation) were all purple. This character was, then, the "dominant" trait and white the "recessive." Similar dom- inance or recessiveness was observed for many other alternative traits. When two members of the F, generation were crossed, he observed that about three-fourths of the progeny in the F, generation were purple and one-fourth white. These experiments suggested that any particular character-determining unit of heredity exists in two forms and that these "aZZeZic" forms do not blend but maintain their identity throughout the life of the F, organisms to separate later in the fol- lowing generation. The units of heredity were subsequently named "genes,, by Johanssen in 1911. An organism, like the F, peas of Mendel, which contains both allelic forms is said to be a heterozy- gote, and those possessing a double dose of one or the other allele is a homozygote. We refer, genetically, to the former as Rr and to the latter as RR or rr (homozygous for the dominant and recessive forms respectively). F, generation Mendel's experiment, summarized in Figure 9, illustrates the "law of segregation." The frequency of occurrence of purple and white flowered plants in the F, generation (3: 1) is to be expected if the two allelic forms of this particular color-determining gene, one dominant over the other, segregate to yield equal numbers of R and r units during the formation of germ cells and then proceed to recom- bine at random in the new generation. Mendel checked this hy- and the superficial characteristics acquired as the result of environmental pres- sures will not be inherited. Figure 9. Mendel's first law, the law of segregation; R stands for the gene for purple and I for the gene for white flower color. Black rings and white rings symbolize purple and white-flowered plants, respectively. Purple color is domi- nant over white. John Wiley & Sons, Redrawn from T. Dobzhansky, Eoolution, Genetics, and Man, 1955. 16 THE MOLECULAR BASIS OF EVOLUTION GENES AS DETERMINANTS OF HEREDITY 17 Parent generation 4 generation F2 generation AABB mbh \ AB x AaBb Figure IO. Mendel's second law, the law of independent assortment; A and a rep- resent the genes for yellow and green colors, respectively, and B and b those for smooth and wrinkled seed surfaces. Yellow is dominant over green and round is dominant over wrinkled. Redrawn from T. Dobzhansky, Euolutfon, Genetics, and Man, John Wiley & Sons, 1955. pothesis by allowing the purple-flowered plants in the F, generation to produce an F, generation. One-third of the F, plants (the RR strain) produced only purple-flowered progeny, whereas two-thirds (the Rr variety) produced either white- or purple-flowered progeny in the ratio 1:3 as predicted by the principle of segregation. In some of his experiments Mendel crossed peas which differed in two or more traits. Thus, as summarized in Figure 10, be crossed peas having yellow, smooth seeds with others having green, wrinkled seeds; he knew in advance that the gene for yellow color was dominant over that for green and the gene for round seeds was dominant over that for wrinkled. The F, generation had seeds which were yellow and smooth, since both dominant traits were present in this hybrid and determined the phenotype. In the F, generation, however, the phenotype was determined by a random combination of the four segregated traits as shown in the figure. Seeds of the F, progeny showed all the four possible combinations of phenotype but, because of the dominance of yellow and smooth over green and wrinkled, these appeared in a ratio of 9:3:3: 1 with only one-sixteenth of the seeds having the double recessive characteristics. This phe- nomenon, independent assortment of genetic traits, is the second basic "law" growing out of Mendel's studies. The simplicity of Mendel's experiments and their ease of interpre- tation were really due to his good fortune in choosing sets of traits which segregated and recombined to give the theoretical 3: 1 ratio. In many instances this ratio is not obtained, and instead certain sets of genes may segregate together to yield what are termed "linked" traits. To understand the linkage of genes we must first consider the phenomena of mitosis and meiosis. Cytologists have been aware for over a hundred years of chromo- somes as visible rod or thread-like structures that appear in the nucleus during cell division. The number of chromosomes per nucleus is a characteristic constant for any given species. The genetic information present in a cell is accurately perpetuated in each of the daughter cells by the process of mitosis. The stages in mitosis are shown in Figure 11 as they are observed in the root tips of the common onion. The simplified drawing on the left side of the figure depicts the behavior of a single chromosome of this plant. The centromeres are represented, in this figure, by open circles. These specialized structures within each chromosomal strand act as points of attachment for the fibers which bind the chromosomes to the pole of the spindle during subdivision of the cell. The centro- mere is replicated during the division cycle, as shown. Occasional GENES AS DETERMINANTS OF HEREDITY 19 II) THE MOLECULAR BASIS OF EVOLUTION Figure 11. Mitotic cell division in the common onion: A, interphase; B, prophase; C, metaphase; D, anaphase; E, telophase; F, daughter cells. From T. Dobzhan- sky, Eoolutfon, Genetfcs, and Man, John Wiley &I Sons, 1955. cells containing chromosomes which lack a centromere, or which have more than one, do not survive. The genetically significant event is the exact duplication of each chromosomal daughter-strand during the period between stages F and B, whereby hereditary con- stancy is insured in all the somatic cells of an organism during its growth and development. The nucleus of the somatic cell (diploid) contains twice as many chromosomal strands as the germ cells or gametes (haploid). The complement of chromosomal strands in a gamete is the same as that of somatic cells immediately following mitosis, before the ma- chinery of the cell has had an opportunity to bring about duplica- tion of each strand. That is, each gamete contains only a single aIlelic form of each gene. When two sex celIs unite, the resulting diploid zygote contains the hereditary units of both parents arranged in such a way that the corresponding chromosomal strands are paired with each set of allelic genes in exact physical complementarity. When the time comes for the cells of the reproductive tract to produce gametes, there occurs a process termed meiosis, which is summarized schematically in Figure 12. The sets of chromosomes first enter a stage resembling prophase in mitosis. The corresponding maternal and paternal chromosome sets then proceed to find one another by a miraculous procedure in which each bit of cytologically discernible detail along the maternal strand pairs with its opposite number in the paternal strand. Each of the two strands then sub- divides into two, and, in most organisms, the pairs of strands are bound together at one or more points by "chiasmata" (Figure 120). The further stages of meiosis lead to the formation of gametes containing only one chromosome of each kind. As shown schemati- cally in the figure, the centromere divides during the second meiotic division. The details of these latter stages of meiosis are somewhat different in different organisms, but the end result, haploid sex cells, is the same. Early in this century cytologists recognized that the phenomena of independent assortment and segregation of heritable characteristics were consistent with the behavior of chromosomes during cell divi- sion. Direct evidence for such a correlation was soon forthcoming, largely through the efforts and imagination of T. H. Morgan. Mor- gan chose as his experimental object the fruit fly, Drosophila mekzno- guster, which contains extremely large chromosomes in the cells of its salivary gIands. This organism possessed a number of important advantages for genetic research, including a high rate of multipli- cation and a genetic apparatus having only four pairs of chromosomes. GENES AS DETERMINANTS OF HEREDITY 21 -- @ Figure 12. Schematic design of the stages of meiosis. Only a single pair of chromosomes is shown. The paternal chromosomes are in black and the maternal in white. #The centromeres are shown as white circles. After T. Dobzhansky, Euolution, Genettcs, and Man, John Wiley & Sons, 1955. By crossing strains of flies which showed different inherited traits, Morgan demonstrated that many of these traits behaved according to the principles of Mendelian genetics. He soon observed, however, that a number of traits did not show independent assortment but were frequently transmitted from parent to progeny as though they were linked together in a genetic bundle. A consideration of the scheme in Figure 12 will make clear the (correct) explanation put forward 22 THE MOLECULAR BASIS OF EVOLUTION by Morgan for these observations. Except for the segments of each chromosomal strand that may be exchanged for their counterparts in the course of the formation of chiasmata, the total genetic information in each chromosome appears in any specific gamete as a unit. Thus, two closely linked genes (and we may think of this linkage, in physical terms, as distance along the strand) are not likely to become separated from one another during meiosis. Morgan and his scien- tific followers in the field soon found that the traits with which they dealt fell into four linkage groups and concluded that each cor- responded to one of the four chromosomes. This conclusion was completely supported when subsequent studies on the giant salivary gland chromosomes of Drosophila made possible the direct com- parison of gene mutations as detected by genetic analysis with visible morphological changes in the individual chromosomes them- selves (Figure 13). Genes that are linked together frequently do show independent assortment, in spite of their location on the same chromosome. This separation is explainable in terms of the exchange of chromo- somal segments that takes place between the two strands during the formation of chiasma. (S ee t ransfers indicated in Figure 120.) Morgan suggested that the frequency of separation, or of recombina- tion, of two linked genes is a function of the linear distance separating them. Stated in other terms, the probability of a chiasma occurring between two distant genes would be much greater than the proba- bility of one occurring between two genes which are close to one another. His hypothesis has been amply confirmed by a vast amount of data on the recombination of linked genes in a variety of organisms and, although there exist numerous examples of quantitative devi- ation from the rule, frequency of recombination is in general a relia- ble measure of the separation between genes. At this juncture it may be wise to introduce an aside directed toward the novice in genetics. The picture we have drawn of the development of the fundamental concepts of genetics has been made purposely rosy for simplicity's sake. In this discussion, and in what follows, we are interested in getting across only the most basic con- ceptual framework of the subject and cannot consider the many reservations and qualifications to be found in any adequate text- book. (For example, in male Drosophila no chiasmata are formed during the process of spermatogenesis, and consequently no linked genes can undergo recombination in the progenies of hybrid males. In the reproduction of bacteriophage, a matter we shall discuss at greater length in later chapters, recombination of linked genes GENES AS DETERMINANTS OF HEREDITY 23 takes place, from a statistical point of uiew, in a manner quite analo- gous to recombination in higher organisms. Estimates of the dis- tances between two genes on the phage "chromosome" may be based on the same general sort of calculation that we employ for studies on sweet peas, in spite of the fact that classical reciprocal crossover does not occur; that is, wild-type and double recombinant phages do not, both, generally result from a single mating event.) If genes may be thought of as being arranged in a linear fashion along the chromosomal strand, and if the distances between them may be estimated by linkage analysis, it is clear that a "map" can be constructed expressing their physical relation to one another. Such maps have been prepared for a number of species of higher organisms and more recently for bacteria and viruses as well. A map of some of the genes that have been studied in Drosophila mel- anognster is shown in Figure 14. In general, the distances indicated behveen genes can be shown to be qualitatively correct by internal checks. Thus, in a series of crosses involving three genes A, B, and C, if it is found that the distance between A and B is x units and behveen B and C is y units, the distance between A and C will be found to be approximately x plus y units. The units used here are "units of recombination" and are merely the percentage of the prog- eny from any particular cross that is different from either parent genotype. For a variety of reasons, the "genetic distances" indicated on maps such as that shown in Figure 13 bear only a rough corre- spondence to the actual physical parameters of the chromosomal strand. One factor responsible for such deviations is the apparent greater potentiality of some parts of the chromosome to crossover than others. Another factor involves the occurrence of multiple crossovers. As the length between two genes becomes larger and larger, the chance of multiple crossovers will increase and, in the limit, there will be an equal chance of an even number and an odd number of crossovers. Thus with widely separated genes and with random crossover, the `map distance" would approach 50 recombi- nation units rather than 100. Genetic maps appear, in general, to be a reliable representation of the relative order of genes, confirming the concept of a linear arrangement. But it must be recognized that the frequency of crossover varies from point to point along the chromosome, and from species to species, and has great influence on the additivity of distances and on the total apparent map length. In the vast majority of cases, the translation of phenotype into the language of genetics follows the simple rules we have attempted to summarize. The difficulties experienced by nonspecialists in the GENES AS DETERMINANTS OF HEREDITY 25 24 THE MOLECULAR BASIS OF EVOLUTION d ID lx * m : yellow(B) al `27.7 lozenge (E) d 59.0 vermilion (E) a.1 mtitun(W) J 43.0 uble (B) u.4 garnet(E) b d\ ii4 /56.7 forked(H) B/ -57.0 bar(E) En 59.5 flued hy 13.0 dumpy 0) 16.5 clot(E) b ee h 81.0 dacha(B) 41.0 jammed(W) ; th rt US black(B) D 161.0 reduced(H) ,$ ,54.5 purple W) 2s btitlc (H) `55.0 light(E) sb 57.5 cinnabar (I?) u 620 enpiled (B)$ 61.0 vestigial ,w,L H. 72.0 lobe(E) ' 75.5 curved(W) aJ m w.a h=Py(B' 100.7 brown(E) 107.0 o pc&(B) M' 192 jwelin (H) so e+(E) 26.5 hairy (Ii) ,dichMb (H) -i;,&&(E) &O eculet 47.5 &formed(E) `46.0 pink(E) 50.0 curled(W) ,5&z o tuhble (H) -56.5 lpiaalev (H) `58.7 bithow 62.0 ettQe(Bl `63.0 @em(E) 661 delta(V) a.5 haike 70.7 &my(B) 74.7 cmdhd(El 91.1 rourh (E' 100.7 duet(E) ma minute-#(H) Figure 14. Genetic maps of the chromosomes of Drosophila melanogaster. After C. Bridges, from Mary J. Guthrie and John M. Anderson, General Zoology, John Wiley & Sons, 1957. course of reading genetic literature arise from the terminology which has been needed by experts to categorize the abnormal. A gene is recognized only because it can be modified and appear in an ab- normal allelic form which determines some unusual phenotypic char- acter. We refer to such changes in genes as mutations, but we must be constantly aware of the fact that the word has a multiplicity of meanings and that true understanding of genie modification can only be reached when the genetics becomes describable in chemical terms. The appearance of a new phenotypic character may be due to a change in the gene itself, chemical or configurational, to a deletion or reduplication of the gene, or to one of a number of "position ef- fects" involving the inversion or translocation of genes to new posi- tions along the chromosome. As stated by T. Dobzhansky,2 `A chromosome is not just a container for genes but a harmonious sys- tem of interacting genes. The arrangement of genes in a chromosome has developed gradually during the evolution of the organism to which the chromosome belongs; the structure of a chromosome, like the structure of any organ, is a product of adaptive evolution." It is to be hoped that the foregoing discussion of the simplest elements of genetics will be sufficiently irritating in its compactness (and in- completeness) to cause some readers of this book to look into a few of the volumes listed at the end of this chapter. Most of what follows in this book will be concerned with what genes do, and we approach the subject in terms as chemical as pos- sible within the limits of our present knowledge of nucleic acid and protein structure. In the classical sense, the term "gene" has a purely operational meaning. It may be applied to any unit of heredity that can undergo a mutation and be detected by a change in phenotype. As the determined distances between genes on chromosome maps be- come less and less, the maximum size of the chemical unit which de- termines a gene must be thought of as being smaller and smaller: Our impression of the size of a gene, from genetic information alone, depends entirely on the sensitivity of the methods available for the detection of extremely infrequent crossovers. It is precisely within this twilight zone of detectability that the classical definition of the gene begins to break down; here contemporary research in genetics and chemistry finds common ground. Estimates of the size of a gene (as an operational unit) have been made by several methods which together more or less define the upper and lower limits. One sort of estimate is possible from crossover data. Muller and Prokofyeva,3 for example, localized four genes on the giant salivary gland chromo- somes of Drosophila within a distance of 0.5 micron and concluded GENES AS DETERMINANTS OF HEREDITY 17 26 THE MOLECULAR BASIS OF EVOLUTION that the upper mean limit of length for each must therefore be 1250 A. Other estimates, derived from studies of the effects of ionizing radia- tion on the frequency of mutation, indicate that a single gene may occupy a volume corresponding to a sphere with a diameter as small as 10 to 100 A. The discrepancy between crossover and radiation data is considered too large to be due to experimental or interpreta- tive error and suggests that two different aspects of gene structure are being measured by the two techniques, one having to do with the crossover of the entire, intact gene (that is, a .functional unit of genetics) and one with the modification of chemical fine structure within its macromolecular architecture. This conclusion appears to be supported by recent developments in genetic fine-structure analysis, a few of which we shall review subse- quently. To establish a bridge between the more classical concepts of genetics and the rather revolutionary findings of the contemporary microbial geneticist, it is instructive to consider an example of the apparent subdivision of a single gene in the genetic material of Drosophila. In the course of linkage analysis, certain genetic units called "pseudoalleles" have been detected which appear to be concerned with the same, or at least with a closely related, function. One such set of pseudoalleles makes up the "lozenge genes" of Drosophila melano- gaster. A mutation in the "lozenge" region causes changes in the pigmentation of the eyes and also certain other morphological changes. The mutant forms are recessive to the normal allelic form of the gene; that is, heterozygotes show normal pigmentation. Green and Green' have studied three mutational loci within this region of the genetic map, all of which have "lozenge" characteristics. From an analysis of crossover data, they have determined that all three loci fall within a genetic distance of less than 0.1 units of recombination. They were further able to show that &n&e heterozygotes, in which the two mutant alleles were on the same chromosomal strand, showed the wild-type character, whereas an arrangement in which the two mutants appeared on different strands of the same chromosome pro- duced the mutant phenotype. The phenotypic consequences of the various arrangements of two mutant loci are shown in Figure 15. Two explanations for these observations have been offered. One suggests that each of the individual loci controls a different enzymatic activity which is in close physical association with the genetic locus itself. These enzymes are pictured as components of a series of consecutive reactions leading to the formation of an essential chem- ical material. Such a situation might apply if the individual re- 28 THE MOLECULAR BASIS OF EVOLUTION Mutant phenotype (trams) Wild phenotype (cd Flgure 15. Schematic diagram of the cb and truns arrangements of the pseudo- allelic "lozenge" genes in Drosophilu melunogaster. After M. M. Green and K. C. Green, Proc. Natl. Acad. Sci. U.S., 35, 586 ( 1949). actions were of such a nature that the operation of the reaction chain depended on certain minimum concentrations of intermediates and would be interrupted should diffusion (from one chromosomal strand in the "mutant" heterozygotes of Figure 15 to another, for example) lead to a suboptimal concentration level for any of the intermediates. This explanation for pseudoallelism clearly involves a number of rather large assumptions and seems less likely, at the moment, than the second alternative, namely, that each pseudoallelic mutation, although distinguishable, like any "gene," by crossover, is really a change in the stcbstructure of the functional parent gene. Thus, we may postulate that a mutation at any of the three loci of the lozenge gene might equally impair its function and that only with the cis arrangement, in which one complete unmarred strand carries the load, can the normal phenotype be expressed. To anticipate some of our later discussion, this idea has been used by Benzer as the basis for the coining of a new genetic term, the "cistron," by which is meant a genetic unit of function subdivisible by genetic GENES AS DETERMINANTS OF HEREDITY 29 Both cistrons functional Only cistron A functional Both cistrons functional Both cistrons functional I 0 Cistron A 1;d Cis Trans arrangement arrangement Figure 16. The subdivision of a functional gene into "cistrons," both of which must cooperate to produce an expression in the phenotype. When two mutations occur in the same cistron, normal function can only be expressed when the loci are in the ck arrangement, but not when they are in the truns arrangement. Based on the suggestions of Seymour Benzer, The ChemlcaZ Bash of Heredity, Johns Hop- kins Press, 1957. tests into ultimate units of recombination termed the "recon." In this system of terminology, two recons would belong to the same cistron when the cb arrangement of two mutant loci in a double heterozygote (Figure 16) results in functional adequacy and the tram arrangement does not. The demonstration that genes are made up of blocks of very closely linked subunits which may be differentiated by crossover has been a tremendous stimulus to bio- chemists interested in "genetic chemistry." The mutational effect of ionizing radiation on a bundle of genetic matter having an esti- mated diameter of 10 A. or so becomes a much more tangible phenomenon when we can compare such a distance with equivalent chemical distances, such as the separation of side chains on a polypeptide or the molecular dimensions of a dinucleotide. As we shall discuss in later chapters, the ultimate mutable units of genetics do, indeed, appear to be about this size, and it is possible that we may soon be able to equate them with individual nucleotide residues along the polynucleotide strands of deoxyribonucleic acid. 30 THE MOLECULAR BASIS OF EVOLUTION An Introduction to the Concept of "Biochemical Genetics" No summary of genetic principles would be complete without some discussion of heredity in Neurospora. Neurospora occupies a special niche in genetics because a great deal of the evidence relating genetic constitution to biochemical behavior has been obtained through its study. It has long been evident that mutations are reflected as changes in biochemical properties. This is essentially a paraphrase of the state- ment that mutations are detected only because of the difference in phenotype which they induce, the phenotype of an organism pre- sumably being the sum of its biochemical potentialities. The studies on the genetic control of the structure of flower pigments by Law- rence,5 Scott-Moncrieff, and their colleagues helped establish the fact that individual genes determine the exact chemical structure of these pigments by regulating the extent of methqxylation, hydroxyla- tion, or conjugation with carbohydrate of certain heterocyclic com- pounds called anthocyans. These studies already began to suggest that the modification of a single gene leads to a change in some specific biosynthetic process. Wild strains of Neurosporn may be selected which will grow well on an extremely simple culture medium consisting essentially of sugar, salts, and a single vitamin, biotin. By exposing such cultures to some mutagenic agent (e.g. X-rays), we obtain mutants that no longer grow on the minimal medium but require the addition of nutritional additives like yeast extract and hydrolyzed proteins and nucleic acids. By systematic dissection of the additive mixture, it may be determined which single nutritional requirement has been induced by mutation. The isolation of mutant forms having clear-cut nutri- tional requirements is not always simple, and many have been isolated which undergo spontaneous reversion to the wild type or which continue to grow on a minimal medium, although at a much reduced rate. However, a large number of stable, full-blown mutants that require a single nutritional additive for growth has now been isolated. These nutritional substances include a variety of amino acids, purines and pyrimidines, and vitamins. Because of the conventional chromosomal system of inheritance, the position of these mutant loci in Neurosporu may be established by orthodox crossing over methods. The experimental approach to mapping is indicated by a consideration of the natural history of Neurosporu, the main points of which are shown in Figure 17. In asexual reproduction, the haploid conidia germinate to produce GENES AS DETERMINANTS OF HEREDITY 31 Conidia Conidia Ascospo germinat Ascospore germination Ascus sac with ascospores 4&d Figure 17. The life cycle of Neurosporu crassa. The genetic events which occur during the first and second meiotic divisions are illustrated in greater detail in Figure 18. Redrawn, in part, from R. P. Wagner and H. K. Mitchell, Genetics and Metabolism, John Wiley & Sons, 1955. more haploid mycelia. Increase in mass also takes place by simple growth of existing mycelia through mitosis and the utilization of nutrients from the culture medium. In sexual reproduction cross- fertilization takes place between two mating types, variously referred to as A and a, or + and -. The conidia of these two types appear to differ only in a single genetic locus on one of the chromosomes. In a cross, the haploid nuclei of the two mating types become as- sociated within a common cytoplasm. In subsequent events (Figure 17) the nuclei of both mating types undergo numerous equational divisions (a,b) and subsequently fuse, side by side, into a diploid pair 32 THE MOLECULAR BASIS OF EVOLUTION (cd). This zygote (d) th en undergoes two rounds of meiosis (e,f) to produce four haploid nuclei (f) which then divide mitotically, to yield eight ascospores (g). When these ascospores are exposed to heat or to certain other stimuli (furfural), germination is induced. One of the advantages of Neurospora as an experimental tool in genetics is the fact that the order of events during meiosis is faith- fully mirrored in the final asci. As summarized in Figure 17, the upper and lower sets of two nuclei at the four-nucleate stage are derived from the upper and lower nuclei of the binucleate state, and a similar regularity is preserved after the subsequent mitotic division (stage g). The individual ascospores may be dissected out by hand, in order. With some mutations, which cause a visible difference in the appearance of the final ascospore, we may estimate, without testing the individual spores, the frequency of crossing over during meiosis, and thus the map position of the locus in question in relation to the centromere as a zero point. This procedure is illustrated in an elegant way by an example taken from the work of D. R. Stadler" on an unusual lysine-requiring mutant. This mutant, one of a num- ber of lysine-requiring strains studied by N. Good in 1951, exhibits delayed ascospore formation, and mutant spores may be detected within the ascus by their colorless appearance. Perpetuation of this abnormal strain is possible, in spite of the arrested maturation, because the vegetative mycelium can be cultivated indefinitely without the necessity for sexual reproduction and also because an occasional mutant spore will mature upon aging. The photograph in Figure 18 shows the typical appearance of the asci that are produced when the mutant is crossed with a wild-type strain. The critical stages in meiosis following the cross are shown schc- matically in Figure 19. The two haploid conidia first fuse to form a zygote a,. (This zyg t o e is known to be in a double-stranded form (a,) at the start of the first meiotic division.) During this first meiotic division crossover may or may not occur between the two sets of parental strands. In Neurospora, the centromeres from each parental chromosome do not divide during the first meiotic step, and the crossed-over pairs of strands remain attached as shown in the figure (b and c). The frequency of crossing over of a given allele during the first meiosis is assumed to be a function of the distance of this locus from the centromere. During the second meiotic division each nucleus yields two daugh- ter nuclei to give a total of four, arranged in a row, the upper and lower set derived by division of the upper and lower of the two GENES AS DETERMINANTS OF HEREDITY 33 nuclei in the binucleate cell. If no crossover has occurred, the order shown in d develops, whereas with crossover four different arrangements may be obtained (e). When the four-nucleate cells undergo subsequent mitosis, various asci are produced as shown in the photograph. The spores containing the mutant locus are easily distinguished by their colorless appearance. Inspection of the photograph (Figure 18) indicates that in nine of the fourteen mature ascospores no crossover has occurred; that is, the normal and the mutant forms of the locus in question have segregated at the first meiotic division. Figure 18. Appearance of asci produced upon crossing a wild-type strain of Neurosporu with a lysine-requiring mutant which exhibits delayed maturation. As discussed in the text, the approximate location of the mutant locus on its chromosome may be deduced from the relative frequencies of first- and second- division segregation. This photograph was obtained through the kindness of Dr. David Et. Stadler of the University of Washington. Five ascospores show a pattern consistent with second division segre- gation, one alternating as in Figure 19, e, and e,, and four symmetri- cal as in e2 and e,. Therefore, five-fourteenths of the mature asco- spores, during development from zygotes, have undergone crossover. Assuming linearity of genes, a direct relationship between crossover frequency and linear distance, and the absence of centromere division in the first meiosis, the mutant locus would be calculated to be 5/14 X 109 or 36 per cent of the distance from the centromere to the end of the chromosomal strand. (Actually this map distance is to be divided by a factor of two since the unit of mapping in Neurospom is defined as one-half of this ratio.) 34 THE MOLECULAR BASIS OF EVOLUTION GENES AS DETERMINANTS OF HEREDITY 35 , Meiosis # 1 Centromere does not divide I Centromere divides I or 1 + + 1 fed or + 1 1 t k3) or + 1 + 1 fed) Figure 19. A schematic diagram of the genetic events which occur during the development of an ascus from a zygote in Neurospora. The left side of the dia- gram shows the results of first division segregation, and the right side, those of second division segregation of the two alleles, 1 and +. The crossover frequency values obtained from cross to cross were found by Stadler to vary over a considerable range, as is frequently observed in genetic practice. Accurate mapping must always involve a series of crosses between three separate markers or two markers and the centromere, so that additivity may be used as a check. This example is included here because it illustrates how an approximate estimate may be made of the location of a mutant locus in Neuro- spora, even without exhaustive crossing of progeny, when the muta- tion produces a visible change in the convenient ascospore "re- cording system." The great value of the Neurospora mutant technique as a tool for relating genetics to biochemistry will be evident from a consideration of the following example. Three genetically distinct mutants, which will grow on the minimal medium when this is supplemented with one or more of the three amino acids, arginine, citrulline, and orni- thine, have been isolated. Mutant 1 can grow only when supplied arginine and cannot utilize citrulline or ornithine. Mutant 2 can use both citrulline and arginine, and mutant 3 can manage on any one of the three nutritional additives. These observations suggest that arginine may be produced through the sequence of reactions shown in Figure 20. Assuming the correctness of this biochemical hypothe- sis, we may propose that the mutant loci in the three mutants each affect a specific enzymatic process in the reaction chain leading to the synthesis of arginine. The correctness of this proposition is indicated by the fact that nutritional mutants will, in general, utilize and grow on intermediates that come after the `block" but not those that precede it. Indeed, in most instances, there is an accumu- lation of intermediate metabolites preceding the block. The particular reaction sequence leading to arginine formation is a well-established one for many organisms. The study of the three Neurospora mutants is, thus, mainly a confirmatory one, but it has great historical interest since it was one of the earlier clean-cut examples of the direct relation between the enzymatic potential of an organism and its heredity. In many later investigations results derived from the study of other mutants have frequently served as the first wedge in the elucidation of new metabolic pathways. Perhaps the most significant development growing out of the study of the inheritance of nutritional requirements in Neurospora has been the enunciation of the "one gene-one enzyme" hypothesis by G. W. Beadle and E. L. Tatum and their collaborators. This hypothesis, which proposes that a single gene controls the synthesis of only one enzyme or other specific cellular protein, can be made quite flexible by the proper choice of semantics. The breadth of interpretation is 36 THE MOLECULAR BASIS OF EVOLUTION Compounds Utilized by .Mutanta - - + Omithine - + t Citrulline + -I- t Arginine k-C-C--c-COOH Urea \ -Y( 0 0 II NH,-C-NH NH, C-C-C-C-COOH f 0 NH -6,, 2 I C-C-C-C-COOH Figure 20. A series of biochemical reactions in the biosynthesis of arginine, the order of which could b e established by the study of the nutritional requirements of three mutants of Neurospora. From the work of A. M. Srb. and N. H. Horo- witz, J. Biol. Chem., 154, 129 (1944). directly dependent on the definition we choose to give to the word "gene." Thus, as is true of the pseudoalleles of the "lozenge" gene in Drosophila, finer and finer genetic analysis begins to discriminate between loci which are part of the same functional unit. In a relatively coarse analysis, such as the study of the three mutants in the arginine pathway, we are not able to say with certainty whether the blocked step in mutant 2, for example, is immediately prior to citrulline or whether one of a number of intermediate steps between omithine and citrulline is blocked instead. At the other extreme, an exhaustive genetic analysis might permit the detection of two genetic loci separated by so small a distance along the genetic strand that they would be part of the same functional unit. Mutation of either of these might alter or abolish the biological activity of the same protein molecule. This situation has, indeed, been observed for a number of microorganisms and bacteriophages, and much of what follows in this book will deal with this theme. One excellent example of a direct relationship between a single protein and a single gene is the case of the two types of tyrosinase in Neurospora. Horowitz7 and his colleagues have shown that the mutation of a single genetic locus causes the formation of a heat- GENES AS DETERMINANTS OF HEREDITY 37 labile tyrosinase which is indistinguishable from the usual, heat- stable enzyme in all other physical and kinetic properties. The two forms of the enzyme may be isolated in quite pure form, and there can be no doubt that the genetic modification affects a single protein molecule. The difference between the two forms of the enzyme is inherited in a strictly Mendelian way; that is, a given pure strain of Neurosporu produces only one form of the enzyme, and the progeny of a cross between the two strains are identical with one or the other parent strain in equal proportion. The possibilities suggested by this and other similar gene-protein relationships are among the most intriguing in the whole of biology. Clearly, if slight modifications in protein structure can ultimately be equated with equally slight changes in the molecular structure of genetic material, there will be opened to us a whole new area of research and speculation on the most basic aspects of the evolutionary process. REFERENCES 1. J. Krafka, J. Gen. Physiol., 2, 409 ( 1920). 2. T. Dobzhansky, Evolution, Genetics, and Man, John Wiley & Sons, New York, 1955. 3. H. J. Muller and H. A. Prokofyeva, Compt. rend. acad. sci., U.R.S.S., 4, 74 (1934). 4. M. M. Green and K. C. Green, Proc. Natl. Acad. Sci. U.S., 35, 588 ( 1949). 5. W. J. C. Lawrence, in Blochemkal Sac. Symposia, Cambridge, Engl., No. 4 (1950). 6. D. R. Stadler, Genetics, 41, No. 4, 528 ( 1958). 7. N. H. Horowitz and M. Fling, in Enzymes: Units of Biological Structure and Function, (0. G. Gaebler, editor), Academic Press, New York, p. 139, 1956. SUGGESTIONS FOR FURTHER READING Catcheside, D. G., The Genetics of Micro-Organizms, 1951, Pitman Publishing Corporation, New York. The Chemical Basis of Heredity (W. D. McElroy and B. Glass, editors), Johns Hopkins Press, Baltimore, 1957. Pontecorvo, G., in Advances in Enzymology, volume 13, Interscience Publish- ers, New York, p. 121, 1952. Wagner, Ft. P., and H. K. Mitchell, Genetics and MetaboZism, 1955, John Wiley & Sons, New York. 38 THE MOLECULAR BASIS OF EVOLUTION chapter 3 THE CHEMICAL NATURE OF GENETIC MATERIAL T here can be little doubt that the major share of the heredity of a strain is carried in the chromosomes of its germ cells. This conclusion is based firmly on the observations of the cytologist and the geneticist that specific mutations may be directly related to localized morphological changes in chromosomes. It serves as the starting point for one of the most thriving enterprises in modern bio- logical research, namely, the identification and chemical description of genetic material. Progress has been rapid and we can now state with some assurance that the substance most directly associated with the storage and perpetuation of hereditary information is the deoxy- ribonucleic acid (DNA) of the chromosomal strands. Aside from the circumstantial evidence furnished by the cyto- chemical localization of DNA in chromosomes, there are a number of other lines of evidence which give more explicit information bear- ing on this idea. It was demonstrated by Boivin, Vendrely, and Vendrelyl in 1948, for example, that the DNA content of somatic cells (diploid) was constant from tissue to tissue in a single species but that sperm cells, .(haploid) contained exactly half as much. i : 39 These observations were extended to a number of other species by Mirsky2 and his collaborators. The latter group also showed that the distribution and quantities of various other chemical components of nuclei generally did not correlate in a way to be expected for genetically critical material. We know that the nucleus contains, in addition to DNA, ribonucleoprotein, various arginine-rich protamines and histones, a tryptophan-rich protein fraction, and a small amount of lipid. None of these substances (with the exception of the prota- mine-histone fraction, which may be directly associated with DNA) appears to have the constancy of distribution from cell type to cell type within a species of the sort exhibited by the DNA component. Another type of experimental observation which suggests a genetic role for DNA is concerned with the effects of mutagenic agents, such as ultraviolet radiation and certain chemicals. It has been shown that the efficiency spectrum of ultraviolet light in producing mutations is closely related to the absorption spectrum of nucleic acid, Such experiments are not completely convincing in themselves since the absorption of ultraviolet photons by the nucleic acid mole- cule might conceivably be only the first step in a chain of reactions in which the final target could reside in molecules of a rather different chemical nature. Experiments on the mutagenic effect of such agents as mustard gas are similarly inconclusive since, although the nucleic acids do appear to be much more chemically reactive to these sub- stances in vitro than are the proteins, we cannot discount the special sensitivity of a particularly important member of the latter class of compounds. In spite of these possible objections, there is a certain amount of direct evidence which indicates that when we tamper with the chemical structure of DNA, be it by radiation or by chemical techniques, the rate of mutation is increased. Zamen- hof3 and his colleagues have shown that when the thymine analogue, %bromouracil, is incorporated by a cell into the structure of its DNA, the frequency of mutation is greatly increased. The reasons for this stimulation of the mutation rate may be related to the pres- ence of the abnormal pyrimidine base in the polynucleotide sequence of the DNA molecule, or it may equally well be the result of some aberration in the kinetics of the biosynthesis of DNA. Whatever the reason, we may at least conclude that the DNA molecule is impli- cated in this chemically induced mutagenesis. The metabolic stability of DNA also supports the close association of DNA with genes. In spite of a number of early misleading reports, the consensus now clearly indicates that the DNA content of chromo- somes does not change during the stages of division, nor do the 40 THE MOLECULAR BASIS OF EVOLUTION subcomponents of its structure undergo equilibration with extra- nuclear sources of DNA precursors. The most clear-cut support for this conclusion comes from cytochemical and radiochemical studies on the nuclei and chromosomes of growing tissues, particu- larly of plants. Howard and Pelt,' for example, attacked the problem by growing roots of the English broad bean, Vi& fuba, in the pres- ence of radioactive orthophosphate. Autoradiographs of squash preparations of the root tissue were then prepared by the stripping film technique. An analysis of the way in which radioactivity was associated with the nuclei in various stages of division and in the resting stage indicated that incorporation of the isotope takes place only in the resting, interphase nucleus and that prophase and meta- phase nuclei are not actively synthesizing nucleic acid. Autoradio- graphs of root tips which had been incubated with isotope for more extended periods permitted the further conclusion that the tagged nucleic acids of the chromosomes are passed on to daughter cells without intermediate degradation and resynthesis. These experi- ments, and the resulting conclusions, have been greatly refined, both as the result of improvements in the technique of chromosome auto- radiography and by the study of purified isotopically labeled DNA from various sources. We shall return to these more recent studies after we have first considered the chemistry of DNA and the organ- ization of the chromosome in more detail. However, the experiments of Howard and Pelt, even without embellishment, are quite satisfy- ing from the genetic point of view since they suggest conservation of DNA and the physical continuity of the gene during cell repli- cation. Some of the best evidence for the central importance of DNA in genetics comes from studies on "transformation." This phenomenon discovered in pneumococcus by F. Griffith in 1928, has since beed extended to a number of other microorganisms. It involves the change in the genotype of one strain of cells that is produced by exposure to extracts of cells of a different strain. Pneumococci generally exist in two forms. One forms "smooth" colonies on agar plates, possesses a type-specific polysaccharide cap- sular substance, and is virulent. The other forms "rough" colonies and lacks both the virulence and the polysaccharide of the smooth form. The smooth forms are genetically stable, and a strain char- acterized by a Type II polysaccharide, for example, does not spon- taneously mutate to Type III. "Smooth" organisms do, however, mutate to rough forms, and this conversion appears to be irreversi- ble. Griffith showed that when mice were subjected to mixed THE CHEMICAL NATURE OF GENETIC MATERIAL 41 injections of living non-encapsulated "rough" organisms and killed "smooth" organisms, living encapsulated bacteria could be isolated from the animal. The progeny of these transformed bacteria were also encapsulated, and the specific polysaccharide was shown to persist indefinitely through successive generations until spontaneous mutation to a rough form occurred. Avery and his collaborators," and later Hotchkiss, Zamenhof, and others, have shown that the substance in Griffith's extracts which is responsible for the transformation has the chemical characteristics of DNA.g The evidence for this is now very convincing, and the trans- formation of organisms for a host of genetic markers in addition to that controlling encapsulation can now be attributed, with relative certainty, to the DNA molecule. The transformation is definitely not caused by a protein contaminant. Many of the DNA-borne characters were originally selected by , exposure of bacteria to sink-or-swim situations (Figure 21). For example, when pneumococci are grown in the presence of strepto- mycin, essentially all the cells are killed by the drug. A very few cells, however, survive and continue to multiply, producing a culture which is resistant to the levels of streptomycin employed. These organisms have acquired, by a chance mutation, the physiological characteristics which permit them to occupy a new "ecological niche" in nature (although a rather unnatural one). Not only do the cells which survive streptomycin behave as a new and constant phenotype, but samples of DNA prepared from them possess the ability to trans- form other cells to a state of streptomycin resistance. The genetic stability of transformable traits is highly suggestive of true gene transfer. It is difficult, however, to prove unequivocally that actual chromosomal material is being transferred by this process from cell to cell. The chemistry and cytology of bacterial nuclei is still quite obscure and, to make matters more difficult, genetic anal- ysis of the sort that can be done with sweet peas or Drosophila is not easy with bacteria because multiplication takes place most com- monly by mother-daughter division rather than by sexual mating. Sexual mating does take place fairly frequently in some microorgan- isms, and it seems likely that the transmission of transformed char- acters as unit particles of heredity could be studied by some direct hybridization technique if the proper choice of bacterium and other transformable traits were combined in one experimental system. To my knowledge, however, such a study has not been carried out for any of the traits that can be transferred by a purified DNA prep- aration. 42 THE MOLECULAR BASIS OF EVOLUTION Parent culture %a3 )f<> ((`, II' ~8 lb @ Culture + rare mutant, (e.g. - ze c resistant, etc., 1 in 10') I Klective environment Mutant culture (or stock variant, e.g. encapsulated) Purified mutant DNA (transforming agent) + Parent culture $51 @ < J 08, Its %ii r 1, x Culture containing transformed $33 3 D cells (1 in 1O'to 103) * ' Figure 21. Experimental steps in the transformation of a bacterial culture. Rr- drawn from R. Hotchkiss, The Nucleic Acids, volume 2 (E. Chargaff and j. N. Davidson, editors ) , Academic Press, 1954. THE CHEMICAL NATURE OF GENETIC MATERIAL 43 There are, however, in addition to genetic stability, several other features of transformation that support the notion that the transfer of unit characters by DNA is closely analogous to the normal genetic process occurring during cell division. It has been shown, for ex- ample, that the DNA prepared from strains of bacterial cells which have been transformed for two transformable markers can occasion- ally produce simultaneous transformation for both traits, as though these were linked on the same "chromosomal" fragment, Thus, Mar- mur and Hotchkiss' have selected, from a strain of streptomycin- resistant pneumococci, mutants that have also undergone a mutation which permits them to ferment mannitol. When DNA preparations from such doubly labeled cells were added to a culture of "wild- type" pneumococci, three distinct types of transformed bacteria could be isolated. The majority were either transformed for streptomycin resistance or for mannitol utilization, but some individual cells had clearly been transformed for both characters. The frequency of double transformation was considerably greater than the product of the frequencies of the two single transformations, an observation which is genetically interpretable only in terms of the linked transfer of two unit characters by a single event. This observation, so remi- niscent of linkage in the chromosomes of higher organisms, certainly suggests that a single fragment of DNA can contain the information for the elaboration of two distinct physiological systems and that this fragment represents a piece of the normal genetic material of the bacterial cell from which it was derived. These studies have since been extended by Hotchkiss and his col- leagues to cells that contain three transformable markers. They have shown that, during transformation with DNA, these markers may re- main linked, be separated, or be reassembled by recombination in a manner completely analogous to that observed with doubly marked cells. In spite of our ignorance of the details of the process, we may conclude with reasonable certainty that the mechanical transfer of genetic information from cell to cell by purified DNA represents a good model for some of the events that occur during conventional gene transfer in dividing cells. Chemical Structure of DNA One of the most fascinating recent developments in biochemistry has been the study of the structure of deoxyribonucleic acid, both from the standpoint of its molecular composition and in terms of the 44 THE MOLECULAR BASIS OF EVOLUTION arrangements of its component parts in three dimensions. If DNA is to be established as the substance responsible for the transmission of heredity at the molecular level, we should be able to demonstrate that the morphological behavior of chromosomes can be explained as a function of the structure and metabolism of DNA. Amazingly enough, such a picture has begun to emerge as the result of a series of very skillful experiments and deductions. There are the usual inconsistencies and hopeful extrapolations, but the present outlook is certainly an optimistic one. In 1948 the conception of DNA as a regular array of repeating tetranucleotide units was seriously shaken by the fundamental studies8 of Vischer and Chargaff and of Hotchkiss, who showed by chromato- graphic techniques that the four heterocyclic bases in DNA are not present in equal amounts and that there are actually more than four such bases in some samples of DNA. Since these studies, the list of naturally occurring purines and pyrimidines in the DNA molecule has grown to seven, and a complete reappraisal of the structural details of DNA has taken place. By an examination of the products produced from DNA by acid or enzymatic hydrolysis we may deduce that the successive stages of degradation are as follows: * DNA + nucleotides -+ nucleosides + phosphoric acid + purine and pyrimidine bases + deoxyribose The chemical structure of these substances is indicated in Figure 22. In most samples of DNA, four heterocyclic bases predominate- the purines adenine and guanine and the pyrimidines thymine and cytosine. However, some samples of DNA (e.g. from wheat germ and the grasses in general) contain 5-methylcytosine in large amounts and, indeed, this derivative of cytosine is found in small quantity in preparations from mammalian tissue as well. The DNA prepared from the `: of porcine pituitary tissue are shown in Figure 74 (page 153). The material known as /3-MSH contains a heptapeptide sequence which is identical with residues 4 to 10 of ACTH. An even more striking ex- ample of recurring sequence is a-MSH, which has a structure identi- cal with the first thirteen residues of ACTH except for the addition of a C-terminal amide group and an N-terminal acyl radical. The similarities in these structures are particularly interesting because of the fact that ACTH is formed in the posterior lobe of the pituitary gland, whereas MSH appears to be synthesized in the pars intermedia. ( 1958). (The reader should be cautioned that a direct relationship be- tween nuclear and cytoplasmic RNA is far from established-see for ex- ample, J. W. Woodward, J. Biophys. B&hem. Cytology, 4, 383 ( 1958 ). 8. Reviewed in a series of papers in Proc. Nat. Aced. Sci. U.S., 44, No. 2 (1958). 9. P. C. Zamecnik and E. B. Keller, J. Btol. Chem., 209, 337 (1954). 10. J. A. Gladner and K. Laki, J. Am. Chem. Sot., 80, 1263 (1958). 11. R. Loftfield, in Progress in Biophysics and Bfochemktry, Pergamon Press, London, 1958. 12. S. Spiegelman in The Chemical Basis of Heredity (W. D. McElroy and B. Glass, editors), Johns Hopkins Press, Baltimore, 1957. 13. J. L. Simkin and T. S. Work, Nature, 179, 1214 (1957). 14. D. Steinberg, M. Vaughan, and C. B. Anfinsen, Science, 124, 389 (1956). 210 THE MOLECULAR BASIS OF EVOLUTION chapter 11 GENES, PROTEINS, AND EVOLUTION 4, . . . we must remember that heredity, development, and evolution are essentially epigenetic and not preformistic. We do not inherit from our ancestors, close or remote, separate characters, functional or vestigial. What we do inherit is, instead, genes which determine the pattern of developmental processes. . . . " T. DOBZHANSKY, Evolution, Genetics, and Man, A s the genes of a species are modified and reshuf- fled, occasional organisms will appear within a restricted population having phenotypic characteristics that enable them to explore desir- able ecological niches which were unattainable by their predecessors. The individual changes are generally quite small. Many generations must come and go, during which forays into formerly forbidden ter- ritory by this developing branch of the population become more fre- GENES, PROTEINS, AND EVOLUTION 211 quent and are of longer duration as the result of further reorganiza- tion of the gene pool by random mutation and natural selection. In time the summation of these changes results in a new species, fully at home in its new environment and su5ciently different in physiology from its distant ancestors that cross-fertilization is no longer possible. We have discussed, in several earlier chapters, the techniques used by the geneticist for the analysis and description of limited portions of such a chain of events. As long as crosses can be made between different family lines, phenotypic changes can generally be related to specific genes and the spread of these genes through a population can be fairly accurately mapped. Thus, the basic assumptions of evolu- tionary theory may be directly tested, and with some precision, when the segment of time under consideration is small, and we are able to describe the process in terms of changes in genotype. When we deal with evolution on a larger scale, however, the tools of the geneticist are no longer applicable. The evolutionist must now rely on the study of relative morphology and ecology as deduced from the fossil record, or on the comparative anatomy and physiology of living rep- resentatives of surviving species. The principal aim of this book has been to examine the basic prin- ciples underlying another possible method for the study of evolution. This method is based on the hypothesis that the individual proteins which characterize a particular species are unique reflections of the genes which control their synthesis. The examination of the chem- istry of a series of homologous proteins is, of course, a purely pheno- typic approach to the problem. Nevertheless, the evidence available to us, even at this early date, suggests that the. structure of proteins may be a relatively direct expression of gene structure and that com- parative protein chemistry may furnish a qualitative view of geno- typic differences and similarities. If we accept the general hypothe- sis, we are led to infer, for example, that the "insulin-determining" genes of the pig and the sperm whale are identical, like the insulins whose structures they determine. Indeed, should several genes be concerned with the synthesis of insulin, the same would also be true for these. Another interesting potentiality of comparative protein chemistry is that it might permit us to determine whether the same phenotypic characteristic, shown by two completely unrelated organisms, is at- tributable to analogous or to homologous genes. For example, both bacteriophage T2 and chicken's eggs contain proteins that have lyso- xyme activity. The genetic material of both coliphage and chickens must be said to contain information that can direct the formation of 212 THE MOLECULAR BASIS OF EVOLUTION proteins with this function. Is it possible that these two organisms contain nearly identical (that is, homologous) stretches of genetic material, or are the genes for lysozyme synthesis, and the lysozymes themselves, entirely different? This would appear to be the sort of question that might be attacked directly by the comparative study of protein structure. As we have already seen, in connection with cyto- chrome c, ribonuclease, hemoglobin, and other proteins, there is ex- cellent evidence which indicates that many homologous genes do ap- pear to have survived happily through long periods of time, some well exceeding the span of the fossil record. A Biochemical `Approach to the Species Problem Th e paleontologist, in estimating the rates and directions of evolu- tion, must depend almost entirely on morphological evidence. Even with this relatively crude sort of yardstick, he can begin to distin- guish patterns of change such as we discussed in Chapter 1, in con- nection with the characteristics of tooth structure in the evolving horses. He is limited, however, to the results of evolution and can never hope to elucidate the underlying physiological changes that participate to produce new phyla. of Although most of the ancient species disappeared, representatives almost all the phyla escaped extinction by adapting to their new environments, thus perpetuating large parts of heredity. We have available to us, then, a contemporary sample of the life of the past from which we should be able to deduce a great deal about the fac- tors that were decisive in phylogenesis long ago. The study of "biochemical evolution" has already been of considerable value in the establishment of biological interrelationships. For example, the oc- currence of melanocyte-stimulating, oxytocic, and vasopressor hor- monal activity in extracts of the neural gland if tunicates furnishes strong evidence in support of the assignment of this subphyllum, the Urochordata, to the direct pathway between the invertebrates (which lack MSH activity) and vertebrates. The presence of both arginine phosphate (an invertebrate phosphagen) and creatine phosphate (the typically vertebrate phosphagen) in tunicates adds additional support to this assignment. We shall not attempt to discuss here the numerous contributions of this sort that biochemistry has made to evolutionary theory. The reader will find this material summarized in a number of comprehen- sive essays and books." 2, 3 Our present concern is primarily with GENES, PROTEINS, AND EVOLUTION 213 Figure 96. An electron photomicrograph of collagen fibrils from bovine skin. Magnification x 42,000. Obtained through the kindness of Dr. Jerome Gross, Massachusetts General Hospital, Harvard University Medical School. 214 THE MOLECULAR BASIS OF EVOLUTION the biochemical changes in protein molecules that are much nearer, in metabolic terms, to the genes themselves than are such products of enzymatic action as the phosphagens, or the eye pigments of Drosophila. As \Vald has put it "It is a truism in biochemistry that each species of animal and plant possesses specifically different pro- teins." The full understanding of speciation must, almost certainly, be sought in the structllre of proteins. To expand this point a bit, let us consider two elegant examples of speciation that are demon- strably related to changes in protein strrlcture. Th e protein collagen is largely responsible for the physical prop- erties of such structural tissues as skin and cartilage. LVhen collagen fibrils (Figure 96) are exposed to heat they change markedly in in- ternal structure and yield the molecular form known iis gelatin. Now recent studies by X-ray crystallography have shown that the collagen molecule is very likely composed of three strands of poly- peptide, cross-linked through a system of hydrogen bonds of con- siderable strength.' The amino acid sequence, Gly.Pro.IIypro, ap- pears fairly frequently along the chains, and the hydroxyl groups on the hydroxyproline residues are presumably major contributors to the hydrogen bond network. \Vhen collagen is heated in solution, the hydrogen bonds become ruptured at a critical temperature, known as the "shrinkage temperature," and the organized structure is quickly disoriented to form the mow globular and amorphous gelatin strnc- ture. Although the exact mechanism of this rearrangement is not known, it is possible, on the basis of the results of current work on the properties of synthetic polyproline and of mixed polymers of proline and glycine, that the shrinkage may be associated with a cis-trclns isomcrization at l)rolil7c-l"olinc Or proline-hydroxyproline bonds," in conjunction with ordinary "entropic" denatrwation. On the basis of these chemical and physical observations we might srqqmsc that collagcw molewlcs, suited to either cold or warm habitats, co~~ld bc devised hy natlu'ca through the introduction or de- letion of hydroxyproline residues. Animals living in climates tending to be very warm would do well to lltilizc collagens with I$.$ shrink- agcl trnll~c,r;lturc,.~, ant1 thaw liviug in cold clirnatcs cor~ltl do with considerably fcwcbr sitw for cross-linkage and with lowc~r shrinkage tcmperatwcs. The studies of K. If. Gustavson and of T. Takahashi on the col- lngens of fishes suggest that this is precisely the mechanism which has been employed." The shrinkage tempcraturcs of cold-\vater fishes arc always lower than those of warm-water fishes, and an amazingly linear relationship exists between shrinkage temperatnre and the con- GENES, PROTEINS, AND EVOLUTION 215 13 , I I I I TABLE 17 40 50 60 70 Shrinkage temperature, z, "C Figure 97. The relationship between the hydroxyproline contents of the collagens of various fishes and their "shrinkage temperature."' tent of hydroxyproline (Figure 97), although the vertebrate collagens are otherwise extremely similar in composition. It is a provocative fact that collagen shrinkage temperatures seem to fall about 15 or 20o above the highest temperatures likely to be encountered by a species, as though this margin of safety were adequate in the ordi- nary course of climatic events. The relationship between the visual pigments of marine fishes and the depths of their habitats is another dramatic example of adapta- tion through modification of protein structure. Denton and War- ren,' Munz,s Wald and his colleagues,D and many other investigators have studied the chemical structure and the spectral properties of a variety of fish rhodopsins. Rhodopsin, composed of a vitamin A derivative complexed with a protein, opsin, constitutes the light- sensitive element of the retinal rods. The vitamin A-like prosthetic group, retinene, responsible for light absorption, has been found to be identical in all the species listed in Table 17. Since opsins do not themselves absorb light in the spectral interval between 480 and 503 rnp., the shifts in the position of the absorption maxima shown in Figure 98 must be attributed to the effects of the opsins on the 216 THE MOLECULAR BASIS OF EVOLUTION Spectral Properties of Rhodopsins from Various Fishes Species Summer flounder (Paralichthys den#atus Linnaeus) Scup (Stenotomus uer~ico20~ Mitchell) Butterfish (Poronotus triacanthus Peck) Barracuda (Sphyraena bore&r DeKay) Cod (Gadus callarias Linnaeus) Cusk (Rrosme brosme Miiller) Lancet-fish (Alepiaarusferoz Lowe) Summer Range of Depth, fathoms a-10 503 0.695 l-20 498 0.686 l-30 499 0.610 l-10 498 0.575 5-76 496 0.530 lo-100 494 0.455 >a00 480 0.%50 E54o/&,ax spectral properties of retinene. The mechanism by which conjuga- tion with opsin can induce a change in the spectral properties of retinene is quite obscure. We have previously discussed a related in- stance of a spectral shift, where the absorption characteristics of the 0.8 up, butterfish, barracuda 500 550 600 Wave length, rnfi Figure 98. Absorption spectra of rhodopsins of marine fishes in 2 per cent aqueous digitonin solution. The maximum absorption (A,,,.=) shifts toward shorter wave- lengths in rough correlation with the depth of habitat. See Table 17. GENES, PROTEINS, AND EVOLUTION 217 tyrosine residue are modified by hydrogen bonding of the hydroxyl group. The shift in this case was small, of the order of a few milli- microns. In the rhodopsin absorption system maxima differ by as much as 23 mp as we move from the summer flounder to the lancet fish. This large shift implies a major change in the nature of the interaction between protein and prosthetic group. The biological observation that makes all this of special interest is the fact that a correlation is observed between the mean depth of habitat of the various species of fishes and the spectral properties of their visual pigments. The correlation is not at all exact and, as the data in Table 17 show, a wide spread of A,,,~, exists at all depths. Nevertheless, the information available is sufficient to form the basis of a strong hypothesis. Over twenty years ago G. L. Clarke observed that the increasing blueness of light with depth in the ocean raises "the question of the possibility of a shift in the sensitivity of the eye of a deep-water fish toward the blue end of the spectrum." This possibility is realized in the spectroscopic observations just listed, and it is now possible to apply the techniques of protein chemistry to the elucidation of the details of this fascinating chapter in biochemical ecology. The study of the structural modifications in opsin which have taken place during the evolution of the fishes will be especially interesting since, as we have seen for the insulins and cytochromes c, large spans of evolutionary time may pass without too extensive a change in a particular protein molecule. The changes in the opsin molecule may be so cleverly contrived and so incisive that extensive alterations in sequence and folding have been unnecessary. On the other hand, if alterations hnve been extensive, we shall be required to rationalize a very complex set of interactions between protein and prosthetic group. Both alternatives are intriguing, to say the least, and the study of the chemistry of the opsins should make a most valuable contribution to the understanding of evolution at the molecu- lar level. The Rate of Evolution As G. G. Simpson has pointed out, the question "How fast has evo- lution occurred?" is meaningless without the addition of the qualifica- tions, "the evolution of what organisms, of which of their structures, and at what time in their history." The opossum, for example, has changed relatively little in the past 80,OOO~OOO years, whereas the 218 THE MOLECULAR BASIS OF EVOLUTION evolution of the horses during the past 60,000,OOO years has involved at least eight distinct genera. Just as the anatomical organization of some organisms has changed much more rapidly than that of others, it seems likely that we shall find a large spread in the rates at which specific protein molecules have been modified during evolution. Although our basis for discus- sion of this point is still very thin, it is already evident that some pro- teins have undergone far greater structural change than others over an equivalent period. Compare, for example, the somatotropins of the sperm whale and the sheep with the insulins of these same species. Although the changes in insulin structure have been restricted to very minor modifications in a limited part of one polypeptide chain, the somatotropins are quite markedly modified in molecular weight, in cystine content, and in the number of polypeptide chains. Equally striking differences in degrees of modification exist between numerous others of the examples discussed in Chapter 7. How are we to plan our experimental approach in attempting to establish some chemical coherence in the tremendous puzzle of specia- tion? We must, it would seem to me, begin with the basic assump- tion that the phenotypic character of a species is primarily determined by its unique spectrum of proteins. We may then proceed to a study of the extent to which each of the individual proteins within any spectrum may be modified without loss of biological function. As we already know, the degree of "violability" of different proteins may vary enormously as judged from the results of in vitro studies on denaturation and chemical modification in relation to function. Even here, however, many of the observed differences in sensitivity may be overemphasized and may depend on the choice of methods used for modification. Even though two proteins may be very similar in regard to the proportion of their total structure that is essential for function, one set of reagents may attack critical parts of one and not seriously alter the other. Amino groups, for example, may be acety- lated with essentially no effect in pepsin, but at least some of these same groups appear to be critical for the activity of lysozyme. A proper comparison of two biologically active proteins thus must de- pend on the use of a wide variety of inactivating reagents, and ulti- mately on the deliberate degradative sort of study that aims to reduce proteins to their minimum, functionally adequate size. Since, however, proteins can be modified without loss of function, it seems certain that the permissible degree of modification, in terms of fractions of their total structure, will vary somewhat from molecu- GENES, PROTEINS, AND EVOLUTION 219 lar species to species. It does not seem too farfetched to think of the proteins of a given organism as being subdivisible into those that have structures quite closely tailored to an essential functional re- quirement, those that are designed with only moderate "efficiency" or whose function is relatively dispensable, and those that are inter- mediate. Once again, illustrations come to mind. Several individuals exhibiting only slight clinical abnormality have been shown to be completely devoid of serum albumin. These individuals, able to lead a normal existence, are living evidence for the dispensability of this protein under the ecological circumstances peculiar to humans. On the other hand, no one will question the inability of most species to survive in the absence of cytochrome c or of the enzymes necessary for oxidative phosphorylation. If we accept these subdivisions of the protein spectrum, we may `express" a species in terms of a hierarchy of protein structures rang- ing in violability from none to very much. The further evolution of this species, involving the usual mutation and natural selection, would then be reflected in a change in its proteins, one end of the spectrum remaining relatively fixed while the other may change con- siderably. Thus the cytochrome c molecule, which we might think of as a relatively "primitive" protein, and indispensable for most life, would be stubbornly perpetuated in the evolving phyla with minimal change, whereas the structure of the serum albumins might fluctuate with the shifting parameters of natural selection. From time to time, entirely new protein structures might arise, as in the dramatic ap- pearance of insulin and of other hormones at the point in evolution when the protovertebrates and vertebrates appeared. The molecu- lar basis for such "explosive" appearance of new protein entities is, of course, completely obscure. Only a thorough understanding of the processes of protein biosynthesis and of genetic information trans- fer will enable us to choose between such alternatives as the de now creation of a whole new gene as opposed to the fortuitous reshuffling of already available genetic units. We may safely predict that the patterns of change observed in the protein spectrum by future biochemists will not always be smooth and tidy. The criteria of natural selection will differ greatly from species to species, from environment to environment, and from period to period, and the survival value of gene mutations in a population, and of their images in the phenotype, will be quite varied. A large number of important aspects of evolution have been omitted from this book. Some of these omissions may be attributed to type- 220 THE MOLECULAR BASIS OF EVOLUTION GENES, PROTEINS, AND EVOLUTION 221 writer fatigue. Most of them, however, have been purposely omitted because of the lack of adequate factual material for discussion, and this book is already well supplied with speculation. We might, for example, have taken up the question of the spatial organization of genes in relation to function. The recent studies on the mapping of genes related to histidine biosynthesis in Salmonella (Figure 99) by Hartmann, Demerec, and others indicate that the various cistrons associated with the series of intermediate enzymes occur in the same region of the genetic strand and that these genetic determinants are arranged on the gene map in the same order as the reaction sequence itself. A schematic representation of this linkage map is given in Figure 99. The evolutionary implication that linked biochemical steps have been added, successively, in sequence along the chromosome is a very exciting one but is clearly not general. In Neurospora, for ex- ample, genetic loci for closely related enzymatic steps are scattered at random throughout the chromosomal apparatus. We might, also, have spent some time on the question of cytoplasmic heredity, which we know to be of importance in many biological sys- tems. Here again the scarcity of published information is a limiting factor. The study of inheritance of traits in a non-Mendelian fashion is likely to be difficult and confusing, and the genetic or biochemical study of such traits might receive disproportionately little attention. As Nanney has recently suggested, "It is perhaps only natural that in- vestigations of `messy' characteristics are discontinued before publica- tion and that investigators move on to traits more readily analyzed." The omnivorous reader will find Nanney's reviewl" of the subject of cytoplasmic heredity in The Chemical Basis of Heredity excellent reading. The list of omissions can be extended. The chemistry of RNA and its genetic properties, the rearrangements of genes within the chromo- some and the phenotypic consequences of such rearrangements, the problem of polyploidy, the interactions of nonallelic genes-many of these might, even now, be discussed with some intelligence in bio- chemical terms. The relationships between genotype and phenotype will, predict- ably, become a major preoccupation of more and more "pure" and medical scientists during the coming years. This book has grown out of my own attempts to arrive at some sort of appreciation of the potentialities of chemical genetics and the evolutionary approach. It will have been well worth the effort if it can help to stimulate the growing interest in evolution as the central theme in the life sciences. 222 THE MOLECULAR BASlS OF EVOLUTION REFERENCES 1. J. B. S. Haldane, The Biochemistry of Genetics, Allen & Unwin, London, second impression 1956. 2. M. Florkin, Biochemical Eoolution, Academic Press, New York, 1949. 3. G. Wald, in Modern Trends in Physiology and Biochemistry, Academic Press, New York, 1952. 4. A. Rich and F. H. C. Crick, Nature, 176,915 (1955). 5. W. F. Harrington, Natrcre, 181, 997 ( 1958). 6. K. H. Gustavson, The Chemistry and Reactioity of Collagen, Academic Press, New York, 1956. The analytical studies of Takahashi are discussed on pages 224-227. 7. E. J. Denton and F. J. Warren, Nature, 178, 1059 (1956). 8. F. W. Munz, Science, 125, 1142 ( 1957). 9. G. Wald, P. K. Brown and P. S. Brown, Nature. 180,969 ( 1957). 10. D. L. Nanney, The Chemical Basis of Heredity (W. D. McElroy and B. Glass, editors), Johns Hopkins Press, Baltimore, 1957. GENES, PROTEINS, AND EVOLUTION 223 INDEX .4cetabularta mediterranea, protein syn- thesis in, 195, 203 ACTH, 115, 116 Activation of amino acids for protein synthesis, 205, 206 "Active centers," chemical nature of, 268 Adrenocorticotropic hormone, active degradation products of, 129 Adrenocorticotropin, structure of, 115, 116 Adrenocorticotropins, species differ- ences in, 152 Alkaline phosphatase, genetic de- termination of, 182 Allelic genes, 16 Allometric change, 10 Amino acid activation, 205, 207 Amino acid analogues, incorporation into proteins, 190 Amino acid sequences, repetition of in proteins, 207-269 Amino acids, abbreviations for, 105 Aristogenesis, 6, 13 Bacteriophage, plaque morphology of, 67-71 r-mutants of, 68-71 Bacteriophage proteins, fractionation of, 179, 182, 183 Bacteriophages, chemistry and enzy- mology of, 71 life cycle and biosynthesis of, 81 morphology of T group, 7.5-77, 180, 181 Biochemical evolution, 213 Biosynthesis of proteins, 195 Carbobenzoxylated polypeptide chains, trypsin digestion of, 108, 109 Carboxypeptidase, use of in protein structure studies, 107 Centromeres, 19,21, 22, 33, 35 Chiasmata, 21, 23 Chromatography, ion exchange, 169, 110 Chromosome, organization of, 59-61 Chromosomes, 19, 21, 24, 2628 Cis-trans test, 29, 30, 88, 89 applied to host range function, 177 Cistron, 29, 36, 89, 93 Code, "commaless," 63, 64 "Codes," genetic, 62-65 Collagen, characteristics of in relation to hydroxyproline content, 215 species differences in, 215, 216 structure of, 214, 215 Configurational isomerism in proteins, 186 a-Corticotropin, porcine, structure of, 130 Cytochrome c, species differences in, 157 Cytoplasmic heredity, 222 Denaturation, 127 Deoxyribonucleic acid, chemical struc- ture of, 4456 conservation of during cell replica- tion, 41, 57-60 in chromosomes, 394.1, 44 molecular dimensions of, 4951, 78-81 synthesis of, 55, 56 X-ray diffraction analysis of, 49 Deuterium exchange, 123-125 225 Differentiation, of teeth, 10 reptilian jaw, 13 Di-isopropylfluorophosphate, 208 Dinitrophenylation, 106, 107 Diploid cells, 21 Disulfide bridges, location of in pro- teins, 113, 114 2%~~ oitd, 6, 13 Endoplasmic reticulum, 196, 197, 199 Entelechy, 6, 13 Enzymatic digestion of polypeptide chains, 106-111, 113 Ergastoplasm, 196 Evolution, rate of, 218 "Fingerprinting" technique for pep- tides, 144 Gamete, 21 Genes, 15, 16 analogous, 142 determinants of protein structure, 164 dominant, 16 homologous, 142 linked, 22, 23 molecuhir size of, 27, 28 recessive, 16 substructure of, 67 Genetic codes, 62-85 Genetic fine structure, 173 Genetic maps, 24, 25, 26 fine structure, 86, 88-95, I73 in bacteriophage, 84, 173-176 Genetic recombination, 16 Genetics of host range in bacterio- phage, 173 Genotype, 15 Growth hormones, species differences in, 158 Gryphaea, evolution of, 7-10 Haploid cells, 21 a-Helical coiling in globular proteins, 118, 119 Helical coiling in proteins, 100, 101 a-Helix, 100-102 dimensions of, 101 226 llcmoglobins, 167-171 abnormal, structural differences in, 16%171 species differences in, 158, 160, 161 Heredity, cytoplasmie, 222 Heterozygote, 16 Homozygote, 16 IIorses, evolution of, 10-12 IIost range genetics in bacteriophage, 173 Hypertensins, species differences in, 155, 157 Immunochemical comparisons of serum proteins, 162, 163 Independent assortment, law of, 18, 19 Insulin, active degradation product of, 128 structure of, 122 Iusulins, species difference in, 154, 155 Law of independent assortment, 18, 19 Law of segregation, 16, 17 Linkage, 22, 23 Linkage groups, 23, 26 Linkage map, for histidine biosynthesis in SaZmotaella, 221, 222 of T4 bacteriophage, 90, 92 Lozenge genes, 28, 29 Lysozyme, of bacteriophage, 73-75, 183. 212 specificity of, 74 Macroevolution, 7, 9, 10 Maps, genetic, in bacteriophage, 84, 173-176 Megaevolution, 7, 9, Meiosis, 19, 21, 22 stages in, 22 Mclanotropins, species differences in, 154 structures of, 153 Membrane, cytoplasmic, 196-199 Mendelian genetics, 16-19 Microevolution, 7 "M icroheterogeneity," 187 Mitosis, 19, 20 stages in, 20 Mlltagenesis, chemical, 40 Mutants, deletion, 86, 89, 91 Mutation, 27 THE MOLECULAR BASIS OF EVOLUTION Mutations, effects on protein structure, 166 Muton, 93 Myoglobin, structure of, 98 Natural selection, 11, 15 Neurospcm, 3138 nutritional mutants of, 31, 32, 34, 36, 37 Neurospora crassa, life cycle of, 32 Nucleus, composition of, 40 "One gene-one enzyme" hypothesis, 36 Opsins, species variations in, 216-218 Optical rotation of proteins, 118, 119 Ostrea, evolution of, 7 Oxidation of disulfide groups in pro- teius, 107, 108 Papain, active degradation products of, 130, 131 Paper chromatography, of amino acids, 150, 151 of peptides, 145, 146 Paper electrophoresis of peptides, 14.5, 146 Peptide "fingerprints," 144 Pcptide patterns as "fingerprints" of proteins, 144 Peptide separation on paper, 144-146 Phenotype, 15 Phosphoserine, in proteins, 208 Phylogenetic relationships, -3-6 l'olypeptide chain, dimensions of, 100 Preadaptation, 13 Prolactins, species differences in, 158, 160 Protamines, conjugation of with DNA, 200, 201 Protein biosynthesis, 195 in Acetdmlaria mediterruneu, 195, 203 in ruptured-cell preparations, 205 Protein structure, configurational iso- merism in, 186 sequential isomerism in, 18G species variation in, 142 Proteins, bacteriophage, fractionation of, 179, 182, 183 biological activity of, in relation to structure, 126 INDEX Proteins, heterogeneity of, 185 Pseudoalleles, 28, 29 "Quantum evolution," 11 Radiation, adaptive, 10 mutational effect of, 28, 29 Ilcct)lllbination frccluency, 23 Recombination units, 25 Recon, 30, 93 Replication, and structure of deoxy- ribonucleic acid, 55 "copy-choice," 86, 87 Reversible denaturation, of papain, 131 of ribonuclease, 136, 137 Hibonuclease, action of, 103, 104 composition of, 104, 105 partial reductidn with retention of activity, 134, 135 pepsin digestion of, 133, 135, 137, 138 peptidc "fingerprint" of, 144, 14G, 146-151 photooxidation of, 132, 133 reduction of, 133-135 reversible denaturation of, 136, 137 structure-function relationships, 131 structure of, 112, 115 subtilisin digestion of, 132, 135, 137-140 synthetic substrate for, 104 Ribonucleases, beef and sheep, compo- sitions of, 147 species differences in structures of, 149 Ribonucleic acid, chemical structure of, 4Fj, 46 nuclear, turnover of, 201, 202 role in protein synthesis, 203, 204, 207 Suln~onelka, chemical genetics of, 221, 222 Saltation, 7 Secondary structure, 117 "Scgrcgation, law of," 16, 17 Sq~cntial isomerism in proteins, 186 Serum albumin, absence from sera of certain illdividuals, 220 Scrllm proteins, species differences in, 161-163 227 Species variation in protein structure, 142 Spectroscopic properties of proteins, 119 Structure, of proteins, 99 in relation to function, 126 primary, 99 secondary, 99-102 tertiary, 99, 102 Teeth, differentiation of, 10 Tertiary structure, 117 Three-factor cross, 175 Transduction, 95 22% - Transformation, by DNA, 41-44 of linked genes, 44 Tyrosine, anomolous light absorption of, in proteins, 119, 120 -carboxylate interactions, 120, 121 Urea, effect of on protein structure, 120, a4 Visual pigments, species differences in, 216218 Zygote, 21, 32, 33, 35 THE MOLECULAR BASIS OF EVOLUTION