GENETICS V^ Marine Biological Laboratory Library Woods Hole, Mass. Presented by little, Brown and Co, College Dept, Boston, Mass. SSE o CD GENETICS GENETICS IRWIN H. HERSKOWITZ Saint Louis University t,. Little, Brown and Company TORONTO COPYRIGHT © 1962, BY LITTLE, BROWN AND COMPANY (iNC.) ALL RIGHTS RESERVED. NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER. LIBRARY OF CONGRESS CATALOG CARD NO. 62-14045 Published simultaneously in Canada by Little, Brown & Company (Canada) Limited PRINTED IN THE UNITED STATES OF AMERICA PREFACE I NTEREST in the study of genes has increased greatly in the past few decades and grows at an ever- increasing pace. This is due both to the rapid advances in our understanding of the gene and to the important applications made of this knowledge. Even more significant has been the recognition of the fundamental im- portance of genetic knowledge for the future advancement of numerous other areas of scientific investigation and for the preserva- tion and improvement of the well-being of mankind in general. The amount of instruction in genetics given in junior and senior high school classes of biology and general science is increasing, and many of the institutes sponsored by the U.S. National Science Foundation for high school teachers of biology give concentrated instruction in genetics. There is also an increasing amount of genetics taught in intro- ductory courses of biology, botany, and zoology at the college level and a separate course in genetics has become a requirement in many colleges for students majoring in biology or agriculture. It is now generally recognized that genetics has become a core discipline, knowledge of which is essential for an understanding of modern and future biology. The impact of the gene is being felt, more- over, not only by the professional teacher and research worker in biology, but also by students of a number of other related, and seemingly unrelated, disciplines. Medical and dental schools are increasingly interested in the genetic training of their students both before and after they start their professional preparation. More and more students whose major interest is in biochemistry, chemistry, psychology, biophysics, physics, statistics, or mathematics find that the study of genes offers new and challenging opportunities in these various fields. Finally, the impor- tance of genetics for life in our atomic and interplanetary age has been recognized by scholars in the humanities and arts as well as by the informed general public. This mounting interest in the gene has led or will doubtless soon lead to considerable modification in the teaching of genetics at the college level. Because the fund of knowl- edge regarding the nature and consequences of genes is growing so rapidly, usually more than one single semester course is required to cover the material that is considered essential knowledge for the professional bi- ologist. One solution has been to have two courses in genetics, of which one is general and the other advanced. Many smaller col- leges are now initiating a course in general genetics for the first time. Because of the interest of the nonprofessional biologist, the general genetics course already given is being modified in numerous colleges so that it can be taken in the earlier rather than the later years of college study. The question is, what should be the content and aim of an introductory course in genetics? There is general agreement on one score, namely, that a first course should provide an understanding of the nature of the gene, for this knowledge is prerequisite for its fruitful application to the solution of prob- lems in biology and all the other fields mentioned. There are a number of textbooks on genetics which help satisfy this require- ment. However, it is my personal convic- tion that there are several other desiderata for such a course, toward whose realization an appropriate textbook can make significant contributions. I shall take this opportunity PREFACE to mention several qualities a general genetics text should have and how the present text attempts to meet these criteria. The text should be so organized that the principles dealing with the nature of the gene are separated as much as possible from the applications or uses of these principles. Too often students finish an elementary course without a clear understanding of the basic principles regarding genes, on the one hand, and the consequences of these prin- ciples, on the other. In the table of contents all the Chapters containing either basic con- cepts of the gene or information necessary for the comprehension of basic concepts are indicated by an asterisk, while Chapters concerned entirely with the applications or consequences of these basic principles are not so marked. The marked Chapters deal- ing with the nature of the gene include all those which define, or delimit, the ge- netic material operationally in terms of its recombinational, mutational, functional, chemical, and replicational properties, while the other Chapters are concerned primarily with the utilization of genetic principles for the elucidation of problems in morphology (traits), in development and physiology of individuals, in populations, and in evolution. While it would seem essential at one time or another during the course to study many or all the Chapters dealing with the nature of the genetic material, any or all of the Chapters concerning applications may be omitted at the discretion of the instructor. A text should be used as a supplement to, and not a substitute for, the teacher. It has become impossible, as well as undesirable, to include in one text how each principle of genetics has been proven in the case of every plant and animal studied. It is even less possible to give examples of the applica- tion of each of these principles to all the diff'erent kinds of organisms. Accordingly, in the present text only one or a few experi- mentally favorable or historically important organisms are usually employed to establish a principle or to illustrate an application. Additional proofs, applications, or examples are left to the instructor who, depending upon his and his students' training and in- terest, can supply other illustrations by means of lectures and laboratory sessions or by means of assignments to detailed accounts in other texts and in the original literature. A text, therefore, should be used to present the fundamentals, while the instructor should serve to clarify, amplify, coordinate, and integrate them. There is Httle value in having the student attend lectures in which all the time is spent listening to the same material that was discussed in the text. Since the study of the gene is an experi- mental rather than a descriptive procedure, the text should, whenever feasible, derive the principles from the results of experiments. The plan has been to build a solid structure of genetic theory upon evidences and reason- ing presented earlier or concurrently. Sen- tences, paragraphs, section or chapter headings, which state conclusions to be ar- rived at sometime later in the text have been avoided. In general the attempt has been made to adhere as much as possible to the following method of presentation: to recog- nize a problem, offer evidence related to it, analyze all reasonable explanations, and draw whatever conclusions are warranted on the basis of the information presented. Di- gressions from the main purposes are avoided, so, for example, a history of pregenetic thought is not presented. Not the least value of a concise text is the challenge the student may be given to utilize the books and journals in the library. The reading of genetic works in the original by uninformed students is relatively unfruitful for the effort expended, but may be a very rewarding experience if done after reading appropriate sections of the text. Accordingly references requiring different degrees of so- phistication are given at the ends of Chapters. PREFACE Part of a letter by G. Mendel and the Nobel Prize Lectures presented by geneticists are included in the book as Supplements. These Supplements should be completely under- standable, or nearly so, if the appropriate Chapters preceding them have been read, and can serve as a review and overview of genetic principles and their applications. The Supplements can also function to bridge the gap between the textbook and the re- search worker, giving the reader some idea of the history of the subject and the person- alities of the people involved. In certain cases it has been considered desirable in the present work to combine the results, obtained by different people at different times using different organisms and methods, into a single organized body of information, rather than to try to prove a principle or illustrate an application using a patchwork of evidences in which each worker is given his due priority. Since the later Chapters deal with recent advances in ge- netics, whose discussion may be absent from already published textbooks, additional in- formation can be gained from reading the original scientific literature. Accordingly, more references are given to particular work- ers in the later than in the earlier Chapters. The citations to the literature included in the Nobel Prize talks should prove especially valuable to those who wish to do additional reading on key topics. It is hoped that the student will benefit, whatever his future course of study, from the numerous opportunities this text provides for him to think scientifically — to study the design of experiments, to analyze the infor- mation these provide, and to reach valid conclusions. Because a genetics course is elected more and more frequently by students who do not wish to specialize in biology and because it should be no longer a course only for upper classmen, I have tried to use simple biological examples and termi- nology whenever possible, and have explained in some detail certain biological phenomena generally understood by students specializing in biology. Because many students in a first course in genetics are deficient in training in chemistry and physics I have also explained certain aspects of these sciences, important for understanding genes and their behavior, in somewhat more detail than is found in the usual text. On the other hand, since elaborate statistical analysis is needed usually only to demonstrate certain complex appli- cations of genetic principles, the mathematics of genetics has been de-emphasized here, leaving it to the individual instructor to elaborate upon, as he sees fit, in the lecture and laboratory periods. Questions and problems are presented after each Chapter. Instructors or students who would like to have additional discussion and examination questions covering much of the material in this book as well as reading assign- ments in other basic textbooks can find these in the Study Guide and Workbook for Genetics (McGraw-Hill Book Co., Inc., 1960) which I prepared. Finally, it should be emphasized that be- cause this textbook of genetics has been pre- pared by a fellow human being it therefore has certain limitations. This book or any other text is subject to a very personal factor in that the author has selected certain infor- mation for presentation and rejected other material considered less essential. Moreover, while each author does the best he can with the knowledge available, what is accepted by him one day is quickly subject to improvement or rejection by him consequent to the dis- coveries of the next day. To foster these im- pressions the present work is written, as much as possible, as a personal discussion between you and me; it is aimed to help you think critically, to challenge the experimental evi- dence I have presented, the reasoning I have used in its evaluation, and the conclusions I have arrived at. You are cordially invited to play this thinking game with me. But since reasoning reigns supreme here do not look for a superfluity of visual material, whose presence sometimes substitutes looking for thinking. I suggest that you keep pencil and paper handy so you can make your own visual aids and provide yourself with ex- perience in using the knowledge contained here. Readers who already have some ideas about genes should note that certain con- cepts of the gene change during the course of this book. At some earlier point in the book the view of the gene may be quite dif- ferent from the one commonly held and/or different from the one presented later. Re- tain an open mind. Be alert to those oc- casions on which I shall draw incorrect conclusions, or what at one point may seem like a valid conclusion but which later, either in this book or in tomorrow's discoveries, proves to be only partially true or even wrong. Suggestions for Use of the Book This book can be used several ways. It con- tains more information than can be covered in the usual one-semester introductory course for undergraduates. A one-semester course (meeting about 31-45 hours) can be based upon (1) the 31 Chapters marked with asterisks, or (2) the first 35 Chapters, omitting at will certain unstarred Chapters before Chapter 35, or (3) the fol- lowing 24 Chapters: 1-4, 6, 31-49, supple- mented as needed with several lectures on material in unread Chapters. A tno-semester course (meeting a total of about 60-90 hours) can be based upon the first 30 Chapters the first semester and the last 19 Chapters the second semester. Or, the 31 Chapters with asterisks can be used the first semester and the remaining 18 un- starred Chapters the second. Making use of the Supplements and refer- ence fists, for additional reading and dis- cussion, the book can be used also in upper class and graduate introductory genetics courses. Acknowledgments Those figures for which credit is not otherwise given were prepared by William J. Briggs. I wish to thank my wife, Reida Postrel Her- skowitz, for preparing the typescript. I am especially indebted to my present and former students for numerous suggestions. IRWIN H. HERSKOWITZ CONTENTS *1 Genetic Material 7 *2 Gene Segregation 8 *3 Mitosis 16 *4 Meiosis 23 *5 Segregation in Man — Multiple Allelism 32 *6 Independent Segregation 39 7 Gene Interaction and Phenotypic Expression 49 8 Gene Interaction and Continuous Traits 56 9 Multiple Alleles and Lethals 62 10 Pleiotropism, Penetrance and Expressivity 68 11 Studies of Human Twins 74 *12 Sex-Linkage 81 13 Sex Determination (I) 94 14 Sex Determination (II) 100 *15 Intergenic Linkage Ill *16 Crossing Over and Chiasma 116 *17 Gene Arrangement and Chiasmata 128 *18 Changes Involving Whole Genomes and Single Whole Chromosomes 137 *19 Structural Changes within Chromosomes 150 20 Cytogenetics of Oenothera 162 21 Natural and Induced Chromosomal Changes 173 11 Position Effect and Allelism in Drosophila 185 *23 Gene and Point Mutations 197 24 Point Mutants — Their Detection and Effects in Individuals 204 *25 The Genetic Control of Mutability 213 26 The Gene Pool in Cross-Fertilizing Populations 224 IX 79873 X CONTENTS 27 Mutation and Selection — Nonrandom Mating and Heterosis 229 *28 Mutational Loads and Their Consequences to Populations 239 29 Races and the Origin of Species 252 30 Developmental Genetics 262 *31 Biochemical Genetics (I) 271 *32 Biochemical Genetics (II) 281 *33 Chemical Nature of Genes 293 *34 Organization, Replication, and Types of DNA in Vivo 306 *35 Replication of DNA in Vitro 320 *36 Bacteria: Clones and Mutation 329 *37 Bacteria: Recombination (I. Transformation and Chain Recombination in Vitro) 340 *38 Bacteria: Recombination (II. Conjugation) 349 *39 Bacteria: Recombination (III. The Episome F) 356 *40 Bacteria: Recombination (IV. Episomes and Nucleotide-Sharing) 366 *41 Bacteria: Recombination (V. Transduction) 374 *42 Viruses: Recombination in Bacteriophage (I) 382 *43 Viruses: Recombination in Bacteriophage (II) 390 *44 Viruses: Bacterial, Animal, and Plant 400 45 Extranuclear Genes and Their Interrelations with Nuclear Genes 409 *46 Gene Action and Operons 421 *47 Gene Action and Amino Acid Coding 427 48 The Biochemical Evolution of Genetic Material 439 *49 Genes — Nature and Consequence 444 Author Index, 453 Subject Index, 457 Supplements: I Part of a Letter {1867) from Gregor Mendel to C. Ncigeli II Nobel Prize Lecture {1934) of Thomas Hunt Morgan III Nobel Prize Lecture {1946) of Hermann Joseph Muller IV Nobel Prize Lecture {1958) of George Wells Beadle V Nobel Prize Lecture {1958) of Edward Lawrie Tatum VI Nobel Prize Lecture {1959) of Arthur Kornberg VII Nobel Prize Lecture {1958) of Joshua Lederberg The essential feature of the operational viewpoint is that an object or phenomenon under experimental investigation cannot usefully be defined in terms of assumed properties beyond experimental determination but rather must be defined in terms of the actual operations that may be applied in dealing with it. . . . What is a gene in operational terms ? L. J. Stadler, "The Gene," Science, 120: 811-819, 1954 Must we geneticists become bacteriologists, physiological chemists and physicists, simultaneously with being zoologists and botanists? Let us hope so. H. J. MuLLER, "Variation Due to Change in the Individual Gene," American Naturalist, 56: 32-50, 1922 Chapter *1 GENETIC MATERIAL S' INCE human beings are curious, you surely have already noticed certain things about yourself. In the first place, you recognize yourself as being the same kind of creature as your par- ents. Your parents gave rise to you. another human — not to a cereal, a fish, or a bird. This is so even though the raw materials from which you were initially constructed and from which you subsequently grew were originally nonhuman. Let us, therefore, start by assuming the existence of some in- trinsic factor which determines that humans shall beget humans. We can call this inborn factor for the genesis of like from like the genetic factor. Since each kind or species of living thing, be it plant or animal, produces offspring of its own kind we can generalize and hypothesize that each species of organ- ism has such a built-in genetic factor. But we must now also admit that the genetic fac- tors for dog, for apple tree, and for man must all differ in some way in order to produce such different organisms as end products. You must have also noticed that, in respect to certain details, you are similar to and dif- ferent from your parents. What is the basis for this? You have already observed from common experience that the environment in which parents and children live can some- times be the cause of similarities and dif- ferences between them. Thus, if the caloric content of the diets of parents and children is similar they will weigh more nearly alike than if their caloric intake is different. Are all similarities and differences among human 1 beings produced by environment? Or, does the intrinsic genetic factor we have invented to be responsible for like begetting like play a role in the production of the detailed simi- larities and differences which we see upon comparing children with parents? This question may be answered after con- sidering the results of studying certain bean plants.' The particular kind of bean plant referred to reproduces as we do, sexually, a difference being that a single plant performs the functions both of male and of female parent. Assume, for the present, that the genetic factor is transmitted from the parent to the offspring, and that the transmitted factor must be the same type as that of the parent. Let us also assume that the genetic factor has a natural rather than a super- natural or spiritual basis. If the genetic factor has a natural basis it ought to have a material basis and have chemical and/or physical properties as have other material things. We are led, therefore, to postulate the exist- tence of genetic material. Let us now con- sider a particular bean seed. When the plant grown from this seed produces offspring bean seeds (Figure 1-1 A), we find that the offspring bean seeds vary from each other in size, some being very small, some small, and some medium. On our hypotheses these seeds must all have the same type of genetic material, genetic constitution, or genotype. The simplest explanation we can offer for the size differences between them is that this variation was caused by environmental dif- ferences which occurred during seed forma- tion. This idea can be tested by growing each of these seeds and scoring the size of seeds that they produce. When this is done it is found that each seed also produces off- spring bean seeds of very small, small, and medium sizes, regardless of the size of the parent seed itself. And this test can be made generation after generation with the same result. We can term such a line of descent, ^ Based upon W. Johannsen's experiments. CHAPTER 1 whose members carry the same genotype, a pure line. The expression of the genotype in traits or characters (bean size in our ex- ample) is called the phenotype. So environ- mental differences have caused the same genotype to produce a variety of phenotypes, and we conclude that the differences between the seeds of a pure line are environmentally produced and are not due to any differences in genotype. But consider next another particular bean seed, of this same species of bean, that gives rise to offspring beans (Figure 1-1 B) which are very large, large, and medium sized. Since each of these produces offspring beans which again show the same range of pheno- types we are clearly dealing with another and different pure line, within which phenotypic variability is attributable to environmental fluctuation. How can we explain the differences be- tween these two different pure lines, one of which can produce very small and small bean seeds while the other can produce very large and large ones? All the beans were grown under the same environmental conditions, so these phenotypic differences cannot be due to environmental differences; instead they must be due to genotypic differences. So we must conclude that the genetic material in these two pure lines is different. How can we explain the fact that some of the seeds in both of these genotypically dif- ferent pure lines are similar — medium sized? In this case different genotypes have produced the same phenotype through the action of the environment. What is the consequence of the fact al- ready mentioned that under similar environ- mental conditions the average size of the beans produced within a pure Hne remains the same regardless of the size of the specific beans planted? Thus in the pure line first described the offspring bean seeds have the same average size whether the very small or the medium seed is used as parent. Similarly the average size of seed produced in the second pure line is the same when either the medium or the very large seed is the parent. In other words selection for bean size within pure lines is futile, as would be expected on our hypothesis that all members of a pure line are genetically identical. Throughout the bean experiments de- scribed, effort was made to keep the envi- ronment the same. This does not mean that the environment did not vary, but that it varied approximately in the same directions and to the same degree for all individuals in the study. In this particular work it hap- pened that phenotypic variability due to the fluctuations of environment was not so great as to completely mask the phenotypic effect of a genetic difference. In any randomly chosen case, however, one cannot predict to what degree any particular phenotype will be influenced by the genotype and by the en- vironment. So theoretically both phenotypic similarities and phenotypic differences be- tween two individuals of the same species could result from each one of the following four combinations: 1. Identical genotypes in near-identical envi- ronments. 2. Different genotypes in near-identical envi- ronments. 3. Identical genotypes in different environ- ments. 4. Different genotypes in different environ- ments. Following are specific examples of how each combination can result in either pheno- typic difference or phenotypic similarity: I. Identical genotypes in near-identical en- vironments: Phenotypic difference — one small and one medium sized bean from the same pure line. Phenotypic similarity — two small sized beans from the same pure line. Genetic Material Typical ^ Offspring Typical ^ 0 0 Offspring /l\ /l\ /l\ PURE LINE I B C I /l\ /l\ /l\ • •• ••• ••• PURE LINE II /l\ /l\ /l\ ••i 1 ••• 4 V / \\ .1. / w 1 / / \ / 1 • • • **^ • NEW MUTANT PURE LINE OLD PURE LINE • •• J FIGURE 1- -1. Rehilive sizes of seeds oblmned j rom self fertilized bean plants CHAPTER 1 2. Different genotypes in near-identical en- vironments: Phenotypic difference — one small and one large bean from genetically different pure lines. Phenotypic similarity — two medium sized beans from genetically different pure lines. 3. Identical genotypes in different environ- ments: Phenotypic difference — one bean plant grown in the light is green while another grown in the dark is white, though both are from the same pure line. Phenotypic similarity — if two rabbits come from a certain pure line (geneti- cally black rabbits), both will have black coats even though one individual grew at high and the other individual grew at low temperatures. 4. Different genotypes in different environ- ments: Phenotypic difference — a rabbit from a genetically black line, grown in a cold climate, has black fur, while a rabbit from a Himalayan line, grown under temperate conditions, is Himalayan, i.e., white except for the extremities (paws, tail, snout, and ears) which are black (see Figure 1-2). Phenotypic similarity — a rabbit from a genetically black line grown at a mod- erate temperature and a rabbit from a genetically Himalayan line grown at a cold temperature are both black furred. The case of coat color in rabbits is instruc- tive in another respect. The rabbit that is genetically black will always produce a black coat no matter what the temperature, so long as it is not lethal. For this genotype there seems to be no range of pheno- typic expression with respect to temperature variations. In the Himalayan strain, how- ever, the situation is different, as already de- scribed in part. If grown at very high tem- peratures such rabbits would have coats that are entirely white. In this case the pheno- typic range of reaction, or norm of reaction. ■^H FIGURE 1-2. Male Siamese cat, grown under temperate conditions, slwwing tlie same pigmentation pattern as the Himalayan rabbit. {After C. E. Keeler and V. Cobb.) Genetic Material of the genotype is relatively great, varying with increasing temperature from completely black through the Himalayan pattern to completely white. We are now in a position to answer the question concerned with the basis of simi- larities and differences between children or between them and their parents. Extending the principles just described for beans and rabbits to all other kinds of organisms, in- cluding man, we conclude that not only is the genetic material in different species of organisms different, but it can differ from one organism to another in the same species. Phenotypic similarities between individuals may occur when they are carrying the same or different genotypes, and phenotypic dif- ferences between individuals may or may not be accompanied by genotypic differences. Having agreed that genetic variation exists within as well as between species, we may ask: how does genetic variation arise? If you breed a pure line of large beans for many generations you will find on rare occasions a very small bean which will give rise to offspring beans ranging from tiny to small, and which clearly make up a new, different, pure line (Figure 1-lC). What apparently has happened is that the genetic material in the pure line of large beans has somehow changed to another transmissible form which henceforth causes the production on the av- erage of very small beans. Such a change in the genotype that is transmitted to progeny may be said to be produced by the process of mutation, while the new type of individual may be called a mutant. Just as it is easy to ascribe differences be- tween dogs and cats to genetic differences, so it is often simple to tell that certain dif- ferences between lines of the same species have a genetic basis. There are many strains or breeds of pigeons, dogs, cattle, and of other domesticated animals each of which differs from the other in phenotype. That many of these differences are due to genetic differences has been established by the re- tention of these phenotypic differences even after the different breeds are grown genera- tion after generation in essentially identical environments. Revealed in this way, the genotypes within a species are of immense variety. We should keep this already present genetic variation in mind in seeking to learn something more about the nature of the genetic material. In order to learn more about the genetic material we should examine more closely the material things comprising organisms, par- ticularly those materials which are trans- mitted from parent to offspring. Most types of organisms are composed of usually micro- scopic building blocks called cells plus sub- stances which have been manufactured by cells. These organisms start life either as a single cell, or by the fusion of two cells into one, or as a group of nonfusing cells de- rived from their parents. In those cases where the new individual starts life as one or a group of nonfusing cells derived from a single parent, reproduction is said to be asexual, while in cases where two parents contribute cells, reproduction is said to be sexual. In sexual reproduction two cells, called gametes, fuse in the process of fertiliza- tion into one cell, called the zygote, which is the start of a new individual. In higher animals these gametes are called egg (female) and sperm (male), and the zygote the ferti- lized egg. In the bean plant, male and female gametes are produced in the same in- dividual which, as already mentioned, serves as both parents, and self-fertilization nor- mally occurs; in human beings the two kinds of gametes are produced in separate indi- viduals of different sex, so that cross-fertiliza- tion always occurs. When is this hypothetical inborn genetic material transferred? Is it transferred simul- taneously with the very inception of the new individual or is it transferred sometime after? Is the transfer accomplished only once, sev- CHAPTER 1 eral, many times, or continuously? We may get some answer by considering certain or- ganisms, composed of but a single cell, which reproduce asexually by dividing into two cells. In this process the parent becomes ex- tinct, so to speak, its individuality being re- placed by two daughter cells of the same kind. Once formed, the two daughters often separate, never to meet again. In such a case, at least, the genetic material, whatever it is, must have been transmitted before the com- pletion of cell division. .Accordingly, we should examine this process of cell division in some detail for clues concerning the physical basis of the genetic factor. The preceding reasoning has led us to postulate the existence of genetic material, which is transmissible and mutable (capable of mutation), and which together with the en- vironment determines phenotypes. But be- fore we examine cell division for additional evidence of some physical basis for the genetic material, let us consider some work which may provide us with more information with regard to the transmissive properties of the genetic material. SUMMARY AND CONCLUSIONS Organisms are assumed to contain an intrinsic genetic factor which is responsible for like reproducing like. This genetic factor is presumed to have a material basis. Accordingly, the genetic material must be different in different species of organisms and may be different in different lines or breeds of the same species. Variations in phenotype may be due to either or both genetic and environmental differences. The contribution of one of these two factors to phenotypic variability may be detected by avoiding variability in the other of these two factors. Genotypic differences arise by the process of mutation. The genetic material is presumably transmitted from parents to offspring by means of the cellular bridge between generations. REFERENCES Johannsen, W., 1909. Elemente der exakten Erblichkeitslehre. Jena. See also a translation of the summary and conclusions of his 1903 paper, "Hered- ity in Populations and Pure Lines," in Classic Papers in Genetics, Peters, J. A. (Ed.), Englewood Cliffs, N.J., Prentice- Hall, 1959, pp. 20-26. WiLHELM LUDWIG JOHANNSEN (1861-1926). (By permission of Genetics, Inc., vol. 8, p. 1, 1923.) Genetic Material Phenotypic variation among associates in the Department oj Zoology, Columbia Uni- versity, 1954. Phenotypic similarity is reflected in the interest of numbered individ- uals in genetical-cytological matters. 1. John A. Moore 2. Arthur W. Pollister 3. Howard Levene 4. Francis J. Ryan 5. Franz Schrader 6. LesUe C. Dunn 7. Theodosius Dobzhanksy ©(D(DO@000 0®00®0® QUESTIONS FOR DISCUSSION 1.1. Has the phenotype of one generation any effect upon the genotype of the next? Ex- plain. 1.2. What do you think of the thesis that the genotype is more important to organisms than is the environment? 1.3. Is the environment for two organisms ever identical? Explain. 1.4. What is meant by an operational definition? 1.5. Define the genetic factor. Have you given an operational or a nonoperational defi- nition? Explain. 1.6. When the same similarities or differences in phenotype can be produced by either the environment or the genotype, can one ever be sure which is the determining factor? Explain. 1.7. What evidence can you give to support the view that the genetic material is transmitted from parent to ofi'spring? Do you think this evidence constitutes conclusive proof of transmission? Explain. 1.8. What conclusions can you come to regarding the genetic factor in Himalayan rabbits and in Siamese cats? 1.9. Assume the genetic factor has a supernatural basis. Could we learn anything about it from the use of the scientific method of investigation? Explain. 1.10. Do you think human beings provide good material for the study of the genetic factor? Explain. 1.11. What size limitations can you give to the genetic material? 1.12. Is the existence of genetic material presumed or proven? Explain. Chapter *2 GENE SEGREGATION WE HAVE been led to postulate the existence of genetic ma- terial from observations re- garding the similarities and the differences in phenotypes which occur among offspring and between them and their parents. Hypo- thetically, this material is transmitted from generation to generation, and we can hope to learn more about it by studying the oc- currence of traits in lines of descent. Perhaps this procedure will reveal additional trans- mission properties of the genetic material. We shall, therefore, continue to study what we may call transmission genetics. There is a choice to be made at this point. We could investigate the genetic material either in lines reproducing asexually or in lines, like the beans already discussed, which reproduce sexually by self-fertilization. In both cases we would be dealing with pure lines. However, instead of taking either of those paths of investigation let us turn our attention to the study of the genetic material in organisms reproducing sexually by cross- fertilization. In the experimental work de- scribed henceforth, it can be assumed, unless stated to the contrary, that appropriate pre- caution has been taken to assure that the phenotypic similarities and differences de- scribed are genotypic in origin and are not the result of varying environmental condi- tions. It is possible to obtain different strains of a cross-fertilizing animal or plant which show phenotypic differences with respect to a given trait. For example, for the trait height one line might be short, the other tall; or for the trait color one line might be red and the other white. The question raised now is what will happen phenotypically in the offspring if two lines showing different alternatives for the same trait are crossed? Can we, by studying the results of the first and subse- quent generations, learn anything regarding the genetic material? Let us consider some specific experiments like this which can be performed with the garden pea,^ first with respect to what should be done and why it should be done. Then we can examine the results obtained and discuss what they reveal regarding the genetic material. The garden pea plant is a favorable or- ganism for these studies because it is simple and inexpensive to raise and the length of a generation is short enough to permit the study of a number of generations in succes- sion. Although garden peas are normally self-fertilizing, it is possible also to cross- fertilize them; in fact the experimenter can control all mating by simple and appropriate techniques. Moreover there exist a num- ber of strains differing phenotypically with regard to different traits. It is necessary, of course, to breed these strains by self-fertiliza- tion for several generations and to observe the phenotypes, to make sure that pure lines have indeed been obtained. Which pure lines should we cross together? Since we do not know what phenotype to expect in the offspring it would be unwise to use as parents two lines whose flowers differ, say, in shades of pink or whose seeds differ only in average size. For such traits might be subject to variation due to the action of the environment which could cause the phe- notypes in the two parental strains to overlap. This would confuse our deciding from the phenotypes what genotypes were present. So we should first select for use only strains which show a sharp, nonoverlapping, easily ^ Based upon G. Mendel's experiments. Gene Segregation detected, difference. Second, to avoid un- necessary complexity in following the results of the matings, we should use only strains having a single major difference. Third, we should use only lines which can be successfully cross-fertilized in both di- rections; that is, where matings can be made reciprocally — the line furnishing the male gamete in some crosses with a second line also provides the female gametes in other crosses with this second line. This is a de- sirable step whose purpose is to determine whether it makes any difference upon which parental line the offspring start their develop- ment (as pea seeds formed on the maternal parent). Fourth, all crosses should be fully fertile; that is, the parental lines should be hardy plants growing vigorously and producing full sets of seed capable of growing to maturity, not only when self-fertilized but when crossed to each other reciprocally. If this precaution is not taken it is possible that insuflficient numbers of offspring will be obtained or, more important, that the offspring observed will be an incomplete sample of those whose development started. Deaths that occur be- tween the time of fertilization and the time that we make our observations regarding the phenotype of the offspring may lead to serious bias. Differential viability for different geno- types could cause us to miss, or underestimate the frequency of, certain phenotypes; this would give us misleading results with regard to genotypes, especially on the view that the genetic material is transmitted at the time the new organism starts its existence, i.e., at the time of fertilization. Two strains of garden pea, one producing colored flowers and the other colorless flow- ers, satisfy the prerequisites discussed. The breeding procedure followed and the ob- servations made are now described, accurate records of parental and offspring phenotypes having been kept, of course. Cross-fertilizations were made reciprocally between pure line colored flowers and pure line colorless flowers, these individuals serv- ing as the parents of the first generation (Pi). The offspring seeds were planted and the color of their flowers scored. All these off- spring, which comprise what we may call the first filial generation (Fi), were phenotypically uniform, having colored flowers just like one of the Pi. The Fi results were the same for the reciprocal matings. In the discussion which follows in this Chapter and subsequent ones, it will be correct to assume that all crosses were made reciprocally and produced identical results, unless a statement to the con- trary is made. What can we conclude about the genetic material from these results? Let us use sym- bols as a shorthand method of representing the genetic material — C for the genetic ma- terial whose effect produces colored flowers, present in all members of the colored flowered pure line, and c for the genetic material producing colorless flowers, present in all the colorless flowered pure line individuals. All Fi individuals must contain C since they pro- duce colored flowers. What has happened to c? Has it failed to be transmitted? We may learn more by permitting the Fi colored to serve as P2 (second parental gen- eration) and reproduce by self-fertilization to yield F2 progeny. When this is done, and large enough numbers of F2 are obtained from each P2 plant, it is found that among the offspring of every P2 some are colored and some are white. In terms of genetic material, then, these F2 must carry, respec- tively, C or c. It is no surprise that some F2 contain C, but where did the c come from which is necessary for colorless F2? One could at first suppose that in these cases c either arose spontaneously from some non- genetic origin or that C mutated to c. We can bypass the first possible explanation by assuming that genetic material can arise only from pre-existing genetic material and that this material is self-reproducing {self-replicating). 10 CHAPTER 2 The second explanation can be eliminated by the fact that in the pure line containing C, mutations to c are found to be thousands of times more rare than the occurrence of c among the Fo. So, if the P2 (Fi) were geno- typically like pure line C individuals, as we have assumed, mutation could not be the explanation for the difference in breeding behavior between Pi C and P2 C. In the absence of a simpler explanation, we are faced with the necessity of postulating that the genetic material is not always com- posed of a single indivisible unit. The appear- ance of c in F2 can be explained by making the more complex assumption that each P2 (Fi) contains not only C but c as well; in other words, that in some individuals the genetic material contains two units. Let us use the word gene to refer to a unit or restricted portion of the genetic material. But, if we assume that there is a pair of genes in the P2, we shall have to apply this rule to all other individuals in our experiment. For, in sci- ence, we obey the law of parsimony {Occam's rule) which states that we must not multiply hypotheses or assumptions needlessly. So, instead of having some individuals with paired genes and others with these singly, we shall require all to have a pair of genes in their genetic material. Accordingly, the two pure lines and the Pi must have been CC and cc, and all Fi must have been Cc. Those F2 which are colorless must be cc. Now your attention is called to the indi- viduals in F2 that are cc. These have color- less flowers that are phenotypically identical with those of the original pure line of color- less used in the Pi. And, in fact, crosses of F2 colorless individuals either with themselves or with any other colorless individual (F2, or pure line) produce all colorless progeny. In other words, F2 cc individuals are geno- typically just as pure with respect to the trait under consideration as are pure line indi- viduals. This is true despite the fact that both c's in the F2 had been carried in Fi individuals where C was the other member of the pair of genes. We conclude, therefore, that when c is transmitted to the F2 it is uncontaminated, or untainted, by having been in the presence of C in the Fi even though it had not been expressed in any noticeable way in the pheno- type of those individuals. We can generalize this conclusion and state that the nature and transmission of any gene is uninfluenced by whatever its partner gene (allele) may be. Since each P2 produced colored and white F2 offspring, each P2 had the genotype Cc composed necessarily of C from the CC Pi and c from the cc Pi. This specifies that one and only one member of a pair of genes in a parent is transmitted to each individual off- spring, so that in the transmission process the members of a parental pair of genes must become separated, or segregated, from each other. The paired, or diploid, condition of the genes, then, becomes an unpaired, single, or haploid (monoploid) one during transmis- sion, but diploidy is restored in the offspring because a haploid genotype is contributed to it by each parent. Accepting the hypothesis that paired genes are segregated at the time they are transmitted to progeny, are the two alleles in a parent equally likely to be transmitted to offspring? We already know, from the F2 produced by self-fertilization of Fi Cc, that both genes of a given individual are transmissible. Let us test the hypothesis that both members of a pair of alleles are equally transmissible. If so, then, the male parent (or part) would con- tribute C half the time and c half the time; similarly 50% of the time C and 50% of the time c would be contributed by the female parent (or part). Finally, assume diploidy is restored at random; that is, the haploid gene from one parent enters an offspring without regard to the haploid gene contrib- uted by the other parent. Accordingly, an offspring which receives C from the female (50% of offspring) will have an equal chance of receiving C or c from the male, so that of Gene Segregation 11 all offspring 25% will be CC and 25% Co. Those offspring receiving c from the female (50% of offspring) will have an equal chance of receiving C or c from the male, so that the contribution to all the offspring genotypes will be 25% Co and 25% cc from this source. On this basis the F2 would be predicted to contain 25% of individuals that are CC, 50% that are Cc, and 25% cc. This expectation can be expressed as relative frequencies in several ways: K CC : ji Cc : ji cc, or \ CC : 2 Cc : I cc, or .25 CC : .50 Cc : .25 cc. As already reasoned CC and Cc are pheno- typically indistinguishable, having colored flowers, so that phenotypically 75% of the F2 would be colored and 25% would be color- less. What is their relative frequency in the Fo actually observed? Although a penny has in theory a 50% chance of falling head up and a 50% chance of falling tail up, you realize that a sufficiently large number of tosses is required to actually obtain approximately 50% heads, 50% tails. So, in the present case, an accurate test of the theoretical expectation of 75% colored and 25% colorless will be obtained only if a sufficiently large sample of offspring is scored. Accordingly, instead of scoring just the off- spring of one P2 we shall total the results for the offspring of all P2. And when this is done, it turns out that the actual results (among 929 plants, 75.9% were colored and 24.1% colorless) are very close to expectation. It should be emphasized that obtaining or not obtaining the phenotypic ratio % colored to K colorless is a critical test neither for genes being paired, nor of their untaintability, nor of their segregation — these properties of genes having been previously established on other grounds. The ratio merely tests the ideas that segregation of paired uncontaminable genes results in an equal chance for offspring to receive either haploid product of segrega- tion from a parent and that the haploid prod- ucts from different parents come together at random to restore the diploid condition. If all the assumptions so far made are correct, the 75% of F2 which are colored should be of two genotypes, % CC, breeding like pure line CC individuals, and % breed- ing like the Fi Cc individuals. Accordingly, each F2 colored plant is permitted to self- fertilize and, in fact, very nearly % produce only colored F3 whereas % produce F3 of both colored and colorless types. The theo- retical genotypic ratio expected in the F2, % CC : H Cc : % cc, is, in this way, fully confirmed in experience. The gene model we have proposed to explain these pheno- typic results is summarized in Figure 2-1. It is convenient to introduce two additional terms at this time. A homozygote is an indi- vidual that is pure with respect to the genes in question, like CC or cc, while a hetero- zygote, or hybrid, is impure in this respect, like Cc. An independent test of all the hypotheses presented in this Chapter can be made in the following way. Fi colored plants are crossed to colorless plants, this cross being symbolized genetically: Fi Cc X cc. As the result of segregation half of the off- spring should receive C and half c from the Cc parent, and all should receive a c from the cc parent. So, the genotypes of the off- spring from this cross should be, theoret- ally, Cc 50% of the time and cc 50% of the time, and the expected phenotypic ratio should be, then, )i colored : ){ colorless. This expectation is actually observed (85 colored : 81 colorless). The next question we may ask is, are the principles we have established generally applicable? Thus far they apply strictly only to the genie determination of flower color in garden peas. Now it is possible to test all these ideas six additional times, using six other traits each of which occurs in two clear-cut alternatives and fulfills the pre- requisites for suitability already described. In each case, when two appropriate pure lines were crossed the Fi hybrids produced 12 CHAPTER 2 FIGURE 2-1. Genotypic model pro- posed to explain the phenotypic results of certain crosses involving colored and colorless flowered pea plants. CC X cc (Cross -fertilization) I I all C all c (Gametes) all Cc p> Cc X Cc (Self-fertilization of F, ) /\ /\ G. Vn C, 72 c Vi C, Vzc f. Male gametes 72 C 72 c Female '/» C gametfes y^ ^ '/4 CC 74 Cc V4 Cc 74 cc or 74 CC 72 Cc 74 cc P3 like like breeds like P,CC if^^ , P,« O^ were phenotypically uniform, as before. Moreover, self-fertilization of the Fi gave F2 which were proven to occur in the expected 1:2:1 genotypic ratio. Recall that the Cc phenotype is indis- tinguishable from CC. In Cc individuals the phenotypic expression of c is masked by the expression of C. The ability of a gene to express itself phenotypically in the presence of a different allele is described in terms of dominance. In the present case C is said to be the dominant gene when present with c, which is called, accordingly, recessive. It should also be noted particularly that the concept of the gene so far developed has no dependency upon the occurrence or non- occurrence of dominance. Indeed, testing our postulates has been made more compli- cated by the fact that C is, for all intents and purposes, completely dominant to c, since our Fi Cc showed only the expression of C and the presence of c was detected only upon breeding Fi individuals. Also, only upon further breeding of colored F2 were we able to determine that )i. were CC and 73 Cc. Dominance, then, refers to the phenotypic Gene Segregatic 13 expression of genes in diploid condition and has no relation to their mechanism of trans- mission. It turned out, for the six other traits used to test the general applicability of our gene concept, that in each case one allele was dominant to the other alternative one in the hybrid. From this, one might be led to con- clude that dominance is a universal phenome- non since it was found in each of seven differ- ent studies on the occurrence and transmis- sion of traits based on genes in the garden pea. However, before making this decision examine the results of breeding certain chickens. Here black X white produces blue- gray Fi. Mating two blue-gray Fi produces in Fo /4 black, ]n blue-gray, and K white. You see in this case that dominance does not occur at all, so that dominance is not a rule for the phenotypic expression of alleles in hetero- zygotes. Note that when dominance is ab- sent, genotypes can be written with certainty from a knowledge of phenotypes. It should be realized that it was cross- fertilization that made it possible to show that genes occur as pairs, which after segre- gation become unpaired, then recombine to form pairs in the offspring. In other words, the view that the genetic material contains separable paired units is based upon the re- combination which these units undergo in cross-fertihzation. At this point we ought to consider the meaning of the term genetic recombination. You will agree that the genetic units themselves are not required to undergo novel changes (mutations) when undergoing recombination. That is, the types of genes present in a genetically recombinant individual had already existed before re- combination. Given an individual whose gene pair is AA\ segregation followed by self- fertilization may produce AA' again. But, this genotype is not considered a genetic recombination, but rather a reconstitution of the original arrangement of the units. However, the self-fertilization under discus- sion may also produce AA or A' A' . These represent two new genetic combinations, and are considered to be genetic recombinations. Accordingly, when events lead to the pro- duction of "old" combinations and "new" combinations of genes, it is only the latter type of grouping which is called genetic re- combination. This usage is reasonable in view of the importance that new combinations have in our understanding of genetic material (we were able to derive the principle of segre- gation only because new combinations of genes were produced by sexuality). Accord- ingly, genetic recombination should be identi- fied with the reassortment or regrouping of genes as a consequence of which new arrange- ments of them are produced. Any process that has the potential of producing new arrangements of genetic units is, therefore, a mechanism for genetic recombination. The phenotypic results of the experiments discussed in this and the preceding Chapter have led us to hypothesize the existence of genetic material which is self-replicating, mutable, and transmissible. The pea plant experiments reveal that the genetic material can be partitioned into a pair of units by means of the operation or technique of genetic recombination. There may be techniques or operations other than recombination which may be employed to study the nature of the genetic material. Should these different operations reveal that the total genetic ma- terial is divisible into smaller units, this would not necessarily imply an equivalence among the units. Thus, to use a nongenetic analogy, a book (equivalent to the total genetic ma- terial) can be partitioned operationally in terms of chapters, pages, paragraphs, words, letters, illustrations, and so forth. Each operation reveals something about the book, but the different units by which it is described are necessarily neither identical nor mutually inclusive. The present Chapter has revealed that the genetic material contains a pair of genes. 14 CHAPTER 2 But you should recognize that the properties of these genes must be expressed in terms of recombination, the operation by which they were detected. It is conceivable that other operations will also partition the genetic material into units, whose characteristics will have to be expressed in terms of the opera- tions employed for their detection. However, until it is found that the different units re- vealed by different operations are not equiva- lent, we shall use the term gene to refer to any unit of the genetic material, regardless of the operation by which the unit was discovered. In most of the genetic hterature to be re- ferred to subsequently, as well as in the greater portion of this book, it is usually simple for the reader to determine from the context of the discussion which operations are involved when the term gene is used. The possibility exists that the genetic ma- terial may be shown, via the study of genetic recombination, to contain more than two genes, in which case the maximum size of a gene would be reduced. Henceforth, we will be interested in any attempts to learn to what extent the genetic material can be par- titioned into genes, for such work leads us to a better understanding of the nature of the genetic material in terms of its recombina- tional properties. Remember that near the beginning of this Chapter we chose to inves- tigate the genetic material from its recombina- tional properties, and have postponed the study of the nature and consequence of the genetic material as revealed by other, non- recombinational, techniques or operations. SUMMARY AND CONCLUSIONS Genetic material is assumed to be self-replicating and to arise only from pre-existing genetic material. The term gene is used to refer to a unit or restricted portion of the total genetic ma- terial as discovered via any operational procedure. The genes discovered in the present Chapter were revealed by recombination. Genes occur in pairs. When they are transmitted in sexual reproduction the members of a pair segregate so that any offspring receives only one member of a pair from each parent. The gene is uncontaminated by the type of gene that is its allele prior to segregation, and enters the new individual uninfluenced by the type of gene being contributed from the other parent. REFERENCES Mendel, G., 1866. "Experiments in Plant Hybridization," translated in Sinnott, E. W., L. C. Dunn, and Th. Dobzhansky, Principles of Genetics, 5th Ed., New York, McGraw-Hill, 1958, pp. 419-443; also in Dodson, E. O., Genetics, the Modern Science of Heredity, Philadelphia, Saunders, 1956, pp. 285 311; also in Classic Papers in Genetics, Peters, J. A. (Ed.), Englewood Cliffs, N.J., Prentice-Hall, 1959, pp. 1-20. The Birth of Genetics, Supplement to Genetics, 35, No. 5, Part 2, 1950, 47 pp. Contains English translation of G. Mendel's letters to C. Nageli (1866-1873), and papers by H. De Vries, by C. Correns, and by E. Tschermak published in 1900. QUESTIONS FOR DISCUSSION 2.1. How would you recognize that a line of garden peas had become genotypically pure for a given trait? 2.2. Criticize the assumption that genes come only from pre-existing genes and do not arise de novo. Gene Segregation 15 2.3. Does a parent lose its genetic material when it is transmitted to progeny? Defend your answer. 2.4. Is it necessary to assume that genes are able to reproduce themselves? Explain. 2.5. List all the assumptions required to explain a 3 : 1 ratio in Fo on a genetic basis. 2.6. A black and a white guinea pig are mated and produce all black offspring. Two such offspring when mated produce mostly black but some white progeny. Explain these results genetically. 2.7. A cross of two pink flowered plants produces offspring whose flowers are either red, pink, or white. Defining your genetic symbols, give all the different kinds of genotypes involved and the phenotypes they represent. 2.8. What operation was employed in studying the gene in the present Chapter? Define a gene in terms of size. 2.9. Discuss the role of dominance in the study of genes. 2.10. Do organisms that reproduce asexually have genes? 2.11. What relation has a gene to the phenotypic effect with which it is associated? 2.12. Do you agree with the statement on p. 10 that a cross between two colorless pea plants results in "all colorless progeny"? Why? 2.13. Throughout this book the use of the word "heredity" and its derivatives has been avoided. Why do you think this is, or is not, justified? Chapter *3 MITOSIS I: "n looking for the biological basis for the transmission of genetic -material from parents to progeny your attention has been called (p. 5) to the cellular bridge between generations. Re- member that it is only via this bridge that genie transmission may take place, at least in single-celled organisms for whom cell divi- sion is equivalent to reproduction. More- over, all cellular organisms are remarkably similar in the way that they accomplish cell division. To initiate our present search for the material basis of genes let us examine briefly certain general features of cell structure and the appearance of cells undergoing division, as seen under the microscope. There are two major parts of the cell, a peripheral portion called the cytosome, con- taining substances making up cytoplasm, and a more central portion called the nucleus, containing nucleoplasm. In the final stages of cell division in higher plants, the cytoplasm is divided by the formation of a cell plate, which starts internally and proceeds toward the periphery until the separation into two daughter cells is complete. In the case of animal cells, a furrow starts peripherally and proceeds inward, until the parent cell is cleaved into two. The degree to which the two daughter cells are identical with re- spect to cytoplasmic components depends upon the position of the cell plate or furrow in the parent cell. In some cases these occur in the middle of the cell, but in many other cases they are located off-center, so that the two daughter cells contain very different 16 amounts of cytoplasm. Although cytoplas- mic components often may be distributed unequally between daughter cells, this is not true for the nuclear contents. Nuclear divi- sion usually directly precedes cytosomal division. But the nucleus does not simply separate into two parts by forming a furrow or cell plate. Instead of simply and directly separating into two parts, the nucleus un- dergoes a remarkable series of preparatory activities before it divides; this process of indirect nuclear division is called mitosis. Even though a nucleus shows no visible evidence that it is going to undergo mitosis, it is known to be very active chemically. In appearance (Figure 3-1 A), it is bounded by a nuclear membrane and is filled by a more or less homogeneous-appearing ground sub- stance in which one or more small bodies called nucleoli are located. The first indication that the nucleus is going to divide is the appearance in its ground substance of a mass of separate fibers (Figure 3-1 B), some of which seem to be associated with the nucleoli. These fibers are called chromosomes. This appearance marks the start of the first phase of mitosis, or prophase. Careful cytological observation reveals that each chromosome is in turn composed of two delicate threads irregularly coiled about each other. Each of the paired threads within a chromosome is called a chromatid. As pro- phase continues the chromatids within each chromosome become shorter and thicker and untwist from each other (Figure 3-lC). The nucleoli become smaller and it is believed that some of the material incorporated to thicken the chromatids is derived from the nucleoli. By the end of prophase (Figure 3-1 D) the nucleoh and nuclear membrane have disappeared and the chromatids have formed thick rods which begin to move ac- tively for the first time. Active motility is not the property of the entire chromosome, but is restricted to a particular region of it called the centromere. Mitosis 17 18 CHAPTER 3 This movement of centromeres occurs in a particular direction relative to a structure called the spindle which has been in the process of formation throughout prophase. When completed, the spindle has an appear- ance similar to what you will see upon spread- ing your fingers and touching corresponding fingertips together. Your wrists serve as the poles of the spindle and your fingers as spindle fibers. The chromosomes migrate from what- ever position in the spindle region they may have, so that their centromeres come to lie in a single plane perpendicular to the axis between the poles, that is, at the equator of the spindle, which is represented by the plane formed where your fingertips touch. Now the rest of the chromosome, being pas- sive, can be in any plane in the spindle. Once all the centromeres have arrived at the equa- torial plane of the spindle, mitosis has reached the middle phase, or metaphase (Figure 3-1 E). Until now the chromatids of a chromosome are still attached to each other at or near the centromere, although elsewhere they are largely free. They next also separate at the centromeres, the two centromeres suddenly moving apart, one going toward one pole of the spindle, the other toward the other pole, with the rest of each chromatid, now called a chromosome, being passively dragged along. This stage, in which the chromatids separate, move toward and arrive at the poles as chro- mosomes, is called anaphase (Figure 3-1 F). Once the chromosomes are at the poles, the last stage, or telophase (Figure 3-lG), occurs, in which the events that follow ap- pear to be the reverse of those that happened in prophase. Specifically, the spindle disin- tegrates, a new nuclear membrane is formed around the chromosomes, and nucleoli re- appear containing material probably drained away from the chromosomes. The chromo- somes become thinner and longer and then can be seen to consist of two delicate threads (chromatids) wound one about the other. Finally, as the chromosomes lose their visible identity the nucleus enters the intenniiotic, interphase, or metabolic stage. A general view of mitosis in the onion root tip is shown in Figure 3-2. FIGURE 3-2. Mitosis in t/ie onion root tip — general view. {Courtesy of R. E. Cleland.) In reading this generalized account of the mitotic phases, you may have gained the impression that, in one respect, it was either incomplete or misleading. For it was stated that the prophase chromosome is composed of two chromatids, that metaphase puts these into position for separation at anaphase, and that once separated their newly attained in- dividuality is recognized by calling them chromosomes. But chromosomes were de- fined as containing two visible threads! The question rightly asked is, does the anaphase chromosome contain the two threads or chromatids that are seen at telophase? This would be true if each chromatid somehow visibly reproduced itself between the time it was seen relatively uncoiled at prophase Mitosis 19 and the next time it was seen relatively un- coiled, at telophase. Note that we have been discussing the replication of chromatids as detected by microscopic observation. We can also study chromosome and chromatid replication using other operations. Accord- ingly, let us consider some evidence regarding chromosome replication at the chemical level, in the hope that this will help us under- stand its replication at the visible level. Chromosomes ("colored bodies") are unique in the cell since they are the only objects which stain purple by a procedure called the Feulgen-Rossenbeck technique. It is possible to measure the amount of chromosomal material by the amount of purple stain taken up by the chromosomes. It is found that the amount of chromosomal material does not change between prophase and telophase, but doubles over a period of hours during the intermitotic stage. At the start of prophase, therefore, each chromo- some has already replicated chemically. At the visible level, however, this is not yet appar- ent, so that each of the two visible chromatids in a chromosome also contains the chemical materials for an identical chromatid which is still invisible under the microscope. This new material is submicroscopic either because it has not yet assumed a proper chromatid form or has done so but is so tightly paired with its sister chromatid that together they appear as one strand. However, before the next occasion when unwound threads can be seen, that is, at the telophase of the same mitosis, this replication at the visible level has already been accomplished. So, the sub- microscopic chemical reproduction which takes place in a given interphase stage is not visible in chromatid form until the next telophase. What are the consequences of mitosis? Speaking in terms of visible structures, the chromosomal content of the parent nucleus has become repeated in each daughter nu- cleus, so that the subsequent division of the cytoplasm produces daughter cells whose chromosomal composition is identical to each other and to the parent cell from which they were derived. The cells of different species are different in that either they have different numbers of chromosomes per nu- cleus or the chromosomes differ in appear- ance, or both. Different chromosomes may differ from each other in their size, their stainability with various dyes, and in the location of their centromeres. Almost all chromosomes have a subterminal centromere which separates the chromosome into two parts (arms); all chromosomes are linearly arranged and unbranched. When very care- ful analyses are made, any two species can be shown to differ in their chromosome com- plements. Examination at metaphase of the kinds of chromosomes within the nucleus of sexually reproducing organisms typically shows that for each chromosome which arrives at the equatorial plane there is another chromosome very similar or identical to it in appearance which also takes a position independently in this plane. Chromosomes thus occur as pairs, and the members of a pair are called Iwmolo- gous c/iromosomes, or homologs, whereas chromosomes of different pairs are non- homologous, or non homologs. It should be repeated that the members of a pair of homo- logs assume their position at mitotic meta- phase independently of each other. The number of chromosomes seen in typical mitosis of the garden pea is 14 (2N, or the diploid number of chromosomes) or 7 pairs; in Indian corn (maize) there are 10 pairs, in human beings 23. Whatever number of chromosomes is present in the zygote, then, other things being equal, the same number of chromosomes will be found in every cell of a multicellular organism descended from the zygote by cell divisions in which mitosis has occurred. From the information presented, we are now in a position to hypothesize what may be the 20 CHAPTER 3 material basis for genes. While it is possible that we may find other kinds of genes, using other criteria or operations, the kind that we have identified by recombination possesses certain specific properties including a very regular mode of transmission (Chapter 2). One regular cellular component transmitted by all cells to daughter cells is the chromo- somes. These have several properties of interest with respect to genes: chromosomes reproduce themselves and are transmitted equally to the daughter cells so that these are identical, in this respect, to each other and to their parent cell. We shall make the reasonable assumptions that genes arise only by a process which in- volves the replication of pre-existing genes, and also that diff"erent alleles arise only from each other by mutation, that is, by a gene changing to an alternative form of that gene v/hich in turn is involved in reproducing the alternative form until it mutates. It is found that chromosomes may occasionally become visibly altered in certain ways. In these cases all chromosomes mitotically de- rived from such a modified chromosome have exactly the same alteration. Therefore, both genes and chromosomes are capable of mu- tation and are subsequently involved in replicating their new form. Another property of the gene is the reten- tion of its individuality regardless of the nature of its allelic gene. One indirect evi- dence has already been cited for believing this is true also for the chromosomes. This is the independent way that each chromo- some arrives at metaphase. It might be thought, when the chromosomes "disappear" during interphase, that their individuality is lost and even that their contents are dispersed. Evidence that the nuclear material is not dis- persed into the cytoplasm between mitoses al- ready has been presented in the retention of the full amount of chromosomal material within the nucleus during interphase, insofar as revealed by the Feulgen-Rossenbeck chromosome-staining procedure. Neverthe- less, it is possible that those components of chromosomes which remain intranuclear may become scrambled during interphase and later resynthesize their proper form during the next prophase. Four lines of evidence can be mentioned bearing on this question. The first three come from studying the appearance of successive mitoses. It is possible to observe the positions of chromo- somes at late anaphase or telophase and again observe the position of the chromo- somes as they enter the next prophase. When this is done it is found that the chromosomes have held the same relative positions, as ex- pected had they retained their integrity dur- ing interphase. Second, since the nucleolar material does not disperse during interphase, those parts of the chromosomes with which the nucleolus is associated probably remain associated during that interval. Third, it sometimes happens that two originally identi- cal homologs are modified by mutation so that one is changed in one respect and the other is changed in a diff'erent respect. The finding that, mitosis after mitosis, these two homologs retain their separate diff'erences is evidence that each homolog, like each allele, has retained its individuality cell generation after cell generation. Finally, there is more direct evidence on the retention of chromo- somal individuality during interphase from cells of the larval salivary glands of certain fruit flies. For these cells have interphase nuclei, and it is possible, because of their giant size, to squash them open and show that they contain the correct number of relatively uncoiled chromosomes linearly identical to the chromosomes seen during mitosis. You may already be impressed with the points of similarity between genes and chro- mosomes. However, your attention is called to what is an apparent disparity in behavior between the two. If all nuclei divide by mitosis, then a gamete should contain the same number of chromosomes as the other Mitosis 21 cells derived from the original zygote, and number. Instead each contains a complete, since the zygote of any generation combines unpaired, haploid, set of chromosomes. The two gametes, the number of chromosomes zygote therefore has the diploid chromosome should increase in the zygotes of successive constitution restored because each gamete generations. Yet we know that paired genes furnishes a haploid set of chromosomes, one in one generation remain paired genes in the set contributed by the sperm from the father, next sexual generation, so that their diploid and another set by the egg from the mother, condition is maintained generation after In this way chromosomes remain as pairs, generation via segregation and fertilization. sexual generation after sexual generation. We are led to assume, therefore, that some- and the number of chromosomes remains thing paralleling segregation of genes must unchanged. Clearly, then, the cell divisions take place for chromosomes. This is reason- preceding gamete formation cannot be invari- able in view of the statement already made ably mitotic, but must involve at some point that all individuals of a species have a char- a special mechanism for reducing chromo- acteristic chromosomal composition. In some number. The nature of this special actual fact, as expected, the gametes do not kind of nuclear behavior is investigated in contain the paired, diploid, chromosome the next Chapter. SUMMARY AND CONCLUSIONS Studies of cell division in which nuclei divide mitotically reveal that, of all cellular com- ponents, the chromosomes are the structures most likely to serve as the material basis for genes. This hypothesis receives support from several of the properties of chromosomes which parallel established or assumed properties of genes. Chromosomes come only from pre-existing chromosomes; chromosomes can mutate on occasion, the mutant chromosome then replicating the mutant form; different species have different chromosomal composi- tions; the chromosome content is identical both quantitatively and qualitatively in each cell of a line produced by asexual reproduction; chromosomes are unpaired in gametes and paired in zygotes; each chromosome retains its individuality, mitotic cell generation after mitotic cell generation, regardless of the nature of its homologous chromosome. REFERENCES Flemming, W., 1 879. "Contributions to the Knowledge of the Cell and its Life Phenomena," as abridged and translated in Great Experiments in Biology, Gabriel, M. L., and S. Fogel (Eds.), Englewood Cliffs, N.J., Prentice-Hall, 1955,'pp. 240-245. Schrader, F., Mitosis: the Movement ofCliromosomes in Cell Division, New York, Columbia University, 1953. Scientific American, Sept. 1961, Vol. 205, No. 3, "The Living Cell," articles by J. Brachet and by D. Mazia. Swanson, C. P., Cytology and Cytogenetics, Englewood Cliffs, N.J., Prentice-Hall, 1957. QUESTIONS FOR DISCUSSION 3.1. What are the consequences of mitosis? 3.2. Including those properties of chromosomes listed in the Summary and Conclusions, state the corresponding gene property and whether it is one which is proved or as- sumed. Give evidence or reasons for accepting or rejecting these as genie properties. 22 CHAPTER 3 3.3. If the chromosomes serve as the material basis for genes, each cell of the body derived by mitosis should carry the same genotype. Using a multicellular plant, describe how you would test this idea. 3.4. What are the advantages or disadvantages of chromosome coiling? 3.5. Can you imagine a spindle which is too small for normal cell division? Explain. 3.6. Suppose certain nuclei normally do not divide with the aid of a spindle. In what respect would this affect your ideas about genes? 3.7. Is it scientifically correct to assume at this point that; (1) all genes recombine, (2) chromosomes are equal to genes? Explain. 3.8. Discuss the statement that all cell divisions are normally mitotic. 3.9. Differentiate between replication of chromatids and of chromosomal material. 3.10. List the events that must take place before a given telophase chromosome can give rise to a chromosome made entirely of chromosomal material not yet synthesized. Chapter *4 MEIOSIS B; Y WHAT kind of process do both male and female gametes 'come to contain only one set of chromosomes, composed of one member of each pair of chromosomes found in the nucleus of an ordinary body, or somatic, cell? Gametes cannot be produced by regular mitotic division or they would be diploid. The reduction from two sets to one has been found to be brought about by another type of indirect nuclear process, called meiosis, which actually requires two successive nuclear divisions to accomplish this result. Since the diploid number of chromosomes is maintained generation after generation in sexually reproducing forms, it is not surpris- ing that meiosis always occurs at some time in the life cycle of each sexually reproducing individual. In most animals meiosis com- prises the last two nuclear divisions before the mature sperm or egg is produced. Meiosis occurs at different times in the life history of different plants, but almost never immedi- ately before the formation of gametes. There are minor variations in different species in the details with which meiosis is carried out; what is presented here is a generalized account of its main features. In order that the cytological description of the meiotic process may be more meaningful to you, several assumptions will be made. Suppose that the processes which direct the nucleus to divide act especially early in the case of meiosis, before the chromosomes have attained the degree of coiling first seen in mitotic prophase. Suppose further that a 23 relatively more uncoiled state of the chromo- some is, under these conditions, associated with an especially strong attraction of like chromosome parts for Uke parts and that this attractive force extends over considerable, though still microscopic, distances. Then, with one additional novelty which will be described later, the meiotic process will occur in a predictable way when the chromosomes undergo in sucession two divisions of a mi- totic nature. In prophase of the first meiotic division, just as in the case of mitotic prophase, each chromosome would contain two chromatids plus an equal amount of chromosomal ma- terial not yet visible as chromatids (Chapter 3). But, now, because of the early onset of nuclear division, homologous chromosomes would pair point for corresponding point (making a bundle of four chromatids plus an equal amount of future chromatid material). Once paired, the chromosomes would pro- ceed as pairs to the equator of the spindle for the metaphase. (Recall that in mitosis, on the other hand, each chromosome of the two sets present behaves independently of its homolo- gous chromosome in going to the spindle's equator.) At anaphase the members of a pair would separate and go to opposite poles (note each anaphase chromosome would still contain two chromatids plus an equivalent amount of future chromatid material). In the interphase which follows the first telo- phase, no synthesis of future chromatid ma- terial would take place since what was made in the previous interphase had not been used to make visible chromatids in the first meiotic division. So, the second meiotic division could now take place at any time and proceed as a typical mitosis. In the second meiotic prophase each chromosome would contain two chromatids and two future chromatids. Each chromosome would proceed to meta- phase independently; at anaphase the two chromatids would separate and go to oppo- site poles of the spindle (once separated the 24 CHAPTER 4 chromatids may be called chromosomes, as mentioned in the previous Chapter). By telophase the future chromatid would become visible so that each telophase chromosome contains two chromatids. While mitosis always involves chromosome duplication and separation alternately, in meiosis one duplication is followed by two separations. The result is the maintenance of the diploid chromosome condition in mitosis, but a reduction from the diploid to the haploid (monoploid) condition when meiosis is completed. Let us now examine the actual meiotic process in some detail (Figure 4-1), after which a more complete discussion will be made of specific cytological phenomena and their genetic implications. Prophase of the first meiotic division {prophase I) is of long duration, as compared to mitotic prophase, and is divided into several substages each of which has its own distinguishing character- istics. 1. As they emerge from the intermitotic phase the chromosomes are long and thin, more so than in the earliest prophase of mitosis. This is the leptonema (thin thread) stage of prophase I. 2. Next the thin threads pair with each other in a process called synapsis. This pair- ing is very exact, being not merely between homologous chromosomes, but between ex- actly corresponding individual points of the homologs. Synapsis proceeds zipperwise until the two homologs are completely apposed. This is the zygonema (joining thread) stage. 3. The apposition of homologs becomes so tight that it is difficult to identify two separate chromosomes {pachynema, thick thread, stage) (Figure 4-2 A). 4. Following this, the tight pairing of the pachynema is relaxed and then it can be clearly seen that each pair of synapsed chromosomes contains four threads, two vis- ible chromatids for each chromosome (Figure FIGURE 4-1. Meiosis in t/ie lily — general view. {Courtesy of R. E. Cleland.) 4-2B, C). A pair of synapsed chromosomes is called a bivalent when referring to chromo- somes, but is called tetrad when referring to chromatids. Although the chromatids sepa- rate from each other in pairs here and there, they are still all in close contact with each other at other places along their length. Each place where the four chromatids are still held together is called a chiasma (cross; the plural is chiasmata) (Figure 4-3 A). A chiasma is characterized by the fact that the two chroma- tids which synapse to make a pair on one side of the point of contact separate at the point of contact and synapse with other partners FIGURE 4-2. {Opposite). Meiosis in the lily. The leptonema and zygonema stages of prophase I have been omitted. {Courtesy of R. E. Cleland.) {By permission of McGraw-Hill Book Co., Inc., from Study Guide and Workbook for Genetics, by I. H. Herskowitz, copyright 1960.) Meiosis 25 have been excluded from our sample, since both children will be normally pigmented. Our sample will include the following, how- ever: all those families whose first child is normal (X) and second child is albino ('!). making up %& (% of %) of all families; those families where the reverse is true (% of K), comprising another Y^ of all families; and those families in which both children are al- bino (V4 of %), which make up Me of all fami- lies. So every seven albino-containing families scored will give, on the average, 6 normal children (three from each of the two kinds of families containing one albino) and 8 albinos (three from each of the two kinds of families containing one albino and two from each family containing two albinos), so that the ratio expected is 3 : 4 as nonalbino : albino. Offspring Line Offspring, in order of birth ( I. to r. ) Dizygotic Twins* Monozygotic Twins* * See Chapter 11. It may be noted that the observed propor- tions of nonalbino and albino children in families of three, or of four, or of more children from normal parents also fit the expected proportions calculated in a similar manner. 4. Marriages between two albinos produce only albino children, as expected genetically from aa X aa. 5. Twins arising from the same zygote (monozygotic or identical twins) are either both albino or nonalbino. Since such twins are genetically identical they would be ex- pected to be both normal, AA or Aa, or both albino, aa. These evidences offer clear proof that human albinism is usually the result of a single pair of genes. 34 CHAPTER 5 D O Oj<^~5 a ^y^-o nhit] 6tU 6 6r{^ 6 cVd 6 d 6^ i5**55c57) fl5i 665& 6S& FIGURE 5-2. /i pedigree of albinism in man. The anomaly of woolly hair is a rare trait in Norwegians and can be attributed, after a study of pedigrees, to the presence of a rare dominant gene, call it W. For, when woolly- haired individuals (Ww) marry normal- haired individuals (uu), it is predicted and found that approximately 509c of children have woolly and 509c normal hair. Note that the affected parent is represented as a hetero- zygote, the trait being so rare that the homo- zygote WW is probably nonexistent, since, barring mutation, an individual with this genotype would have to have parents both of whom had woolly hair. A study of pedigrees was instrumental in clarifying the nature of certain ataxias, which involve a lack of neuromuscular coordination, found in certain famihes in Sweden. In some families, affected people had parents who were apparently unrelated, whereas other affected people had parents who were first cousins. From the rarity of this disease it was suggested that the ataxia was being caused by the presence of a dominant gene in heterozygous condition in those cases where the parents were unrelated, and by the pres- ence of a recessive gene in homozygous condition in those cases where the parents were related. When careful clinical tests were made by a neurologist it was found that, indeed, there were differences in symptoms in the cases where the parents were and were not related. In this way a combination of pedigree and medical studies established the genetic basis and nature of two kinds of ataxia. Numerous family studies have been made regarding blood type. However, before dis- cussing their genetic meaning, we shall need to know just what is meant by a blood type or blood group. Human blood contains red blood corpus- cles (cells) carried in a fluid medium, the plasma. The corpuscles carry on their sur- faces substances called antigens, while the plasma contains, or may form, substances called antibodies. An antibody is a very specific kind of molecule, which is capable of reacting with and binding a specific antigen. This reaction may be visualized as a lock (antibody) which holds or binds a particular key (antigen). If a rabbit is injected with a suitable antigenic material, in the form of foreign red blood cells to which it has never before been exposed, certain antibody-pro- ducing cells of the rabbit will manufacture an abundance of antibodies which will appear in its plasma, some of which will be used to react specifically with the antigenic compo- nent of the foreign red blood cells. If, on some later occasion, the same antigen is injected into the rabbit's blood stream there will be, Segregation in Man — Multiple AUelism 35 now, already present, specific antibodies to bind the antigen. The antigen-antibody complex then formed often causes the blood to clump or agglutinate. It is simple to ar- range the procedure so that this reaction may be observed in a test tube or on a glass slide. What is done ^ is to inject red blood corpus- cles from different people into different rabbits, with the result that the rabbits form antibodies against the antigens introduced. The rabbit's blood, devoid of cells, can then serve as an antiserum, containing antibodies, which will clump any red blood cells added to it carrying the original types of antigens. It is found that two very distinct antisera are formed by these rabbits, and that any per- son's blood cells tested with these two anti- sera can react in one of three ways: the red blood cells are agglutinated or clumped either in one antiserum (arbitrarily called anti-M), or in the other (called anti-N), or in both of these antisera. So all people can be classified by their blood cell antigens as belonging to either M, or N, or MN blood group, re- spectively. When parents and their offspring are tested for "MTV" blood type such family studies give the results shown in Figure 5-3. Parents of type 6 give offspring which are in the proportion of 1 : 2 : 1 for M : MN : N blood types. This suggests these blood types are the phenotypic consequence of the pres- ence of a pair of segregating genes. If we let M = the gene for blood group antigen M, M' = the allelic gene which produces the N blood group antigen, mating 6 must be, genetically, MM' X MM' and the offspring \MM : IMM' : \M'M'. Note that these al- leles show no dominance of one over the other phenotypically, MM' individuals showing both M and N blood group phenotype. All the other family results also are consistent with the genetic explanation proposed for MN blood groups. PARENTS CHILDREN 1 M X M 2 N X N 3 M X N 4 MN X N 5 MN X M M MN N ALL — — — — ALL — ALL — Va 6 MN X MN Va V2 1/4 FIGURE 5-3. Distribution of MN blood group phenotypes in different human families. Still two other antisera, called anti-A and anti-B, can be prepared.' When blood from different people is tested using these antisera it is found to be of one of four types: clumped in anti-A (blood type A), clumped in anti-B (blood type B), clumped in both (type AB), and clumped in neither (O). Family studies of "'ABO"' blood types give the phenotypic results shown in Figure 5-4. Note there that two kinds of results are ob- tained from A X O and also from B X O parents. In each case one result (marriage types 9 and 1 1) may be explained by assuming that the non-O parent is a heterozygote in which the gene for O is recessive. Let / be the gene for O blood group type and /^ the gene for A blood, the latter being dominant. Then the parents are: in marriage type 9 /'/ X //, in type 10 I^I^ X //, and in 13/7 X //. In order to explain 1 1 and 12 we shall have to assume the presence of a gene P for B blood group, which is also a dominant allele of / and from which it segregates. Then mating 1 1 is Pi X it and 12 is PP X //. Note that we have made a new supposition with regard to genes. In the former case the alternative ^ Based upon K. Landsteiner's work. ^ Based upon work of K. Landsteiner and P. Levine. 13 36 PARENTS CHAPTER 5 7 AB X AB 8 AB X O 9* A X O 10* A X O n* B X O 12* B X O O X O CHILDREN AB 74 Vt Va — V2 — Vj — V2 Al 1 — — y^ ALL — V^ V2 — — ALL — „_ „_ _ ALL FIGURE 5^. Distribution of ABO blood group pheno- types in different human families. In some families. allelic form of / is /^ while in the latter case it is P. This means that we are making the additional assumption that the gene can exist in more than one alternative condition, so that a gene can have multiple, different, alleles. Of course, while any individual has only one pair of genes, these may be of the same or of different types. Since the heterozygote /V^ shows no dominance, and appears as AB blood type, all the results indicated in the table are explained. (Some readers may have suspected that multiple allelism was possible from the fact that three different genes were involved in the presence and absence of the ataxias already described.) There are a number of other ways to type blood. One of these involves the presence or absence of what is called the Rhesus or Rh factor. Red blood cells from Rhesus monkeys may be injected into rabbits; if a second in- jection of Rhesus blood is given some time later, it will be clumped. This is explained by the presence of an antigen carried by Rhesus red blood cells against which the rabbit had manufactured antibodies before its second exposure to Rhesus blood. The antigen involved here is called Rh. Instead of injecting Rhesus red blood cells into a rabbit which has anti-Rh antibodies in its serum, suppose human blood is injected. In this event it turns out that 85% of people have blood which is clumped, these people having what is called the Rhesus-positive (or Rh-posi- tive) blood type, whereas 15% of people have blood which is not clumped, and have the Rhesus-negative (or Rh-negative) blood type. Accordingly, 85% of humans have the same Rh antigen as have Rhesus monkeys, while 15%, do not. A combination of family and pedigree studies shows that presence of Rh antigen in human beings is controlled by a dominant gene, call it R, and its absence to a recessive allele, say r, their distribution fol- lowing the principle of segregation. Finally, let us consider the genetic basis for certain kinds of anemia. Among Italians who live in Italy or who have emigrated, there may be an anemia of two special kinds. One type, severe and usually fatal in childhood, is called Cooley's anemia or thalassemia major; Segregation in Man — Multiple Allelism 37 the other type, a more moderate anemia, is phenotypic expression may involve complete, called microcytemia, or thalassemia minor. partial, or no dominance, in no case does Pedigree and family studies all support the this have an effect either on the genes them- hypothesis that t. major children are homo- selves or upon their segregation and recom- zygotes (//) for a pair of genes. Both their bination. parents have t. minor and are heterozygotes Another important point to remember is {Tt) for this gene. Analysis on a population that initially someone had to collect the level has resulted in the classification of more phenotypic data in pedigree and family stud- than 100,000 people in Italy as TT, Tt, or //. ies, and then apply the principles known Notice that in the case of thalassemia, neither about genes to explain these data genetically, r nor / is completely dominant (nor recessive). using the simplest suitable explanations in You have seen, therefore, that some much the same way as was illustrated here for morphological and some chemical character- albinism and for MN and ABO blood types, istics of man are based upon segregating Sometimes the data are insufficient and the genes. While the relation between the alleles investigator is left with several equally prob- in the heterozygote with respect to their able genetic explanations. SUMMARY AND CONCLUSIONS Data furnished in pedigree and family studies provide evidence that there are a number of human traits whose occurrence is based upon the effect of a pair of genes. These traits are of a morphological as well as of a chemical nature, the alleles sometimes showing com- plete, partial, or no phenotypic dominance in the heterozygote. It was necessary to postulate that a gene can exist in any one of more than two alternative states. REFERENCES Mohr, O. L., "Woolly Hair a Dominant Mutant Character in Man," J. Hered., 23 : 345-352, 1932. Neel, J. v., and Schull, W. J., Human Heredity, University of Chicago, 1954, pp. 83-86, 89-91,240 241. Stern, C, Principles of Human Genetics, San Francisco, Freeman, 1961. QUESTIONS FOR DISCUSSION 5.1. What is the difference between the pedigree and family methods of investigation? 5.2. What evidence is there that pigmentation (albinism vs. nonalbinism) is due to genes that are segregating? 5.3. Two nonalbinos marry and have an albino child. What is the chance that the next child is albino? nonalbino? that of the next two children, both are albinos? non- albinos? one is albino and one nonalbino? 5.4. What proportion of three-child families, whose parents are both heterozygous for albinism, have (a) no albino children? (b) all albino children? (c) at least one albino child? 5.5. Would you conclude that the gene for woolly hair is completely dominant to non- woolly hair? Explain. 38 CHAPTER 5 5.6. Discuss the occurrence of dominance with respect to blood group types. 5.7. Why was it necessary to assume that a gene may have more than two allelic forms? 5.8. A baby has blood type AB. What can you tell about the genotypes of its parents? What would you predict about the blood \ypes of children it will later produce? 5.9. If one parent is A. blood type and the other is B, give their respective genotypes if they produced a large number of children whose blood types were: a. All AB. b. Half AB, half B. c. Half AB, half A. d. 'i AB, '^ A, •:, B, 'i O. 5.10. Give examples in man of complete, partial, and no dominance. 5.11. Is the occurrence of complete dominance helpful in determining the genie basis of alternatives for a given trait? Explain. 5.12. What do you suppose is meant by multiple allelism? 5.13. Have you learned anything new about genie properties from this Chapter? Justify your answer. Chapter *6 INDEPENDENT SEGREGATION I: N THE preceding Chapters we have discussed the transmission -genetics of akernative genes for a single trait and have found that a single pair of genes could explain the data in each case. The question asked now is, what will be the genetic unit of transmission when two or more different traits are followed simul- taneously in breeding experiments? The answer to this may lie in the results of some additional experiments performed with the garden pea.^ Previous studies had already shown that, like the flower color trait described in Chapter 2, seed shape and seed color were each due to a single pair of genes. That is, a Pi of pure line round X pure line wrinkled seeds gave round Fi, round being dominant. Self-fertilizing the Fi round gave F2 which were in the proportion of 3 round : 1 wrinkled. Similarly, a Pi of pure line yellow X pure line green seeds gave yel- low F], yellow being dominant, and self-ferti- lization of the yellow Fi gave 3 yellow : 1 green in F2. The question presented above may be asked relative to the seed traits of shape and color. What will happen when individuals are crossed that simultaneously difi'er with regard to both of these traits? Suppose in Pi a round yellow strain is crossed with a wrinkled green strain, these strains being available as pure lines. In Fi only round yel- low seeds are obtained (Figure 6-1). This result is what would be expected had we been studying shape and color of seeds separately. We find in this case, therefore, that there is ^ Based upon experiments of G. Mendel. 39 ■^ I y Round Yellow x Wrinkled Green ALL Round Yellow F, Round Yellow x F, Round Yellow PHENOTYPE NUMBER RATIO Round Yellow Round Green Wrinkled Yellow Wrinkled Green 315 101 108 32 9.06 2.9 3.1 T^ ^^^m ^ FIGURE 6-1. Phenotypic results from studying two traits simultaneously. no phenotypic efi'ect of the dominance of one trait upon the phenotypic expression of the other trait. Self-fertilization of the round yellow Fi gives offspring which, when counted in suffi- ciently large numbers, occur in the relative frequencies of 9 round yellow : 3 round green : 3 wrinkled yellow : 1 wrinkled green. Notice that segregation and recombination have occurred for each trait, as revealed in F2 by 12 round : 4 wrinkled and by 12 yel- low : 4 green. So, in this case there is also no effect of one trait upon the segregation- recombination behavior of the genetic ma- terial for a different trait. 40 CHAPTER 6 From these results what else can we decide regarding the gene? Until now, we have been able to explain all the experimental data pre- sented by supposing that each sexually re- producing individual contains only a single pair of genes. Accordingly, we shall still consider that each Pi individual carries but a single pair of genes, but require each gene to have two simultaneous effects, one on seed shape and the other on seed color. The re- sults obtained are consistent with this expec- tation in the following respect: the Fi are round yellow, and the F2 give a 3 : 1 ratio for yellow vs. green and also for round vs. wrinkled. But, on this hypothesis, the F2 would be of only two types — 3 round yel- low : 1 wrinkled green! The facts are that in F2 not only these grandparental (Pi) combina- tions are found but two new, recombinational classes of offspring appear, namely, round green and wrinkled yellow! Apparently, then, what is genetically transmitted is not composed of a single pair of indivisible units, but is com- posed of separable pairs of units, each pair capable of undergoing segregation and recom- bination. Let us assume, therefore, that each sexually reproducing organism contains more than one pair of genes. In the present case, let R (round) and r (wrinkled) be the alleles of one pair of genes while Y (yellow) and y (green) are the alleles of the second pair. The Pi, then, would be RR YY (round yellow) and rr yy (wrinkled green). Each pair of genes would undergo segregation so that a gamete would contain only one member of each pair. In this manner the former parent would produce only RY gametes and the latter one only ry, and all Fi would be Rr Yy (round yellow) as observed. On the current hypothesis, the gametes formed by the Fi would contain either R or r, and, moreover, would contain either Y or y. Since R and Y do not always go to- gether into a gamete, nor do r and y, there must be four genotypes possible in gametes, RY, Ry, rY, ry. Since these possible haploid genotypes will be found both in male and in female gametes, it is expected that the F2 would contain the diploid genotypes and their corresponding phenotypes indicated in Figure 6 2. Note that nine different geno- types are possible in F2, four giving the round yellow phenotype, two giving round green, two wrinkled yellow, and one wrinkled green. This is consistent with the fact that four phenotypes were actually found in F2, sub- stantiating our hypothesis that the genetic material transmitted in a gamete is composed of subunits, each of which has the properties of a gene. How can we account for the fact that the observed F2 phenotypes occurred in the rela- tive proportions of 9 : 3 : 3 : 1 , respectively? This can be done by making the simple assumption that the segregation of the mem- bers of one pair of genes occurs independently of the segregation of the members of another pair of genes. As a result of this (see Fig- ure 6-3) half of all gametes receive R, of which half will carry 7 and half>'; the other half of all gametes receive r, of which half will carry Y and half y. Thus the male gamete population in the P2 will be 25% RY, 25% Ry, 25% rY, and 25%o ry. The Po also produce female gametes of the same four genotypes in the same relative frequencies. Since fertilization has already been assumed to occur at random the F2 expected are shown in Figure 6-4. The branching track in Figure 6-4 can be read beginning at the top: % of female gametes are RY and are fertilized % of the time by RY male gametes (producing Ks of all offspring as RR YY), % of the time fertiliza- tion is by Ry male gametes (so that Ke of all offspring are RR Yy from this origin), etc. By summing up like classes, the kinds and relative numbers of genotypes and of pheno- types are obtained as shown in the chart. The branching track may be used to obtain the 9 : 3 : 3 : 1 phenotypic ratio more simply. I 41 FIGURE 6-2 {Left). Expected genotypes and phenotypes in F2 following segregation. R R Y Y '^'^^^^ Round I R r Y Y 'r Y®l'ow R r Yy ^ fr R R y y . R^und ^ Rryy ▼ ^reen -V rrYy Wrinkled Yellow r r V V 4 Wrinkled ' ' ^ Green p /2 R V^r FIGURE 6-3 (Right). Genotypes of gametes formed by a di hybrid, Rr Yy, undergoing inde- pendent segregation. Vi Y i- 1/4 R Y 1/2 y ^ 1/, p y y, Y ^ Va r Y Va y *► V4 r y U -D 42 CHAPTER 6 TYPES AND RELATIVE FREQUENCY OF GAMETES ? cT T y 1/4 RY Ry rY V4 rY V4 ry 0 RY Ry rY ry /4 RY 0 U Ry r\ /4 rY ly 'A ry A RY '/4 Ry r\ '/4 rY ly '/4 ry Genotypes /16 RRYY /16 RRYy /1 6 RrYY /1 6 RrYy /I 6 RRYy /16 RRyy /1 6 RrYy /16 Rryy /1 6 RrYY /1 6 RrYy /1 6 rrYY /1 6 rrYy /16 RrYy /16 Rryy /1 6 rrYy /16 rryy OFFSPRING Genotypic Frequency X 1/16 RRYY RRYy RrYY RrYy RRyy Rryy rrYY rrYy rryy Phenotypic Frequency X 1/16 9 Round Yellow 3 Round Green 3 Wrinkled Yellow 1 Wrinkled Green FIGURE 6-4. Independent segre- gation and random fertilization in a cross between identical dihy- brids. You know that crossing together two mono- hybrids (heterozygotes for one pair of genes) yields a 3 : 1 phenotypic ratio of domi- nant : recessive trait. If the recombinational activity of one pair of genes is independent of the same behavior by another pair of genes, both being heterozygous and showing dominance, then the two independent 3 : 1 ratios may be combined in the progeny as in Figure 6-5. That chart may be read : among the offspring, the X which are round (because of segregation, random fertiHzation, and the dominance of R in the cross Rr X Rr) will be % of the time also yellow and V^ of the time will be also green (because of segrega- tion, random fertilization, and the dominance of Y in the cross Yy X Yy)\ so, of all progeny %f, will be round yellow and Ke round green, etc. You see, then, that independent segrega- tion by two different pairs of genes results in the formation of gametes whose gene combi- nations, new and old, are in equal frequency. In the present case, the dihybrid (hetero- zygote for two pairs of genes) received both R and Y from one parent and both r and y from the other, its gametic recombinations being Ry and rY. Had the dihybrid received Ry from one parent and rY from the other, the gametic recombinations would have been Independent Segregation 43 RY and ry, and the old combinations Ry and rY. So, regardless of how the genes enter the individual, the dihybrid forms four, equally frequent, genetically different gametes. The types and frequencies of gametes formed by the Rr Yy dihybrid can be de- termined more easily after mating it with a double recessive individual, i.e., an individual homozygous recessive for both pairs of genes concerned. In the cross of Rr Yy X rr yy, the double recessive parent produces only ry gametes while the dihybrid produces four different and equally frequent types, RY, Ry, rY, ry. Accordingly, the finding that among the offspring of this cross (Figure 6-6) very nearly 25% are round yellow (55 offspring), 25% round green (51 offspring), 25% wrin- kled yellow (49 offspring), and 25% wrinkled green (52 offspring) is a direct confirmation both of segregation by the members of a single pair of genes and of independent segre- gation by different pairs of genes. Whenever one is dealing with complete dominance, a cross to an individual recessive for the pairs of genes involved will always serve to identify the genotype of the other parent, since the phenotypic types and fre- quencies of the offspring will correspond to the genotypic types and frequencies occurring in the gametes of the latter. This kind of cross is therefore called a test cross, and is also called a hackcross if one of the parents in the series of crosses had been homozygous recessive for the genes under study. ^™ I PARENTS Rr Yy x Rr Yy OFFSPRING % Round A Wrinkled <; <: 'A Yellow 9/16 Round Yellow 'A Green ^ 3/16 Round Green '/4 Yellow ^ 3/16 Wrinkled Green Va Green 1/16 Wrinkled Green FIGURE 6-5. Phenotypic results of a cross between identical dihybrids. \k 44 CHAPTER 6 GAMETES 9 V4 RY 74 Ry V4 rY 74 ry Try 1 ry 1 ry Iry GENOTYPES 'A Rr Yy k 74 Rr yy k T V4 rr Yy T V4 rr yy PHENOTYPES V4 Round Yellow V4 Round Green V4 Wrinkled Yellow Va Wrinkled Green FIGURE 6-6. Test cross or backer oss of the F, di hybrid (Rr Yy) with the double recessive indi- vidual (rr yy). We are now in a position to return to a consideration of the material basis for genes. If one gene pair is to be physically associated with the corresponding short regions on a pair of homologous chromosomes within which a chiasma cannot occur, the question is, where in relation to one pair of genes is a second pair located? Two possibilities occur — either both pairs are on the same chromosome pair or they are on different, nonhomologous chromosome pairs. Let us examine the assumption that different pairs of genes are located on different pairs of chro- mosomes. If this is true, then there are several different arrangements that the parts of different pairs of chromosomes may take relative to each other at metaphase I of meiosis (Figure 6-7). It has been established that different pairs of chromosomes line up at metaphase I independently of each other. Moreover, it is entirely reasonable that the way cen- tromeres in a tetrad orient toward the poles at metaphase I would be uninfluenced by the presence or absence of chiasmata in that tetrad. If, then, as in Case A, there is no chiasma between the centromere and gene pair Aa nor between the centromere and gene pair Bb, alignments I and II, being equally frequent, will result in four different, Independent Segregation 45 CASE A No chiasma Haploid Meiotic Pole -*- Metaphase I -^ Pole Products at Telophase II A a I B B bU U ..H b I AB, AB, ab, ab II bjJUbBllB II Ab, Ab, aB, aB CASE B After one chiasma "^ in one pair r u fui A 1 Ia all Ua .H..I •' 11 •' II b U 1 B b U 1 B II Ab, AB, ab, oB CASE C After one < chiasma in each pair aI Ua aI Ua I B 1 U b B 1 U b I AB, ab, AB, ab . U or II bUlBbLllB II Ab, aB, Ab, aB FIGURE 6-7. Meiotic fate of gene pairs located in nonhomologous chromosomes. 46 CHAPTER 6 equally frequent types of gametes at the end of meiosis. The same result is also obtained either when there is a chiasma between the centromere and the gene in question in one tetrad but not the other (Case B), or when a chiasma occurs in each of the tetrads (Case C). Therefore, independent segregation of different pairs of chromosomes can serve as the physical basis for independent segregation of different pairs of genes, regardless of chiasma formation. Let us examine, now, the consequences of assuming that A and B are on one and the same chromosome while a and b are on the homologous chromosome of the pair (Fig- ure 6-8). When there is no chiasma. Case A, only the old (maternal and paternal) combi- nations are found in the gametes. When there is a chiasma between the two different pairs of genes, Case B, then all four classes occur with equal frequency (two old and two new combinational types). But, unless every tetrad has a chiasma in the region between linked genes, the number of old gene combi- nations found among the gametes will exceed the new combinations. Although a tetrad usually contains one or more chiasma, there are numerous points along the chromosome where a chiasma can arise. It would necessi- tate an additional hypothesis to require that each tetrad have a chiasma within a given interval. Moreover, we have no knowledge as to the genie interval, that is, the distance between genes presumed to be on the same chromosome. Accordingly, we shall neglect, for the time being, the possibility that genes on the same chromosome pair could form old and new combinations with equal fre- quency— that is, we shall assume that two pairs of genes which do so, and are therefore segregating independently of each other, must be located on different pairs of chromosomes. Evidence that is at least consistent with this presumption is obtained from studies with FIGURE 6-8. Meiotic fate of gene pairs located in the same pair of chromosomes. Pole^« Metaphase I ►Pole Haploid Meiotic Products at Telophase II CASE A No chiasma *l 1' a between gene pairs .1 I. b AB, AB, ab, ab CASE B After one chiasma between gene pairs I1::fl AB, Ab, aB, ab Independent Segregation 47 garden peas. From the breeding behavior tion shows that the garden pea possesses a of hybrids it is possible to estabhsh the exist- diploid number of seven pairs of chromo- ence of seven different pairs of genes (each somes, so that the number of chromosome happening to show dominance in the hybrid pairs is large enough for each pair of genes condition), each pair segregating independ- to be located on a different pair of chromo- ently of all the others. Cytological observa- somes. SUMMARY AND CONCLUSIONS When two different traits were studied separately, in each case the phenotypic alternatives were found to be due to the presence of a single pair of genes. Studies were then made of the distribution of phenotypes in successive generations when these two pairs of traits were followed simultaneously in the same individuals. The data obtained showed that each trait is due to the presence of a different pair of genes, proving that the genetic material is made not of a single indivisible pair of genes but of a number of pairs. The results, moreover, are best explained on the principle that the segre- gation of one pair of genes is at random with respect to the segregation of all other pairs tested. The simplest hypothesis for the physical basis of independent segregation is that different pairs of genes reside in different pairs of chromosomes. REFERENCES Mendel, G. See references at the end of Chapter 2. Supplement I (at the end of this book). QUESTIONS FOR DISCUSSION 6.1. Make genetic diagrams for the crosses and progeny discussed in the second paragraph on p. 39. Be sure to define your symbols. 6.2. Is a test cross or backcross used to determine genotypes from phenotypes in cases of no dominance? Explain. 6.3. What types and frequencies of gametes are formed by the following genotypes, all gene pairs segregating independently? a. Aa Bb CC b. AA BB Cc DD c. Aa Bb Cc d. Mm Nn Oo Pp 6.4. How many different genotypes are possible in offspring from crosses in which both parents are undergoing independent segregation for the following numbers of pairs of heterozygous genes — 1, 2, 3, 4, n? 6.5. What conclusions could you reach about the parents if the offspring had phenotypes in the following proportions? a. 3:1 b. 1 :1 c. 9:3:3:1 d. 1:1:1:1 6.6. Would you be justified in concluding that a pair of chromosomes can contain but a single pair of genes? Explain. 48 CHAPTER 6 6.7. A father of blood group types M and O has a child of MN and B blood types. What genotypes are possible for the mother? 6.8. What proportion of the offspring of the following crosses, involving independent segregation, will be completely homozygous? a. Aa Bb X Aa Bb b. AA BB CC X AA bb cc c. Aa BB Cc X AA Bb cc d. AA' X A" A'" 6.9. Why, following independent segregation, would you expect that gametes fertilize at random with respect to their genotypes? 6.10. Discuss the particulate nature of the genetic material. 6.11. Does the discovery of independent segregation affect your concept of gene size? Explain. 6.12. Discuss your current understanding of the term "genetic recombination." Chapter 7 GENE INTERACTION AND PHENOTYPIC EXPRESSION Yi "ou ARE already familiar with some of the phenotypic con- sequences of gene interaction, in that the phenotype of a heterozygote may show the effects of only one allele, or some of the effects of both alleles, or the complete effects of both alleles. These phenotypic consequences have already been called com- plete, partial, and no dominance, respec- tively. In the garden pea hybrids already discussed, complete dominance was respon- sible for the 3:1 phenotypic ratio obtained from crossing two monohybrids. This necessitated the extra labor of testing the off- spring possessing the dominant phenotype in order to identify the 1:2:1 genotypic ratio predicted from such crosses. Had no dominance obtained the phenotypic ratio would have been the same as the genotypic one. Nevertheless, in all cases genes were segregating, and the specific ratios observed depended only upon the dominance relation within the gene pair, that is, the relation between the expression of one allele and that of its partner. You have seen also that complete domi- nance had no influence upon the indepen- dence of the segregation of different pairs of genes within a given individual. The geno- typic ratio expected from crossing two par- ticular dihybrids has already been derived (Chapter 6). Let us rederive this ratio, em- ploying more general symbols for genes, for reasons soon to be apparent, using the branching track method in a still slightly different way. Let A and A' be one pair of alleles and B and B' another. Mating AA' BB' by AA' BB' will give the genotypic ratio shown in Figure 7-1. Note that among every 16 offspring, on the average, there would be 9 different genotypes: 1 with all unprimed gene symbols, 1 with all primed gene symbols, and 7 others having 3 primed or 3 unprimed or 2 primed gene symbols. Let us re-examine how this geno- typic ratio gave rise to the 9 : 3 : 3 : 1 pheno- typic ratio in crosses between dihybrid garden peas. Two factors were responsible. One was the occurrence of dominance within each pair of alleles, the other was the fact that the trait determined by one pair of genes was un- associated with the trait determined by the other pair of genes. In what way does the phenotypic ratio expected from crosses be- tween dihybrids for unrelated traits depend upon whether dominance obtains for neither, one, or both pairs of genes? This can be answered with the aid of the left side of Figure 7-2. In any column in Figure 7-2, boxes filled with the same symbol have identical pheno- types. Note that in DI the genotypic and phenotypic ratios are identical, and that the other D columns have two or more geno- types represented by a single phenotype. DI could be exemplified by the phenotypic ex- pectation from matings between two people both having AB and MN blood types (see Chapter 4). DII could be exemplified by the phenotypic expectation from matings be- tween two people both of MN blood type and heterozygous for albinism (see Chapter 4). We have already discussed Dili in Chapter 6. It should be recalled that the genes for round-wrinkled and the genes for yellow- green though affecting the same part of a pea plant, the seed, act on different traits — tex- ture and color, there being no obvious rela- tionship between the two. So in this and the other cases under D a particular part CHAPTER 7 AA' X AA' BB' X BB' AA'BB' X AA'BB' x 1/16 y4AA % AA' 74 A'A' V4BB Vi BB' y4 B'B' V4BB Va BB' Va B'B' 'ABB Vj BB' V4 B'B' 0 1 AABB 2 AABB' 1 AAB'B' 2 AA'BB 4 AA'BB' 2 AA'B'B' 1 A'A'BB 2 A'A' BB' piGURE 7-1. Recombination 1 A'A' B'B' frequencies. of an organism is capable of showing the presence of any phenotype due to one pair of genes at the same time as it shows any phenotype of another pair. Under D, then, the two pairs of genes produce effects which are independently distinguishable because they do not impose upon each other's ex- pression, i.e., they do not superpose. What phenotypic ratios are expected when the two different pairs of genes affect the same trait in the same direction (Figure 7-2, S)? The ratio in SI would follow if any un- primed gene contributed an equal and cumu- lative effect on the phenotype, say by forming melanin pigment, the primed genes con- tributing none of this effect. SII would fol- low for cumulative effects when A A, AA' , and BB' each produces equal phenotypic effect, say on height, BB produces twice this effect, and A'A' and B'B' produces none of this effect. SIII would follow if either A 01 B gives the full phenotypic effect, say on flower color, only A'A' B'B' producing none of this effect. In each of the examples under S the ratios obtained were simplifications of the corres- ponding ratios found under D, due to the fact that different combinations of alleles from two different pairs of genes acting in the same direction gave the same phenotypic effect. In these cases, then, different pairs of genes have a common phenotypic background on which their effects superpose, the effect of one gene interfering with the detection of the effect of the other pair. What may the phenotypic ratios from crosses between identical dihybrids become, when both pairs of genes show dominance Gene Interaction and Phenotypic Expressi 51 :§ ^ < u> S - u. o to Z O ^ 1. << /\ ^< c ^ O - c c 0) ._ .- ■£ Q) 0) .— WW ^r>vm^5o/Drosophila melanogaster. {Courtesy ofC. Stern; by permission of Genetics, Inc., vol. 28, p. 443, 1943.) Multiple Alleles and Lethals 65 these populations is completely dominant to ci. But the hybrid of ci with wild flies from population 3, c/+^ ci, shows the cubitus vein interrupted! Moreover, the lack of domi- nance of c/+'^ over ci can be shown to be an effect of this gene pair rather than a modify- ing effect of some other gene pair. Appar- ently, then, the c/+ allele in population 3 is different from the one in populations 1 and 2. Thus, alleles which at first seem alike may prove to be different when tested further. Such alleles are said to be isoalleles. Some other techniques which may be employed to detect isoalleles include the response of the tested alleles to the presence of nonallelic genes, to environmental changes as of tem- perature and humidity, and to agents which modify mutation rates. The number of alleles that can be proven isoallelic will depend upon how many different phenotypic criteria you employ to compare alleles, and how small a phenotypic difference you are able to recog- nize. The more delicate the tests and the larger their number, the greater is the chance for demonstrating isoallelism. Although we have described isoalleles among the genes normally expressed in indi- viduals living in the wild (wild type isoalleles), there are also isoalleles for mutant genes (mutant isoalleles). For instance, it has been proven that the mutant gene w, producing white eye in different strains of Drosophila, actually comprises a series of multiple iso- alleles {w\ w~, w^, etc.). Lethals In the snapdragon (Antirrhinum) one can find two kinds of full grown plants, those which are green and those which are a paler green called auria. Green crossed by green pro- duces only green, but auria by auria produces seedhngs of which 25% are green (AA), 50% auria (Aa), and 25% white (aa). The latter die, after exhausting the food in the seed from which they grew, because they lack chlorophyll and cannot synthesize food. So, among the grown plants, the phenotypic ratio observed is % green : % auria. In this case, then, lack of dominance gives a 1:2:1 ratio among seedlings, characteristic of a cross between monohybrids, which because of lethality becomes a 2 : 1 ratio among the older survivors. These ratios were discovered in the reverse order in a case in mice. In this case, in which genetic lethality was first demonstrated, crosses between two yellow mice never gave all yellow progeny, but always gave 2 yel- low : 1 nonyellow. It was then shown that from such matings % of the fertilized eggs which should have completed development failed to do so and aborted early. Those dy- ing were clearly the homozygotes of one type, with nonyellows being the other homozygous type, since crosses between nonyellows pro- duced only nonyellows. Note that the gene symbols usually employed will not be satis- factory here. For now we have two effects to describe for each gene — one effect on color and one on viabihty. Moreover, the allele which is dominant for the one effect is recessive for the other, and vice versa. This problem is solved by using base letters with superscripts for each gene (Figure 9-3), where the base letter refers to one trait and the superscript refers to the other trait. Let the superscript / be the recessive lethal eff"ect of the gene dominant for yellow, Y, and L be the superscript for the dominant normal viability of the allele recessive for nonyellow, y. Then the Fi from crossing two yellow mice (ry^ X Y'y^) are I FT' (dies) : 2 Y'y^ (yellow) : 1 y^y^ (nonyellow). In both the snapdragon and mouse cases described, death resulted from the presence of an allele in homozygous condition. Those alleles which kill the individual before it can reproduce are called lethal genes or lethals — those producing this effect only when homozygous are recessive lethals, while those acting this way when heterozygous are domi- nant lethals. 66 CHAPTER 9 G, yellow X yellow ylyL y' yL V2Y' , Viy^ V2Y' , V2Y^ y4Y'Y' 'ayV^ VaV^ y^ dies yellow nonyellow L J FIGURE 9 3. Results of matings between yellow mice. From the biological standpoint, lethality is characterized not by the absence of an individual or a class of offspring, but by its inability to reproduce. So, for example, a genotype which causes complete sterility is genically lethal, even should its possessor live forever. Lethals which actually kill the organism may act very early or very late in development, or at any stage in between. Sometimes a lethal effect is produced not by one gene or a pair, but by the combined effect of several nonallelic genes. In this case, some of the nonalleles are contributed by each parent, and the offspring dies because the nonalleles, viable when separate, are lethal when present together. Different alleles, recessive or dominant, have been shown to affect viability to differ- ent degrees. These effects cover the entire spectrum — ranging from those which are lethal, to those which are greatly or slightly detrimental, to those which are apparently neutral or even beneficial (Figure 9^). When there is differential viability for different non- alleles or alleles, phenotypic ratios may be significantly modified from those expected. The importance of the precautions to be taken, relative to the viability and fertility of the individuals bred in experiments designed to establish principles of transmission genet- ics, has been discussed in Chapter 2, and is by now obvious. SUPRA-VITAL (beneficial) 1.3 1^ 1.2 NORMAL VIABILITY I 1^ 1.1 SUB-VITAL (detrimental) J^ I r 1.0 .9 .8 .7 .6 .5 RELATIVE VIABILITY t I SUB-LETHAL (semi-lethal) I LETHAL I I I ' ^A| FIGURE 9-4. Classification of effects that mutants have on viability. Multiple Alleles and Lethals 57 SUMMARY AND CONCLUSIONS The diflFerent alternative states which a gene may assume in a multiple allelic series may produce different degrees of effect upon a quantitative phenotypic result, or may involve apparently different qualitative effects, or both. Dominance is absent when the alleles in the hybrid produce qualitatively different effects, and may or may not obtain when purely quantitative effects are involved. The establishment of isoallelism in any given case is largely a matter of the precision and variety of the testing procedures employed. Different alleles may produce detectable differences upon viability by acting at any stage in the life history of individuals, and may modify the expected phenotypic ratio so that certain classes of offspring are in excess, or in reduced frequency, or are absent. The effect mentioned last is produced by dominant and (homozygous) recessive lethal genes. REFERENCES Hadorn, E., Developmental Genetics and Lethal Factors, New York, Wiley, 1961. Race, R. R., and Sanger, R., Blood Groups in Man, 3rd Ed., Springfield, 111., C. C Thomas 1959. Wiener, A. S., and Wexler, I. B., Heredity of the Blood Groups, New York, Grune & Stratton, 1958. QUESTIONS FOR DISCUSSION 9.1. How would you prove that you were dealing with multiple alleles, rather than multiple pairs of genes? 9.2. How many different genotypes are possible when there are four different alleles of a single gene? 9.3. Does the discussion in the text imply that: (a) there is an infinite variety of isoalleles? (b) no two genes are ever identical? Explain. 9.4. Describe how you would proceed to test whether the genes for white eye in two different populations of Drosophila were alleles, isoalleles, or nonalleles. 9.5. An agouti rabbit crossed to a chinchilla rabbit produced an agouti offspring. What genotypic and phenotypic results would you expect from crossing the Fi agouti with an albino? 9.6. For each of the following matings involving Nicotiana give the percentage of aborted pollen tubes and the genotypes of the offspring. o^ 9 a. si s2 X si s3 b. si s3 X s2 s4 c. si s4 X si s4 d. s3 s4 X s2 s3 9.7. Two curly-winged stubble-bristled Drosophila are mated. Among a large number of adult progeny scored there are 4 curly stubble : 2 curly only : 2 stubble only : 1 neither curly nor stubble (which were therefore normal, wild-type). Explain these results genetically. 9.8. Could you prove the existence of multiple allelism in an organism that reproduces asexually only? Explain. 9.9. Discuss the factors which can modify the expected phenotypic ratio. Chapter 10 PLEIOTROPISM, PENETRANCE AND EXPRESSIVITY Pleiotropism h: "ow MANY phenotypic effects does a gene have? In com- . paring the phenotypes of two genetically different lines of rabbits, one chin- chilla (c''''c'''') and one white {cc) there is only one apparent phenotypic difference — the presence vs. the absence of coat pigment. Saying that the c"'' gene has many effects — to produce pigment on the ears, on the trunk, on the limbs, on the tail, and to produce no pigment in the intestine, none in the pancreas, etc., complicates the description without add- ing any more meaning. In the case of Himalayan rabbits (cV), the coat itself is usually variegated, being black at the extremi- ties and white elsewhere (Chapters 1 and 9). Does that mean this allele has a different kind of action in different parts of the coat? No. For the pigment differences are attributed rather to the effect of temperature upon the action (at less than 34° C) or inaction (at more than 34° C) of an enzyme, produced by this genotype, which transforms nonpig- mented into pigmented material. Thus, where the body temperature is less than 34° C, as it is at the extremities, pigment is produced, while on the warm parts of the body no pig- ment is formed because of heat inactivation of this enzyme. The Himalayan pattern is attributed, then, to an allele whose single effect is modified by differences in the environment. In discussing the MN blood groups (Chap- ters), it was stated that M produces M antigen while M' produces N antigen. In this case 68 it might be thought first that the gene has two effects, since one allele produces M but not N antigen and the other produces N but not M antigen. But, again, the lack of an effect cannot be counted as an effect, and it is simpler to think of this gene as having the ability to produce a single antigen whose specific nature depends upon the particular allele that is present. This, then, is a case in which one trait may be affected in different qualitative ways. In Chapter 9 we discussed a multiple allelic series for eye color in Drosophila. There also we were dealing with one trait, in that case eye color pigment, differ- ent alleles affecting it in an apparently quanti- tative manner. In the last Chapter we also considered a case of inheritance in the snap- dragon. But there again we were dealing not with two different effects of a gene, one on pigmentation and the other on viability, but rather on the single activity, chlorophyll production, which had lethality as the con- sequence of its failure. The question posed refers then to whether or not a gene has effects upon two or more traits which are apparently independent of, or unrelated to, each other in their origin. Such effects of a gene we can call multiple, manifold, or pleiotropic effects. None of the examples just mentioned dealt with such multiple effects. However, we have already discussed a case which seems to fulfill these requirements. This is the case of the yellow mouse. The allele which produces yellow coat color as a dominant effect also has a recessive lethal effect. On the presump- tion that homozygotes for this allele would have had yellow body color had they survived, and on the basis that there is no obvious relation between coat color and viability, it could be concluded that this is a case where the gene shows pleiotropism. Studies have been made to test the idea that, in general, genes are pleiotropic. The procedure in one of these studies ^ was as ^ Based upon Th. Dobzhansky's work. Pleiotropism, Penetrance and Expressivity 69 follows: two strains of Drosophila were ob- tained that were practically identical genetic- ally (isogenic) except that one was pure for the gene for dull red eye color (h'+) and the other was pure for its allele white (vr). Then some other trait was chosen for examination in these two strains, a trait which is appar- ently unconnected with that for color of eyes. The trait selected was the shape of an organ, located internally, called a sperma- theca, which is found in females and is used to store the sperm that they receive. The ratio of the diameter to the height of this organ was determined for the two strains. This index of shape was found to be signifi- cantly different in the dull red as compared to the white strain. From this result it can be concluded that the eye color gene studied is pleiotropic. The results of other studies have shown that many different genes are morphologically pleiotropic. Another example ^ may be taken from Drosophila. There is a recessive lethal gene ^ Based upon E. Hadom's work. called lethal-translucida which causes pupae to become translucent and die. Using suit- able techniques, it is possible to compare the kinds and amounts of chemical substances in the blood fluid of normal larvae and pupae, with those found in the recessive lethal homo- zygotes (Figure 10-1). When this is done, some substances are found to be equal in amount in both genotypes (peptide III), others are more abundant in the lethal than in the normal individual (peptide I, peptide II, and proline), still others are less abundant (glu- tamine) or absent (cystine) in the lethal. This case illustrates that pleiotropism can occur at the biochemical level. One of the most instructive studies of pleiotropism involves the genetic disease in man called sickle cell anemia. Homozygotes for a certain allele show the following differ- ent effects, either singly or in any combina- tion: anemia, enlarged spleen, heart trouble, paralysis from brain damage, kidney trouble, and skin lesions. As a consequence, homo- zygotes for the gene for sickling usually die FIGURE 10-1. Pleiotropism at the biochemical level. {After E. Hadorn.) PEPTIDE I PEPTIDE II CYSTINE PEPTIDE III GLUTAMINE PROLINE 70 CHAPTER 10 as adolescents or young adults; this allele, therefore, almost always functions as a re- cessive lethal. It is found that the red blood cells of these homozygotes may become sickle-shaped in- stead of being disc -shaped (Figure 10-2). Sickle-shaped cells may clump and clog blood I • • 9 vessels in various parts of the body leading to the malfunctions of all the organs already mentioned; in addition, since these cor- puscles are defective, they are destroyed by the body, which as a consequence becomes anemic. We see, then, that the wide variety of appar- ently unrelated phenotypic effects of the gene for sickling are but consequences of the sick- ling of red blood cells. Moreover, studies at the biochemical level show that the sickling behavior itself is the result of the presence of an abnormal type of hemoglobin which sickle cell homozygotes carry in their red blood cells. There is, then, a pedigree of causes for the multiple effects of the gene for sickling. The first cause is the gene, the second is the abnormal hemoglobin it produces, the third is the sickling that follows, the fourth is the subsequent red cell clumping and destruction which produce gross organic defects and anemia. In this case all the multiple effects of the gene are attributed to a single or unitary effect which is of a biochemical, perhaps enzymatic, nature. This single effect then affects many varied chemical reactions which are involved in the production of different, at first appar- ently unrelated, traits. We may even hy- pothesize that most, if not all, genes have a single primary phenotypic effect. It may yet be found that the pleiotropic effects de- scribed in the mouse and Drosophila are tertiary or even further removed effects in a pedigree of causes, whose primary cause is genie and whose single secondary cause is still undetermined. Replying to the question with which this section started, the simplest hypothesis is that most, if not all, genes have one primary phenotypic effect following which a pedigree of causes ends in pleiotropism. FIGURE 10-2. Silhouettes showing various types of human red blood cells : normal, in normal homo- zygote (A), sickle cell trait, in mutant heterozygote (B), sickle cell disease, in mutant homozygote (C). Pleiotropism, Penetrance and Expressivity 71 5.5 6.6 oh5^6 o fl^k 6 FIGURE 10-3. A pedigree of Polydactyly in man. 6.6 5.5 6.6 6.6 6.6 5.5 5.5 5.6 6.7 i5c55i 6.6 6.6 Penetrance and Expressivity One of the reasons for the ease with which the principles of transmission genetics were estabhshed is the fact that each of the geno- types used expressed itself repeatedly in approximately the same way, despite the nor- mal fluctuations of the environment. We shall refer to the ability of a genotype or of its parts to be expressed phenotypically in one way or another as penetrance. Most genes studied up to now are fully penetrant. Consider a pedigree for Polydactyly (Figure 10-3), a rare condition in human beings in which individuals may have more than five digits on a limb. The topmost female was aff"ected, having five fingers on each hand, but six toes on each foot. Her husband was normal with respect to this trait. This couple had five children, of whom three were affected. This result suggests that Polydactyly is due to a single dominant gene, P, so that the par- ents would be, then, mother Pp, father pp. Consistent with this hypothesis is the result of the marriage of one of their aff'ected daughters to a normal man which produced two sons, one of whom was aff"ected, and this affected son, in turn, had five children including some aff'ected and some unaffected. But examine now the left side of this pedi- gree. Shown here is the first-born son who was unaffected, yet had an affected daughter. How may this be explained? It might be supposed that this son was genotypically pp and that his daughter was a mutant individual, Pp, derived from an egg or a sperm in which the p gene underwent mutation to the P allele. The following reasoning argues against this interpretation, however. It was noted already that Polydactyly is rare, so that mutations from p to P must be still more rare. Therefore, the chance that such a mu- tation will occur in a sex cell of one of two normal parents in this pedigree is very small. Examination of other pedigrees for Polydac- tyly also reveals other cases in which two normal individuals have an affected child. It is extremely improbable, then, that such a rare mutation, if it occurs at random among normal individuals, would occur so often among the normal ones in Polydactyly pedi- grees. A different explanation is that the first- born son was in fact Pp but that the P was not penetrant in him, though it was in his daughter. This interpretation is supported by the kind of expression the P gene produced 72 CHAPTER 10 in the affected individuals in this pedigree. These individuals may have the normal num- ber of fingers but have extra toes, or they may have the reverse ; they may have different numbers of toes on the two feet, or they may have extra fingers on one hand and the normal number on the other. The expression of Polydactyly, so far as the number of extra digits is concerned, is clearly quite variable. Accordingly, since it is possible to have no expression on one limb of an individual known to be Pp it must also occur that, on occasion, expression fails on all four limbs of an individual with this genotype. The P gene, therefore, has a penetrance of less than 100%, sometimes failing to produce any detectable phenotypic effect when pres- ent. So, while a polydactylous person is certain to carry P, a normal phenotype can represent either the Pp or pp genotype. Since Polydactyly is rare it is usually quite safe to score as pp the genotype of a normal indi- vidual who marries into a line of descent con- taining P. It has already been mentioned that the way that P is expressed in an individual is quite variable with respect to the number and posi- tion of extra digits. Further variabiUty of expression is demonstrated by the different degrees of development which the extra digits show. The term expressivity is used to refer to the kind of effect produced by a genotype when it is penetrant. So, in individuals where P is nonpenetrant there is no expres- sivity, and when P is penetrant its expressivity is variable. What factors are involved in the production of variable penetrance, or, in cases of pene- trance, of variable expressivity? A study of a genetically uniform line of guinea pigs showed that Polydactyly occurred more frequently in the litters from younger than from older mothers. In this case the physiological changes accompanying age modified pene- trance. In another case, a genetically uni- form line of Drosophila flies showed a greater percent of penetrance of an abnormal abdo- men phenotype when moisture content dur- ing development was high than when it was low. These are both examples of how varia- tions in penetrance can be produced by varia- tions in the environment of different indi- viduals of essentially identical genotype. You are already familar with the effect of variations in genotype upon penetrance, under essentially constant environmental conditions. Remember that the penetrance of an allele may depend upon the nature of its partner allele in cases of complete or par- tial dominance, and that the penetrance of one or a pair of alleles may be modified by its epistatic-hypostatic relations to nonalleHc genes (Chapter 7). Similarly, it can be shown that variable expressivity may be the conse- quence of differences in either or both the environment and the genotype. Several additional points should be made. The terms penetrance and expressivity were used to compare the phenotypic events which occur in different individuals. That is, once any phenotypic expression occurred within an individual, the genotype was said to be penetrant, and all other phenotypic compari- sons between penetrant individuals were considered matters of expressivity. In fact, however, one can also correctly speak about penetrance within an individual in those cases where the particular genotype has two or more occasions to express itself. Thus, for example, the gene for Polydactyly has two apparently equal chances to be penetrant in the case of the hands, and two apparently equal chances to be penetrant in the case of the feet. So the genotype may be penetrant in one hand (six fingers) and not in the other (five fingers), it may be penetrant in the feet (6.6) and not in the hands (5.5). When differ- ences in penetrance (or expressivity) are shown by essentially duplicate parts of the same individual (one hand having seven and the other six digits, or one hand having one large extra digit and the other, one small ex- Pleiotropism, Penetrance and Expressivity 73 tra one), you can be reasonably certain that to attribute, with assurance, similarities or these differences have an environmental and differences among them to genotype or to not a genetic basis. However, when different environment, if both of these factors are individuals are compared with respect to pen- varying in uncontrolled ways (as already dis- etrance or expressivity, it is often impossible cussed in Chapter 1). SUMMARY AND CONCLUSIONS A gene usually produces effects upon a wide variety of morphological and biochemical traits. These pleiotropic effects are the consequence of a pedigree of causes traceable, in some cases, to a single effect on the part of the gene. It is hypothesized that most, if not all, genes have a single primary phenotypic effect of a biochemical nature. Penetrance and expressivity depend upon both the genotype and the environment. The most practicable traits for the study of transmission genetics are those whose penetrance is 100^ and whose expressivity is uniform when subjected to the normal variations of en- vironment. REFERENCES Dobzhansky, Th., and Holtz, A. M., "A Re-examination of Manifold Effects of Genes in Drosophila melanogaster" Genetics, 28:295-303, 1943. Hadorn, E., "Patterns of Development and Biochemical Pleiotropy," Cold Spring Harbor Symp. on Quant. Biol., 21:363-374, 1956. Goldschmidt, R. B., Theoretical Genetics, Berkeley and Los Angeles, University of California, 1955. QUESTIONS FOR DISCUSSION 10.1. In what respects are the terms penetrance and dominance similar and in what respects are they different? 10.2. Is it the gene for dull red eye color which is pleiotropic in Drosophila, or is it the allele for white eye color? Explain. 10.3. Most of the genes studied in Drosophila affect the exoskeleton of the fly. Do you suppose these genes also have effects on the internal organs? Why? 10.4. Would you expect to find individuals that are homozygous for Polydactyly? Explain. What phenotype would you expect them to have? Why? 10.5. Why are genes whose penetrance is 100% and expressivity is uniform particularly valuable in a study of gene properties? 10.6. Two normal people marry and have a single child who is polydactylous on one hand only. How can you explain this? 10.7. A certain type of baldness is due to a gene which is dominant in men and recessive in women. A nonbald man marries a bald woman and they have a bald son. Give the genotypes of all individuals and discuss the penetrance of the genes involved. 10.8. A man has one brown eye and one blue eye. Explain. 10.9. How could you distinguish whether a given phenotype was due to a rare dominant gene with complete penetrance or a rare recessive gene of low penetrance? Chapter 11 STUDIES OF HUMAN TWINS 1 N THE preceding Chapter it was concluded that, in general, pene- . trance and expressivity may be modified by the environment or by the geno- type, or both. In organisms other than man, it is possible to standardize conditions experi- mentally, so that a standard genotype exposed to different environments would show to what extent environment was responsible for phenotypic variability, whereas a standard environment to which different genotypes were exposed would reveal to what extent these genotypes produced different pheno- types (cf. p. 6). Since neither the environ- ment nor the genotypes of human beings are subject to experimental control, the question may be asked, how can it be determined to what extent a particular human trait is con- trolled by genotype (nature) and by environ- ment (nurture)? Fortunately, this nature- nurture problem can be studied using the results of certain naturally occurring experi- ments. What are these? An individual contains many different parts, all of which can be presumed to have the identical genotype. Accordingly, as men- tioned in the last Chapter, one can attribute to nurture any phenotypic differences in ex- pressivity or penetrance found among parts which are essentially duplicates of each other. So, for example, a Polydactyly heterozygote with six fingers on one hand and five on the other illustrates the extent to which environ- ment can affect this trait. When, however, a trait appears which involves the entire individual, or which occurs either in several 74 nonduplicated parts of the body or in a single part, the contribution of nurture can be learned only from comparisons of different individuals who have identical genotypes. What is the probability that following sex- ual reproduction of human beings two indi- viduals of identical genotype will be produced? On the assumption that the members of each pair of chromosomes in the two parents are genetically different in one respect, then, the chance of two offspring being genically identi- cal is )r^ X }^", or K'*^. This is so because the chance a gamete will carry the same geno- type as another gamete of that individual is }<2^^, since chromosome pairs segregate inde- pendently, and because gametes fertilize at random. Since each human individual is heterozygous for numerous genes, the chance of obtaining genetic identity in two siblings (brothers and/or sisters of the same parents) is, in effect, infinitely small. However, two or more siblings with identi- cal genotypes may be produced as a conse- quence of asexual reproduction in man. This kind of reproduction occurs in the following manner. A single fertilized egg starts its development normally by undergoing a series of mitotic cell divisions. At some time, how- ever, the cells produced fail to adhere to each other, as they would normally do to form a single developing unit, but instead become separated into two or more parts, each of which may be capable of forming a complete individual. Each individual produced this way is genetically identical to all others formed from the same fertilized egg. The separation referred to may occur at the two- cell stage or it may occur later, at which time the number of cells may be unequal in the two or more groups formed. It is even possi- ble for these separations to occur twice, at different times in the development of a partic- ular zygote. Individuals produced this way are called identical or monozygotic twins, trip- lets, quadruplets, etc. We need only consider identical twins here, since multiple births of Studies of Human Twins 75 greater number are usually too infrequent to be useful for a general study of the nature- nurture problem. Multiple human births may occur also as a consequence of sexual reproduction. In this case the twins produced start with two separate eggs, each fertilized by a separate sperm. Such twins are genetically different, being, in this respect, no more similar than siblings conceived at different times, and are, therefore, called nonidentical or dizygotic {fraternal) twins. These two kinds of twins provide another natural experiment for determining the rela- tive influence of genotype and environment upon the phenotype. For monozygotic twins furnish the identical genotype in two indi- viduals, and both kinds of twins share very similar environments before birth and, when raised together, after birth. Accordingly, the phenotypic differences be- tween identical twins reared together are, barring the rare event of mutation, purely the consequence of environment (Figure 11-1). One can compare the average of these differ- ences between identical twins with the aver- age of the differences between identical twins who, for one reason or another, were reared apart, usually in separate families. This would yield information regarding the influence upon the phenotype of greater, as compared with lesser, environmental differ- ences. Since nonidentical or identical twins reared together are exposed to environments which vary to the same extent on the average, a comparison of the average difference be- tween identical twins and the average differ- ence between nonidentical twins will give an index of the role of the genotype in causing the differences observed. However, in order to collect data from twin studies, you can see how essential it is to be able to recognize in each case whether the twins are monozygotic or dizygotic in origin. The best way to identify twins as non- identical is to compare the two individuals phenotypically. They should be compared with regard to a large number of traits known to have a basis in genes that are 100% pene- trant and of fairly uniform expressivity. These would include such traits as sex, eye color, ABO, MN, Rh, and other blood group types. Naturally, only those traits for which at least one parent is heterozygous can be of use in testing the dizygotic origin of twins. Ignor- ing the rare event of mutation, any single difference in such traits would prove the twins nonidentical. Of course, two such differences would make your decision infallible for all intents and purposes, since two mutations occurring in the limited number of traits being followed in a pair of identical twins would be so rare as to be beyond any reason- able probability of occurrence. With these criteria, twins of opposite sex may be classi- fied immediately as nonidentical. Classification of twins as identical is based on the same procedure except that the greater the number of traits for which no difference at all is shown, the greater is the probability they are identical. For if the number of traits serving to test the genotypes of twins is sufliciently large, it becomes nearly certain that had they been dizygotic in origin they would have shown one or more differences, their failure to show any difference being attributable to their genotypes being identical because they were both derived from a single zygote. We are now in a position to study the rela- tive roles of genotype and environment in producing specific traits. What is done is to score the percentage of pairs of twins, reared together, in which one or both twins have the trait under consideration. Let us outline the procedure which we might actually follow. Suppose we wished to study the blood group AB m this respect. What we would do first is to eliminate from consideration all pairs of twins in which neither individual was type AB. We would have remaining, then, twins which had at least one member of AB 76 blood type. Then, we would determine the percentage of concordance, that is, the per- centage of cases where, given one twin to be of the specified phenotype, the other one is also. Now in the case of identical twins the concordance for AB blood type is found to be 100%. In determining concordance for non- identical twins, we should usually include in our data only those cases where the twins are of the same sex. This is desirable because the postnatal environment of twins of oppo- site sex is likely to be more different than the environment of identical twins — which must be necessarily of identical sex on our criteria. (Were both genotype and environment differ- ent for the two kinds of twins, we would not be able to specify the cause of a given pheno- typic difference which is greater among non- FiGURE 11-1. Identical twins, Ira and Joel, at 3^ months, at 8 years, and at 16 years of age. {Courtesy of Mrs. Reida Postrel Herskowitz, July 14th, 1946.) Studies of Human Twins 77 identicals than identicals.) The precaution of using only twins of the same sex has been taken in all the twin studies discussed here. The concordance of AB blood type is determined as approximately 64% for non- identicals. What conclusions can we draw? Had concordance been the same for both types of twins we would conclude that there is no genetic difference for AB blood group in the two types of twins. The concordances observed do differ, however, and do so in a particular direction. The 100% concord- ance for identicals is taken to mean that this trait is determined genetically with a pene- trance of 100% despite the environmental fluctuations normally occurring between identical twins. The lower percentage of concordance for nonidenticals cannot be at- tributed in any part to environment, since an equivalent amount of environmental fluctuation caused no differences in the case of identicals. This lower concordance must be attributed, therefore, to the differences in genotype which nonidenticals have in this respect. Of course, we could have predicted such results from our previous discussion (Chapter 5), where it was shown that AB blood type is genetically determined and is known to have complete penetrance. The lower concordance for nonidenticals, there- fore, must be due to their receiving different genotypes from parents, one or both of whom were heterozygous for I^ or P. It should be noted that it is theoretically possible to obtain a result in which concord- ance is lower for identicals than it is for non- identicals. Such a difference in concordance, if found, could be ascribed to environmental differences not being equivalent for both kinds of twins, being in fact greater among the identicals than among the nonidenticals. Let us discuss the results of concordance studies for some physical traits in twins (Figure 11-2). Concordance for clubfoot is 32% for identicals, but only 3% for non- identicals. The extra concordance of 29% (32^ 3%) found among identicals must be attributed to their identical genotype. The 3% concordance found among nonidenticals might be due entirely to similarity in genotype or entirely to the environment, or to some combination of these two factors. Since we cannot decide this from these data, it is con- cluded that in twins or other individuals exposed to the same general environment as are twins, the occurrence of clubfoot can be attributed to genotype approximately 29% of the time, with 32% as the approximate upper limit. In the case of the identicals, 68% of the time the second twin failed to have clubfoot when the first twin did. This failure of con- cordance is called discordance. The 68% discordance between identicals is attributable to differences in environment occurring be- tween the partners of a set of twins. It is IDENTICAL NON-IDENTICAL ABO BLOOD GROUP CLUBFOOT TUBERCULOSIS PARALYTIC POLIOMYELITIS 100 33 74 36 64 28 Figure 11-2. Discordance {umliaded) and percentage concordance {shaded) for various physical traits in twins reared together. 78 CHAPTER 11 concluded, then, that in twins or other indi- viduals exposed to the same general environ- ment as are twins, the occurrence of clubfoot is the result of the environment approximately 68% of the time, with 71% as the approxi- mate upper limit. Concordance-discordance studies reveal only the relative contributions of genotype and environment to a particular phenotype (clubfoot, in the case just discussed). Such studies do not teach us anything about the kinds of environment involved when the geno- type determines the phenotype under con- sideration, nor do they teach us anything about the genotypes involved when the en- vironment decides the phenotype. The twin studies just discussed also offer no informa- tion on the effect upon penetrance of clubfoot caused by environmental differences greater than those found between twins reared to- gether. In the case of tuberculosis, concordance is 74% for identicals and 28% for nonidenti- cals. Accepting the supposition that both types of twins have the same average exposure to the tubercle bacillus, the susceptibility to this disease is determined genetically 46-74% of the time and environmentally 26-54% of the time. In support of the view that the extra concordance among identicals has a genetic basis are the findings that concordant identicals usually have the same form of this disease, attacking the same place, with the same severity, whereas these similarities are less frequent among concordant nonidenticals. Paralytic poliomyelitis is 36% concordant for identicals and 6% concordant for non- identicals. Here, as in the case of tubercu- losis, the occurrence of the disease does not depend upon the infective organisms, because most human beings are exposed to these normally. Accordingly, the occurrence of this disease depends upon the rest of the environment 64-70% of the time and the genotype 30-36% of the time. In the case of measles, the fact that concordance is very high among both types of twins simply means that any genetic basis for susceptibility to this disease is quite uniform throughout the popu- lation from which the twin samples were obtained. The relative contributions of genotype and environment to personality and other mental traits may also be studied by the twin method. If a metronome is run at a series of different speeds, the tempo chosen as preferable will be different for different people. This tempo preference may be considered to be one aspect of the general personality. When tests are made to compare the preferred tempo of identical twins, the difference in their scores is found to be 7.8 of the units employed (Figure 11-3). This is, as might be expected, not significantly different from the difference in score of 8.7 units that is obtained by test- ing a given individual on different occasions. INDIVIDUALS DIFFERENCE IN SCORE Same person on different occasions 8.7 Monozygotic twins 7.8 Dizygotic twins 15.0 Siblings 14.5 Unrelated 19.5 FIGURE 11-3. Variation in tempo preference. {After C. Stern.) However, nonidenticals have a difference in score of 15 which is significantly different, being about twice that of the identicals. Since siblings born at different times have a difference in score of 14.5, they prove to be as similar in this respect as are nonidentical twins. Finally, unrelated persons show dif- ferences in score of 19.5 units. Since the greater the genetic similarity the smaller the difference in score, it may be concluded that Studies of Human Twins 79 there is a genotypic contribution to this per- sonaHty trait. Studies of twins for the mental disease schizophrenia show concordance of 86% for identicals and 14% for nonidenticals. How- ever, it is likely that the environment is not the same for both types of twins, more dis- cordance being produced by differences in social environment in the case of nonidenti- cals than in the case of identicals. Neverthe- less, in support of the view that not all the concordance for identicals is attributable to their similar environment, and that there is some genotypic basis for concordance, are two cases of identical twins who were sepa- rated, grew up in different environments, yet were concordant at about the same age. You are doubtless familiar with the fact that different people score differently on I.Q. examinations. We can use the differences in ability to answer questions on these examinations as a measure of what may be called test intelligence. While the scores of nonsiblings vary widely above and below 100, the difference between the scores of twins reared together is only 3.1 for identicals but is 8.5 for nonidenticals. Clearly identity in genotype makes for greater similarity in score. Tests of identicals reared apart show their scores differ by 6. In this case the greater difference in environment makes for a greater difference in performance of identi- cals, but this is still not so great a difference as is obtained between nonidenticals reared together. There are, therefore, both geno- typic and environmental factors affecting the trait test intelligence. Note that in the case of AB blood group we had previously discussed the nature of the genetic factors involved in the determination of the phenotype. We have not done this for the other traits studied in this Chapter. It should be re-emphasized, therefore, that though the twin methods used here tell whether there are genotypic differences asso- ciated with the occurrence and nonoccurrence of the phenotype under consideration, they do not offer any information regarding the nature of these gene differences. Whether or not the genotypic alternatives have any capacity for recombination, or whether or not they recombine in a regular predictable manner, cannot be determined from the data pre- sented. SUMMARY AND CONCLUSIONS In human beings, the occurrence of essentially duplicate parts within an individual, and of identical and nonidentical twins, offers the opportunity to test the effect of environment and of genotype upon the appearance of a given phenotypic alternative. A considerable number of physical and mental traits has been shown to be determined by the joint action of genotype and environment, sometimes the one and at other times the other having the greater influence. The twin methods described do not study the transmissive properties of the genotypes involved. They do not, therefore, reveal anything regarding the recombinational properties of the genetic factors studied. REFERENCES Kallman, F. J., Heredity in Health and Mental Disorder, New York, Norton, 1953. Montagu, A., Human Heredity, Cleveland, World, 1959. Newman, H. H., Multiple Human Births, New York, Doubleday, Doran, 1940. Osborn, F., Preface to Eugenics, Rev. Ed., New York, Harper, 1951. Osborn, R. H., and De George, F. V., Genetic Basis of Morphological Variation, Cam- bridge, Mass., Harvard, 1959. 80 CHAPTER 11 QUESTIONS FOR DISCUSSION 11.1. In determining whether or not twins are dizygotic, why must traits be studied for which one or both parents are heterozygotes? 11.2. Are mistakes ever made in classifying twins as being dizygotic in origin? Why? 11.3. When nonidentical twins are discordant for AB blood type, why must one or both parents have been heterozygous for /"^ or /^? 11.4. Invent a particular situation which would result in greater discordance for identical than for nonidentical twins. 11.5. What would be the probability of twins being dizygotic in origin if both had the genotype aa Bb CC Dd Ee Ff, each pair of alleles segregating independently, if the parents were genotypically Aa Bb CC DD Ee Ff and Aa BB CC dd ee FFl 11.6. How would you proceed to test whether, in women, there is a genetic basis for the maturation of more than one egg at a time? 11.7. In what way can you imagine that the paternal genotype could influence the frequency of twinning? 11.8. Is tuberculosis inherited? Explain, 11.9. What can twin studies by themselves tell you about genes? about genetic recombi- nation? 11.10. Is it valid to apply the conclusions from twin studies to nontwin members of the population? Explain. Chapter *12 SEX-LINKAGE WE HAVE already found that different pairs of genes seg- regate independently, and have hypothesized that this behavior is due to different pairs of genes being located in differ- ent pairs of chromosomes. You may now ask what the genetic basis for sex is. In the case of the garden pea we cannot obtain the answer from a study of just the two alternatives, male- ness and femaleness, since all the pea plants dealt with were bisexual. So long as there are only two alternatives for the sex trait and both occur in every individual, there can be no phenotypic differences produced by segrega- tion and recombination, and a genie basis for sex cannot be determined. We can, however, attempt a study of the genetic basis for sex, say in Drosophila, where the typical individual is either male or female (Figure 12-1). When normal males and females are mated, their progeny are in the approximate pheno- typic ratio of male : female as 1:1. This permits the hypothesis that sex is determined by a single gene pair, and that one of the sexes of Drosophila is a homozygote and the other is a heterozygote. At the moment, however, we cannot say which sex carries which genotype. In accordance with our view that chromosomes contain the genes, there should be one pair of chromosomes con- cerned with sex. Let us call the homologous pair of chromosomes, which the homozygote for the sex genes carries, the XX pair, and the pair carried by the heterozygote, the XY pair. Segregation and random fertilization will pro- duce equal numbers of XX and XY individ- 81 FIGURE 12-1. Normal (wild-type) Drosophila me\- anogaster male (A) and female (B). (Drawn by E. M. Wallace.) uals. Since the X and the Y chromosomes carry the genes for sex, these can be called sex chromosomes, while the other chromo- somes which an individual carries can be called autosomes (A). Since Drosophila melanogaster has a diploid chromosome num- ber of four pairs, each individual can now be represented as either XX -f 3AA or XY -f 3AA. Consider first crosses involving the reces- sives cubitus interruptus, ci, and ebony body color, e, and their dominant alleles c/+ (normal wing venation) and e+ (gray body color). Let one cross be c/+c/ e+e X ci ci e e, in which one parent is dihybrid and the other parent is the double recessive (Figure 12-2). 82 CHAPTER 12 The offspring appear in a 1 : 1 : 1 : 1 ratio, demonstrating that the two pairs of genes are segregating independently. The same result and conclusions obtain from the cross of ci+ci e e X ci ci e^e. Consider next crosses in which sex and wing venation are studied simultaneously both in ci^ci XX by ci ci XY and in ci+ci XY by ci ci XX. The result found in both cases is a 1 : 1 : 1 : 1 ratio of cubitus male, cubitus female, normal male, normal female. Here, then, the sex genes segregate independently of the genes for cubitus. Therefore, by our hypothesis, the ci alleles are located autosomally. Similarly, e+e XX by e e XY or e+e XY by e e XX also gives a 1 : 1 : 1 : 1 ratio, show- ing that ebony is located autosomally. It may be concluded also, since ebony and cubitus are found to segregate independently of each other, that they are located on different pairs of autosomes. Note that the last two types of crosses could be described as backcrosses of a mono- hybrid which wei-e made reciprocally, that is, one time the male was the hybrid parent, and the other time the female was, even though we cannot yet specify that male is XX or XY. In either case a 1 : 1 ratio is found among the FIGURE 12-2. Results of buckcrossing a dihybrid. + + ci ci e e x CI CI e e ^, 7i sons, and a 1 : I ratio among the daughters. We may now reconsider the meaning of the statement made earlier (p. 9), that all crosses gave the same results when made reciprocally. This meant that the observed phenotypes and their proportions were the same for sons as for daughters, even though the crosses were made reciprocally. So, for example, a cross of the dihybrids ci+ci e+e X ci+ci e+e gave a 9 : 3 : 3 : 1 ratio among the sons and a 9:3:3:1 ratio among the daughters be- Sex-Linkage 83 d^^'Ox white c/ B I "^'dc/ 9? d red dull red m 99 white dull red FIGURE 12-3. Phenotypic re- sults of reciprocal matings in- volving eye color. v.-.-/ cause the sex genes were located on the sex chromosomes while the other genes happened to be autosomal. Therefore, in all previous work, we were dealing with autosomal trans- mission. This always showed independent segregation from the sex genes and permitted the statement that sex did not influence the results; that is, the results were the same among the sons as among the daughters even though reciprocal matings were made. But consider now the results of crosses involving the dull red (h'+) and white {w) eye color alleles. Dull red 9 X white cf (Figure 12-3 A) produces all dull red sons and daugh- ters in Fi, as expected, w+ being dominant. However, the reciprocal cross (Figure 12-3B) of white 9 X dull red cf gave only white sons and dull red daughters. Note that the first cross produced the same result for sons as for daughters, but the second, reciprocal, cross gave different results, sons resembling their mothers, and daughters resembling their fathers. Because of this diff"erence in result from reciprocal matings, we must conclude that w+ and its alleles are not located auto- somally. Let us assume that the gene for white eye is located in the sex chromosomes, being therefore sex-linked, and see what conse- quences this would have for its transmission relative to the sex phenotype.^ In the rest of this Chapter we will not be particularly con- cerned with learning the genetic basis of sex in any greater detail, but will utilize princi- pally the hypothesis that the two sexes are XX and XY. ^SeeT. H. Morgan (1910). 84 CHAPTER 12 Assume first that females are XY and males XX. The first cross is then dull red 99 (fe- males) X"'"Y«'* by white cf cT (males) X"'X'^ (Figure 12-4, A-1), and the Fi expected are X"'*X"' sons and X"'Y'"'^ daughters, all dull red-eyed, as observed. The reciprocal cross (Figure 12-4, B-1) is, then, white 99 X'-'Y"- by dull red d" & X'" X"'\ The Fi daughters (X'^'Y'") are expected to be dull red-eyed, as observed. However, the Fi sons (X"'"X"') are expected to be dull red-eyed, whereas they are actually white-eyed. Therefore, we must reject this particular hypothesis for correlat- ing sex chromosomes and eye color genes. Let us assume next the reverse situation — that females are XX and males XY. The same crosses are represented now as dull red 99 X'-'^X"" by white d^d X^-Y"- producing X-^X- (dull red) daughters and X-^Y'" (dull red) sons (Figure 12-4, A-2); reciprocally, white 99 X-X- by dull red dd x-^Y-* gives X'""X«' (dull red) daughters and X"'Y«'^ (dull red) sons (Figure 12-4, B-2). The expected phenotype given last is contrary to fact, the phenotype of the Fi sons being white, not dull red. Since we cannot explain the observations merely by identifying maleness with XX or XY, we shall have to increase the number of assumptions used in an attempt to accom- plish this. Let us test two hypotheses simul- taneously, namely that Drosophila males are XY and that the Y chromosome can carry only w, and cannot carry w+. The genotypes and results of the first cross given in the last para- graph remain the same (Figure 12-4, A-3). The reciprocal cross (Figure 12-4, B-3) be- comes white 99 X'^X"' by dull red d& X'"'Y"' to produce X«"X«' (dull red) daughters and X"'Y"' (white) sons, as observed. Since these hypotheses fit the observations we may accept them. There are a large number of other traits which, hke white eyes, can be studied one at a time in Drosophila. Their transmission genetics also proves to be based upon a pair of genes on the sex chromosomes, each case giving different results in Fi when pure lines of the two alternatives are crossed recipro- cally. Moreover, each case can be explained by assuming that females are XX, males XY, with the Y carrying the most recessive and least effective allele known for the gene pair under test, as is the case for white. The finding, in dozens of different cases, that the Y chromosome always behaves as though it contains the least influential allele of the gene pair, tempts the hypothesis that for the gene pair under test the Y in fact contains no allele at all! The very fact that a partially or com- pletely dominant allele of such a gene is normally never found on the Y of Drosophila must mean that such alleles cannot be formed there by mutation of the most recessive allele, most simply because this recessive allele does not exist on the Y. Accordingly, the Y can routinely be considered to lack an allele of a gene located on the X, and Figure 12-4, A-3 and B-3 should have Y substituted for each Y-. In all the cases where the Y carries no allele of a gene on the X, because sons receive their single X from their mother, they will show phenotypically whatever is contributed in the X they receive from their mother. With re- gard to these genes, therefore, a female is being test crossed whenever (or to whomever) she mates, since her genotype can be determined directly from the phenotypes of her sons. Genes present on the X chromosome and absent on the Y are said to be hemizygous in the Drosophila male, because half of the zygotes he produces will receive these alleles in the X he contributes, while the other half will not because they receive the Y. Note that the X of a Drosophila male is obtained from his mother and is transmitted to each of his daughters. In the case of chickens, nonbarred feather 99 X barred feather cf cf produces offspring which are all barred — barred (B) being domi- nant to nonbarred (b) (Figure 12-5 A). In the Sex-Linkage A-l B-l w w X Y 99 x'"y"99 A-2 B-2 p, XX yxxvu p, xxyxXvU F, F^ i x">99 x">9? w w X X A-3 9 x->99 w w X X Y ^ w w X X B-3 + 9w w X X Y c/ W W X Y c/c/ *x-99 FIGURE 12-4. Three attempts (A-l and B-l, A-2 and B-2, A-3 and B-3) to represent matings A and B in Figure 12-3 genotypically. Shaded genotypes must be incorrect. reciprocal cross (Figure I2-5B) of barred 99 X nonbarred cf cf all sons are barred and all daughters nonbarred. Here also the re- sults of reciprocal matings differ, so that we are dealing again with sex-linkage. Note from the second cross that the exceptional individuals are the Fi that show the recessive trait, as was the case in Drosophila. But in the present case the sex is opposite, since the Fi which are nonbarred are females, whereas the exceptional Fi Drosophila were white-eyed males. In order to explain these results we can assume, as in Drosophila, that sex is determined by XX vs. XY, that the X chro- mosome does, and the Y chromosome does not, contain a gene for barred or nonbarred feathers, but that, contrary to the situation in Drosophila, males are XX and females XY. 86 CHAPTER 12 F^ Nonbarred Q x Barred Barred (J O 99 Barred c/. B Barred Q x Nonbarred Barred (30 Nonbarred c/ 99 A-l B b p) B B/-^ P, XYVx XX*^ X Y ^^ X Y FIGURE 12-5. Phenotypie {A and B) and genotypic (A-l and B-1) results of reciprocal matings involving barred and nonbarred feathers in chickens. B p. b b^ P, XYVx XX^ ^^ 99 B b X X The genotypes of the bird crosses are, on these hypotheses, X''Y (nonbarred 9 ) by X^X^ (barred (f) producing X^Y (barred 9 ) and X^X* (barred cf) in Fi; the reciprocal mating of X«Y (barred 9 ) by X''X'' (non- barred cf ) produces X'-Y (nonbarred 9) and X^X* (barred d") in Fi, all as observed (Figure 12-5, A-l and B-1). You may be wondering if any clues can be obtained, from cytological observation of chromosomes, for the absence of alleles on the Y which are present on the X, both in the case of Drosophila and of poultry. It would be reasonable to expect, if the gene content for X and Y is so different, that this might be reflected in a difference in the cytological appearance of the two kinds of sex chromo- somes. Note, however, our explanation of sex -linkage has been made independently of any cytological expectation. It is found cytologically (Figure 12-6) in Drosophila that three pairs of chromosomes are the same in the male and the female, homologous chromosomes being very similar morphologically. In the female the homo- logs of the fourth pair are also similar mor- phologically, but in the male, while one member of the fourth pair is just like the homologs of the fourth pair in the female, its partner chromosome has a distinctly different morphology. Thus, the distinctive cytological appearance of this last chromosome is con- sistent with the genetic expectation for a Y chromosome, being present once in the male and not at all in the female. The other homo- log in the male is then called the X, and is Sex-Linkage 87 FEMALE JJK X X MALE 4^ FIGURE 12-6. Silhouettes of chromosomes of Drosophila melanogaster as seen at mitotic meta- phase. present twice in the female. Moreover, the reverse cytological picture is observed in poultry; here the males show that the homo- logs are similar for each pair of chromosomes, while the female has one pair that is hetero- morphic, that is, one whose members are dif- ferent from each other in appearance, one member being similar to, and one different from, the corresponding pair in the male. In moths, as in birds, it is also found that males are XX and females XY. In human beings, genetic and cytological evidence shows XY to be male and XX to be female, as in Drosophila. It might be mentioned that in man a certain kind of red-green color-blind- ness is sex-linked, due to a recessive allele, c, present on the X and absent on the Y. Ac- cordingly, color-blind women, X'X% who marry normal men, X^Y, have normal daugh- ters, X^X^ and color-blind sons, X'^Y. The classical bleeders' disease in human beings, hemophilia type A, is also due to an X-linked recessive gene, /?, absent from the Y. This is a rare disease usually occurring in males; recently, however, a few hemophilic women have been discovered in England. These homozygotes are viable; they are extremely infrequent because they must have for parents a hemophilic father, X''Y, and a heterozygous mother, X^X'' (Figure 12-7). Let us consider certain additional experi- ments performed with the sex-linked gene for white eye in Drosophila} When white 99 (X«'X«') are crossed to dull red d" d" (X-^'Y) and large numbers of progeny are scored, almost all Fi are white sons (X"'Y) and dull red daughters (X"'"X"'), as explained. But one or two Fi per thousand do not show this typical result of sex-linkage, but are exceptional in being dull red-eyed sons or white-eyed daughters (Figure 12-8 A). These exceptional flies cannot be explained as the result of careless scoring of phenotypes or contamination by strange flies. Moreover, they cannot be explained as being due to mu- tation, since the mutation rate from m'+ to w or the reverse is several orders of magnitude lower in frequency than that with which the two kinds of exceptional flies are obtained. ^ Based upon work of C. B. Bridges. FIGURE 12-7. Pedigree showing a woman homo- zygous for the Iwmophilia gene. / 4-riii ^h-D 6 6 A. PHENOTYPES R White CHAPTER 12 Red F, TYPICAL white L S Idull ''* Female | i. ■ .j diploid 3 2 3 2 ^ haploid 1 1 Intersex 2 3 Normal male 1 2 Supermale 1 3 SEX INDEX No. X's No. A sets 1.5 1.0 1.0 1.0 1.0 0.67 0.50 0.33 Sex Determination (If) 105 ried IX + lA set (being haploid) have been found. As expected from their sex index of 1.0, these individuals or parts were female. Since all known facts support the exact cor- respondence between chromosomal consti- tution and sexual types, we can accept chromosome balance as the typical basis of sex determination in Drosophila. What is the relationship between X-auto- some balance and tra, the sex transforming gene? Sex is determined by X-autosome balance when the individuals carry trci+, which they normally do. On those rare occasions when tra tra is present, the balance view does not apply, 2X + 2A sets, you recall, producing male. We have seen in Drosophila that determina- tion of sex is primarily genetic and that the sex type differentiated in different parts of an individual depends only upon the genotype carried by these parts (Chapter 13). Autono- mous sexual development of each part of this organism is possible because of the absence of hormones affecting sexual differ- entiation. What is the situation in man? In human beings sexual type is determined at fertilization, Y-bearing zygotes becoming males, Y-less zygotes females. The manner in which the appropriate sex phenotype is produced can be described briefly as follows, liarly in its development, the gonad is neu- tral, i.e., there is no macroscopic indication whether it will later form testis or ovary. This early gonad has two regions, an outer one called the cortex and an inner one called the medulla. As development proceeds, the cortex degenerates in those individuals that carry a Y (males) and the medulla forms testis, while in those individuals genetically destined to be females the medulla degener- ates and the cortex forms ovary. Once the testis and ovary are formed, they take over the regulation of further sexual differentiation by means of the hormones they produce, so that these hormones direct the development or degeneration of various sexual ducts, the formation of genitalia, and other sexual characteristics. Since sexual dif- ferentiation is largely under the control of the sex hormones it should not be surprising to find genetically normal individuals who are morphologically abnormal with regard to sex. For any abnormal condition in the environment, which can upset the production of sex hormone and/or the response of tissues to it, may produce effects during development which result in an abnormal sex phenotype. Such abnormal sex phenotypes due to en- vironmental factors could be intersexual, and theoretically could be produced either from genotypic males who have developed partially in the direction of female or from genotypic females partially differentiated in the direc- tion of male. Some intersexes in humans prove to be due to an abnormal genotype present at fertilization, as is true for all inter- sexual Drosophila; other human intersexes result from genotypic mosaicism, which pro- duces gynandromorphs in Drosophila where sex hormones are absent. Of course you realize that the phenotypes normally con- sidered male and female show some vari- ability. So, while it is sometimes easy to classify an individual as being an intersex, because that person is clearly between the two sex norms, other individuals at the ex- tremes of normality may not readily be labeled as normal, or intersex, or supersex. It is debatable whether human beings that are XO or XXY, but otherwise diploid, should be called intersexes, or whether they should be considered of normal but of under- developed sex. Since we have just been discussing how the genotype may be related to the production of intersexes, consider how the genotype is related to the sex ratio, that is, the relative numbers of males and females born. On the average, 106 boys are born for each 100 girls. At first this might surprise you, since half of sperm are expected to carry X, and half Y, and all eggs an X, so that the sex ratio 106 CHAPTER 14 expected is 1 : 1 as boy : girl. Even if the four meiotic products of spermatogenesis are usually X, X, Y, Y, the possibility exists that during or after spermiogenesis (conversion of the telophase II cell into a sperm) some X- bearing sperm are lost. This is supported by a report that human ejaculates contain sperm heads of two sizes and shapes, the smaller type being in sufficient excess to explain an excess of males at fertilization, if the smaller sperm contains the smaller Y chromosome and the larger sperm carries the larger X chromosome. It is likely, then, that at conception males and females are unequal in number. A study of the sex ratio at birth has shown that the ratio of 1.067 : 1 is found only among young parents, and that it decreases steadily until it is about 1.036 : 1 among the children of older parents. How might this significant decrease be explained? It might be due to a greater chance for male babies to abort in older mothers. It might be due, in part, to the increased likelihood, among older mothers, for a chromosome to be lost, in the earliest mitotic divisions of the fertilized egg, -by failing to be included in one of the daughter nuclei. For, if the chro- mosome lost was an X, and the zygote was XY, then the loss would be expected to be lethal, and what would have been a boy would be aborted, while if the zygote losing an X was XX, a girl would still be born. Moreover, if the chromosome lost in the XY individual was a Y, a girl might be born instead of a boy. Part of the effect must be due to the increase in meiotic nondisjunction with maternal age (zygotes of XXX type form viable females, while zygotes of YO type are expected to be lost before birth). We must not preclude the possibility that the fathers are somewhat responsible for this shift in sex ratio. There may be an increase, with paternal age, in postmeiotic selection against Y-carrying sperm. Perhaps, as fathers become older, there is an increased chance for certain abnormal events to take place during the meiotic divisions preceding sperm formation. How could this be of sig- nificance? Suppose the XY tetrad undergoes nondisjunction in such a way that from a given prophase I cell, the four meiotic prod- ucts, each forming a sperm, contain, re- spectively, X, X, YY, 0. The first two sperm listed would produce normal daughters, the last one an underdeveloped XO daughter, while only the YY would be capable of pro- ducing maleness. Moreover, this "male" individual would be XYY and we do not know if such a genotype is viable. While still other genetic and nongenetic explanations for the shift in sex ratio with age are possible, the present discussion will suffice to demon- strate how the basic facts of sex determina- tion, chromosome loss, and nondisjunction may be used to set up various explanatory hypotheses whose validity may subsequently be subject to test. The sex ratio has been found abnormal in another respect. When pedigrees are ex- amined for sex ratio, it occasionally is found that a dozen consecutive births are of the same sex. This could happen purely as a matter of chance if enough pedigrees are scored, just as it is possible (but unlikely) that you could have a coin fall with the same side up in a dozen consecutive tosses. But one family is reported to have had only boys in 47 births, and in another well substantiated case a family has had 72 births, all girls. In both these cases the result is so improbable that it is not reasonable to consider it to be merely due to chance! While we do not know the basis for such results in man, we can ex- amine two different cases in Drosophila in which almost only female progeny are pro- duced; these might suggest an explanation for those human pedigrees in which only one sex occurs in the progeny. In the first case in Drosophila, the males were found to be responsible for the almost exclusive production of daughters. These males are XY but carry a gene called "sex Sex Determination {II) 107 FIGURE 14-6. Head shapes in human sperm. Round-headed sperm are reported to be smaller and more numerous than oval-headed sperm, suggesting these carry the Y and X chromosomes, respectively. {Courtesy of L. B. Shettles.) ratio." Because of this gene, almost all Y chromosomes degenerate during meiosis and almost all sperm carry an X. In the second case, the females were the important factor, for it was found that these females transmit a spirochaete microorganism to their offspring through their eggs. Such females, when mated to normal males, produce zygotes which in- itiate development. But very early in devel- opment the spirochaete kills XY individuals, so that almost all survivors are female. Finally, when one talks about sex deter- mination the question often comes up: Can sex be predetermined? In theory the answer 108 CHAPTER 14 is yes, in the sense that if we could control the genotypes of zygotes we would determine ahead of time what sex would be formed. And again, since X- and Y-bearing sperm of men apparently differ in cytological appear- ance (Figure 14-6; see also Figure 14-7), it should be theoretically possible to separate these, thereby leading to the control of the sex of progeny. Using various animal forms, ex- periments along these lines have been per- formed with some success by Russian, U.S., and Swedish workers using electric currents or centrifugation. Though these methods have given encouraging results, the results are not yet consistent and the techniques not yet suitable for practical use. FIGURE 14-7. According to L. B. Shettles, these phase-contrast photo- graphs show a concentric arrangement of the chromosomes present in rounded and elongated types of sperm head. A, B, C, and D from different men. The light, lobulated chains are stained by the Feulgen-Rossenbeck procedure. SUMMARY AND CONCLUSIONS Genes responsible for sex determination are located not only in the sex chromosomes, but in the autosomes as well. Although sex type may be changed through the action of a single pair of genes, a given sex is usually the result of the interaction of several, and probably many, pairs of genes. Sex is, therefore, a polygenic trait (Chapter 8), in which the usual chromosomal differences found among zygotes serve as visible manifestations of differences in the balance of genes concerned with sex, and by which one balance produces male and another female. Whenever, as in female Drosophila, genie balance is unaffected by the addition or subtraction of whole sets of chromosomes, sex also is unaffected. However, changes in chromosome content which make for intermediate genie balances produce inter- mediate sex types — intersexes; those which make the balance more extreme than normal produce extreme sex types — supersexes. Sex Determination (II) 109 These principles of sex determination presumably apply also to human beings. In man and many other organisms, a large part of sexual differentiation is under the control of sex hormones produced by the gonads. While this type of control makes the occurrence of individuals who are typically male in one part and typically female in another part very unlikely, it may lead to the formation of abnormal sex types for nongenetic reasons. REFERENCES Bangham, A. D., "Electrophoretic Characteristics of Ram and Rabbit Spermatozoa," Proc. Roy. Soc, Ser. B, 155 : 292-305, 1961. Bridges, C. B., "Sex in Relation to Chromosomes and Genes," Amer. Nat., 59:127-137, 1925. Reprinted in Classic Papers in Generics, Peters, J. A. (Ed.), Englewood ClitTs, N.J., Prentice-Hall, 1959, pp. 117-123. Goldschmidt, R. B., Theoretical Genet- ics, Berkeley and Los Angeles, University of California Press, 1955. Shettles, L. B., "Nuclear Morphology of Human Spermatozoa," Na- ture, London, 186:648-649, 1960. Shettles, L. B., "Nuclear Structure of Human Spermatozoa," Nature, London, 188:918-919, 1960. Sturtevant, A. H., "A Gene in Droso- pliila Melanogaster that Trans- forms Females into Males," Genetics, 30:297 299, 1945. Alfred H. Sturtevant (//; 1945). QUESTIONS FOR DISCUSSION 14.1. Does a gene have to have an alternative allele before it can be discovered? Explain. 14.2. Make a schematic representation of a trivalent as it might appear during synapsis. Suppose each carried a different allele, a\ a~, a\ of the same gene. Show diagrammatically the chromosomal and genetic content of the four meiotic products you might obtain from the trivalent you drew. 14.3. Ignoring chiasma formation, how many chromosomally-different kinds of eggs can a triploid Drosophila female produce? How many of these eggs have more than a 5% chance of occurring? 110 CHAPTER 14 14.4. What explanations can you offer, other than those presented, which may pertain to the shift in sex ratio with age of human parents? 14.5. No YO human beings are known. Is this chromosomalconstitution considered lethal? Why? 14.6. List the types of human zygotes formed following maternal nondisjunction of the X chromosome. What are the phenotypes expected for each of the zygotes these may form in turn? 14.7. List specific causes for the production of abnormal sex types in human beings. 14.8. How can you account for the fact that one XO individual is known who has had a successful pregnancy, whereas most known XO's are sterile? 14.9. Discuss the general applicability of the chromosomal balance theory of sex determi- nation. 14.10. In Drosophila, why are gynanders not intersexes? Is this true in man also? Explain. 14.11. What chromosomal constitution can you give for a triploid human being who is "male"? "female"? Which of these chromosomal constitutions would you expect to deviate most from its normal sex? Explain. 14.12. A non-hemophilic man and woman have a son who proves to be a hemophilic Klinefelter male. Describe the chromosomal content and genotypes of all three individuals mentioned. Chapter *15 INTERGENIC LINKAGE I 't was reported in Chapter 6 (pp. 46-41) that each of the first .seven pairs of genes studied in the garden pea segregated independently. This can be attributed to each pair of genes being located on a different pair of the seven pairs of chromosomes which this organism carries. What we should consider now are the results obtained when an eighth pair of genes, show- ing dominance and affecting an unrelated trait, is studied simultaneously with each of the other seven pairs in turn. When a dihy- brid for a particular one of the seven and for the eighth gene pairs is made, a 9 : 3 : 3 : 1 phenotypic ratio is obtained when the dihy- brid is self-fertilized, and a 1 : 1 : 1 : 1 phe- notypic ratio is obtained when this individual is backcrossed to the double recessive. This demonstrates in two independent tests that the two pairs of genes involved are segregat- ing independently and are located on non- homologous chromosomes. The same ratios are obtained, from the same types of crosses, with dihybrids made with each of five other genes and this eighth gene pair, so that the same conclusion also applies for them. How- ever, the results obtained from the dihybrid made with the remaining gene pair and the eighth will be described in some detail, since these results are radically different. The seventh pair of genes happens to be the one concerned with the seed coat, a trait we have already studied in Chapter 6, whose allelic alternatives are round (R) and wrinkled (r). As mentioned earlier, we may use vari- ous symbols to represent genes. In the pres- ent case, the first letter (or so) of the pheno- type produced by the dominant allele (the one normally found in nature) has been used in upper and lower case to represent the domi- nant and recessive alleles, respectively. In other conventions (see Figure 15-1), the first letter (or so) of the recessive mutant (not normally found) allele is used in lower case for the recessive allele (wrinkled = h-), while the normal dominant allele is given either the same symbol in upper case, or is given a + symbol as a superscript or base to the lower case symbol, or is given as + alone (so that round may be, respectively, W, w+, +«', +). Sometimes the hybrid +1^ is represented as = or — or +/vv to show that these alleles w w are on different members of a pair of homol- ogous chromosomes. W w w w + — +/ w w FIGURE 15-1. Various ways of representing t/ie round-wrinkled hybrid by gene symbols. The eighth pair of genes deals with the presence (+) and absence (/) of tendrils, threadlike structures used for attachment as the plant climbs. When a double recessive pea plant (wrinkled, no tendrils = w w 1 1) is crossed to a pure double dominant (round, tendrils = + -f + +), all Fi are round with tendrils (+ vv + /), as expected. (Notice we are using the + system for symbols here.) When the Fi are self-fertilized (dihybrid X dihybrid) the following results are obtained in F2: Phenotype No. Individuals round, tendrils 319 round, no tendrils 4 wrinkled, tendrils 3 wrinkled, no tendrils 123 Note that each gene pair shows segregation in the Fo since round : wrinkled as 323 : 126 (a 3 : 1 ratio) and tendrils : no tendrils as 112 CHAPTER 15 322 : 127 (a 3:1 ratio). Yet these 3 : 1 ratios are not produced independently of each other, for if they were they would give a 9 : 3 : 3 : 1 ratio, which is certainly not found here. Instead, there are in F2 relatively too many plants phenotypically like the Pi parents (wrinkled, no tendrils; round, ten- drils), and relatively too few, new recombina- tional types (round, no tendrils; wrinkled, tendrils). Examine also the phenotypic results ob- tained from backcrossing the dihybrid in question (-{- w -\- t X w w 1 1): Phenotypes No. Individuals round, tendrils 516 round, no tendrils 9 wrinkled, tendrils 7 wrinkled, no tendrils 492 The expectation according to independent segregation is a 1 : 1 : 1 : 1 ratio for each of the types obtained. Actually, there are relatively excessive numbers of gametes, pro- duced by the dihybrid, containing the old combinations (+ + and w t) — that is, the combinations which came from the parents to form the dihybrid — and relatively too few new combinational or recombinational types, just as is true in the case where two of these dihybrids are crossed. We conclude again, therefore, that independent segregation does not obtain here. The fact that we get some recombinational types in the present case confirms the earlier statement (which was really an assumption) that we are actually dealing with two separate pairs of genes. Let us assume that the two pairs of genes involved are located on the same pair of homologous chromosomes, a possibility which was introduced in Chapter 6 (p. 46). In this event, the genes are said to be linked to each other because they are on the same chromosome. Recall that the phe- nomenon of sex-linkage, already discussed in Chapter 12, dealt with a single gene (such as that for white eye) and its location on or linkage to a particular chromosome (the X chromosome). We are now concerned with studying intergenic linkage, which involves all the nonallelic genes presumed to be located on the same chromosome. (Evidence bearing on this can only be obtained by studying the transmission genetics of more than one trait, other than sex, at a time.) For the first time, then, we shall be testing the hypothesis that a chromosome contains more than one gene. Let us re-examine, by means of Figures 15-2 and 15-3, respectively, the results of the two kinds of crosses described. In these Figures we will use a horizontal line to repre- sent a chromosome and indicate the presence of one member of each gene pair on each chromosome. In those cases where the genes could be either the normal or the mutant allele, a question mark is placed in the appro- priate position. If linkage had been complete, that is, if the chromosome carrying w t or H — \- was forever unchangeable (except for the rare event of mutation), then all results in Figure 15-2 down through the genotypes of the P2 would be consistent with this view. However, the occurrence of seven recombina- tional individuals shows that linkage is not complete. These recombinational individuals have a chromosome which has kept one allele and received the nonallele present in the homologous chromosome. Moreover, the reciprocal type of recombinant is apparently equally frequent, as though a given pair of genes had switched positions in the homologs — that is, as though they had reciprocally crossed over. For this reason such recom- binational individuals are said to carry a crossover chromosome produced by a process called crossing over. So, complete linkage between genes is prevented by a genetic proc- ess, crossing over, which produces genetic recombinations called crossovers. What other rules, if any, can we establish for the crossing-over process and the cross- overs it produces? Among the progeny ob- tained from backcrossing the dihybrid (Figure 15-3), 16 received crossovers in the gametes Wrinkled, no tendrils wt X Round, tendrils 113 wt Round, tendrils wt + + FIGURE 1 5-2 {Left) . Linkage between nonallelic genes in the garden pea. Pj F, Round, tendrils (self-fertilized) + + + + wt wt Round, tendrils + + ?? 319 Round, no tendrils + t ?t 4 Wrinkled, tendrils wt 3 w? Wrinkled, no tendrils wt wt 123 TOTAL 449 FIGURE 15-3 (Right). Linkage be- tween nonallelic genes in the garden pea. The dihybrid parent was ob- tained from the Fi in Fig. 15-2. F Round, tendrils x Wrinkled, no tendrils wt w t w t contributed by the dihybrid and 1008 did not. The reciprocal crossover classes are appar- ently equally frequent. So approximately one crossover was produced for each 63 non- cross9vers. The F2 results in Figure 15-2 are consistent with this proportion, as you will find if you work it out. It is also possible to make a dihybrid for these genes which receives one mutant Round, tendrils Round, no tendrils Wrinkled, tendrils Wrinkled, no tendrils + + 516 w t + t 9 wt mO^ 7 wt wt 492 wt - TOTAL 1024 114 CHAPTER 15 and one normal (w +) from one parent, and one normal and one mutant (+ /) from the other parent. When this dihybrid is back- crossed, the crossovers (w t or -\- +) and non- crossovers (u' + or + /) also occur in the pro- portion 1 : 63. Apparently crossing over is a genetic phenomenon which occurs with the same frequency whether the two mutant genes enter the dihybrid from the same or from dif- ferent parents. Crossovers, therefore, occur in the gametes of an individual with a fre- quency that is constant and independent of the specific way the alleles were carried by the gametes that fused to initiate that individual. If this is so, then it must follow that even in -f +/+ + and w t/w t individuals, one gamete in each 64 produced is a crossover for these genes, but is undetected because it carries no new combination of alleles. We see then that when two linked mutants enter a zygote together, because they are located on the same chromosome, they tend to stay together when transmitted to the next gen- eration {coupling), but if they enter the zygote separately, being located on different mem- bers of a pair of homologous chromosomes, they tend to be transmitted separately to the next generation {repulsion). In another species, the sweet pea, it is found that the trait purple flowers is due to a single gene (+) whose recessive allele (/•) produces red flowers. Long pollen (+) is dominant to round pollen {ro). A pure line of purple long (+ +/ + +) crossed to red round {r ro/r ro) produces Fi that are all purple long (+ -\-/rro). Since self-fertilization of the Fi produces too many Pi phenotypes and too few, new, recombinational types (purple round and red long) for these two pairs of genes to be segregating independently, these genes also must be linked. But, as before, linkage is not complete. In this case, the crossovers obtained can be accounted for if the P2 (Fi) dihybrid forms gametes in the relative proportions 10-f -I- : lOrro : 1 +ro : 1 r+. This rate of crossing over is obtained no matter how the genes enter the dihybrid. Notice, however, that this constant rate (Ki) diff"ers from the rate observed in the garden pea example previously discussed O^). Consider also the rate of crossing over in two other cases. You recall, in Drosophila, that the mutant gene, w, for white eye is X-linked. So also is a different mutant gene which produces miniature wings {m). Using pure lines, a white-eyed, long-winged fly is crossed to a dull red-eyed, miniature-winged one. The Fi female carries two X's and is, therefore, w +/+ m. This female is then mated to any male and the sons are scored phenotypically. Any male can be used as parent, since any male will transmit to his sons a Y chromosome that is devoid of the genes under consideration. In fact, it can be shown that the Y is lacking most of the genes known to be present on the X, the gene bobbed bristles, bb, being a notable excep- tion. (Besides a bb allele, the Y contains several genes for male fertility which have no alleles on the X.) Since the sons will re- ceive their X from their mother, they will show by their phenotype directly which of these alleles each received in the gamete she provided. It is found among the sons of this mating that about one crossover type appears for every two that are noncrossovers. Finally, in man, color-blindness (c) and hemophilia type A {h) are recessive X-linked mutant genes absent on the Y chromosome. Though rare, there are some women with the genotype + h/c +, i.e., having one of these mutants on each X. Available data indicate that crossover {c h or -\ — [-) and non- crossover {-\- h or c +) sons occur in the approximate ratio of 1 : 9. These results show that when linkage be- tween genes fails to be complete, the per- centages of crossovers formed between any two given pairs of genes is constant, but that this frequency can be quite different in cases studied in different organisms. Intergenic Linkage 115 SUMMARY AND CONCLUSIONS When independent segregation fails, the nonallelic genes involved tend to be inherited in a linked manner. Recombination between linked genes offers proof that a chromosome con- tains more than one gene. Just as linkage is an exception to independent segregation, crossing over is an exception to linkage and causes linkage to be incomplete. In any given case, the degree to which linkage is incomplete, that is, the rate of crossing over, is constant and independent both of the specific alleles which are present for the two different genes and of the combinations these were in when they entered the individual forming the gametes. Moreover, reciprocal crossover types are equally frequent. The crossover frequency in different cases, as studied in different organisms, is found to vary considerably. REFERENCES Morgan, T. H., "Random Segregation Versus Coupling in Mendelian Inheritance," Science, 34:384, 1911. Reprinted in Great Experiments in Biology, Gabriel, M. L., and S. Fogel (Eds.), Englewood Cliffs, N.J., Prentice-Hall, 1955, pp. 257-259. QUESTIONS FOR DISCUSSION 15.1. Distinguish between sex linkage and the linkage of nonalleles. 15.2. Does the linkage of two pairs of genes prove that they are located on the same chro- mosome? Explain. 15.3. Discuss the advantages and disadvantages of linkage and crossing over with respect to the fitness of individuals carrying certain genotypes. 15.4. In Drosoplula, y and spl are X-linked. A female genotypically -\ — \-/y spl produces sons of which 3.0% carry either v + or + spl. What are the genotypes and relative frequencies of gametes produced by the mother? Is the father's genotype important? Explain. 15.5. Name all the processes so far discussed which lead to genetic recombination. 15.6. Do you think that one of the main principles demonstrated in the present Chapter is that chromosomes contain more than one gene? Explain. 15.7. How would you proceed to state a "law of independent segregation" in the light of your present knowledge? 15.8. What evidence do you have that crossing over does not involve the unilateral move- ment of one gene, from its position in one chromosome to a position in the homologous chromosome? 15.9. Does crossing over always result in genetic recombination? Explain. 15.10. In what respect do you think the development of the principles of genetics in this text would have been affected had the first two pairs of genes, simultaneously studied in crosses, been linked? 15.11. Assume the gene for woolly hair (p. 34) is located autosomally. A nonwoolly haired nonhemophilic man marries a woolly haired nonhemophilic woman. They have a woolly haired hemophilic son. Give the genotypes of all three individuals. Give the genotypes and frequencies of the gametes usually produced by the son. Chapter *16 CROSSING OVER AND CHIASMA Yi "ou HAVE already seen how the study of cross-fertihzing species demonstrated that the genetic material was divisible first into a single pair of genes (because of segregation), then into a number of gene pairs (because of inde- pendent segregation), and then into still more genes (because those in the same chromosome are not forever bound together, or completely linked, but become separated and may pro- duce new combinations). Linked genes may form these new combinations when the genes of a pair mutually switch their position in a pair of homologous chromosomes by a proc- ess named crossing over (Chapter 15). The strength of linkage was found to vary in different cases studied in different organisms. Let us continue to study linkage and crossing over by means of a number of examples taken from the same organism, Drosophila melano- gaster. Two mutants b (black body color) and vg (vestigial wings) are studied simultaneously. A Pi cross between vg +/vg + (vestigial ^) 99 and + b/-\- b (black) cf cf produces all normal Fi, vg+/-\-b. As shown in Figure 16-1 A, a backcross of the Fi female {vg -\- / -\- b 9 X vg b/vg b cf ) produces only 20% of F2 with recombinant chromosomes (all F2 carry vg b from the father, their maternal chromosome being 40% of the time vg +, 40%o + b, \0% -f- +, 10% vg b). Since these results are in- dependent of sex, we conclude that b and vg are linked autosomally. (For comparison, recall from the last Chapter that the X-linked genes m and w showed 33%, crossing over.) When, however, the reciprocal cross is made with the Fi, the dihybrid being the male (vg +/+ ^ c^ X vg b/vg b 9), 50% of off- spring are vg -\-/vgb (vestigial) and 50% + b/vg b (black) (Figure 16-1^8). This dem- onstrates no offspring with crossovers, so that linkage is complete for these genes in the male Drosophila. Of course, had linkage been complete in the female also, we should not have had any evidence that vg and b are separable and are therefore two genes instead of one. It develops, moreover, that in Dro- sophila any genes which show incomplete link- age in the female show complete linkage in the male, so this sex, therefore, does not undergo the process of crossing over.^ It may be noted that in animals, in general, the hetero- gametic sex has reduced or no crossing over. What will be the consequence of crossing two Drosophila which are dihybrid vg +/+ b"? The female will produce gametes in the follow- ing frequencies: .4 vg +, A -\- b, A -\ — \-, .1 vg b; the male .5 vg +, .5 + b. Combin- ing these by means of a branched track pro- duces the results shown in Figure 16-2. The 2:1:1:0 phenotypic ratio obtained is easy to distinguish experimentally from a 9:3:3:1 ratio, which would be expected according to independent segregation. We can generalize the results to be expected in Drosophila. Let the two Hnked mutants be a and b and each dihybrid a +/+ b. Let 2p equal the frequency of noncrossover eggs (a + plus + b), and 1 — 2p the frequency of crossover eggs (a b plus + +). When linkage is incomplete, p < .5. The results are shown in Figure 16-3. We see there that no matter what value, below .5, p is permitted to have, the phenotypic ratio will be 2 : 1 : 1 : 0. If ^ The convention used here, and usually on subsequent occasions, is to describe the phenotypes of individuals only with respect to the appearance of mutant traits, all traits not mentioned being of the normal type. 116 2 A special kind of "crossing over" does occur on very rare occasions in the germ line (see p. 118) of male Drosophila, but is not of the kind that typically occurs in females. Crossing Over and Chiasma 117 ^i vg + ^ . + b / , 2i± (/c/an< ?? vg b vg b B ? vg + b ..d' 40% vg b 50% vg + ^ ^ vg + + b vg b FIGURE 16-1. Results of reciprocal crosses in- volving black body color (b) and vestigial (vg) wings. the dihybrids are ah/-\- +, however, this ratio is not obtained. Thus far, two cases in Drosophila have been studied and different crossover rates found in each, one case involving the X, the other an autosome. The question may now be asked: What is the strength of linkage between a given gene and several others located in the same chromosome? This can be studied readily for certain X-linked genes. Females are constructed with the genotypes shown in Figure 16-4. The frequencies of crossover combinations, as detected in their sons, are shown in the other column of the Figure. The recombination rates given are those found between the gene for yellow body color (y) in each instance and for white eye (m'), crossveinless wings (cv), cut wings (ct), minia- 118 FIGURE 16-2. Derivation of the pheiiotypic ratio obtained in Drosophila from mating together two dihybrids for linked genes. EGGS .4 vg + .4 + b .1 + + .1 vg b SPERM .5 vg + .5 + b .5 vg + 5 5 .5 + b .5 vg + .5 + b CHAPTER 16 + bpK.2+b/ + b A vg + Ly/ .05 + + / vg + Ly .05 + + / + b .05 vg b / vg + .05 vg b / + b .2 black .05 normal .05 normal .05 vestigial .05 black ture wings (/?z), and forked bristles (/). For example, the female dihybrid for y and cv produces about 13 eggs of each 100 which carry crossovers {+-^ovycv). What does this value, and what do the other still different linkage values, mean in terms of meiosis? Recall from what was discussed in the pres- ent Chapter and the one preceding that no commitment was made as to where or when the genetic event of crossing over takes place. Since we have been concerned with complete and incomplete linkage as studied in succes- sive generations of individuals, let us consider only crossing over that occurs in the cell line 2 Normal 1 Vestigial 1 Black 0 Vestigial black giving rise to the gametes (the germ line), ignoring the possibiHty that crossing over occurs in somatic (nongerminal, p. 23) cells. Although the possibilities still remain that crossing over is premeiotic, meiotic, and postmeiotic in occurrence, we shall presume that all crossovers are produced during meio- sis. We have already discussed (pp. 44^6) the theoretical genetic consequences of a chiasma between two pairs of genes during meiosis, and we shall suppose that a chiasma represents physical cytological evidence that a genetic crossing over has occurred. The genetic events theoretically associated Crossing Over and Chiasma 119 FIGURE 16-3 {Left). General- ized form of Figure 16-2. See text for details. EGGS SPERM ZYGOTES Frequency I Genotype Frequency i Genotype Phenotype o + / a + .50 " + + a + / + b .25 " a + .5-p .5-p V a + / + b .25 " + b + b / + b 0 a b / a + a b/ + b + + / a + + + / + b \. FEMALES ^ + / + w y + / + cv y + / + ct y + / + m y + / + f a b \ % CROSSOVER CHROMOSOMES AMONG SONS 1.5 ^' ■ u 20 • \ 34 48 FIGURE 16-4 (Right). Crossover percentages between one gene and others linked to it. 120 CHAPTER 16 with a chiasma are depicted in Figure 16-5 in somewhat more detail than those which are shown in Figure 6-8B (p. 46). Stage I shows a pair of homologous chromosomes, one member (hollow bar) carrying the reces- sives a and b, and the other (solid bar) carrying their normal alleles. The black dots represent centromeres. The homologs syn- apse, form a tetrad (each univalent now repre- sented by two sister strands), and give the appearance at diplonema depicted in stage II. Here there is a chiasma between the a and b loci (the places in a chromosome containing the genes). Note, when the univalents are initially identical in appearance, that a chi- asma results in the physical exchange of exactly equivalent segments between two non- sister strands of a tetrad, the strands exchang- ing segments being just as long after the ex- change as they were before. Stage III shows the dyads present after the first meiotic divi- sion is completed. Note that the upper cell contains one -\ — \- noncrossover strand and one + b crossover strand, while the bottom cell contains the reciprocal crossover strand a + and the noncrossover strand a b. Stage IV shows the four haploid cells produced after the dyads form monads and the second meiotic division is completed. Notice that if one chiasma occurs in any position between the loci of a and b, two haploid nuclei are produced, containing noncrossover, parental FIGURE 16-5. The genetic consequences expected following a cliiasma between linked genes. Ill Crossing Over and Chiasma 111 combinations, while the other two haploid nuclei contain crossover, nonparental re- combinations. Evidence that the crossovers found in gametes have this origin is ordinarily difficult to obtain. This is so because, in females, typically only one of the four haploid prod- ucts, from each nucleus starting the meiotic divisions, is retained as the nucleus of a func- tional gamete, the others being lost (often as polar body nuclei) . Even in those cases where each of the four haploid products becomes a gamete, as in sperm or pollen formation, the four gametes, produced from a cell containing a givenchiasma,mixwith those produced from other meiotic cells which may or may not have had a similar chiasma. As a consequence, therefore, only one of the four meiotic prod- ucts from a given chiasma is observed or identified at a time. Note that the chiasma ex- planation for crossing over would provide equal numbers of the two reciprocal kinds of crossovers, and also equal numbers of the two types of noncrossovers, as is required from the crossover data already presented. How- ever, crossing over during the two-stranded stage I would also satisfy these require- ments. Can we obtain genetic evidence as to whether crossing over occurs in the two- strand or four-strand (tetrad) stage? One possible way to determine this would be to use a genetic system whereby not one but two strands of the four in a tetrad are retained in a single gamete. If such a gamete carried one strand which is a noncrossover and an- other homologous one which is a crossover, this would support the four-strand hypothesis exclusively. This possibility becomes a re- ality through the use of Drosophila females whose two X's are not free to segregate from each other because they share a single centro- mere. Such attached-X's are V-shaped at anaphase. During meiosis the attached-X replicates once and the four arms synapse to form a tetrad, yielding two meiotic products each of which carries attached-X's and two devoid of X chromosomes. From a genetic analysis of the female progeny, of females whose attached-X's are dihybrid, evidence favorable to the four-strand view has been obtained (Figure 16-6). In pursuit of additional evidence regarding the time crossing over takes place during meiosis, the red bread mold Neurospora can be investigated. Neurospora ("nerve spore") is usually haploid and comes in two different sexes. In the sexual process, so-called fruit- ing bodies are formed, composed of cells each containing two haploid nuclei, each of which was derived originally from a different parent (Figure 16-7). Two such haploid nuclei fuse to form a diploid nucleus containing seven pairs of chromosomes, and the cell elongates to form a sac. The diploid nucleus immedi- ately undergoes meiosis, in the manner shown, so that at completion of meiosis the four haploid products are arranged in tandem, i.e., the topmost two nuclei come from one first division nucleus, the bottom two from the other first division nucleus. Subse- quently, each haploid nucleus divides once mitotically, in tandem, so that each meiotic product is present in duplicate within the elongated sac, and each of the eight nuclei becomes encased to form a football-shaped spore. Each haploid spore (ascospore) can be removed from the sac (ascus), grown indi- vidually, and its genotype determined directly. You see, therefore, that these events and pro- cedures make it possible to obtain and iden- tify all of the products of meiosis derived from a single diploid nucleus. Using the same symbols as were used in Figure 16-5, let us follow, in Figure 16-8, the genetic consequences of a single chiasma that occurs between the loci under study. This will provide us with the results expected from four-strand stage crossing over. Since only one pair of the seven pairs of chromosomes present are being traced, the others are omitted from the Figure. As a consequence 122 CHAPTER 16 METAPHASE I w f w i w« f34b': wQ f34b B w f w V. \ wO f34b/ TELOPHASE II light apricot weak forked light apricot weak forked white weak forked apricot weak forked FIGURE 16-6. Genotypic and phenotypic consequences of no chiasma {A) and of one type of chiasma (B) between marker genes in an attuched-X female of Drosophila. Crossing Over and Chiasma 123 t TWO HAPLOID NUCLEI DIPLOID NUCLEUS O J DIPLOID NUCLEUS FIRST MEIOTIC DIVISION SECOND MEIOTIC DIVISION MITOTIC DIVISION AND ir^ SPORE FORMATION liSj FIGURE 16-7 {Above). Meiosis in Neurospora. FIGURE 16-8 (Riglit). Cliiasma and crossing over in Neurospora. DIPLOID NUCLEUS ¥ I ^ DIPLONEMA t ^ AFTER FIRST DIVISION + + 3 FOUR MEIOTIC PRODUCTS EIGHT SPORES 124 CHAPTER 16 exactly two of the four meiotic products are crossovers and exactly two are not. Note on the other hand, that had crossing over occurred in the two-strand stage (in the top- most nucleus), all the meiotic products in a single sac would be recombinant. The result actually observed in some cases is that either none of the eight spores in an ascus is a crossover between a and b, or exactly four of the eight are; never are all eight spores, from a single sac, crossovers. This demonstrates conclusively that crossing over occurs in the four-strand stage, as shown in Figure 16-8. It has already been implied that chiasma formation is a normal part of meiosis (p. 26). One of the consequences of chiasma forma- tion is to prevent the premature separation of dyads by holding them together as a tetrad until anaphase I. (There is usually at least one, and there may be as many as six chias- mata, per tetrad.) Therefore, crossing over too is a normal part of meiosis. The fact that there are numerous places along a chromosome where a chiasma can form has a very interesting consequence. A single chiasma formed at any position between two loci will result in crossovers for these loci. Accordingly, it is reasonable to believe that the greater the distance between two loci, the greater will be the chance for a chiasma to occur between them, and the greater will be the frequency of crossovers for them. Re- ciprocally, the closer two loci are, the smaller will be the chance a chiasma will occur be- tween them, and the smaller will be the num- ber of crossovers for them. According to this view, the frequency of crossovers can be used as an indication of the relative dis- tances between loci. (The results presented in Figure 16-4 should now have additional meaning for you.) Suppose, in our model, that no chiasma occurs in 80% of spore sacs in the genetically marked region we have been considering. These sacs would produce 80% of the total number of spores carrying only parental, noncrossover genotypes. From the 20% of spore sacs which do contain such a chiasma, one would find half of the spores to be of the parental types and half to be recombina- tional. So, a chiasma frequency of 20% would result in 10% of all spores being of crossover type. We can express the distance between the loci of a and b as being 10 cross- over map units, where a map unit is that dis- tance which gives one crossover per hundred (spores, in the present case). It is generally true, then, that in the simple case (where the genes are sufficiently close together, as in the present example), crossover frequency (map distance) is just one-half chiasma frequency. What procedures may be used to measure crossover frequency in Neurosporal There are several. First, this can be done in terms of spore sacs. As many spores are tested from each sac as are required to decide whether or not the sac carries a crossover in the region tested. (No more than five spores need to be tested per sac.) Speaking in terms of the a-b model already discussed, 20% of sacs would have crossovers, 80% would not. And since each sac containing crossovers has four spores that are crossovers and four that are not, crossing over frequency would be 10%. Second, all the spores from many sacs are mixed and then a random sample of spores is taken and tested. This method would give 10%, recombination with our model, and is like the sampling procedure involved in deter- mining crossover frequency in animal sperm. A third procedure would be to test one ran- domly chosen spore from each sac and dis- card the others. Again, 10% recombination would be obtained. This method resembles the situation in many females (including Dro- sophila and human beings) where, normally, one random product of meiosis enters the egg, the others being lost. In what has been discussed relating chias- mata and crossing over, no study was de- scribed that directly correlated a genetically 125 detected crossover with some cytologically detectable event involving a particular chro- mosome region. Such a connection cannot be made if both members of a pair of homologous chromosomes are identical in cytological ap- pearance. This is true because a crossover strand, having exchanged a cytologically identical segment with its homolog, would appear the same as a noncrossover strand (cf. p. 120). But it is possible to construct a dihybrid for linked genes in which one homo- log differs cytologically from its partner on both sides of the loci being tested. Such a genetic dihybrid is, simultaneously, cytologi- cally dihybrid in the way specified (Figure 16-9). In this case, it is possible to collect noncrossover progeny and show cytologically that they invariably retain the original chro- mosomal arrangement, while crossovers al- ways show cytologically a new chromosome arrangement that is explained by a mutual exchange between the homologs of specific chromosome regions.^ ^ In such a way, genetic crossovers were correlated exactly with cytological crossovers by Stern (1931) using Diosophila, and by Creighton and McClintock (1931) using maize. TETRAD WITH ONE CHIASMA =^31 X FIGURE 16-9. Correlation between genet iced and cytological crossovers. tiZ MEIOTIC PRODUCTS ^m ^ ^ B A - b^ a A B a >^ — Noncrossover Crossover Crossover Noncrossover SUMMARY AND CONCLUSIONS A crossover chromosome carried by a gamete is derived from a tetrad in which a chiasma, involving only two of the four strands, has occurred between the linked genes showing recombination. For closely linked genes, crossover frequency is one-half the frequency with which a chiasma occurs between their loci. It is hypothesized that crossover frequency is directly related to distances between genes on a chromosome. One unit of crossover distance between genes is defined as one crossover per one hundred postmeiotic cells (spores or gametes). Since different genes linked to the same gene show different percentages of crossing over with this gene, they are presumably different distances from it. 126 CHAPTER 16 Maize workers (/. to r.) at Cornell University in 1929: C. R. Burnham, M. M. Rhoades, G. W. Beadle {crouched), R. A. Emerson, and Barbara McClintock. REFERENCES Creighton, H. S., and McClintock, B., "A Correlation of Cytological and Genetical Crossing-over in Zea Mays," Proc. Nat. Acad. Sci., UtS., 17:492-497, 1931. Re- printed in Classic Papers in Genetics, Peters, J. A. (Ed.), Englewood Cliffs, N.J., Prentice-Hall, 1959, pp. 155- 160; also in Great Experiments in Biology, Gabriel, M. L., and S. Fogel (Eds.), Englewood Cliffs, N.J., Prentice-Hall, 1955,^" pp. 267-272. Stern, C, "Zytologisch-genetische Untersuchungen als Beweise fur die Morgansche Theorie des Faktorenaus- tauschs," Biol. Zbl., 51:547-587, 1931. Curt Stern, /// the early 1930' s. Crossing Over and Chiasma j27 QUESTIONS FOR DISCUSSION 16.1. Does the Summary and Conclusions section contain one or more statements which have not been made in essentially the same terms earlier in the Chapter? If your answer is yes, give an example. 16.2. In a species where both sexes undergo crossing over with equal frequency, what is the percentage of crossing over between two loci if a mating between identical dihybrids {Ab/aB) gives four equally viable classes of offspring, the smallest class comnrisine 1% of all offspring? ^ ^ 16.3. How would you proceed to prove genetically that the last division in a spore sac of Neurospora is a mitotic one? 16.4. Could you determine, in the absence of crossing over, whether the alternatives for two different traits were due to a single pair of genes or to two pairs of linked genes'^ Explain. 16.5. Draw an attached-X chromosome of Drosophila heterozygous both for y and for m. Show the kinds of gametes which could be obtained following: a. No chiasma. b. One chiasma between the nonallelic genes. c. One chiasma not between the genes mentioned. 16.6. Suppose a plant has a long pair of chromosomes, one member of which has a large knob at one end of the chromosome while the other member has a small knob at the opposite end of the chromosome. Suppose, moreover, there is also a shorter pair of homologs, one member terminating with a large knob, while the other member termi- nates at the other end with a small knob. With respect to these chromosomes, what combinations and configurations would you expect to find readily in the gametes of this individual? 16.7. What reasons can you present for believing that germ-line crossing over is based neither upon premeiotic nor upon postmeiotic events? 16.8. Translate into English the title of the article written by C. Stern. Chapter *17 GENE ARRANGEMENT AND CHIASMATA I 't was found in the preceding Chapter that the frequency of .crossing over between genes may be considered in terms of distance measured in map units. Different genes Hnked to a given gene were found to have different cross- over distances from it. The question arises, how are these different genes related to each other spatially? It may be that the crossover distances between a number of linked genes represent physical distances from one gene to another which would describe either some three-dimensional configuration (like a sphere, cube, prism, or polyhedron) or some two- dimensional one (like a circle, ellipse, tri- angle, or line). In order to determine if this is so, it is necessary to determine all the map distances for a minimum of three linked loci. (The crossover distance between two genes defines only two points; two points are insufficient to tell us whether linked genes occur in a specific geometrical arrangement.) The arrangement of linked loci can be in- vestigated in Drosophila using the three X- linked genes, y (for yellow body color), iv (for white eyes), and spl (for split bristles). Dihy- brid females of each of the following types are obtained, y w/-\- -f , y spl/-{- +, w spl/-\- +, and each is backcrossed to the appropriate double recessive male. The crossover per- centages, or map distances, obtained from these crosses are, respectively, y io w \.5,y to spl 3.0, and w to spl 1 .5. These data describe a simple arrangement for these three genes, namely a linear one, since the crossover dis- 128 tance between the end genes j and spl equals the sum of the crossover distances from the middle gene w to the end ones. We shall presume, henceforth, that crossover distance is proportional to physical hnear distance between genes. It is also possible to study a fourth and then all other X-linked genes, and to map their positions relative to these three. When this is done, it is found that all are arranged in a linear order. In such a crossover or genetic map, y is arbitrarily assigned the position, or locus, 0. A standard crossover map for the Drosophila X would have the genes y, w, spl, cv, ct, m, and / lined up at the respective positions, 0, 1.5, 3.0, 13, 21, 36, 56.7. From this standard map one can read that ct and m are 15 map units apart (36-21). Since one map unit equals one crossover per hundred gametes, the dihybrid for ct and m (Fig- ure 17-1) should produce 15% crossovers (7.5% + + and 7.5%, ct m). However, such a result is obtained only when certain condi- tions are met. The crossover rates actually observed will depend upon several factors. One of these is the number of individuals making up the sample. Standard distances are arrived at only after scoring large numbers of progeny. In small samples it is very likely that, by chance, the observed values will deviate con- siderably from the standard map distance in both directions. The larger the size of the sample, the closer will the observed value approach the standard one. Another factor influencing observed cross- over rates concerns the fact that different phenotypic classes may have different viabili- ties (cf. p. 9). Usually the phenotypic ex- pression of a + allele is more viable than that of its mutant forms. So, in Figure 17-1, for example, phenotypically ct m sons are not as viable as normal (wild-type) sons, and though both types are equally frequent as zygotes, the former fail to complete develop- ment more often than the latter, and are, Gene Arrangement and Chiasmata therefore, relatively less frequent among the adults scored for crossovers. It should be noted also that zygotes destined to become either miniature or cut are also discriminated against more than are zygotes destined to produce the wild -type. Whenever pheno- types are scored after some long develop- + m ct + 9 - .-/ 129 ANY d FIGURE 17-1. Crossover rate for two X-linked genes in Drosophila. Sons: + m / Y 42.5 % ct + / Y 42.5 % + + / Y 7.5 % ct m / Y 7.5 % ^^=^. mental period, much of the error due to differential viability may be avoided by pro- viding optimal culture conditions. Another way to avoid most of this kind of error is to obtain progeny carrying the chromosome to be scored for crossovers, in which the homolo- gous chromosome contains the normal alleles of all genes under crossover test. Since such progeny are phenotypically normal they will all have approximately the same viability and can be scored as to chromosome type from the offspring each produces when individually test crossed. Thus, for example, the female in Figure 17-1 is crossed to wild-type males and the daughters (all phenotypically normal) are individually mated to any male. Daugh- ters which carry, in addition to + + on one X obtained from their father, a homologous X of one of the following types, + m, ct -\-, -|- -|-, ctm, will produce sons, of which, respectively, some are miniature but none cut, some cut but none miniature, all normal and some miniature and cut. In this way the generation being tested for crossover rate is protected from differential viability and its genotypes are detected in the generation fol- lowing. For some purposes the extra labor entailed by the use of this method is justified. 130 CHAPTER 17 Variability in crossover rates may be due also to factors influencing the process of crossing over itself. Such factors include temperature, nutrition, age of the female, and the presence of specific genes. In order to understand better the relation- ships between crossing over and chiasmata, consider the properties of the following, over- simplified model (Figure 17-2). Assume that a chromosome (ignoring the centromere) is divided into five equal segments marked by ^ Hybrid Studied MEIOTIC 40 PRODUCTS 40 FIGURE 17-2. Crossover con- sequences following a single chiasma. PER CENT OF ALL PRODUCTS 45 45 six genes. Assume further that each tetrad with this chromosome contains one, and only one, chiasma which can occur at random among these segments. What would be ob- served if only the region between a and b was followed in the hexahybrid shown in the Figure? The chance that the chiasma will occur in the a-b region is 20%. From each 25 tetrads 100 haploid meiotic products are produced. Of each 25 tetrads, 20%, or five tetrads, will have the chiasma in the a-b region. These will produce 10 crossover and 10 noncrossover strands. The latter 10, when added to the 80 noncrossover strands from the other 20 tetrads, give 90 noncrossover strands. So, in this region, 20% chiasmata gives 10% crossovers, as explained in Chap- ter 16. Similarly, were only the b-c region followed, 10% crossovers would be observed. If now only the a-c region is followed, the chiasma will fall within it 40% of the time and 20% of all haploid meiotic products will be crossovers between a and c. Note that in this model the sum of the distances a-b and b-c equals the distance as measured between a and c directly. So the model aligns the genes linearly, just as was observed in the experiment described near the beginning of this Chapter. Note also, from the way the model was described, that the occurrence of a chiasma in one region automatically excludes its occurrence in some other region. We find, then, that the chance for a chiasma in the a-c region is equal to the sum of the separate chances for a chiasma in the a-b and b-c re- gions. It is a general rule that the total probability that any one of a series of mutually exclusive events will occur is equal to the sum of their separate probabilities of occurrence. Accordingly, the chance of a chiasma occur- ring between a and /is, of course, 100 (20 + 20 + 20 + 20 + 20)%, so that recombination is 50%, and the num- ber of map units in our model is fifty. Gene Arrangement and Chiasmata 131 You should be dissatisfied, however, with the abiHty of this model to represent reality, in view of the fact, previously noted, that a given tetrad usually contains more than one chiasma. This complication prompts the fol- lowing question: What is the relationship between two chiasmata in the same tetrad? Two possible relationships come to mind. First, the frequency with which two chiasmata occur simultaneously within a certain region may be larger or smaller than that expected by chance. Second, the frequency with which the two chiasmata involve the same two strands of the tetrad may be larger or smaller than that which would be expected by chance. Consider the second relationship first. Let us specifiy the strands in the tetrad of the model as 1,2, 3, 4, where 1 and 2 are the normal sister strands and 3 and 4 the mutant sister strands (Figure 17-3). Suppose one chiasma occurs between strands 2 and 3 in the a-b region. There are six combinations of strands possible for a second chiasma which occurs in the b-c region, namely, 1 with 2, 3 with 4, 2 with 3, 2 with 4, 1 with 3, and 1 with 4. These positions are indicated in the Figure. Note, for this second chiasma, that the first two combinations listed would involve sister strands which are, naturally, genetically identical. Since sister-strand crossing over would have an effect upon the production of new genetic combinations only under other, very special, circumstances, such crossing over need not be considered further for our purposes. Each of the last four types of chiasma involves nonsister strands in the b-c region, and together with the single chiasma in the a-b region, forms double chiasmata of three types, respectively: 2- strand (thQ same two strands exchange in both chiasmata), 3-strand (one of the two strands of the first chiasma exchanges in the second; there are two ways this can occur), and 4- strand (the strands exchanging in the second chiasma are those which did not exchange in the first). Restricting our attention to the a-c region, let us examine the genetic consequences of the nonsister double chiasmata described. Figure 17-4 shows, at the left, the configura- tions of these four nonsister types of double chiasmata. The middle column shows the meiotic products of each, and the right col- umn describes whether these products are noncrossovers, single crossovers, or double crossovers for the a-c region. Notice that following 2-strand double chiasmata two of the four meiotic products are genetic non- crossovers (+ + + and a b c) and two are double crossovers {-\- b -\- and a -\- c) recog- nizable because the middle gene is switched in position relative to the end genes. A 3- strand double chiasmata produces one double crossover, two single crossovers (recognizable because each has one end gene switched), and one noncrossover. The 4-strand double chiasmata produces four single crossover strands. Two things may be noted, namely, that each type of double chiasmata produces some strands with a new genetic combination. FIGURE 17-3, Chromatid combinations possible in a double chiasmata. {See text for details.) I 132 CHAPTER 17 i.e., which are crossover, and that each of the three different types produces a character- istic pattern of noncrossover and crossover types. Note further that the genetic conse- quences of these double chiasmata are differ- ent from what would be obtained following a single chiasma (which, you remember, produces two noncrossovers and two singles). In view of the preceding discussion, it should be possible by using an organism like Neurospora, in which all the products of meiosis are recoverable and testable, to learn from the genotypes of the meiotic products the relative frequency with which the four types of double chiasmata occur. If all four types occur with equal frequency, this would mean that the strands forming one chiasma are uninfluenced by those which form an adjacent chiasma. Indeed, work performed with Neurospora shows that all four types do occur. In some experiments the four types occur with equal frequency, and, for our purposes, we can accept the view that there is usually no chromatid interference in chiasma formation, i.e., the chromatids which enter into a chiasma do so uninfluenced by which strands have or have not undergone chiasma formation in an adjacent region. Let us consider now the second possible relationship, already mentioned, between ad- jacent chiasmata. Does the occurrence of one chiasma increase or decrease the prob- ability that an adjacent chiasma will occur, regardless of which strands are involved? Suppose, in a genetic system as in Figure 17-4, the probability of a single chiasma be- tween a and b is 0.2 and between b and c is also 0.2. Having presumed that each of two regions under observation has a 20% chance of forming a single chiasma, let us again make use of the observation that more than one chiasma can form in a tetrad. If the occurrence of a chiasma in the a-b region is uninfluenced or independent of a chiasma in the b-c region, then, of all tetrads, 20% of the 20% with an a-b chiasma will simul- taneously have a b-c chiasma, or 4% will contain double chiasmata. (And from what has been discussed before these 4% will be comprised of the four nonsister types in equal frequency.) It is a general rule that the probability for the simultaneous occurrence of two or more independently occurring events is obtained by multiplying together their sepa- rate probabilities. If an experiment studying these regions actually gave 4% double chiasmata, one would conclude there was no chiasma inter- ference; that is, the fact that homologous chromosomes form one chiasma has no effect upon the hkelihood of their forming another one in an adjacent region. If, on the other hand, only 2% double chiasmata were ob- served, this would represent interference of one chiasma with the formation of another in an adjacent region. The degree of chiasma interference can be expressed by the fraction: double chiasmata observed _ .02 double chiasmata expected .04 = .5 This fraction is called the coejficient oj co- incidence and expresses the frequency with which the expected coincidence of two chi- asmata is actually observed. So, a coefficient of coincidence of 0 would mean one chiasma completely prevented the other one from oc- curring, while a value of 1 would mean that the one chiasma did not interfere at all with the occurrence of the other. In practice, however, one does not deter- mine the actual rates of occurrence and the positions of double chiasmata, since it is not feasible cytologically to score chiasmata in these ways. We are led, therefore, to examine the genetic event of crossing over to see whether it can be used to measure interfer- ence. Since we can be sure that each double crossover observed came from a double chiasmata, let us see if such crossovers can be used to calculate the coefficient of coin- cidence. The expected frequency of double Gene Arrangement and Chiasmal a 133 crossovers in our example can be calculated in the following way: since each region has a 0.2 chance for a single chiasma, each has a 0.1 chance for a single crossover, and the expected chance for a double crossover is 0.1 X 0.1, or 0.01. If, as before, the actual double chiasmata in our example occurred with a frequency of .02, then, since only 4 of the 16 meiotic products appear as double crossovers (Figure 17-4), the observed fre- CHIASMATA quency of double crossovers would be .02/4 = .005. The coefficient of coincidence determined from double crossovers (ob- served frequency /expected frequency) would be .005/.01, or .5, which is the same as the value previously obtained using chiasmata frequencies. In practice, therefore, one can determine the coefficient of coincidence by dividing the observed frequency of double crossovers by their expected frequency, the MEIOTIC PRODUCTS CROSSOVERS 2 Doubles 2 Noncrossovers b c c 1 Double c 2 Singles 4. 1 Noncrossover 3-STRAND ^ 1 Double + 2 Singles c 1 Noncrossover FIGURE 17-4. Double chiasmata types and their genetic consequences. ^+ + + 4-STRAND a +_ a b 4 Singles 134 CHAPTER 17 latter value equaling the product of the ob- served frequencies of single crossovers in the two adjacent regions. It is found, in general, that the coefficient of coincidence is 0 for short map distances and becomes larger as the distances do. This means that a tetrad in which one chiasma forms is somehow inhibited from having a second one occur in a region close by, this inhibition becoming less the farther away this second region. In Drosophila, for example, the coefficient of coincidence is 0 for map distances up to 10-15 map units, so that no double chiasmata (hence no double cross- overs) occur within such distances. As the distance increases beyond 15 map units, the coefficient increases gradually to 1, at which time there is no interference with the forma- tion of double chiasmata. You remember that, if every tetrad in our model had a single chiasma located some- where between a and /, the maximum fre- quency of recombination for these end genes would be 50%. What happens to the fre- quency of recombination for the end genes when our model is also permitted to have double chiasmata? If, now, each tetrad has one or more chiasmata (and hence cross- overs), you might think, at first, that the end genes would form new combinations more than 50% of the time. However, examination of Figure 17-4 will show you, because each type of double chiasmata is equally likely, that, on the average, there will be eight products which would switch the end genes (being those which are single crossovers) and eight products which would not (being com- posed of four double crossovers, in which an interstitial gene switches but end genes retain their original order, plus four noncrossovers). Accordingly, even if every tetrad has double chiasmata, the maximum recombination for the end genes is again 50%. If four genes are studied and three chias- mata occur in each tetrad, one in each region, it turns out that per each 64 meiotic prod- ucts 32 will be recombinational for the end genes and 32 will not. One can work out the fact that for cases where there are four or more chiasmata between end genes, the num- ber of meiotic products bearing odd numbers of crossovers (1, 3, 5, etc.) is 50%> In each of these cases one end gene is shifted relative to the other. However, the remaining strands contain either even numbers of crossovers (which do not cause the end genes to shift relative to each other) or no crossovers. Accordingly, there is still a maximum of 50% recombination for the endmost genes (and, therefore, of course, for any genes between them). If two genes are located far apart in a chromosome and multiple chiasmata normal- ly fall between them, their rate of recombina- tion will be near 50%. Since 50% recombina- tion is taken to mean independent segregation of gene pairs, you might not be able to con- clude from the recombination rate that these two genes are linked (cf. p. 46). Whenever genes are known to be linked, however, their correct order relative to each other may be determined without concern about the cor- rectness of the distances involved. Moreover, this may be decided from the results of a single cross. Suppose the trihybrid + + +/ a b c is test crossed, and the frequencies of phenotypes in the progeny are as shown at the left in Figure 17-5. These frequencies, you recall, represent the frequencies of the corresponding genotypes in the gametes of the trihybrid. The middle gene would be the one which switches least often from the original gene combinations (H — | — \- and a be), for only it requires two chiasmata to switch. Accordingly, this gene is identified as c, and the actual gene order is ac b (or bed). You may understand this more readily by examining the data when the genes are listed in their correct order, as shown at the right in Figure 17-5. Here the frequency of observed crossovers between the a and c loci is 30%, and between e and b 10^ (The Gene Arrangement and Chiasniata 135 + + + 0.31 + + + 0.31 a b c 0.31 a c b 0.31 + be 0.14 + c b 0.14 a + + 0.14 a + + 0.14 + + c 0.01 + c + 0.01 a b + 0.01 a + b 0.01 + b + 0.04 + + b 0.04 a + c 0.04 a c + 0.04 1.00 1.00 FIGURE 17-5. Determination of gene order from a test crossed trihybrid. expected frequency of double crossovers would be 0.3 X 0.1, or 0.03, so that the coefficient of coincidence is .02/.03, or 0.66.) For the a-b region the percentage of cross- overs is 40% (the double crossovers are counted twice since each represents two crossovers between the end genes). Having established the order of these genes, would it be satisfactory to use these data to construct a standard map for the distances between these genes, assuming large numbers of progeny had been scored and standard experimental conditions had obtained? The observed distance between c and b is acceptable for this purpose, since in such a short distance only a single chiasma can be produced. However, the situation is otherwise for the a-c region, which is at least 30 map units long. For in such a distance double chiasmata could occur and the double crossovers these produce would be unde- tected, since there are no genetic markers between a and c whose switch would enable us to detect them. You see, then, from this and from what has been discussed previously, why the crossover rates observed for large distances are less than the standard map distances. For standard map distances are always obtained by the summation of short distances within which only a single chiasma can occur. You should also realize, even though end genes can show at most 50% recombination, that the length of the crossover map may be more than 50 units. For example, if each tetrad contained an average of two chiasmata (see Figure 17-4), there would be a total of 100 crossovers among 100 meiotic products and the map length would be 100 units (even though end genes recombined 50% of the time). In fact, the length of the standard map can be predicted to be equal to the mean number of chiasmata per tetrad X 50. SUMMARY AND CONCLUSIONS Using crossover frequency as an indication of distance, it is found that linked genes are arranged linearly. Observed crossover rates may fluctuate because of sample size and because of factors acting either after crossing over (differential viability), or on the cross- over process itself (temperature, age, nutrition, genotype). Standard crossover maps are made under standard conditions. The presence of one chiasma interferes with the occurrence of a second one nearby in the same tetrad, this chiasma interference diminishing as the distance between the two regions increases. Whenever double chiasmata do occur, the chromatids of one chiasma typically have no influence upon which chromatids form the other chiasma, so that there is no chromatid interference. Recombination between end genes is 50% maximally, no matter how many chiasmata occur per tetrad. While the order of linked genes is easily determined by test crossing trihybrids, the distance between two genes will be underestimated when they are far apart. 136 CHAPTER 17 REFERENCES Sturtevant, A. H., "The Linear Arrangement of Six Sex-Linked Factors in Drosophila, as Shown by Their Mode of Association," J. Exp. Zool., 14 : 43-59, 1913. Reprinted in Classic Papers in Genetics, J. A. Peters (Ed.), Englewood ClifTs, N.J., Prentice-Hall, 1959, pp. 67-78. See Supplement IL QUESTIONS FOR DISCUSSION 17.1. Does the linear arrangement of the genes offer any evidence for or against the view that the chromosomes are carriers of genes? Explain. 17.2. How many gene pairs must be heterozygous to detect (1) a single crossover; (2) a double crossover in Drosophila! in Neurosporal 17.3. Suppose a pair of homologs in Neurospora had the genotypes AB/a b. Draw an ascus, containing 8 spores, derived from a cell that had: a. No chiasma between these homologs. b. One chiasma between the centromere and the gene pair closest to it. c. One chiasma between the two pairs of genes. d. One two-strand double chiasmata between the two pairs of genes. 17.4. What advantages has Neurospora over Drosophila as material for genetic studies? 17.5. Under what conditions would segregation of a pair of alleles occur during the first meiotic division? the second meiotic division? 17.6. What indications would you have that dilTerential viability was having a role in modifying the crossover distances obtained experimentally? 17.7. For how many linked gene pairs must an individual of Drosophila and of Neurospora be hybrid, in order to determine from the crossovers it produces whether these genes are arranged in a linear sequence? Explain. 17.8. A test cross proves that one of the parents produced gametes of the following geno- types: 42.4% PZ, 6.9% Pz, 7.0% pZ, and 43.7% pz. List all the genetic conclu- sions you can reach from these data. 17.9. The trihybrid Aa Bb Cc is backcrossed to the triple recessive, aa bb cc. The following phenotypic results were obtained: abc 64, abC 2, aBc 11, aBC 18, AbC 14, Abe 17, ABc 3, ABC 71. a. Which of these loci are linked? Why? b. Rewrite the genotypes of both parents. c. Determine the observed map distances between all the different pairs of linked genes 17.10. Describe one practical use of the fact that linked genes are arranged linearly. 17.11. How can you determine the position of a centromere in a linkage group of Neuro- spora? 17.12. Discuss the statement on p. 124 that "never are all eight spores, from a single sac, crossovers." Chapter *18 CHANGES INVOLVING WHOLE GENOMES AND SINGLE WHOLE CHROMOSOMES I: N THE preceding Chapters marked by asterisks we used recombina- tion to study the genetic materiak This operation permitted us to divide the genetic material into genes whose properties are expressed in terms of their recombina- tional behavior. The present Chapter and others to follow (especially Chapters 19, 23, 25) deal with the study of the genetic material by means of the operation of mutation. We shall be especially interested to learn to what extent the genetic material can be partitioned into mutational units; that is, we will be concerned with the possibility of describing a gene in terms of its mutational behavior. It has been possible for us to learn the recombinational properties of genes only be- cause the genetic material exists in more than one alternative state and is apparently capable of replicating itself and certain of its modifi- cations. You can readily see that a gene, which is present in homozygous condition in all organisms, is not detectable, since all individuals would have the same genotype, and, therefore, the same range of phenotypic expression. A gene can be detected only if it occurs either in different numbers in differ- ent individuals, or if it has an alternate allele, or both, provided such a genetic differ- ence produces a detectable phenotypic change. A great deal of genetic variation exists among living organisms (Chapter 1). We have seen that some of the phenotypic varia- 137 tion attributable to genes arises via sexuality, by means of which new combinations of already present genes may be produced by segregation, independent segregation, cross- ing over, and fertilization. These mecha- nisms of recombination shuffle the genes, just as shuffling a deck of playing cards produces the great variety of different combinations obtained with the same cards. However, the genetic differences found in a population to- day were not always present in it. What we are concerned with now is how the genetic differences arise whose shuffling produces phenotypic variation by recombina- tion. Before we can study this, however, we must have some way to distinguish the origin of mutants, really new genetic forms, pro- duced by the process of mutation, from the recombination of old, pre-existent genes. Consider a case, involving Drosophila, that illustrates how this distinction may be made. None of the flies in laboratory strains of Drosophila, regardless of origin and cross- breeding, have an appendage on the anterior- dorsal part of the thorax. Suppose a single fly occurs with an appendage in this region (Figure 18-1), and this trait appears in ap- proximately one half of this fly's progeny. How is the new phenotypic variation, called Hexaptera, to be explained? It cannot be due to environmental factors alone. Hex- aptera cannot be due to the interaction of particular members of a pair of genes, already present in the population, which happened to become combined in the same zygote at fertilization. For if the occurrence of Hex- aptera depended upon such a combination, this would have to be so rare that, following segregation, this phenotype would not be expected to appear in any appreciable num- ber of the progeny. Hexaptera cannot be due to the rare combination of two previously existing unlinked nonalleles, since at most only one quarter of the progeny would have the novel phenotype. So, neither segregation nor independent segregation is responsible 138 CHAPTER 18 FIGURE 18-1. The Hexapteni p/ienolype in D. melanogaster. {By permission of Genetics, Inc., vol. 34, p. 13, 1949.) for Hexaptera. However, such a new pheno- type could have arisen by genetic recombina- tion, due to the occurrence of a crossover chromosome, which was very rare because the nonallelic genes involved were extremely close together. Once produced, this combi- nation of linked genes would remain intact and be transmitted to one half of the progeny. But suppose also that the chromosomes of the parents of the first Hexaptera were suit- ably marked with genes, and it was found that the chromosome region whose presence is essential for the production of the new pheno- type was of a noncrossover type. Then crossing over would not explain the results. The only reasonable explanation remaining would be that a novel change had occurred in the genetic material, a mutation, which produced a dominant phenotypic effect. You see, then, that under certain circumstances it may be possible, without great difficulty, to identify a novel phehotype as being due to mutation rather than to genetic recombina- tion, when the mutant produces a dominant phenotypic effect. When, however, the novel phenotype re- quires the mating of two particular individ- uals for its appearance in progeny, it is much more difficult to decide whether the genetic recombination required for its appearance involves old genes or a recently arisen mutant which is apparently completely recessive. Note, after the mutational origin of a com- pletely recessive autosomal gene, that its detection is postponed for that number of generations which is required for two hetero- zygotes to mate and produce a mutant homo- zygote. Under certain conditions, many generations may elapse before the recessive mutant becomes homozygous, during which period the mutant allele may become rela- tively widespread in the population in hetero- zygous condition. In this event, one cannot decide when the mutant first arose, and it may be considered part of the old pool of genes present in the population. It would be easier to identify a phenotype as the result of a recessive mutation if the genotype of the population was known to be uniform prior to this. You see, therefore, why the detection of mutants, both of recessive and of dominant types, is made relatively easy by the employ- ment of pure lines. The pure line procedure for detecting mutants was used with self- fertilizing beans, as described in Chapter 1. As mentioned there, sudden phenotypic changes, not due to environmental fluctua- tion, are occasionally found which clearly represent mutations, not recombinations from genotypically different parents. In cases where completely pure lines cannot be ob- tained because self-fertilization does not occur, detection of mutations is facilitated when they involve genes for which both parents are homozygous or completely known genotypically. Changes Involving Genomes and Chromosomes 139 ♦ f IN 2N 3N FIGURE 18-2. Ploidv in Datura (A^ = 12) (sil/joiiettes). 4N Once a phenotype is proved to result from a mutation, the basis for the genetic change has still to be determined. Let us start our study of the basis and character of mutation with some examples taken from plants. In the evening primrose, Oenothera, a giant type called gigas is found to be a mutant. Other Oenothera, like most sexually reproducing species, are diploid, having two sets of chromosomes, or genomes — one genome having been contributed by each of the gametes. In the gigas type, cyto- logical examination shows that there are three genomes, so that such individuals are triploid. Studies of other groups of diploid plants have revealed related types which prove to have four genomes and so are tetraploid, others may have six sets (hexaploids) or eight (octaploids). Triploidy has been found even in human beings. The occurrence of any ploidy greater than diploidy is called poly- ploidy, although this term may also be used for multiples of the haploid number when monoploidy is the normal condition. Different forms of the Jimson weed. Datura, are found to carry different ploidies.^ Some are haploid, others diploid, triploid, or tetra- ploid. The appearance of the flowers each type produces is shown in Figure 18-2 line B, and their respective seed capsules are shown above in line A of the Figure. Note that the flower size increases with ploidy. The seed capsules illustrated are those which might ^ Based upon work of A. F. Blakeslee and J. Belling. 140 CHAPTER i; have been obtained had the individual under test been fertilized by pollen from a diploid. (In this case each pollen grain would con- tribute one genome to the development of the zygote in the seed.) The differences in size of the seed capsules is due partly to the number of seeds that have set or developed. Polyploidy is also found in animals, for example, in Drosophila. Female Drosophila have been found that are triploid (3X + 3 sets of A) and tetraploid (4X + 4 sets of A) (cf. pp. 102-104). Parts of individuals have been found to be haploid (IX + 1 A set). You recall that triploid females produce gametes many of which are genomically abnormal. Since two tetraploid Datura can be crossed, and a sufficient number of fertile seeds are set to form a tetraploid race, the question arises, can a tetraploid race of Drosophila be produced? The tetraploid Drosophila female forms gametes that more often contain complete genomes than do the gametes of triploids. (The number of genomes present must be even, not odd, if each chromosome is to have a partner at meiosis. So, in tetraploids, where genome number is even, the four ho- mologous chromosomes often segregate 2 and 2, but sometimes segregate 3 and 1.) So this sex presents no difficulty for the continuity of a tetraploid race. However, the tetraploid male would have to carry 2X + 2Y + 4 sets of A in order to be of normal sex. Un- fortunately, during meiosis in such a male the X's would synapse with each other and so would the Y's, so that after meiosis each sperm would carry IX and lY in addition to 2 A sets. Such sperm fertilizing eggs of 2X + 2 sets of A constitution, from tetra- ploid females, would produce zygotes with 3X + lY + 4 sets of A which would develop as sterile intersexes. Thus, a tetraploid race of Drosophila, which is self-maintaining, can- not be established. In fact, it becomes clear that any species containing a heteromorphic pair of sex chromosomes (like X and Y) cannot form polyploid races, since the correct balance between sex chromosomes and auto- somes will be upset by the meiotic divisions. This factor probably explains why polyploid races and species are rarer among animals than among plants, where sexuality is often not associated with such chromosomal differ- ences (Chapter 13). Nevertheless, polyploid races of animals are sometimes found. For example, tetra- ploids of the water shrimp, Arteinia, of the sea urchin. Echinus, and of the roundworm, Ascaris, are known. In the moth, Solenobia, some females may produce haploid eggs and others diploid eggs. Both types of eggs start development without fertilization, i.e., start development parthenogenetically. During de- velopment, however, nuclei of the respective individuals fuse in pairs to establish the diploid and tetraploid conditions. Poly- ploid larvae of salamanders and of frogs also have been obtained, although races are not formed. One way that ploidy can increase is by the addition of genomes of the same kind as are present, that is, by autopoJyploidy, as was the case in the Datura discussed. Autopoly- ploidy can arise several different ways. An organism capable of asexual reproduction may fail to have a normal mitotic anaphase so that the doubled number of chromosomes becomes incorporated into a single nucleus, which thereafter divides normally to produce daughter polyploid nuclei and eventually polyploid progeny. Sometimes two of the haploid nuclei produced by meiosis may fuse to form a diploid gamete which, after fertiliza- tion with a haploid gamete, forms a triploid zygote. Or haploid individuals may undergo meiosis, and, while this usually results in gametes containing only part of a genome, a complete haploid gamete may sometimes be produced which, upon fertilization with another haploid gamete, forms a diploid zygote. By interfering with mitosis and meiosis. Changes Involving Genomes and Chromosomes 141 drugs like colchicine (which destroy the spindle, so that the anaphase movement of chromosomes is prevented) and environ- mental stresses like starvation and cold can artificially induce autopolyploidy. We have already mentioned the normal occurrence of parthenogenesis in Solenobia and the sub- sequent fusion of haploid and of diploid nuclei to establish diploidy and tetraploidy, respectively. In other organisms, partheno- genesis may be artificially induced and haploid development initiated. In such cases, de- velopment as a haploid, of an individual that is ordinarily diploid, is usually abnormal. The abnormality produced must be due some- times to the expression of detrimental genes which would not be expressed in a diploid because of the presence of their normal alleles on homologous chromosomes. That this is not always the case is evidenced by the fact that if chromosome doubling, naturally or artificially induced, occurs at an early stage, a normal diploid (and homozygous) embryo may be produced. Such a chromosome doubling has produced parthenogenetic sala- manders and rabbits (which are female). In these cases, at least, abnormal development when haploid must often have its basis in quite a different factor. This factor probably involves the surface-volume relationships within the nucleus and between the nucleus and the cytosome, which are disturbed when cells adapted to contain a diploid number of chromosomes carry only a haploid set. Autopolyploidy occurs normally in certain somatic cells, such as liver cells in man, and may be induced by the irradiation of cells in tissue culture. In the autopolyploid cells we have so far discussed, each chromosome lies separately in the nucleus and proceeds to the mitotic metaphase independently. There are other cases of autopolyploidy in which all homologous chromosomes are synapsed even though the cell is part of somatic tissue. Let us examine an example of this as found in the giant salivary gland cells of Drosophila larvae. Note, first, that in the usual cell of Drosoph- ila, the metaphase chromosome is sausage- shaped, containing chromatids coiled tightly in a series of spirals like those in a lamp filament, and that during interphase the chromatids have largely unwound. The chromosomes in the salivary gland cell nucleus are also in an unwound state, perhaps even more so than in ordinary interphase, and have undergone three special changes. First, each chromosome present has rep- licated synchronously a number of times in succession, so that one chromosome produced two, two produced four, four produced eight, eight formed 16, 16 formed 32, etc. This replication can occur as many as nine times, producing 512 chromosomes. Second, all sister strands, instead of separating, remain in contact with the homologous loci apposed, giving the appearance of a many-threaded, polytene, cable. Third, since the original members of a pair of homologous chromo- somes are paired at homologous points, by what is called somatic synapsis, a double cable is formed which can contain as many as 1024 chromosomes. When seen under the microscope (Figure 18-3), these double cables have a cross-banded appearance. Be- cause of differences in density along the length of the unwound chromosome, a band is formed by a dense part of one chromosome being synapsed to the homologous region of all the other strands of that type (Figure 18-4). The pattern of bands is sufficiently constant and so characteristic that it is possible to identify not only each chromosome but dif- ferent regions within a chromosome (Figure 18-5). The giant size of salivary chromo- somes, long because they are unwound, and thick because of synapsed polytenes, offers a unique opportunity to correlate genetical and cytological events. While we have so far discussed only auto- polyploidy, there is a different way by which ploidy may increase. It is sometimes found that the genomes in a given species have been 142 CHAPTER 11 .3L •.-, \ . '''-«> .--^^^-'^i^-^^r 2L >*'., "% 1: FIGURE 18-3. Salircirv gland chromosomes of a female larva of D. melanogaster. {Courtesy of B. P. Kaitfmann; by permission of The American Genetic Association, Journal of Heredity, Frontispiece, vol. 30, No. 5, May, 1939.) FIGURE 18 4. A band (at top) cmd interband {below) region of a stretched Drosophila salivary gland chromosome. Photographed with the electron micro- scope at a magnification of approxi- mately I2,200X. Present enlargement is about ]3,000X. {By permission of The American Genetic Association, Journal of Heredity, vol. 43, p. 231, 1952.) FIGURE 18-5. The pair of fourth chro- oUALL |-^ 3 /-< >| mosomes, drawn to the same scale, as seen in salivary gland nuclei {each homo- log is highly polytene) and at mitotic metaphase {arrow). {By permission of The American Genetic Association, C. B. Bridges, '^Salivary Chromosome ^ \ i Maps,'' Journal of Heredity, vol. 26, Jl^-r^-" ^'' p. 62, 1935.) li:|\HV^' \> Changes Involving Genomes and Chromosomes 143 derived from different species. These cases represent examples of amphiploidy, or allo- polyploidy, in which two or more genomes have come from each of the different species. Cultivated wheat is an amphiploid. Amphi- ploids often show a combination of char- acteristics of their different parent species, just as you would expect. This type of poly- ploidy is discussed in more detail in Chap- ter 29. Changes in genome number represent the class of mutational events which involves the largest amounts of genetic material. While many plants are polyploid, this type of mu- tation does not occur many times in succes- sion, for chromosome number would become unwieldy in nuclear division. It should be noted also that certain other classes of muta- tion, like those involving a single locus, would have a greater difficulty expressing them- selves in polyploids than they would have in haploids or diploids where there is no other, or just one other, homologous locus capable of masking the mutant effect. Changes in genome number preserve the same ratios that genes or chromosomes have to each other in the normal diploid. Such changes are said to be euploid ("right-fold"). The next category of mutations to be dis- cussed in the present Chapter involves the addition or subtraction, not of whole chro- mosome sets, but of single whole chromo- somes. Such mutations upset the normal balance referred to and produce aneuploid ("not right-fold") genetic (chromosomal) constitutions. By what mechanisms can single whole chromosomes be added to or subtracted from a genome? We have already discussed two ways in previous Chapters, in the phenomena of chromosomal nondis- junction in normal diploids (Chapter 12) and of chromosomal segregation in autopoly- ploids (triploids in Chapter 14). You recall that nondisjunction in the germ line of Drosophila can produce offspring, otherwise diploid, that are XO, XXX, and XXY. Nondisjunction of the small fourth chromosome can lead to the production of individuals with one fourth chromosome (haplo-IV individuals) or three (triplo-IV individuals), as pictured in Figure 18-6. Even though addition or subtraction of a chromo- some IV makes visible phenotypic changes from the normal diploid condition, as you can see by referring to the Figure, both changes are viable. This is not true for FIGURE 18-6. Haplo-IV (left) and triplo-IV {right) females of D. melanogaster. The haplo-IV is smaller than the wild-tvpe female shown in Fig. 1 2-1. {Drawn by E. M. Wallace.) 144 CHAPTER 1! individuals that are haploid or triploid with respect to either one of the two large auto- somes, such individuals dying during the egg stage before they can hatch as a larva. In these cases, death is attributable to the genetic imbalance of having the numerous genes pres- ent in a long autosome in excess in individ- uals triploid in this respect, or to the de- ficiency of these genes in individuals haploid in this respect. We have seen (Chapter 14) how the diploid individual contains, in its two sets of chro- mosomes, a proper balance of genes for the determination of male and female sex. There is, therefore, every reason to expect that the production of other phenotypic traits is also dependent upon proper genie balance. It is not surprising, then, that a haploid animal mated to a diploid produces very few progeny, since after fertilization most zygotes are chro- mosomally unbalanced by the absence of one or more chromosomes needed to make two complete genomes. The triploid animal mated to a diploid also produces zygotes that are imbalanced, but in the opposite direction, having one or more chromosomes in excess of two genomes. However, in matings with diploids, the triploid animal usually produces more off- spring than the haploid one. This can be explained as the result of the lesser imbalance wrought by the addition of chromosomes to the diploid condition than by the subtraction of chromosomes from it. You can visualize this by comparing how far from normality (diploidy) each of the two abnormal condi- tions is. In the former case, in which one chromosome is in excess, the abnormal chro- mosome number of three is one and a half times larger than the normal number of two. In the latter case, in which one chromosome is missing, the abnormal chromosome num- ber of one is two times smaller than the nor- mal number. Thus, the addition of a chro- mosome makes for a less drastic change in balance than does its subtraction. (Accord- ingly, knowing in Drosophila that the triple dose of a large autosome is lethal, we would have predicted the single dose would be also.) Chromosome addition and subtraction can also be studied in Datura^ The haploid number of chromosomes here is 12. It is ^ Based upon work of A. F. Blakeslee and J. Belling. FIGURE 18-7. Silhouettes of the capsules formed by the twelve kinds of trisomic Datura. II Normal f f f Rolled Glossy Buckling f f f Elongate Echinus Cocklebur fit Microcarpic Reduced Poinsettia Spinach Globe Ilex Changes Involving Genomes and Chromosomes 145 possible to obtain 12 different kinds of indi- viduals, each having a different one of the 12 chromosomes in addition to the diploid num- ber. Each of these kinds is said to be trisomic for a different chromosome (the triplo-IV Drosophila being trisomic for chromosome IV), and is given a different name. The normal seed capsule and those produced by these 12 trisomies are shown in Figure 18-7. It is also possible to obtain viable plants that are diploid but missing one chromosome of a pair; these are monosomies or haplosomics (just as is a haplo-IV Drosophila). Individuals that have two extra chromosomes of the same type (tetrasomics) or have two extra chro- mosomes of different types (doubly trisomic) are also found. Datura permits us to test again our ideas relative to the phenotypic consequences of disturbing the normal balance among chro- mosomes. Compare, by means of Figure 18-8, the seed capsules produced by the nor- mal diploid (2N), with those of dip- loids having either one extra chromosome (2N + 1), of the type producing Globe (Figure 18-7), or two of these (2N + 2). The latter two are polysomics, which can be called trisomic diploid and tetrasomic diploid, respectively. Although the tetrasomic is more stable chromosomally (each chromo- some having a partner at meiosis) than is the trisomic, the tetrasomic phenotype is too ab- normal to establish a race, since it has a still greater genetic imbalance than the trisomic; and produces a still greater deviation from the normal diploid phenotype. In comparison, the tetraploid (4N) indi- vidual is almost like the diploid pheno- typically, as might be expected since chro- mosomal balance is undisturbed. The tetra- ploid which has the "Globe" chromosome extra once (4N + 1, making it a pentasomic tetraploid) deviates from the tetraploid in the same direction as the 2N + 1 already men- tioned deviates from 2N, but does so less extremely. Hexasomic tetraploids (4N + 2) deviate from 4N just about as much as 2N + 1 deviates from 2N. You see, then, that addition of a single chromosome to a tetraploid has less phenotypic effect than its addition to a diploid, since there is a rela- tively smaller shift in balance between chro- FIGURE 1 8-8. Effect upon the capsule of Datura of the presence of one or more extra ''Globe' {Figure 18-7) chromosomes. DIPLOID 2N 2N + 1 (Globe) 2N + 2 ^^^ TETRAPLOID 4N Jd iH # 4N + 1 4N + 2 4N + 3 146 ^ it ^ I. FIGURE 18-9. The chromosomal complement of a normal human female. Cell was in mitotic metaphase {hence chromosomes appear double) when squashed and photographed. {Courtesy of T. C. Hsu.) mosomes in the former than in the latter case. Thus, polyploids can stand whole chromo- some additions and subtractions better than can diploids. We have already mentioned in Chapter 13 some of the phenotypic consequences of whole chromosome subtraction and addition in human beings — otherwise diploid indi- viduals may be XO (Turner's type female) or XXY (Klinefelter's type male). Mongolian idiocy is known to be sometimes the result of a trisomic diploid chromosomal constitu- tion. In this case, the trisomic is the third smallest of all human chromosomes (the smallest being the Y) (Figures 18-9, 18-10). Trisomies for seven other autosomes are also known, each producing its own characteristic set of congenital abnormalities. It is a reason- able expectation that the haploid condition of these or of any other autosome would be lethal before birth, in line with the view that chromosome subtraction is even more detri- mental than chromosome addition. Although we have not always specified the particular manner in which each of the genomes aneuploid for whole chromosomes originated, you will recall that we have already indicated two general mechanisms by which such aneuploidy may arise in the germ line. It should also be mentioned that nondis- junction may also occur in somatic cells, as occurs when both daughter chromosomes proceed to the same pole at mitotic anaphase. This produces, in a diploid, one daughter cell Changes Involving Genomes and Chromosomes 147 \) It n 1 2 3 n H n 4 5 6 X n If n 7 8 9 it If 10 11 w 12 < are composed lengthwise of identical halves, each being termed, therefore, an isochromo- some. This diagram shows these chromo- somes contracting preparatory to metaphase. Diagram 5 shows that in mitotic anaphase the acentric is not pulled to either pole while the dicentric is pulled toward both poles at once. The acentric chromosome is, therefore, not included in either daughter nucleus and so is lost to both. (The acentric pieces in diagram 3 will be lost in any subsequent division, whether they join to each other or do not join at all.) The dicentric isochromo- some, being pulled to both poles at once, forms a bridge that may prevent any of the chromosome from entering either daughter nucleus, so that the dicentric is lost. Alter- natively, the centric regions of the dicentric piece may enter the daughter nuclei, and either the bridge may snap at any one of a number of places between the centromeres, so that the daughter nuclei are free of each other, or the bridge may persist so that the daughter nuclei are joined together. The amount of phenotypic detriment that a single nonrestituting break will produce in the daughter cells and their progeny cells will depend upon the particular chromosome in- volved, the place of breakage, and the fate of the dicentric piece. Suppose first, for ex- ample, that chromosome IV of Drosophila 151 FIGURE 19-1. Conse- quences of a single non- restituting ciiromosome brea/<. (which you recall is often viable as a haplo-IV individual) is the chromosome involved. The break may occur at any locus in IV and the loss of the genes in the acentric piece, though detrimental, will not usually cause invia- bility, nor will the loss of the entire dicentric fragment if excluded from both daughter nuclei. The phenotypic consequences would probably be the same should the bridge be- tween daughter nuclei snap (in which case, you recall, each daughter nucleus certainly will be deficient at least for the genes in the acentric piece). However, note what hap- pens when a bridge, involving a dicentric isochromosome linearly differentiated as a.bcddcb.a, snaps other than between the d segments. If it snaps between b and c, one fragment would be still more deficient, yet viable in the present example, while the other would contain an extra dose of the genes in the cd segment (most probably viable). Regardless of where the bridge snaps, how- ever, both daughter nuclei would carry an unjoined centric fragment which, after repli- cating, would usually form a new dicentric isochromosome which, at the next mitotic division, would again form a bridge. It is possible, then, to have a cycle of bridge- breakage-fusion-bridge events in the course of successive mitotic divisions. If, however, the bridge does not break, and 152 CHAPTER 19 the two daughter nuclei are tied together, the entanglement of the nuclei may interfere with subsequent attempts at nuclear division, even though, as in our example, the presence or absence of the genes located in the bridge may not be of paramount importance to the functioning of these cells. Suppose, next, that the chromosome broken is one of the large autosomes of Drosophila. Detriment or death to one or both daughter cells may occur because of the genes lost, when either the acentric piece or the dicentric fragment is left out of one or both daughter nuclei. In addition, successive bridge-break- age-fusion-bridge cycles may harm future cell generations via the aneuploidy produced as the result of the off-center breakage of di- centric isochromosomes. It would be ex- pected, other things being equal, that shorter dicentrics would usually break and that longer dicentrics would be more likely not to. Of course, any bridge between nuclei that does not break would be expected to have the effect already mentioned. Single chromosome breaks may occur either in the somatic or in the germ line. In the latter case, aneuploid gametes may be pro- duced. In the case of animals, since the genes have been found to be physiologically inactive in the gametes, aneuploid genomes can enter the egg and sperm without impair- ing their functioning. Accordingly, in ani- mals, aneuploid genomes can be carried by unaffected gametes into the zygote, which subsequently may suffer dominant detrimental or lethal effects. In most plants, however, the products of meiosis (pollen, for instance) perform certain physiological functions that require the action of the haploid genome they contain. So, in this case, aneuploidy is usu- ally more lethal or detrimental before fertili- zation than it is after. Having completed our discussion of the consequences of a single nonrestituting break, let us now consider the consequences of two breaks that occur in the same chromosome. Such breaks can be located either para- centrically, in which case both are to one side of the centromere, or pericenin'cally, where the centromere is between the breaks (Figure 19-2). Consider a chromosome linearly differ- entiated as ABCDEFG.HIJ, the centromere being between G and H. When the breaks are paracentric in position (between A and B, and F and G), the fragments may unite to produce a centric chromosome (AG.HIJ, Figure 19-2a) deficient for the acentric interstitial piece (BCDEF). The ends of the latter may join to produce a ring chromosome, which, in any event, is lost in the next nuclear division. When the breaks are pericentric (between D and E, and H and I), the end pieces are lost, whether or not they join together (Figure 19-2c). The centric middle piece can survive if its ends join to form a ring, and if the deficient sections are not extensive. Even if a ring can survive because it is not too hypoploid (the aneuploid condition in which genes or chromosomal regions are missing), it is at a disadvantage in that a single chiasma either with a nonring (called a rod) homolog or with another ring results in a dicentric rod or ring, respectively, as you can see by draw- ing the configurations for these situations yourself. Chromosomes with small deficiencies may act as recessive lethals and may have a lesser detriment than this when heterozygous ; those with large deficiencies usually act as dominant lethals in the next cell generation. Of course, the nucleus, in which breakage or other events occur which lead to a deficient chromosome or, for that matter, any other structural change, is still euploid. It is only after a nuclear division that the daughter cells be- come hypoploid or hyperploid (aneuploid be- cause of an excess of genes or chromosome parts). The preceding portion of this para- graph should be reread with this considera- tion in mind. It should be realized, in describing the Structural Changes Within Chromosomes 153 PARACENTRIC BREAKS ABCDEFGHIJ 1 r— A G H I J Deficiency B C D E F or B C^ £ F (a) lost AFEDCBG H Inversion (b) PERICENTRIC BREAKS ABCDEFGHIJ A B C D I J [ost IaBCDHGFEIJ -^— — — I I n I \^J Deficiency | G F Inversion (0 (d) FIGURE 19-2. Some consequences of two breaks in the same chromosome. production of deficiencies, that we have ignored the usual consequence of two breaks, which is that the ends from both breaks resti- tute. Again, only the consequences of the failure of breaks to restitute will be described in the discussion that follows regarding the production of other types of structural change. Another structural consequence of two breaks in the same chromosome is repre- sented in Figure 19-2b and d. In this case, the middle piece is inverted relative to the end pieces and undergoes exchange unions with them. The result is either a paracentric or a pericentric inversion (Figure 19-2b and d, re- spectively), which can occur by having the middle portion move while the ends are rela- tively stationary, or the reverse. Note that an inversion is a euploid rearrangement, and is of no phenotypic consequence from this standpoint. How would you expect an inversion to be- have during meiosis? If an inversion occurs by mutation and is retained in the germ line of a population, it may become homozygous in some individuals. In these individuals, meiotic behavior will be normal whether or not the tetrad is involved in chiasma forma- tion, since all the strands involved are identi- cally inverted. Other individuals, however, may possess one inverted homolog and a 154 CHAPTER 19 noninverted one, being heterozygous for the inversion. If the inversion is very small the homologs will pair properly everywhere but in the inverted region. Here, because the homologs cannot, in so short a region, twist enough to make homologous loci meet, they will fail to synapse, no chiasma will be formed and no crossing over will occur. Insofar as crossing over can lead to more adaptive recombinants, such inversion heterozygotes are at a disadvantage from this standpoint, as compared to noninversion or inversion homozygotes, because of the absence of re- combination among genes within the inverted region. Nevertheless, very small inversions may survive in any species. But consider now the meiotic behavior of heterozygotes for larger paracentric inver- sions. In this case (Figure 19-3 A), synapsis between homologs is made possible in all regions, with the exception of the parts near the points of breakage, by one homolog looping in the inverted region while the other does not. It happens that this Figure shows the inverted chromosome looping, but the reverse is equally likely to occur. Note that only two nonsister strands are shown; the other two naturally do not have a chiasma where these two have formed one. If one or more chiasmata occur outside the inverted region the four meiotic products will each be eucentric (having one centromere), as usual. If, however, one chiasma occurs anywhere within the inverted region, as shown between C and D, two strands of the tetrad will be eucentric (one with and one without the in- version, being those strands of the tetrad not shown in the Figure) and two will be aneu- centric (having no centromere or more than one). One of the aneucentrics will be acentric (duplicated for A and deficient for G.HIJ) while the other will be dicentric (having duplicated and deficient segments that are the complement of the acentric's). If the inversion is only moderately long, only one chiasma may occur within it, but if it is sufficiently large, some 2-strand double chias- mata may occur within it, in which case the crossover strands will be eucentric. Regardless of the length of a paracentric inversion, a single chiasma within the inverted region in an inversion heterozygote will pro- duce two aneucentric, aneuploid meiotic prod- ucts, as we have just seen. In animals that undergo crossing over each of these products will enter a gamete, which will function but usually have a dominant lethal effect after fertilization. This means that such individ- uals are at reproductive disadvantage, and this often leads to the elimination of the inversion from the population soon after it arises as a mutant. In certain species where there is no crossing over in one sex (the Drosophila male, for example), any homolog, inverted or not, has the same chance of being included in the gametes produced by that sex. There is a special factor to consider, however, with regard to meiosis in the Drosophila fe- male (the sex in which crossing over does occur), where it has been found that the two meiotic divisions occur in tandem, just as in Neurospora (see p. 123). In the Drosophila oocyte heterozygous for a paracentric inver- sion, a single chiasma within the inverted region produces a dicentric at anaphase I which orients the dyads at metaphase II so that the two eucentric monads proceed to the outermost of the four poles at anaphase II. At the end of telophase II, the four meiotic products, therefore, are arranged in a row: eucentric, part of dicentric, remainder of dicentric, eucentric — one of the two end eucentric-containing nuclei becoming the egg nucleus, the others degenerating. In this way the dicentric strand is shunted away from the egg nucleus, which therefore receives one of the two eucentric, noncrossover strands. In Drosophila, therefore, paracentric inversions of any size rarely cause aneuploid gametes in either sex and can become established in nature. Note, however, that in this species, the production of crossover-containing 155 G H I J A. PARACENTRIC INVERSION A B C D I J B. PERICENTRIC INVERSION gametes is suppressed in the paracentric in- version heterozygote female either because of nonsynapsis, or, in cases of synapsis, be- cause of the exckision from the egg of cross- overs within the inverted region. What is the effect of a chiasma within the inverted region in a heterozygote for a larger pericentric inversion? As seen in Fig- ure 19-3B, a single chiasma, such as between F and G, produces four eucentric strands: two are noncrossovers (one with, and one without, the inversion, these being, again, those strands of the tetrad not included in the FIGURE 19-3. A single chiasma in an inversion heterozygote. {See text for explanation.) Figure), one has a duplication (for ABCD) and a deficiency (for IJ), the other has a deficiency and a duplication of these same regions, respectively. These two aneuploid strands will enter the gametes of males, if crossing over occurs in the male. They will also be present in the gametes of females that have crossing over, even in the case of Drosophila, since all meiotic products are eucentric and there is, therefore, no shunting of euploid strands into the egg nucleus. So, the aneu- ploidy resulting from crossing over within a pericentric inversion always puts the hetero- 156 CHAPTER 19 zygote at a reproductive disadvantage. For this reason, it is expected, after arising by mutation, that only the smallest pericentric inversions, which do not synapse when hetero- zygous, will usually be able to survive in a population. Following two breaks in the same chromo- some, the last type of possible rearrangement to be discussed is duplication (Figure 19-4). If joining is delayed until after the broken chromosome reproduces, the two interstitial pieces may join, and, with the appropriate end pieces, produce a eutelomeric chromo- some with the interstitial region repeated (neither, either, or both of these regions may be inverted with respect to the original ar- rangement). (The two remaining end pieces may or may not join to form a deficient chromosome.) Provided the duplicated re- gion is small enough and acentric, such a duplication may survive in the population. We shall consider now what is expected to happen when two breaks occur, one in each of two different chromosomes. In the first case to be considered, the two chromosomes may be homologs (ABCDEFG.HIJ). Usu- ally, the breaks will be at different places, say between A and B in one, and D and E in the other. Exchange unions can occur one way to produce a dicentric and an acentric chro- mosome, whose fate you can readily predict. Other exchange unions can produce two eucentric chromosomes in which BCD is deficient in one and duplicated in the other. In the second case of this kind, the two chromosomes broken are nonhomologous (Figure 1 9-5). If the two centric pieces unite, a dicentric is formed. The two acentric pieces are lost in the next division, whether they join each other or do not join at all. If all pieces join as indicated, what is accom- plished is a mutual exchange of segments between nonhomologous chromosomes; this is called a segmental interchange, or usually, a reciprocal translocation, and is of the an- eucentric type. This type often acts as a dominant lethal in a subsequent division, particularly when the dicentric is pulled toward both poles at once. It is often just as likely, however, that union occurs between the centric piece of one chro- mosome and the acentric piece of the non- homolog, with, vice versa, the centric piece of the second joining the acentric piece of the first. This reciprocal translocation is of the eucentric type. In individuals heterozygous for such an exchange, having two nonhomo- logs translocated and two nontranslocated, BREAKAGE A B C D E F G .H J REPLICATION AB CDE FGHIJ AB CDE FGHIJ CROSS-UNION Figure 19-4. Duplication {tandem type). ABCDECDE FG H A B F G H Structural Changes Within Chromosomes 157 gametes will be formed with deficiencies and duplications if segregation results in their receiving one but not both members of the reciprocal translocation. In nuclei in which the chromosomes are compacted in a relatively small volume, no broken end is very distant from any other broken end, and usually if one of the two unions needed for reciprocal translocation occurs, so does the other. This is the situa- tion in the nucleus of the sperm of Drosophila just after it has been involved in fertilization. In oocytes, and probably in other cells that have a relatively large nuclear volume, the distances between the broken ends of non- homologs are so great that reciprocal trans- locations are comparatively rare, and even if one cross union occurs the two other broken ends usually fail to join to each other, so that only half of a reciprocal translocation — • K L M N O 1 • P Q R S T U called a half-translocation — is produced. The loss or behavior of the unjoined frag- ments usually causes descendent cells to die or to be abnormal, as you would expect. Half-translocations are sometimes found as the result of segregation in heterozygotes for a eucentric reciprocal translocation (see the previous paragraph). Half-translocations having this origin are known to occur in human beings, for example. In view of the preceding discussion of trans- locations, you can predict that such mutants would tend to be eliminated from the popu- lation. There is one exception to this, how- ever, involving eucentric reciprocal trans- locations in which both chromosomes are broken close to their centromeres. In these cases, almost a whole arm of each chromo- some has been mutually exchanged. Such whole arm reciprocal translocations when heterozygous in Drosophila, and probably most other species, undergo synapsis and disjunction in such a regular manner that euploid gametes (containing both or no pieces of the translocation) are usually formed, so that translocation heterozygotes of this type are not at an appreciable reproductive dis- advantage. K L M N O FIGURE 19-5. Reciprocal translocation between nonhomologous chromosomes. ANEUCENTRIC TYPE Q R S T U K L Q R S T U P M_N O EUCENTRIC TYPE 158 CHAPTER 19 Structural changes may result also after three breaks. When all three occur in one chromosome, the two interstitial pieces may exchange positions, producing what is called a shift. Two breaks in one chromosome and one in a nonhomolog can result in the inter- stitial piece of the first chromosome being inserted into the second. This result is called transposition. In this and in the preceding Chapter, it has been pointed out that a change in ploidy may survive in nature when it either causes no shift in chromosome balance (because it deals with whole genomes), or involves eucentric aneuploidy in which small segments of chromosomes are hypo- or hyperploid. For, in the latter case, the deficient or dupli- cated genes are limited in number and produce only moderate phenotypic effects. On the reasonable assumption that the greater the amount of chromosomal material, the greater the complexity possible in an organism, and correspondingly, the greater the possible diversity in its phenotype and adaptiveness, viable changes in ploidy are particularly im- portant in organic evolution. In view of this it becomes desirable to consider the different ways in which small numbers of genes may be added to a genome following breakage. Two methods of increasing gene number following breakage have already been de- scribed. One of these dealt with two breaks in the same chromosome. After breakage, the entire chromosome replicated, after which the broken ends joined so as to form a chro- mosome with the interstitial piece duplicated (cf. p. 156). The other method involved a pair of homologs each having one break in a different region, followed by eucentric cross union (cf. p. 156). A third mechanism in- volves the heterozygote for a shift. If, in this case, the homologs pair in the region of the shift and a chiasma forms within it, you will see, by tracing the resultant strands, that one of the crossovers has a section in duplicate. FIGURE 19-6. Inversion heter- ozygotes in corn {pacliynema) {courtesy of D. T. Morgan, Jr.) and in Drosophila {salivary gland) {courtesy of M. De- merec). Structural Changes Within Chromosomes 159 Finally, it should be mentioned that a chro- mosome containing a transposition may come to be present, in subsequent generations, not with the nonhomologous, deficient, chromo- some from which the piece was transposed, but with two normal chromosomes of that type. In this way an individual is produced containing a pair of normal homologs, part of which is present in hyperploid condition in a nonhomolog. The preceding paragraph illustrates how the same type of structural change (dupli- cation) may result following different types of breakage events. Accordingly, by observ- ing the rearrangement produced, one cannot always specify the particular number of breaks originally involved. Usually, the simplest explanation is proposed. You should also note that cells which are missing an entire chromosome may be produced consequent to breakage; thus, not all such cells are the result of nondisjunction. Breakage events can also produce the monosomies discussed in the preceding Chapter, but not the trisomies. During the course of the present Chapter it is very likely that you have wondered how the structural changes in chromosomes we have discussed are detected. Such mutants may be detected by direct cytological exami- nation of cells. Or they may first be noted by their effects on the phenotype in general, after which genetic tests are made to detect their specific nature and fate in the population. So, identification of the type of structural change involved may be made genetically or cytologically, or by both methods. Deficiencies may sometimes be recognized genetically when heterozygous, since they permit the expression of any allele of all genes present in this region in the nondeficient chromosome. Inversions and translocations may be suspected when mutant heterozygotes show a marked reduction in offspring carry- ing crossovers. Using appropriate marker genes, inversion homozygotes will show certain genes in an order the reverse of nor- mal, while in translocations genes normally not linked, will be found linked. These types of mutants may also be identified cyto- logically; sometimes the cytological method is preceded by genetic studies that indicate which types of structural changes are likely to be involved and/or the particular chromo- somes concerned. Of course, detailed knowl- edge of the appearance of the normal genome is a prerequisite for such cytological work. The prophase of meiosis of some organ- isms, and the giant salivary gland chromo- somes of Diptera, are particularly suited for cytological studies, because synapsis between 0f^ % S FIGURE 19-7. Salivary gland chro- mosomes heterozygous for a shift within the right arm of chromosome 3 of Drosophila melanogaster. A piece from map region "'98''' is in- serted into map region "97." The rightmost buckle is due to the ab- sence of the shifted segment; the leftmost buckle is due to its pres- ence. (Courtesy of B. P. Kauf- mann.) 160 CHAPTER 19 homologs helps locate the presence, absence, or relocation of chromosome parts. For ex- ample, in inversion heterozygotes, there will be one reversed segment which does not pair with its nonreversed homologous segment (if the region is small), or which shows one homolog forming a loop in order to synapse (if the inversion is larger) (Figure 19-6). A deficiency heterozygote will buckle in the region of the deficiency. Since a chromosome with a duplication may also buckle when heterozygous, careful cytological study is needed to distinguish this case from deficiency (see Figure 19-7). Heterozygotes for recipro- cal translocations (Figure 19-8) will show two pairs of nonhomologous chromosomes as- sociated together in synapsis. This discussion should suffice to introduce you to the origin, nature, and consequences of the more common types of structural changes in chromosomes, and to the methods used to identify such mutations. FIGURE 19-8. Heterozygous reciprocal translo- cation in corn {pachynema) {courtesy of M. M. Rhoades) and Drosophila {salivary gland) {cour- tesy of B. P. Kaufniann). SUMMARY AND CONCLUSIONS Structural change in chromosomes is a type of mutation involving the gain, loss, or relocation of chromosome parts. All such mutations are preceded by chromosome breakage. Since proximity of broken ends favors their union, most broken ends restitute. Nonrestitutional unions give rise to structural changes in chromosomes. The occurrence of one, two, or three nonrestituting breaks in one or two chromosomes is discussed in relation to the pro- duction of whole chromosome losses, deficiencies, duplications, inversions, translocations, shifts, and transpositions. Structural Changes Within Chromosomes 151 The chromosomes that have undergon estructural change may be euploid or aneuploid. The cells in which these mutations arise are euploid but may become aneuploid following mitosis, segregation, or crossing over. The structural changes most likely to be retained in the population are the smallest ones, those directly or indirectly causing an increase in gene number being most likely to be important in evolution. REFERENCES Beam, A. G., and German III, J. L., "Chromosomes and Disease," Scient. Amer 205 No. 5:66^76, 1961. Muller, H. J., "The Nature of the Genetic Effects Produced by Radiation," in Radiation Biology, A. Hollaender (Ed.), New York, McGraw-Hill, 1954, Chap. 7, pp. 351-473. QUESTIONS FOR DISCUSSION 19.1. The terms euploid and aneuploid (hypo- or hyperploid) have been applied both to individual chromosomes and to whole nuclei. Give an example of: a. A hypoploid chromosome in a euploid nucleus. b. A hyperploid chromosome in a hyperploid nucleus. c. An aneuploid nucleus containing all structurally normal chromosomes. 19.2. Could a reciprocal translocation occur between homologous chromosomes? Explain. 19.3. What does the term eutelomeric mean, as used on p. 156? 19.4. While most Mongolian idiots are trisomic for a particular small autosome, some are known that have 46 chromosomes. In the latter cases, all chromosomes seem of normal composition except that one of the homologs of a different autosome has one arm that is exceptionally long. Discuss the origin and cause of Mongolian idiocy in these exceptional cases. 19.5. Given the chromosome AB CDE/F.GHI/J, where the period indicates the centro- mere and the slanted lines the positions of three simultaneously produced breakages, draw as many different outcomes as you can. Indicate which one is the most likely to occur. 19.6. The loss of a given chromosome in Drosophila, resulting in monosomy, is approxi- mately 3-5 times as frequent as its gain, resulting in trisomy. Explain. 19.7. Discuss the frequency of monosomies among human zygotes. 19.8. Discuss the detectability, in human chromosomes at mitotic metaphase, of a: a. Paracentric inversion. b. Pericentric inversion, c. Deficiency. d. Duplication. e. Half-translocation. 19.9. What advantages may inversion provide? 19.10. What characteristics of cells undergoing oogenesis favor the production and viable transmission of half-translocations? 19.11. In Drosophila, a male, dihybrid for the mutants hw and st, when backcrossed to bw hw St St, normally produces offspring whose phenotypes are in a 1:1:1:1 ratio. On exceptional occasions, this cross produces offspring which are clearly of only two of the four phenotypes normally obtained. How can you explain such an exception? 19.12. Is the telomere a gene? Why? Chapter 20 CYTOGENETICS OF OENOTHERA Ai T THE close of the last Chapter you were introduced to some .of the genetic and cytological methods for detecting and identifying struc- tural changes in chromosomes. In still earlier Chapters you have learned the char- acteristics and some of the properties of other types of mutational events, as well as the basic principles of transmission genetics. Let us take the opportunity to use this informa- tion in toto in an investigation ^ of a plant called the evening primrose, Oenothera, which has until now been mentioned only on one occasion (p. 139). Oenothera (Figure 20-1) is a common weed found along roadsides, railway embankments, and in abandoned fields. It is self-fertilizing in nature, where it exists in a number of pure breeding strains, each having a characteristic phenotype. These strains can be cross- fertilized in the laboratory, however, and progeny obtained. If the two strains crossed are Lamarckiana and biennis, a surprising result is obtained in Fi. In the first place, the Fi are not all uniform in phenotype, as we would expect from previous experience with crossbreeding two pure lines, but are of three distinct types which we can call A, B, C. In the second place, each of these three Fi types is thereafter pure breeding upon self- fertilization. If the Fi were hybrid, we would expect self-fertilization to produce recombi- nants of more than one phenotype. These ^ Based upon work of H. DeVries, O. Renner, R. E. Cleland, F. Oehlkers, A. F. Blakeslee, J. Belling, S. Emerson, and A. H. Sturtevant. 162 FIGURL 20-1. Oenothera. (Cuurtesy of R. E. Cleland.) two peculiarities are summarized in Figure 20-2, where the typical results obtained from similar crosses with garden peas are indicated side by side. What conclusions can we draw from this information? We must conclude, contrary to any such impression which might have been gained in Chapter 1, that self-fertilizing strains cannot automatically be considered to be pure, homozygous lines. In order to ob- tain three different genotypes in Fi, either Lamarckiana, or biennis, or both, must be heterozygous. Let us make the simplest as- sumption, namely that Lamarckiana is heter- ozygous for a single pair of genes. If so, how can this strain produce only Lamarckiana upon self-fertihzation? This would require that the heterozygote produce only hetero- zygote progeny. But suppose that self- fertilization does, as expected, produce the two homozygotes, but that these are never observed because both types are lethal. (Re- call that for yellow mice, see p. 65, only one Cytogenetics of Oenothera 163 PEAS OENOTHERA Tall Dwarf Lamarckiana Biennis t i 1 + Tall Dwarf Lamarckiana Biennis ♦ ♦ 1 1 Tall Dwarf Lamarckiana Biennis \ / X >^ ^^ F Tall F A B C / \ ' * \ 1 F, 3 Tail- 1 Dwarf F, A B c FIGURE 20-2 {Above). Comparative breeding re- sults from garden peas and Oenothera. FIGURE 2Q-3 (Below). Balanced lethal systems that enforce heterozygosity. ZYGOTIC LETHAL SAMETOPHYTIC LETHAL homozygote is lethal, the other is viable. In the present case the two different alleles would have to act as recessive lethals.) This hy- pothesis would require that of all zygotes, one half die before becoming mature Lamarcki- ana. This view is supported by finding that approximately one half of the ovules regular- ly fail to produce seed upon self-fertilization, and this is evidence that Lamarckiana in nature is a permanent heterozygote in this respect, as the result of a balanced lethal system. In this case, since ovules fail to produce seed, the lethals must kill prior to this stage. In fact, the lethal may kill at the time of fertilization, or very soon thereafter, being in effect a zygotic lethal (Figure 20-3). But, we have ignored another possible time for lethal action. Recall that in some plants, including Oenothera, there is a haploid game- tophyte generation. Permanent heterozygos- ity could be maintained also, if one allele were lethal to the male gametophyte and the other to the female (Figure 20-3). So game- tophytic lethals can also provide a balanced lethal system. Further study shows that half of the ovules fail to produce seed in biennis also, and, in general, in all strains of Oeno- thera found in nature, and that, in fact, both zygotic and gametophytic lethals are involved in these balanced lethal systems. Does the establishment of the existence of a balanced lethal system in Oenothera explain how it is that the phenotype, say of La- marckiana, is the only one produced in the progeny from self-fertilization? Since all sexual organisms so far studied have many pairs of genes, it would not seem reasonable that Oenothera has only a single pair of recessive lethal genes whose pleiotropic (mani- fold) effects produce the entire phenotype. It is more reasonable to believe that there are many gene pairs but that these form a single linkage group, so that the diploid has one genome whose genes are all linked to one recessive lethal, and another genome whose senes are all linked to the allelic lethal. 164 CHAPTER 20 In other words, Lamarckiana behaves as though it contains two complexes of linked genes. Within a strain these genes are com- pletely linked by some mechanism that pre- vents recombination, so that only two kinds of genotypes are found in the gametes. The two gene complexes are so constant in natural populations of a strain that they are given, in the case of Lamarckiana, the names gaudens and velans, so that this strain can be identified as gaudens. velans, whereas biennis can be described by its gene complexes as albi- cans.rubens. Figure 20-4 shows how these bal- anced lethal gene complexes are distributed FIGURE 20-4. Balanced lethal gene complexes in O. biennis and O. Lamarckiana. BIENNIS = LAMARCKIANA = Albicans (A) Gaudens (G) GAMETES PLANT GAMETES ((O)) G ^ PLANT O) A R (•) GAMETES (O) G PLANT / \ / \ Cytogenetics of Oenothera \6S generation after generation in biennis and Lamarckiana. It is a simple matter for you to work out that all the recessive lethal alleles in the one strain cannot be identical to those in the other of these two strains, for the Fi from crossing them would be of only two different phenotypes, whereas three types were actually obtained. We can conclude, therefore, that the balanced lethal system in Oenothera in general involves either a multi- ple allelic series, or several pairs of genes, or both. While each of the three different hybrids obtained in Fi from crossing Lamarckiana and biennis breeds true upon self-fertilization, showing in this way that each contains two completely linked gene complexes, this may or may not be true of the breeding behavior of hybrids obtained from certain other inter- racial crosses. This ambivalence is illus- trated in Figure 20-5. In this Figure, the gene complexes present in the different hy- brids are shown at the left. The distribution in their gametes of the various genetic markers shown at the top of the Figure was determined by breeding tests. So, for example, the cur vans. velans hybrid bred in such a way that all marker genes behaved as though they were still linked (the marker genes and their alleles being distributed in gametes in a total FLAVENS-CURVANS FLAVENS-PERCURVANS FLAVENS-FLECTENS FLAVENS-VELANS RUBENS-FLAVENS RUBENS-CURVANS CURVANS-VELANS RUBENS-VELANS FIGURE 20-5. Linkage groups in liybr ids from interracial crosses. 166 CHAPTER 20 of only two combinations). On the other hand, iheflavens.velans hybrid produced four kinds of gametes, the genes for R, m, and P (all still linked to each other) segregating independently of the genes for B and Sp (both still linked to each other), so that half of the gametes contained one of the two parental combinations, the other half carried one of the two recombinations. In this case, there- fore, genes that belonged to a single linkage group in the parent races behaved as two linkage groups during the gametogenesis of their hybrid. (The fact that 50% recombina- tion occurred in the gametogenesis of the hybrid means that we cannot explain these results by postulating i\\dii flavens (or velans) is always a single linkage group, which cannot crossover with the partner gene complex of the parent race, but which can do so when its partner is velans {or flavens)) Other tests of the hybrid containing jia- vens.curvans showed m and P still linked but separate from B, which was, in turn, separate from Sp and Cu, so that there were now three linkage groups, and perhaps more would have been found had additional genetic markers been employed. In all cases, however, the same hybrid combination always showed the same linkage groups in its gametogenesis. In view of the fact that at least three linkage groups can be identified in certain hybrids (even though under certain conditions these act as one), it is to be expected that the diploid would have at least three pairs of chromo- somes. Cytological examination confirms this genetic expectation, there being seven pairs of chromosomes in all of the Oenothera strains discussed in the present Chapter. (Oenothera gigas, the triploid mentioned in Chapter 18, has 21 chromosomes.) Accord- ing to our assumption that the balanced lethal system is based upon a single pair of genes located on a single pair of homologs, this pair of chromosomes must necessarily remain heterozygous to be viable in Fi. But this would not be expected to be true for the other six pairs of chromosomes, which should "segregate independently. So, for example, gametes of biennis which carry the albicans recessive lethal should be equally likely to carry the riibens or the albicans homolog in each of the other six cases of independent segregation. But this is not found. It is theoretically possible, however, to have seven pairs of chromosomes each heterozygous for a different recessive lethal which would, upon self-fertilization, produce only viable Fi like itself. Since this explanation would predict that only about ^2^ of all ovules would develop as seeds, it cannot be the correct one for Oenothera. FtGURE 20-6. Circle of 14 c/iromosomes in Oeno- thera. Chromosome number is clear in upper cell where the circle has broken open. {Courtesy of R. E. Cleland.) Cytogenetics of Oenothera 167 FIGURE 20-7. Ralph E. Cleland points to zig-zag chromosomal arrangement at the start of anaphase I of an Oenothera having a circle of 14 chromosomes at meta- phase I. {Photograph courtesy of The Calvin Company.) A clue to the orderly segregation of com- plete gene complexes is found in the study of meiosis in Oenothera. Here it is found that the typical Oenothera in nature does not form seven separate bivalents, but, as seen clearly at metaphase I, forms a closed circle of 14 chromosomes, synapsed end to end (Figure 20-6). At anaphase I, moreover, chromo- somes that are adjacent to each other in the circle go to opposite poles of the spindle, so that at the start of the separation the chro- mosomes assume a zigzag arrangement (Figure 20-7). If you make the assumption that paternal and maternal chromosomes alternate in the circle, then all paternal chro- mosomes go to one pole, and all maternal chromosomes to the other. The complete hnkage of all genes in a complex would be explained (if crossing over is rare) by such a manner of chromosome segregation, and the gametes produced by an individual would be identical to those which united to form it (Figure 20-8). If the alternate segregation procedure sepa- rates maternal and paternal genomes, a circle should always contain an even number of chromosomes. Moreover, we could make the prediction that when a genome no longer behaves as a single linkage group it would also no longer form, with the other linkage group, a single circle of 14 chromosomes. Theoretically, there is a total of 15 different ways that 14 chromosomes can be arranged in circles (composed of even numbers of chromosomes) and pairs, as shown in Figure 20-9. And when various race hybrids are made, indeed all 15 types and no others are found in the metaphase I stage, any particular 168 CHAPTER 20 PARENTAL GAMETES ^^^^^^^^^^^^ GAMETES PRODUCED FIGURE 20-8. Manner oj chromosome segregation during meiosis o/ Oenothera. Figure 20-9. Circle and pair arrangements possible for Oenothera chromosomes. / Q 14 Q 10 , 2 Pairs QlO ,0 ^ O 6 ♦O^ ' 2 Pairs G 2 »0 ^ O ^ » ^''°'" Q 12 , 1 Pair O ^ » '^ ^°''"^ Q 8 , Q 4 , 1 Pair O "* » ^ ''°'" Q 6 , Q 6 , 1 Pair 7 Pairs O"* »0 4 »0 ^ » 1 P°'^ QzCIRCLE Cytogenetics of Oenothera 169 hybrid always giving the same meiotic con- figuration. (The top cell in Figure 20-7 shows an inner circle of four and an outer circle of ten chromosomes.) If what has been sup- posed is true, it also ought to follow that while alternate chromosomes within a circle | show complete linkage with each other, such I linkage groups would not be prevented from I segregating independently from those linkage groups made up of chromosomes either in separate circles or in separate pairs. This^ expectation can be tested by comparing the number of linkage groups found in the dif- ferent hybrids of Figure 20-5 with the chro- mosome arrangements seen cytologically during their meiosis. When this is done, it is found that the number of separate groups of chromosomes observed in meiosis is always equal to, or greater than, the number of link- age groups detected genetically. In fact, whenever enough genetic markers are used, the number of linkage groups always equals the number of chromosome groups. While all of the preceding satisfactorily demonstrates that segregation of alternate chromosomes in a circle to the same pole and the presence of balanced lethal systems can explain most of the unusual genetic behavior of Oenothera, other matters still need explana- tion. What causes these chromosomes to form circles in the first place? A clue to this is contained in the observation made near the end of the last Chapter, namely, that two pairs of nonhomologs will be associated to- gether during synapsis if a reciprocal trans- location is present between two of the non- homologs and absent in their homologs, i.e., if these nonhomologs are heterozygous for a reciprocal translocation. This can be il- lustrated in Oenothera by means of Figure 20-10. In Oenothera all chromosomes are small, of the same size, with median centro- meres. To help us identify homologous chromosomes, the ends of each chromosome in a genome are given diff'erent numbers. Suppose, some time in the past, a eucentric reciprocal translocation occurred between the tips marked 2 and 3 (top left of Figure). This rearrangement in heterozygous condition (middle left) would produce an X-shaped configuration at the time of synapsis in pro- phase I (bottom left), and a circular appear- ance at metaphase I — early anaphase I. In this way a circle of four chromosomes would be produced. If now a second reciprocal translocation 2"2 FIGURE 20-10. Heterozygous reciprocal translocations and circle formation. 3 3 2' '2 170 CHAPTER 20 occurred between any chromosome tip in a circle of four and a tip of some other pair of chromosomes, a circle of six chromosomes will form in the heterozygote for both recipro- cal translocations. This is illustrated at the right of Figure 20-10 where the upper dia- gram shows the configuration before tips 4 and 5 exchange, and the lower diagram shows the circle of six following this exchange. Still larger circles can be formed by successive interchanges of this type, six being required to form a circle of 14. The presence of reciprocal translocations in heterozygous condition could explain how various sized circles containing even numbers of chromo- somes are produced in Oenothera. We have not yet completed our analysis of Oenothera, however. One of the questions remaining is: By what mechanism is it ar- ranged that alternate chromosomes in a circle proceed to the same pole during meiosis? The answer to this is unknown. A second question stems from the fact that almost all the different races of Oenothera found in nature form a circle of 14. Are the six trans- locations involved the same in all races? No, for if they were, viable hybrids between races would form either circles of 14 or seven pairs at meiosis. The very fact that all the configurations in Figure 20-9 are found in such hybrids must mean that different gene complexes must differ from each other in the specific ways that their chromosome ends have become translocated. There are many thousands of ways, theoretically, that 14 ends can be arranged in seven groups of two. How can we determine how many of these different arrangements are found in nature? Let us start by taking a particular gene complex, calling it the standard, and labeHng its chromosome ends 1-2, 3-4, 5-6, 7-8, 9-10, 11-12, 13-14. (Normally, i.e., in nature, this complex would form a circle of 14 with the other gene complex, which would therefore have no chromosome with the same pair of ends as has the standard.) Now a series of interracial hybrids is formed with the stand- ard as one of the complexes, and their meiotic arrangements scored. Suppose in one case the hybrid forms 5 pairs and a circle of 4. This must mean that the ends of 5 chromo- somes are in the same order in the complex under test as in the standard, but that they are in a different order in the remaining two chro- mosomes. Until now there was no reason to assign ends 1-2, 3-4 of the standard to any particular chromosomes. But now we can arbitrarily assign these ends to the two stand- ard chromosomes in the circle of 4, and, therefore, the chromosomes in the circle from the complex under test can be called 2-3, 4-1, In this way, the composition of ends of two pairs of chromosomes is specified perma- nently. Figure 20-1 1 (top) shows the standard and tested complexes in our example syn- apsed according to numbers. Let us call the complex just tested A. Sup- pose next another complex, B, is made hybrid both with the standard and with A. You can see that an appropriate result will specify other ends of standard. A, and B. Such procedures can be carried out until all of the standard's chromosomes are specified and the complete order of all 14 ends determined for any other complex. In this manner, it is discovered in nature that a circle of 14 is produced in many different ways, a theoretical and an actual example being shown in the central and lower parts of Figure 20-11. In fact, of 350 complexes analyzed, more than 160 different segmental arrangements have been found. All these results are consistent with the hypothesis that during the course of evolution the ends of Oenothera chromo- somes have been shuffled many times in dif- ferent ways by reciprocal translocation. Finally, a most convincing test of the recipro- cal translocation interpretation would be the ability to predict the meiotic chromosomal arrangement to be found in a hybrid not yet formed. This has been done many times and Cytogenetics of Oenothera 111 i //. A. COMPLEXES DIFFERING BY ONE RECIPROCAL TRANSLOCATION 1.2 3.4 5.6 7.8 9.10 11.12 13.14 ' \ / \ II II I I I \-\ \i< 2.3 4.1 5.6 7.8 9.10 11.12 13.14 I ^ B. COMPLEXES DIFFERING BY SIX RECIPROCAL TRANSLOCATIONS 1.2 3.4. 5.6 7.8 9.10 11.12 13.14 \ / \ / \ / \ / \ / \ / \ 2.3 4.5 6.7 8.9 10.11 12.13 14.1 . I C. MURICATA RACE'S ACTUAL COMPOSITION OvQ\A 1.2 3.4 6.5 13.12 7.11 10.9 8.14 \ / \ / \ / \ / 1 / \ / \ 2.3 4.6 5.13 12.7 11.10 9.8 14.1 A and B are theoretical FIGURE 20-11. Arrangement of chromosome ends in different Oenothera complexes. all such predictions came true. We see, then, that the cytogenetic behavior of Oenothera becomes understandable in view of its (1) long previous history of reciprocal translocation, (2) method of chromosome segregation, (3) balanced lethals, and (4) self-fertilization. At various points in this discussion Oeno- thera seemed to behave exceptionally, ap- parently violating our concepts of pure lines and independent segregation. More com- plete analysis has shown, however, that the failure of Oenothera to behave in the ways expected was due to the operation of other, already known, genetic events. Oenothera is an exception which should be treasured, for in the exact correspondence between its atypical genetics and its atypical cytology, it furnishes an outstanding example of the validity of the chromosome theory of trans- mission genetics. SUMMARY AND CONCLUSIONS Both the genetics and the cytology of Oenothera are exceptional. This, however, is found to be the normal consequence of the simultaneous operation of certain already known cyto- genetic phenomena. In this way, Oenothera provides an outstanding confirmation of the validity of the chromosome theory of transmission genetics. 172 CHAPTER 20 REFERENCES Cleland, R. E., "Some Aspects of Cyto-Genetics of Oenothera," Bot. Rev., 2:316-348, 1936. QUESTIONS FOR DISCUSSION 20.1. What evidence can you present that the genes comprising the balanced lethal system in Lamarckiana are different from those in biennis'} 20.2. Discuss the statement: "All evening primroses in nature are constant hybrids." 20.3. With respect to chromosomes, how does the origin of a circle differ from the origin of a ring? 20.4. Can circles contain an odd number of chromosomes? Explain. 20.5. What new investigations regarding the genetics and/or cytology of Oenothera does the present Chapter suggest to you? 20.6. List the principles of genetics you could have arrived at had Oenothera been the only organism so far studied. 20.7. If this Chapter contains no new principles of genetics, for what purpose do you suppose it was written? 20.8. Curly winged Drosophila mated together always produce some non-curly offspring. Plum eye colored flies mated together always produce some non-plum offspring. But, when flies are mated that are both curly and plum only flies of this type occur among the offspring. Defining your gene symbols, explain all three kinds of results. 20.9. Draw a diagram representing the appearance of a heterozy- gous whole-arm translocation in Drosophila at the time of synapsis. Number the arms of the chromosomes involved. What else would you require in order that a mating of two flies with this configuration pro- duce only offspring of this type? 20.10. Do you suppose that the pres- ervation of heterozygosity has an adaptive advantage in Oeno- thera! In other organisms? Hugo De Vries (1 848- 1 935), pioneer in the study of Oenothera genetics. {By permission of Genetics, Inc., vol. 4, p. 1, 1919.) Chapter 21 NATURAL AND INDUCED CHROMOSOMAL CHANGES HAPTER 19 dealt primarily with different types of structural 'change within chromosomes and the manner in which their origin de- pended upon chromosome breakage. How- ever, little was said there concerning the factors responsible for the production of the key events of breakage and of union. This is one of the matters to be taken up in the present Chapter. Also, in that earlier Chapter, relatively little detail was given con- cerning the types of structural change ac- tually found in nature. We did learn sub- sequently, in Chapter 20, that reciprocal translocations have played an important role in the evolution of Oenothera. It might be claimed, however, that Oenothera does not furnish a representative test of the im- portance of chromosomal rearrangements in evolution, since its cytogenetical behavior is so unorthodox. For the specific reciprocal translocations in Oenothera involve the ends of chromosomes and are retained in natural populations in heterozygous condition. There are hundreds of different species of Drosophila in nature. These species can be compared ecologically, morphologically, physiologically, serologically, and biochemi- cally. They can also be tested for ability to interbreed, and when they do crossbreed, their genetics can be compared ; they can be com- pared relative to the banding patterns of their salivary gland chromosomes and the appear- ance of their chromosomes at metaphase. And when all known information of this kind 173 is taken into account, it becomes possible to arrange these species on a chart so that those closest together are more nearly related in descent, in evolution, than are those farther apart. ^ This has been done in Figure 21-1, which shows the haploid set of chromosomes at metaphase. including the X but not the Y chromosome, for different species or groups of species of Drosophila. For example, the chromosomes of the melanogaster species group are drawn in row 2, column 1, the bot- tom chromosome being the rod-shaped X, the two V's being the two large autosomes (II and III) and the dot representing the tiny chromosome IV. In the other metaphase configurations, chromosomes or their parts which are judged to be homologous are placed in the same relative positions. What can we learn from a comparison of these metaphase plates? Since the amount of detail in a metaphase chromosome is limited to size and shape, we cannot expect to detect any rearrangements of small size at this stage. So, regardless of their importance, small rearrangements involving duplications, deficiencies, shifts, transposi- tions, inversions, and translocations cannot be detected in the Figure. Even a large para- centric inversion is undetected here, since it does not change the shape of the chromo- some. However, other gross structural changes can be detected. In row 4, the chro- mosome patterns in columns 2 and 3 seem identical, except that a pericentric inversion has changed a rod to a V, or the reverse. (Pericentric inversions will always change the relative lengths of arms when the two breaks are different distances from the centromere.) Compare the plate for melanogaster (r.2, c.l) with the plate to its right (r.2, c.2). A V-shaped autosome in melanogaster appears as two rods in its relative. (Note also that the dot chromosome is missing.) In the next plate to the right (r.2, c.3), two rods have combined ^ Based upon work of C. W. Metz and others. 174 X >,< V T CHAPTER 21 (^ •i^ yp* y^p* •f^ ^p >> >> to form a V that is different from either of the two V's in melanogaster. There are other examples in this chart of two rod-shaped chromosomes forming a V- shaped chromosome, or of a V forming two rods. Let us see how these changes can come about. Consider first how a V can originate from two rods (Figure 21-2). It should be recalled that a rod-shaped chromosome has two arms, although one is very short. The short arm may not be noticeable at metaphase or anaphase, but may be demonstrable either cytologically at an earlier or later stage of the nuclear cycle, or by genetic means via the FIGURE 21-1. Chromosome configurations in several Drosophila species. genes it carries. Suppose two rods are broken near their centromeres, one break being in the long arm of one chromosome, the other break being in the short arm of the other chromo- some. If the long acentric arm of the first chromosome joins to the long centric piece of the second chromosome, a V is formed. No- tice that this involves the joining of two whole or almost-whole arms in a eucentric half- translocation. The remaining pieces may join together to form a short eucentric chro- mosome, thereby completing a reciprocal translocation, or they may not join. In either case, if these short pieces are lost in a subse- Natural and Induced Chromosomal Changes / \FI 175 FIGURE 21-2 {Left). Formation of a V- shaped chromosome from two rod-shaped chromosomes. HALF (OR RECIPROCAL) TRANSLOCATION ^\ RECIPROCAL TRANSLOCATION FIGURE 21-3 (Right). Formation of two rod- shaped chromosomes from a V-shaped chromo- some and a Y chromosome. quent nuclear division and if the number of genes lost in these pieces is small enough, the absence of these parts may be tolerated physiologically. The reverse process, the formation of two rods from a V, necessitates the contribution of a centromere from some other chromo- some. This second chromosome may be the Y (Figure 21-3). Suppose the V is broken near its centromere and the Y is broken any- where. If a eucentric reciprocal translocation then occurs, two chromosomes are produced each having one arm that is derived largely from the Y. Then, later, if paracentric dele- PARACENTRIC DELETIONS 176 CHAPTER 21 tions occur in these Y-containing arms, the rod shapes will be attained, completing the change from a V to two rods. Note that al- most all but the centromere of the Y chro- mosome is eventually lost in this process. But this may have little or no disadvantage since the Y carries relatively few loci and is primarily concerned with sperm motility. For example, this series of mutations may be initiated in the male germ line so that two Y-containing rods are produced. Deletion of Y parts can occur without detriment if these chromosomes happen to enter the female germ line, or they may stay in the male germ line provided a regular Y chromosome is added to the chromosome complement in due time. The small IV chromosome in melanogaster, whose monosomy is tolerated in either sex, may also serve to contribute a centromere in changing a V to two rods by an identical or similar series of mutational events. The metaphase plate observations confirm expectation in the case of Drosophila (Chap- ter 20), in that whole arm translocations are capable of persisting in natural populations. Such rearrangements and pericentric inver- sions are very useful in helping us establish evolutionary relationships among different species. But it should be emphasized that this kind of information by itself does not prove either that the formation of different species is a primary consequence of the oc- currence of these rearrangements, or that these rearrangements are of secondary importance in species formation, or that these mutational events occur after species formation is com- pleted. As exemplified by Oenothera and Drosphila, we have seen that gross rearrange- ments of somewhat different types have per- sisted in the course of the evolution of dif- ferent groups of organisms. For this reason it would be prudent to refrain from predict- ing, except in the general way we have done in Chapter 20, which, if any, structural changes would be found associated with the evolution of other particular groups of or- ganisms. Let us turn our attention now to the factors which produce the breaks leading to struc- tural change. Chromosomes do break spon- taneously; that is, they occasionally break during the normal course of events. How- ever, because spontaneous breaks occur rela- tively rarely, the study of them and their con- sequences would be greatly enhanced by the application of external agents capable of pro- ducing breaks in great numbers. One of these agents is radiation, and our attention will be restricted to this agent in the present Chapter (Figure 21-4). The process of break- ing a chromosome is a chemical reaction requiring energy. Radiation may supply energy in three different forms: heat, activa- tion or excitation, and ionization. The bio- chemical effect of a radiation depends upon the type and amount of energy it leaves in tissue, less energetic radiations (like visible light) leaving energy in the form of heat, more energetic radiations (like ultraviolet light) leaving energy also in the form of activations (in which an electron is moved from an inner to an outer orbit). The more energetic the radiation, the greater is the likelihood that the energy absorbed will lead to chemical change. For example, visible light does not produce as many breaks in chromosomes as does ultraviolet light. Radiations of still higher energy (like X rays, gamma rays, and alpha, beta, neutron, proton, and other fast- moving particulate radiations) are most likely to cause breaks. Although such high-energy radiations can heat and activate, most of their energy is left in cells in the form of ioniza- tions, and it is these which produce most of the breaks. Let us consider first what ioniza- tion is and how it is produced, and then how it is connected with the production of breaks. X and gamma rays are electromagnetic waves like visible light, but have relatively shorter wave lengths, and can penetrate tissue more deeply before they are stopped, at Natural and Induced Chromosomal Changes 111 t ^ - f 4 * *> s B which time their energy is absorbed. Energy is transferred into ionization when some of these rays are stopped by an atom which sub- sequently loses an orbital electron. This electron, torn free of the atom, shoots off at great speed and can, in turn, cause other atoms to lose electrons in a similar manner. All atoms losing electrons become positively charged ions. The freed electrons are finally captured by other atoms which become nega- tively charged, or negatively charged ions. Since each electron lost from one atom is eventually gained by another atom, ions oc- cur as pairs. In this way a track of ionizations is produced which often has smaller side branches. The length of a primary track and its side branches, and the density of ion pairs within these, will differ depending upon the type and energy of radiation involved. Suf- fice it to say that all known ionizing radia- FIGURE 21-4. Structural changes X-ray-induced {75-150 r) in normal human male fibroblast-like cells in vitro. Arrows show: {A) broken chromosomes, (B) translocation (center) and dicentric (lower left), (C) ring chromosomes. A, B are in metaphase (see Fig. 18-9), C is late prophase. (Courtesy ofT. T. Puck, Proc. Nat. Acad. Sci., U.S., 44:776-778, 1958.) 178 CHAPTER 21 tions produce clusters of ion pairs within submicroscopic distances. In other words, no amount or kind of high-energy radiation is known at present which produces only single ions, or single pairs of ions, evenly spaced over microscopic (hence relatively large) distances. Since we cannot obtain one ion or a pair sufficiently separated from the next, the genetic effects of ionization must be de- termined from the activity of clusters of ions. Ions undergo chemical reactions to neutral- ize their charge, and, in doing so, clusters of them are capable of producing breaks in chromosomes. The amount of ionization produced by a radiation can be measured in terms of an ionization unit called the roentgen, or r unit, which is equal to about 1.8 X lO'* ion pairs per cubic centimeter of air. A sufficiently penetrating radiation, which produces this 1.8 X 10'^ ion pairs in a cm^ of air, may also produce this amount in successive cm'' of air because only a very small part of the total energy of the incident radiation is left at suc- cessive depths. The amount of energy left at any level depends upon the density of the medium through which the radiation is pass- ing. Thus, in tissue, which is approximately ten times as dense as air, this penetrating high-energy radiation would produce about one thousand times the number of ion pairs per cm^ So, it can be calculated that Ir (always measured in air) produces about 1.5 ion pairs per cubic micron (/x^) of tissue. Since Drosophila sperm heads are about 0.5/i^ Ir would produce, on the average, less than one ion pair in each. But remember that these ions occur in clusters, so that Ir may place dozens of ion pairs in one sperm head, and none of these in dozens of other sperm heads. While the r unit only measures energy left in the form of ionization, another unit, the rad, measures the total amount of energy of the radiation which is absorbed by the medium. In the case of X rays, about 90% of the energy left is in the form of ioniza- tions, the rest as heat and excitations. Ultra- violet radiation can be measured in rads, but not r units, since it is nonionizing. It has been found, for X rays, that the number of chromosome breaks produced in- creases in direct proportion to the dose ex- pressed in r (Figure 21-5). This means that break number increases linearly with X-ray dose. This also means that X rays produce at least some ion clusters which are large enough to cause a break, and that different clusters of ions do not combine their effects to do so. (Had there been such a cooperation between clusters, the break rate at low doses would have been lower than found, due to the waste of smaller clusters having no others with which to cooperate, while the rate at higher doses would have been higher, due to the cooperation among smaller clusters.) Certain radiations, like fast neutrons, pro- duce fewer breaks per r than do X rays. This is explained by the fact that one r of these radiations produces larger (and hence fewer) clusters of ions than do X rays, these larger clusters more often exceeding the size needed to produce a break, and being, there- fore, relatively less efficient in this respect. Ion clusters may produce breaks either directly, by attacking the chromosome itself, or indirectly, by changing oxygen-carrying molecules, which in turn react with the chro- mosomes, or by influencing chemicals which affect oxygen-carrying molecules. In any case, this indirect pathway must be of sub- microscopic dimensions, otherwise there would be cooperation among the chemical effects of different ion clusters to cause breakage. Thus, only ion clusters in or very close to the chromosome can produce breaks in it. This has been visibly demonstrated by the use of beams of radiation of microscopic dimensions. Such a beam passing through a metaphase chromosome breaks it, but fails to do so when directed in the plasm just adja- cent to the chromosome. From what has been stated in the previous Natural and Induced Chromosomal Changes 179 iij6 - q: lli 3 — o q:2 iij Q. I FIGURE 21-5. The relation between X-ray dosage and the frequency of breaks induced in grasshopper chromosomes. {Courtesy of J. G. Carlson, Proc. Nat. Acad Sci., U.S., 27:46, 1941.) A .-r ,^r' .-f-'" "1 I I \ 1 \ \ I \ \ \ \ I 10 20 30 40 50 60 70 80 90 100 110 120 130 DOSAGE IN R UNITS paragraph, it should be no surprise to you that the number of breaks produced by the direct action of a given dose of a particular radiation will depend upon the volume which a chromosome occupies; this volume may be different at different times in the nuclear cycle, and the same chromosome may occupy different volumes in different tissues or sexes. It is also reasonable, in view of the fact that breakage is energy-requiring, that the num- ber of breaks produced indirectly is increased, if during irradiation either the amount of oxygen is increased or the cell's reducing substances are poisoned. As expected, there- fore, replacement of oxygen by nitrogen dur- ing irradiation reduces the number of breaks produced. With this preliminary discussion of some of the factors influencing the production of ra- diation-induced breaks as a background, con- sider next the factors influencing the fate of the ends produced by breakage. Just as breakage involves a chemical reaction, so does the union between two broken ends. It has been shown that the joining of broken ends also requires energy, being enhanced by the presence of oxygen (and prevented by nitrogen) when present after irradiation. So, restitution is prevented if nitrogen has re- placed oxygen after irradiation. This in- creases the time that the ends from the same break stay open, and this often increases their chance for cross-union later in the presence of oxygen. (You see, therefore, that oxygen has two contrary effects on rearrangements, its presence during irradiation increasing the number of breaks and its presence after irradiation increasing restitution.) Since, under a given set of conditions, the number of breaks increases linearly with dose, each part of the dose independently produc- ing its proportional number of breaks, it is clear that the number of breaks produced is independent of the rate at which a given total dose of radiation is administered. It also follows, then, that all structural changes consequent to single breakages are also inde- pendent of the radiation dose rate. Radia- tions, like fast neutrons, which produce tracks that are long and dense with ionizations, may frequently produce two breaks with the same track. In this case, if the same chromosome is broken twice because, having coiled tightly, it lay in the path of the track twice, large and small structural changes of inversion, de- ficiency, and duplication types may be pro- 180 CHAPTER 21 duced. The frequency of these will increase linearly with neutron dose, and be independ- ent of the dose rate. The same tracic of ionizations may, as in the case of fast neu- trons, break two different chromosomes; this would be possible when these chromo- somes are closely packed together, as they are in the sperm head. The fact that the total frequency of reciprocal translocations in- creases linearly with fast neutron dose when sperm are treated is evidence both of this and of the view, already presented in Chapter 20, that proximity of broken ends favors their union. For such a result would be obtained only if both breaks were produced by the same track, and only if the broken ends capable of exchange union were restricted to those located near each other, broken ends produced in the course of different tracks being too far apart. When, however, ordinary X rays are in- volved, the clusters are smaller, and the track of ionizations is shorter. In this case, two breaks will be produced by the same track less frequently, and if they do occur, they will usually be in the same chromosome at places quite close together. For example, the two breaks may occur within submicroscopic dis- tances in successive gyres of a coiled chromo- some, so that only minute to small structural changes will be produced by such breaks. In the case of X rays, two breaks occurring mi- croscopic distances apart in the same nucleus are usually the result of the action of two clusters, each one derived from an independ- ently arising track, so that one break has no dependence upon the other for its produc- tion. For this reason. X-ray-induced gross rearrangements can be dose dependent. For when a low enough dose is given, some nuclei contain only one track and only one break, and no gross two-break rearrangements can occur in them. But when the dose is raised high enough so that these nuclei are traversed by two separate tracks, the two breaks re- quired for such rearrangements can be pro- duced in the same nucleus. The higher the dose of X rays, then, the greater is the ef- ficiency in the utilization of breaks to pro- duce gross rearrangements. Accordingly, for doses causing some cells to have two inde- pendently produced breaks, and for doses higher than this, the frequency of these muta- tions increases, not in direct proportion to the dose, but faster than this. There is, however, a small proportion of single X-ray tracks that will, in the treatment of sperm, for example, cause two breaks, each in a different chromo- some. Therefore, for doses of X rays that produce fewer than two tracks per sperm, gross rearrangement frequency will increase linearly with dose. So, there is actually no dose of X rays to sperm which would not have some chance of producing a gross rearrange- ment, which means that no dose is safe in this respect. X-ray-induced rearrangements, produced by two or more independently arisen breaks, may also be dose-rate-dependent. When a suitably large dose is given over a short inter- val, the ends produced by separate breaks are present simultaneously and can undergo cross-union. But when the same dose of this radiation is given more slowly, the pieces of the first break may restitute before those of the second are produced, in this way elimi- nating the opportunity for cross-union. In this event, the same dose, given in a pro- tracted manner, would produce fewer gross rearrangements than it would when given in a concentrated manner. While this dose-rate- dependence for X rays holds true for most cells, it does not obtain, however, for mature sperm of animals, probably including man. In these cells the broken pieces cannot join each other and are, therefore, accumulated. For this reason, it makes no difference how quickly or slowly the dose is given to the chromosomes in a sperm head, for the breaks remain unjoined until after fertilization when the sperm head swells. We have already mentioned that the spatial Natural and Induced Chromosomal Changes 181 arrangement of chromosomes relative to each other will influence the number of breaks and the kinds of structural changes these produce. It should be noted, specifi- cally, that when the chromosomes are packed into the tiny head of a sperm, the possibilities for multiple breakages and for later joinings may be quite different than they are for chro- mosomes located in a large nucleus. But even within a given type of cell, there are a number of other factors which may influence breakage or rejoining. These include the presence or absence of a restrictive and in- sulating nuclear membrane, the degree of spiralization of the chromosomes, the stress or tension under which the parts of a chromo- some are held, the degree of hydration, the amount of matrix in which the genes are embedded, protoplasmic viscosity and the amount of fluid and particulate movement around the chromosomes, gravity effects, centrifugal forces, and vibrations. One special restriction to the movements of broken pieces occurs in cells whose chromo- somes have just divided, and in meiotic cells where homologs synapse. For under these conditions, the forces, which keep like parts of one strand apposed to the like parts of its sister or homolog, may prevent the pieces newly produced by breakage from moving apart freely, so that the unbroken strand or strands serve as a splint for the broken one, reducing the opportunities for cross-union. All these factors determine to what degree chromosome fragments may move or spring apart; those affecting the distances between different chromosomes, or the parts within a chromosome, may also affect chromosome breakability. The frequencies and types of structural changes will depend also upon the total amount of chromosomal material present in a nucleus and the number and size of the chro- mosomes into which this is partitioned. The rearrangements occurring in different cells of a single individual will depend upon whether the cell is haploid, diploid, or polyploid, and whether or not the chromosomes are in the process of replication and are metabolically active. Finally, there are two different types of chromosomal material and each has a differ- ent capacity to produce structural changes. Most of the chromosomal material in the genome reacts similarly to certain staining procedures, and is said, therefore, to be euchromatic (truly or correctly colored). Other portions of the chromosomes stain either darker or lighter than this, and are said to be heterochromatic. Although some hetero- chromatin may be located at various places along the chromosome arm, it is normally found in both arms adjacent to the centro- mere and, to a lesser extent, near the telo- meres. The genes in heterochromatin func- tion to produce large blocks of chromosomal material in mitotic and meiotic chromosomes, so that their contribution to the size of the chromosome, as seen at metaphase, is rela- tively greater per gene than it is for genes located in euchromatic regions. Besides its special stainability, location, and function, heterochromatin has a fourth characteristic, in that it is less specific in synapsis than is euchromatin; heterochro- matin located at different places along a chromosome or in nonhomologous chromo- somes is often found synapsed. (Thus, for example, in the salivary gland nuclei of Dro- sophila larvae, the heterochromatic regions proximal to the centromeres of all chromo- somes synapse to form one mass, called the chromocenter. Also, the heterochromatic regions near the telomeres are sometimes found in synapsis with the chromocenter.) Relative to the number of loci present, the fifth fact is observed, that radiation-induced structural changes more frequently involve heterochromatic reg'ons than they do eu- chromatic ones. Whether this excess is to be attributed to a greater breakability or lesser restitutability, or both, of heterochro- 182 CHAPTER 21 matin, as compared to euchromatin, cannot be decided. Nevertheless, this means that in many rearrangements, at least one of the breakage points is located in the heterochro- matin near the centromere. It is not unex- pected, therefore, that whole arm reciprocal translocation (which is involved in forming a V-shaped chromosome from two rods) is the most frequent type of reciprocal transloca- tion. Moreover, the likelihood of the change from a V to two rods is enhanced in Dro- sophila by the fact that the Y is almost en- tirely heterochromatic, as is one entire arm of chromosome IV. You should recall that our motivation for studying energetic radiations was based upon their ability to induce many breaks and subse- quently many structural changes. It was be- cause this great supply of rearrangements was readily available that many of the other fac- tors influencing breakage and joining, which we have just been discussing, were originally discovered. Other important discoveries were made possible by the study of structural changes, including, for instance, that the centromere has a genetic basis and that cross- ing over near it is always reduced, that the telomere has a genetic basis, and that there are genetic elements (called coUochores) near the centromere which are especially important for synapsis. Perhaps the most fundamental contribution was the finding, via structural changes, that the genes have the same linear order in the chromosome as they have in crossover maps. However, the spacing of these is different in the two cases (Figure 21- 6). Thus, in view of the reduction of cross- ing over near the centromere, the genes nearest the centromere, which are spaced far apart in the metaphase chromosome, are found to be close together in the crossover map. While we have restricted our attention to the factors influencing the production and fate of breaks produced by ionizing radiation, these factors would be expected to operate upon breaks produced by any other sponta- neously occurring or induced mechanism. For, in general, broken ends produced in different ways all possess the same properties. SUMMARY AND CONCLUSIONS Pericentric inversions and whole arm reciprocal translocations have been frequent in the past evolutionary history of Drosop/iila, the former changing chromosome shape, the latter leading to changes in chromosome number. The components of structural change, breakage and exchange union, are readily studied through the use of ionizing radiations. Such radiations induce breaks primarily by means of the clusters of ion pairs they produce. These clusters form tracks of ionization, whose thickness and length determine the number and location of the breaks. Tracks of ionization must occur very close to, or inside of, the chromosome that is broken. Whether they result from one or from two breaks, all chromosomal rearrangements produced by a single track increase linearly with radiation dose, have no threshold dose, and show no effect of pro- tracting or concentrating the dose. Two-or-more-break structural changes, produced by ion clusters in separate, independently initiated tracks, increase in frequency faster than the dose, and have a threshold dose. If joining of ends produced by breakage can take place during the course of the irradiation, the latter types of rearrangement are reduced in fre- quency by protracting the delivery of the total dose. Since both the breakage and joining processes involve chemical changes, their frequencies 183 X CHROMOSOME cv ct \ ysc br pnwrb C C H CHROMOSOME 0 / I0\20 30 40^50 f 60^70^8.0 90 100 107 al dp b Bl/\cn vg c It tk Centromere px sp C C n CHROMOSOME ' 7" I0\20 30^4,0 St .50^ 60 70 80 90 100 ru ca h D th cu sr e Centromere FIGURE 21-6. Comparison of crossover or genetic (G) and chromosome (C) maps. are modifiable by the metabolic state of tine cell. All types of rearrangement are expected to be affected by the physical and chemical state of the chromosome, the amount and location of its euchromatin and heterochromatin, its position relative to other chromosomes, the number and arrangement of the other chromosomes present, the presence or absence of a nuclear membrane, and the movements of broken ends as influenced by cellular particulates, fluids, and extracellular factors. 184 CHAPTER 21 REFERENCES Bacq, Z. M., and Alexander, P., Fundamentals of Radiobiology, 2nd Ed., New York, Per- gamon Press, 1961. Chu, E. H. Y., Giles, N. H., and Passano, K., "Types and Frequencies of Human Chromo- some Aberrations Induced by X-rays," Proc. Nat. Acad. Sci., U.S., 47:830-839, 1961. "Ionizing Radiation," Scient. Amer., 201 :No. 3 (Sept.), 1959. MuUer, H. J., "General Survey of Mutational Effects of Radiation," in Radiation Biology and Medicine, Claus, W. D. (Ed.), Reading, Mass., Addison-Wesley, Chap. 6, pp. 145-177. Puck, T. T., "Radiation and the Human Cell," Scient. Amer., 202, No. 4:142-153, 1960. Sparrow, A. H., Binnington, J. P., and Pond, V., Bibliography on the Effects of Ionizing Radia- tions on Plants, 1896-1955, Brookhaven Nat. Lab. Publ. 504 (L-103), 1958. QUESTIONS FOR DISCUSSION 21.1. Why should tissue, which is only about ten times as dense as air, have about one thousand times the number of ionizations in it as has air, when both are given the same radiation exposure? 21.2. What evidence can you give to support the view that the ions causing breakage need not always attack the chromosome directly? 21.3. Does the observation that the volume of a chromosome is variable under different conditions mean that it has an inconstant gene content? Explain. 21.4. Do you suppose that chromosomes exposed to X rays are more likely to undergo structural change when they are densely spiralized than when they are relatively uncoiled? Why? 21.5. Discuss the role of heterochromatin in changes involving chromosome number and chromosome shape. 21.6. Do you suppose the oxygen content of a space capsule could affect the mutability of Drosophila passengers? Explain. 21.7. Discuss the relative efficiency, per r, of small doses of X rays and of fast neutrons in producing structural changes in chromosomes. 21.8. Do you suppose that the mutability of ultraviolet light threatens man's survival? Explain. 21.9. Compare the breakages and their consequences caused by the same dose of X rays administered to: a. A polyploid and a diploid liver cell in man. b. A diploid neuron in man and Drosophila. c. A sperm and a spermatogonium in man. 21.10. How could you locate the position of the gene for white eye in the salivary X chromo- some of Drosophila using: a. DitTerent small deletions? b. Overlapping inversions? Chapter 22 POSITION EFFECT AND ALLELISM IN DROSOPHILA WE HAVE already mentioned a number of discoveries made possible by the abundance of structural changes in chromosomes induced by ionizing radiations (Chapter 21). In the course of irradiation studies with Drosophila, the same kind of two-break rearrangement has been produced time and again, in which both of the breakage points have occurred at the same, or nearly the same, positions. In many of these cases it is found that there is a phenotypic change which occurs simultane- ously with the rearrangement, and that the same kind of phenotypic effect is produced by each nearly identical rearrangement. Fur- ther study shows that the phenotypic effect is transmitted whenever the rearrangement is, and that, in many cases, this effect is similar to that of a known allele of a gene located at, or very near, one of the points of breakage. In these cases, then, the change in phenotype seems to be directly connected with the muta- tion of a gene known to be located at or near a point of breakage. To explain this effect, you might at first think that the very fact that a break took place in or adjacent to this gene automatically changed it to this allelic form. But this can- not be the explanation since other breaks at this locus, which go into different kinds of rearrangement, do not produce such a change in phenotype. For the same reason, it is not tenable to presume that the track of ioniza- tions which produced the break simultane- ously produced a minute deficiency or duph- 185 cation of the affected locus. We may con- clude, therefore, that an important feature of this phenotypic change is that it is not based upon what occurs at the time of breakage; this suggests that the change is due to the nature of the broken ends that join. More- over, this change takes place only when the broken end, which joins to the broken end carrying the affected locus, comes from cer- tain specific loci in the genome. So, the gene affected at the broken end is not mutant and will not change its effect if it joins to some broken ends, but will do so, and in the same way, if it joins to certain others. In other words, we can hypothesize that the phenotypic effect, the functioning, of the same gene may be modified when its linear neighbors are changed. This type of pheno- typic change is said to be due to position effect. Would we be justified in calling posi- tion effect a mutation? Mutation was defined on page 5 as a change in the genes, not in their phenotypic expression. Note that all the types of mutation discussed hitherto, from Chapter 18 on, have been based upon material changes in the genome, such as gains, losses, or rearrangements of chromo- somal material, and all novel phenotypic changes so far discussed could be attributed to such mutations. However, in the case of position effect we are presumably dealing with a change in gene effect in which the gene itself has been unchanged! Position effect, there- fore, can be one of the phenotypic conse- quences of mutations involving structural rearrangements, but is not in itself a muta- tion. Position effect is no more to be called mutation than is dominance, although both require a prior mutation for their detection. You may be dissatisfied with our presump- tion that the gene showing position effect is chemically and physically unchanged in any permanent way. You might suppose that when the gene in question joins to certain genes it does so by means of one kind of chemical or physical connection, leading to 186 CHAPTER 22 one type of functioning, whereas, when it joins to other genes, it does so in another way, leading to a different type of function- ing. This kind of explanation would be a mutational one, but is made extremely un- likely by the fact that genes located some dis- tance from a point of breakage sometimes show position effects. You will agree that it is unreasonable to believe that a change in the kind of connection between genes at the point of breakage would somehow spread along the chromosome to affect the configu- ration of a gene whose adjacent genes had not been substituted by others. (This spreading effect is additional reason for dismissing ex- planations of position effect based upon breakage itself or upon other mutational changes connected with tracks of ioniza- tion.) If the physico-chemical nature of a gene showing position effect is unchanged, then two other predictions should prove true. The gene in a position-effect rearrangement should return to its old functioning upon being placed near its old genie neighbors. This can be done in two ways. Rearrange- ment-carrying individuals can be irradiated and progeny examined for structural changes that reverse this rearrangement. Or the gene showing position effect can be moved to a normal chromosome by means of crossing over. In both cases it is found that the gene, returned to its old position, returns to its old way of functioning. A second prediction is that when a normal gene is placed in the re- arranged position via crossing over, it should then cause the position effect. It does. In Drosophila, the organism most studied in this respect, position effects often accom- pany rearrangements that bring genes in euchromatin near those in heterochromatin. Placing a gene normally located in a euchro- matic region near or in a heterochromatic one often produces a special, wavering, posi- tion effect which is expressed in the pheno- type in a mosaic or variegated way. Thus, for example, if the gene for dull-red eye color on the X chromosome, w+, normally located in euchromatin, is placed in the heterochro- matin proximal to the centromere by means of a paracentric inversion, the eye color produced is mottled, being composed of speckles of white and dull red. Such variega- tion is reduced, however, if, by breeding, an extra Y chromosome or another heterochro- matin-rich chromosome is added to the geno- type. It is not yet known how this suppres- sion of variegation is produced. We have seen that the only requirement for the occurrence of position effect is an appro- priate shift in the kind of linear neighbors a gene has. Breakage merely provides us with a way of obtaining such shifts. The possi- bility should be entertained that position effects could also be detected with the aid of some other mechanism for changing the rela- tive positions of genes. We already know that crossing over can do this. Let us see if we can devise a particular crossover system ^ whose operation might produce a position effect. An X-linked mutant is known in Drosoph- ila, which has the effect of reducing the number of facets {ommatidia) in the com- pound eyes. Because it changes the eye from round to a slit shape it is called Bar (B). When the normal and the Bar-con- taining chromosomes are studied in larval salivary gland nuclei, it is found that about six successive bands in the normal chromo- some are duplicated in tandem in the Bar chromosome. Let us designate as 123456 each such region of six bands, so that a nor- mal female would contain 123456/123456 and a homozygous Bar female 123456 123456 /123456 123456. In normal (+/+) females the two homologs would pair with homolo- gous numbers (parts) together, and crossing over could take place between correspond- ing numbers. In homozygous Bar (B/B) ' Based upon investigations of A. H. Sturtevant, H. J. Muller, C. B. Bridges, and others. Position Effect and A 1 lei ism in Drosophila 187 females, proper pairing (eusynapsis) could also occur and normal crossing over follow; but this case potentially offers a different se- quence of events in which synapsis occurs incorrectly (aneusynapsis), the left region in one chromosome pairing with the right region of the second (as in Figure 22-1), leaving the other two regions unpaired. If this incorrect synapsis is followed by normal crossing over anywhere in the paired region (as shown be- tween 2 and 3 in the Figure), the crossover strands will be 123456 and 123456 123456 123456; the former strand has this region only once and will therefore be normal (+), while the latter has this region thrice. If an egg containing the crossover with One region is fertilized by an X-bearing sperm of a nor- mal-eyed male, the zygote would produce a daughter having normal eye shape, demon- strating that Bar has reverted to +. This could also be checked in a subsequent gen- eration by examination of the salivary gland chromosomes. If an egg containing an X with this region FIGURE 22-1. Diagrammatic representation of t/ie normal and the Bar region of tlie X chromo- some and the consequences of crossing over following aneusynapsis. EUSYNAPSIS 12 3 4 5 6 12 3 4 5 6 , PHENOTYPE K ji Normal Female (+/+) EUSYNAPSIS 123456123456 123456123456 123456 12 3456 ANEUSYNAPSIS \ I]X 3456123456 Homozygous \ Bar Female (B/B) Meiotic Products from Crossing Over Indicated 123456123456 123456123456 123456 12 3 4 5 6 Bar ? Normal Bar 188 CHAPTER 22 in triplicate is similarly fertilized, a female would be produced having four of these regions, three in one homolog and one in the other. The question is: What would be the phenotype of such a female? Could it make any phenotypic difference whether these regions are positioned two by two (as in homozygous Bar) or three by one? Note that the genie neighbors of the four regions are different, when two regions are present on each homolog, from what they are when one homolog has three regions and the other has one region. Knowing that position ef- fect does occur, it is possible that the differ- ences in neighbors, when these four regions are arranged in the two different ways, could make for different phenotypic effects. While we do not know precisely what the potential position effect phenotype will be, we can direct our attention to the number of facets in the eye and look for any change from the number expected. The normal round eye of females and males (+/+ and + /Y) contains more than 200 facets, or ommatidia. The homozygous Bar female {B/B) and hemizygous Bar male (5/Y) have about 68 ommatidia per eye. The hetero- zygous female (+/5) has about 150, Bar on one chromosome being incompletely domi- nant to the + condition in the homolog. From the cross +/Y cT X B/B 9, then, the usual Fi females are -\-/B with about 150 ommatidia per eye. Reversions to the + condition, by means of crossing over in an aneusynaptic tetrad, could be detected as a +/+ female, as mentioned. The comple- mentary crossover chromosome having the triple region together with a normal chromo- some would produce a female which might have less than 68 facets, or more than 68 but less than 150 facets, per compound eye. The experimental design is not yet com- plete, however. Since we do not know how often the chromosomes showing the poten- tial position effect will be produced in meiosis, we must eliminate two other possible causes of change in eye shape which would be classi- fied as exceptional. If the cross made is + /Y X B/B, a sperm carrying two X's (because of nondisjunction in the father) fer- tilizing an egg with no X (because of non- disjunction in the female) will produce a + /+ female which would be counted as one of the exceptional types we are seeking. Such zygotes would be extremely rare. Never- theless, we would be able to recognize them if the + chromosome carried the gene for yellow body color, y, as a marker, since such nondisjunctionally produced females would be yellow, not gray, in body color. (In this way we would also recognize any female progeny resulting from the contamination of our cultures by flies of the yellow stock.) So the cross we should make is y +^/Y X >"+ Bly"^ B. For clarity, the genie symbol for round eye is now given as +^. The other event we must eliminate from confusing our results is mutation at or near the B locus. The exceptional phenotypes (round and an unknown shape of eye) we are looking for would always be produced following a crossing over in the region of Bar. All we need do is to make the B/B female dihybrid for genes near to, and on FIGURE 22-2. Compound eye of Drosophila. Left: Ultrabar; center: Bar; right: normal. Position Effect and AUelism in Drosophila 189 either side of, B, near enough (closer together than ten crossover units) so that no double crossovers could occur between them. Bar is located at 57.0, forked bristles (/) at 56.7, and carnation eye color (car) at 62.5 on the X chromosome linkage map. We, therefore, construct females that are y+f+ B car/y+fB car+. Any unusual eye shape that is noncrossover between the loci for /and car can be elimi- nated immediately from consideration. All exceptional phenotypes of interest will be crossovers between /and car; crossovers in this region will normally be present in 5.8% (62.5-56.7) of Fi daughters. Of course, the males used will now have to be yf-{-^ car'Y in order to identify the crossover daughters (which will be either nonforked noncarna- tion, or forked carnation). The cross then is: 7/ + S car, Y c^ X >'+/+ B car/y^fBcar+ 9 . When the experiment is performed it is found that about one female in two thousand is round-eyed and carries a crossover be- tween / and car; a similar number of fe- males, that are crossovers in this region, have very narrow eyes, called Ultrahar (Figure 22- 2), each eye containing about 45 facets. Note that the reciprocal types of exceptional flies are equally frequent, as would be ex- pected if they were the reciprocal products of a crossing over in an aneusynaptic tetrad. Moreover, Ultrabar females can be bred, and the salivary glands of their Fi larvae prove they contained the triple region in one X and a single region in the other X, as predicted. You might still maintain that the Ultrabar individual is a mutant and not a position ef- fect, the production of the mutation somehow being dependent upon a simultaneously occurring crossover. That this is not the case can be demonstrated by taking the two dif- ferent exceptional types of X, placing them together in a female, and occasionally ob- taining perfectly typical Bar chromosomes. These are found to carry two regions pro- duced by crossing over between the single region of one chromosome and the middle region of the triple-dose homolog (Figure 22 3, on p. 190). We conclude, therefore, that four regions, aligned in different positions, produce dif- ferent phenotypes. The alignments were shifted by crossing over and not by mutation- producing chromosome breaks, as was the case in the rearrangement experiments pre- viously described. This discussion should lead you to appreciate the fact that in the ad- vancement of genetic knowledge, while the theory and the preparations to test it experi- mentally are often complicated as in physics, the data obtained are simple and unambigu- ous. One other possibility presents itself for the detection of position effects using crossing over. If the genotype of a Drosophila female were y a b spl ;+ a b spl+, a crossover be- tween a and b would produce no new posi- tions of the a and b genes relative to each other, and no position effect would be ex- pected, or found. But, suppose the genotype was y a+ b spl/y+ a 6+ spl+. In this case both the a and b loci are heterozygous; the mutants are on different homologs, being "across" from each other, or in trans position (Figure 22-4). If crossing over occurs be- CIS TRANS + b a b a + FIGURE 22-4. Cis and trans posi- tions for diliybrid linlced genes. tween these loci we obtain y a+ b+ spl-^ and >'+ a b spl as the crossover chromosomes. If these two crossover chromosomes were pres- ent in the same individual, then both mutants (a and b) would be together on one homolog and their normal alleles both would be on the other homolog, so that these genes now are 190 CHAPTER 22 "Triple + " A 2 5 2 5« y'^f^l 61234561 6 y ULIRABAR + y f 12 3 4 5 6 r f Y 1 ! car "Triple +" y"^ f"^ 12345612345612 3456 <^°'' y+ ^4-r I I 123456 123456 y+ f 123456 123456 ^"''^ MEIOTIC PRODUCTS FIGURE 22-3. Production of Bar chronwsomes by crossing over in Ullrahar females. in the cis position. Note that the trans het- erozygote and cis heterozygote have the same number of genes on each chromosome, but, in the former case, a has b^ for its Hnear neighbor (and a'' has b) whereas in the latter case a has b for its linear neighbor (and a+ has 6+). If the trans dihybrid has one pheno- typic effect and the cis dihybrid, obtained from it by crossing over, has a different phenotypic effect, then position effect will be considered proven if the cis form can, by crossing over, revert to the trans form and restore the old phenotypic effect. This cis-trans test for position effect should Position Effect and AUelism in Drosophila 191 have the best chance of yielding a positive re- sult if the two pairs of genes concerned are adjacent or very close to each other. But this also means that if the genes are very close together crossing over will rarely occur be- tween them, and large numbers of progeny will have to be observed to be sure at least one exchange between them has been in- cluded among the results! What particular genes should we use in our test? Should we use linear neighbors that apparently perform very different functions, or shall we use those having similar effects? If the effect of one gene is to be modified by the effect of its linear neighbor, it would seem reasonable to use neighboring genes that have similar ef- fects, for in this case changing the allele of the neighboring gene might be expected to pro- duce a change in its own effect. Suppose, on the other hand, the two neighbor genes affected totally different traits. In this case, while changing the allele of the neighboring gene would mean that the neighboring allele now present affects the totally different trait in a different way, this would not be expected to have any effect on the functioning of its linear neighbor. The best source of genes which have very similar effects is, of course, the members of a multiple allelic series, for instance, the mem- bers of the white series on the X chromo- some of Drosophila. But you immediately might say that these cannot be used in our ex- periment, since only one is present on each homolog, and we need two pairs of genes to perform a cis-trans test for position effect. Moreover, all these alleles of white are located at 1.5 on the crossover map. When the "trans" hybrid for w {apricot) and w {white) is made, it is found to produce pale apricot eye color. However, such a phenotype does not prove w" and w are alleles. Alleles are alter- natives of the same gene; genes are most properly identified as alleles because they oc- cupy the same site on homologous chromo- somes and cannot be recombined as the con- sequence of crossing over, and because, bar- ring nondisjunction, the members of a pair of alleles always segregate. The statement that w° and w are located at position 1.5 may be incorrect! Suppose these two are really non- allelic but similarly acting genes located close together, one at position 1.49 {w") and one at 1.51 (w). The crossover data, being finite and somewhat variable, could have acciden- tally placed them both at locus 1.5. If vv and w are, in fact, close but not allelic to each other, the trans heterozygote for them should yield the cis heterozygote by crossing over. But if these genes are only .02 of a crossover unit apart, only one such crossover would oc- cur among 5,000 tested chromosomes. If only a few hundred flies are scored in a search for such crossovers, it is very likely none of this type will be found. Such an experiment would have to be done on a very large scale to serve as a test for nonallelism. Let us examine the plan and results of a large scale experiment ^ actually designed to test the nonallelism of h'" and w. Drosophila females were constructed carrying an at- tached-X chromosome containing y w spl on one arm and j+ h-" and 57;/+ on the other. Re- call that the use of attached-X's permits one to recover two strands of the four involved in each crossover (cf. p. 122). The present genetic system even permits both comple- mentary crossover types to appear simul- taneously in the same gamete. The left part of Figure 22-5 shows schematically a portion of this attached-X as it would appear in the tetrad stage at the time of the crossing over, and indicates the standard genetic map loca- tion of the y and spl markers. The female carrying this chromosome has a pale (dilute) apricot phenotype. If such a female is crossed to a 5a/--containing male, the non- Bar Fi daughters (who carry a Y from their father) are usually noncrossovers and have pale apricot-colored eyes like their mother. Crossovers which occur between the white ^ Based upon work of E. B. Lewis. 192 CHAPTER 22 FIGURE 22-5. Crossing over between apricot and white in attached-X chromosomes. + apr + + locus and the centromere produce either white daughters or apricot daughters. Bar- ring mutation, these are the only phenotypes expected if w" and h' are allelic. But if apricot is not allelic to w, it should be distinguished by giving it a new kind of symbol, apr. In this case, if apr lies to the left of w, as shown in the left part of the Figure, a rare crossover could occur between these loci, as indicated there, producing the crossover attached-X shown at the right of the Figure. Note that if apr and w are non- allelic, each must have its own + allele in the other arm of the parental attached-X, so that these female parents are trans heterozy- gotes with respect to these loci. As a result of the crossing over mentioned, these non- allelic genes would be placed in the cis posi- tion. If the cis position also produces light apricot eye color, we will not be able to dis- tinguish such a crossover from any other one between y and spl that does not occur be- tween apr and w, and we will be forced to conclude (for lack of evidence) that apr and w are really alleles. (Two genes are con- sidered allelic, then, when they actually are allelic and cannot be recombined by crossing over, and when they are nonallelic and under- go crossing over but give the same phenotype in both the cis and trans positions.) When large numbers of daughters from the attached-X females were examined, six were found with dull-red eyes. To deter- mine whether these flies were mutant or the H h H h apr w > spl result of a change from the trans to the cis form, the attached-X's found in the dull-red- eyed exceptional flies were detached (by col- lecting the products of the occasional cross- ing over that occurs between the attached-X and the Y in the heterochromatic regions near their centromeres). The genes carried in each detached arm were determined and it was found that one arm always could be repre- sented as y apr+ vv+ 57;/+ and the other arm always as >'+ apr w spl. Such results off"er strong support for the view that the dull-red exceptional females were cis heterozygotes which produced dull-red eye color; they also demonstrate that if apr and w are sepa- rate loci, apr lies to the left of w on the X, as shown in the Figure. (You should work out the distribution of the markers following crossing over between apr and vv on the as- sumption that apr is to the right of w.) Proof that the exceptional dull-red females were the result of a position eff"ect rather than some mutational phenomenon was com- pleted by mating these exceptional females and obtaining occasional daughters which were pale apricot. These new exceptional daughters were then shown to contain the original gene arrangement, this order having been restored by crossing over. The phenotypic diff"erence between pale apricot and dull red is undoubtedly the result of position effect, since the only diff"erence between the cis and trans conditions is the arrangement which the same genetic material Position Effect and Allelism in Drosophila 193 takes. This phenomenon is therefore called a cis-trans position effect. In order to detect such an effect it was necessary to separate two very closely linked genes. The genes used in the experiment had previously been con- sidered alleles because of their closeness on the genetic map and their similar pheno- typic effects. But the fact that the cis and trans positions of them gave different pheno- typic effects made it possible to prove they are nonalleles, occupying different loci. You may next ask about the other genes making up the "white multiple allelic series" (Chap- ter 9). Are some allelic to vr and others allelic to aprl The answer is yes. More- over, some are allelic to neither, and appro- priate crossing over studies have shown that the "white region" on the X is a nest of four (perhaps five) separate, linearly arranged loci with similar effects. In view of the result obtained with the white region, you may correctly ask next whether there are other regions in the ge- nome where two or more genie alternatives have been considered allelic by the same criteria as were originally used in the white region, but which prove to be pseudoallclic, that is, prove to be nonallelic when subjected to the cis-trans test. Again the answer is yes. Numerous examples of such pseudoallelism have been found in diverse organisms includ- ing, for example, cases involving color in cotton, taillessness in mice, lozenge and also vermilion eye colors in Drosophila, and other cases found in Aspergillus, other microorgan- isms, and corn. Another case of pseudoallelism in Drosoph- ila will be discussed ^ briefly, in which the nonalleles differ in their functioning some- what more than do apr and \v. The normal (wild-type) fly (Figure 22-6A) has small club- shaped balancers (halteres) located on the posterior part of the thorax. One of the pseudoalleles, bithorax (bx), converts the haltere into a large wing-like structure ^ Based upon work of E. B. Lewis. (Figure 22-6 B), another called postbithorax ipbx) appears to do much the same thing (Figure 22-6C). But close examination re- veals that these two recessive pseudoalleles really do different things, bithorax convert- ing the front portion, and postbithorax the hind portion, of the haltere into wing-like structure. This is verified by obtaining the double mutant combination {bx pbx, hence the cis form) by crossing over (at a rate of .02%), and observing the phenotypes of flies made homozygous for the double mutant combination. Such flies (Figure 22-6D) have a fully developed second pair of wings. What are the cis-trans effects for bx and pbxl The cis form (+ -\-/bx pbx) has nor- mal balancers, while the trans form {bx -\- / -[- pbx) shows a slight postbithorax effect, providing another example of cis-trans position effect and demonstrating the non- allelism of these genes. You may have already tried to visualize how cis-trans position effects are produced. One model, but not necessarily the only one possible, compares the two homologous chromosomes to assembly lines, each of which makes its products independently. The cis form can make all the products in turn, in the strand containing the two normal alleles. (The strand with both mutants makes less or no end product.) So, over all, much end product is produced by the cis form. In the trans form, however, because each strand contains a mutant (defective machine), the total end product produced is zero or rela- tively little. (It should be noted, however, that detection of a cis-trans position effect requires only that the phenotypes produced by the two arrangements be different — it does not require or specify that cis appear phenotypically normal.) Why should cis-trans position effects be produced? It is reasonable to think that the products of genes which are linear neighbors would be more likely to depend upon each other than they would be to depend upon the 194 CHAPTER 22 FIGURE 22-6. Drosophila melanogaster males: normal {A), bithorax (B), postbitho- rax (C), and bithorax postbithorax (D). (Courtesy of E. B. Lewis; reprinted by per- mission of McGraw-Hill Book Co., Inc., from Study Guide and Workbook for Genetics by I. H. Herskowitz. Copyright, 1960.) products of their alleles in the homologous chromosome, for this homolog is usually located a considerable distance away. Con- sider also cases of position effect brought about by structural changes. The same reasoning could explain position effects fol- lowing shifts in the relative positions of heterochromatin and euchromatin via break- age. In fact, position effects from structural changes would be expected to be particu- larly common in species whose chromosomes or chromosome parts are not located at ran- dom in the nucleus, but take on special posi- tions relative to each other. The facts that during nuclear division Drosophila chromo- somes show somatic synapsis, and that somatic synapsis is found in the interphase nuclei of salivary gland and other cells, sug- gest that at the time of gene action different chromosomes and their parts are arranged so that the products of gene action may be formed or used in particular sequences. Intra- and interchromosomal rearrange- ments which change these sequences might be expected to produce position effects. The fact that Oenothera chromosomes form a circle of 14 during meiosis (Chapter 20) demonstrates a very orderly arrangement of chromosomal parts involving heterozygosity for reciprocal translocations. Here also, a new arrangement of chromosomal parts might be expected to disturb functional se- Position Effect and Allelism in Drosophila 195 quences and produce position effects. And, in fact, position effect is known to occur in Oenothera. Finally, let us consider the morphology of chromosome regions found to contain pseudoalleles. The white series is associated with a double band {doublet) in the salivary gland chromosome; apr may be in one band, w in the other. The vermilion series is asso- ciated with a doublet on the X chromosome, while the bithorax series (composed of five separate pseudoallelic loci) is connected with two doublets. (This indicates, what is proved by other data, that a band may contain more than a single gene.) Since there are many doublets in salivary chromosomes, this suggests that genes located in these regions will be found to be pseudoallelic. How can we explain the origin of appar- ently adjacent loci with similar types of action? There are several possible ways in which this situation may have come about. One explanation is that during the course of evolution, adjacent genes, having different effects, mutated to alleles which performed similar functions, and therefore were ad- vantageous. A second explanation invokes a selective advantage of rearrangements which brought together widely separated non- alleles with similar functions. While both of these mechanisms may explain some of the cases found, it seems more likely that most adjacent and similar genes arose as duplica- tions that occurred once, or more times (as in the bithorax case), in the ways already de- scribed in Chapter 19. Following duplica- tion, the linear array of originally identical genes would become somewhat different from each other functionally because of mu- tation, thereby fostering evolution. SUMMARY AND CONCLUSIONS The same genie matter arranged in different ways may have different phenotypic conse- quences. The shuffling of genes which produces position effects may be accomplished by means of structural changes in chromosomes and by means of crossing over preceded either by aneusynapsis or eusynapsis. While position effect may be one of the consequences of mutation, it itself does not represent mutation. While the decision is usually valid that two genes located in totally different parts of the genome are nonallelic whether or not they have similar or different phenotypic effects (on the presumption that one is not an allele which was transported to a new location via struc- tural change), statements made in earlier Chapters that two genes are allelic may often have been invalid. To properly apply the term allele to two similarly acting genes, apparently at the same map locus, it is necessary to perform exhaustive tests to recombine them via crossing over. Recombination is demonstrated by the detection of a cis-trans position effect, which proves the genes to be pseudoallelic and, therefore, nonallelic. Failure to obtain a cis-trans position effect after exhaustive trials is taken to mean the genes involved are alleles, though this necessarily includes possible cases of separable nonalleles which fail to give this effect. Linear nests of genes with similar effects have probably arisen by one or more duplications in situ of an ancestral gene, followed by mutations that led to differentiation in their effects. Position effect is attributed to some dependency which exists between the gene products of adjacent nonalleles. REFERENCES Bridges, C. B., "The Bar 'Gene' a Duplication," Science, 83:210-211, 1936. Reprinted in Classic Papers in Genetics, Peters, J. A. (Ed.), Englewood Cliffs, N.J., Prentice-Hall, 1959, pp. 163-166. 196 CHAPTER 22 Lewis, E. B., "The Pseudoallelism of White and Apricot in Drosophila Melanogaster," Proc. Nat. Acad. Sci., U.S., 38:953 961, 1952. Muller, H. J., Prokofyeva-Belgovskaya, A. A., and Kossikov, K. V., "Unequal Crossingover in the Bar Mutant as a Result of Duplication of a Minute Chromosome Section," C. R. (Dokl.) Acad. Sci., U.R.S.S., N.S., l(10):87-88, 1936. Sturtevant, A. H., "The Effects of Unequal Crossingover at the Bar Locus in Drosophila," Genetics, 10:117-147, 1925. Reprinted in Classic Papers in Genetics, Peters, J. A. (Ed.), Englewood Cliffs, N.J., Prentice-Hall, 1959, pp. 124-148. QUESTIONS FOR DISCUSSION 22.1. If a novel phenotypic change is associated with a qualitative or quantitative change in the genetic material, how can you decide whether to attribute the effect to mutation or to position effect? 22.2. Do you expect that position effects will be found in most sexually reproducing or- ganisms? Why? 22.3. Is there any evidence that crossing over is ever unequal? Explain. 22.4. Should pseudoalleles be considered to be subgenes (parts of one gene) rather than separate, nonallelic genes? Explain. 22.5. Does position effect require pseudoallelism for its detection? Explain. Is the reverse true? Explain. 22.6. What is the minimum number needed, of genotypic alternatives for a "locus," in order to test whether it shows a cis-trans position effect? Explain. Can one of the alternatives be a deficiency for all or part of the region under investi- gation? Why? 22.7. Can position effect occur in haploids? Why? 22.8. How would you proceed to test whether two recessive mutants in Drosophila that are apparently alleles of the X-linked gene, v+ (normal allele of vermilion eye color), are pseudoalleles? Chapter *23 GENE AND POINT MUTATIONS WHAT have we learned, begin- ning with Chapter 18, about the mutational units of the genetic material? We have seen that the unit of mutation in the genotype may be a whole genome, single chromosomes, and parts of chromosomes. Even though each of these units involves more than one gene, it is possible that our study of these larger units can tell us something about the mutational characteristics of a single gene. It is also possible that our knowledge concerning the recombinational properties of individual genes will shed light in this direction. Ac- cordingly, we shall begin the present discus- sion by considering what can be revealed about single gene mutation by what we have already learned. It has been demonstrated that the genes in a chromosome are arranged linearly. This linear order could be formed in two ways, that is, either by having the genes attach to each other directly, or by having some non- genic material serve to connect adjacent genes. In either event, the fact remains that a chromosome is invariably linear, being either a rod or a ring, but never branched. For not only are all ordinarily observed chromosomes linear, but, even when chro- mosomes have been observed immediately after crossing over, or after they have been broken more than once and the pieces have joined together, no case has ever been ob- served of an authentic branched chromo- some. This is almost conclusive evidence that the gene cannot be joined, directly or 197 indirectly, to other genes at more than two places, and that a mutation of this kind cannot occur either spontaneously or induc- tively in genie material. The fact that this change is never observed, regardless of the organism studied, can be interpreted to mean either that the gene never had this property, or that it is lost to all presently existing genes. We are led to conclude, therefore, that all interstitial (nonterminal) genes are bipolar, and that mutation is in- capable of causing the gene to be more than bipolar. Almost all mutations retain the bipolarity of genes, as evidenced by the "stickiness" of both ends produced by a break in a chromo- some. However, in some relatively rare cases broken ends are known to become per- manently healed, so that mutation from bi- polarity to unipolarity does occur. That mutation must possess this property of chang- ing genes from a bipolar to a unipolar type, or the reverse, is evidenced also by the pres- ence of telomeres, unipolar genes that serve to seal off the normal ends of chromosomes. You may be acquainted with the fact that a change from genie bipolarity to unipolarity is a regular phenomenon in the life history of certain animals, as in certain species of the roundworm Ascaris. Here, in nuclei which remain in the germ line there is a single pair of chromosomes, but in those nuclei which enter the somatic line these chromosomes break up into a number of smaller linear frag- ments, each of which has sealed-off ends and behaves normally during mitosis. (The latter behavior is made possible by the fact that, in these cases, the germ line chromo- some has a number of centromeres along its length, and each fragment of the chromo- some in a somatic cell has one of these centro- meres. This is an exception to our state- ment, on page 19, that normal chromosomes are unicentric, and involves a polycentric chromosome which in the germ line has the action of all but one centromere suppressed.) 198 CHAPTER 23 Because this chromosome fragmentation takes place only in somatic cells, these polarity changes can be ascribed to some physiological difference between cells enter- ing the somatic line and cells remaining in the germ Hne. Should such changes be con- sidered mutational? They should not, be- cause the changes from bipolarity to uni- polarity in Ascaris are numerous and simul- taneous, and lack the novelty required of mutations. It should be noted that while no unambigu- ous case has ever been reported of a muta- tion from unipolarity to bipolarity, the chance of detecting and proving such a change is very small indeed. Accordingly, the capacity of mutation to change a gene in this manner cannot, at present, be denied with any assur- ance. Thus, although there are bipolar and unipolar genes, a change in polarity via muta- tion is clearly known to proceed only from the former to the latter. Does this mean that mutations to nonpolarity do not occur or cannot occur? We cannot give a definite answer at this point, since, thus far, we have restricted our attention to genes all of which have been localized in chromosomes. It must be evident that a unipolar or bipolar gene that mutates to a nonpolar alternative must necessarily drop out of the chromosomal Hne-up. If this happens, then the freed, not- at-all-sticky gene will not be linked to any chromosome. From what has already been presented, there has been no evidence for the existence of genetic material which has been liberated from its chromosomal locus in this way. Since the only kind of gene we have so far identified is the chromosomally located one, we must accept the simplest explana- tion, and conclude that genes cannot occur singly, two of them comprising the smallest group possible. The gene was discovered by studying sexu- ally reproducing individuals, in which many of its recombinational properties were re- vealed because of synapsis and the events consequent to this process. Synapsis is the result of the attraction which exists between corresponding segments of homologous chro- mosomes. It is a remarkable fact that corre- sponding loci located on homologous chro- mosomes do attract each other in synapsis even though the particular alleles contained may be identical or somewhat different. Let us assume that synapsis is directly dependent upon the genie content of a chromosome. Is this synaptic attraction between genes re- stricted to alleles only? It is probable (see Chapter 22) that what are nonallelic genes, at present, were at some time, in the remote past, allelic. Accordingly, mutation must be capable of changing the synaptic specificity of the gene, and it must follow, at least in a general way, that identical genes attract each other more than do nonidentical ones. That different degrees of specific attraction exist between genes is illustrated by the genes located in heterochromatin (cf. p. 181), which synapse much less specifically than those found in euchromatin. Other genes are known (for example, one in maize called asynaptic) which either (1) not only lack synaptic attraction for their alleles but also destroy this attraction between pairs of genes at other loci, or (2) cause general desynapsis. Apparently, the gene is unrestricted muta- tionally in the way it can affect synaptic force. It should be clear from what has been learned (in Chapter 22, for example) that different genes do exist, mutations in them not being explicable merely in terms of their complete loss or inactivation, since each gene can usually mutate to a number of different allelic forms, which comprise a series of mul- tiple alleles. Because different genes exist, it is obvious that mutations occur involving single genes. Since the gene is submicro- scopic (a single band in the salivary gland chromosome of Drosophila can contain more than one gene), so also is a change produced in a single gene. The only way we have of Gene and Point Mutations 199 detecting changes in individual genes, there- fore, is by the phenotypic changes these pro- duce. Since, at present, we recognize a gene operationally as the smallest unit of genetic material whose recombination can be de- tected phenotypically, we must determine the characteristics of mutation in single genes from the phenotypic changes produced in recombinationally detected genes. Accord- ingly, it is clear that we shall not be able to determine from such phenotypic changes whether single gene mutation involves the recombinational gene in toto, or one portion or site within it, or many different sites within it. If gene mutation requires a change in the entire gene, then the material composition of the genes detected by recombination and by mutation would be identical. If, on the other hand, the recombinationally detected gene contains within it one or more sites at which mutation can occur, the basic recombi- national unit of genetic material would be larger than the basic mutational unit of the genetic material. In the absence of critical evidence as to which of these alternatives ob- tains, we shall continue to consider the muta- tional and recombinational genes to be ma- terially equivalent, as we have assumed since page 14, where we invoked the law of parsi- mony. As already mentioned in Chapter 1, any given gene is rather stable, being faithfully replicated many thousands of times before a detectable mutation occurs in it. You should recognize, however, that the greater the sensi- tivity of our tests for detecting mutations, the larger will be the rate of mutation ob- served (recall the detection of isoallelism on p. 65). It remains probable, therefore, that there are transmissible modifications of single genes which escape our present modes of de- tection. Nevertheless, within the limits of our present methods of analysis, the gene ap- pears to be a very stable entity. The studies to be considered next started with the collection of all detectable mutants of genes which were being investigated singly or in combinations. The mutants obtained were then analyzed. Some of the mutations in- volving a given locus proved to be based upon ploidy changes, not involving chromosome breakage; others proved to be associated with gross or small chromosomal rearrange- ments. These types were eliminated from further consideration. Then, sometimes, all genetic and cytological tests known were ap- plied in order also to eliminate the minutest chromosomal rearrangements, including, for example, tiny duplications or deficiencies. The remainder of mutations was then as- sumed, for lack of evidence to the contrary, to comprise in considerable portion mutations involving a single gene (gene mutations) or involving at most only a few genes (intergenic mutations). The mutations remaining, then, behaved as though they occurred at a single point in the genetic and cytological maps, and they were, therefore, called point muta- tions. (Note that gene mutation includes losses of single whole genes.) Let us discuss now those discovered charac- teristics of spontaneous and induced point mutations which are likely to apply to gene mutation. Point mutation can occur in a vast number of different genes, so that this process is not restricted to a very limited type of gene. It might be thought that the con- ditions causing point mutation could be of such a nature that, in the diploid cell, both members of a pair of alleles would tend to respond by mutation. But, when point mu- tation does occur in a diploid cell, it is found that only one gene of the pair present is af- fected. The fact that only one member of a pair of genes mutates, though both are lo- cated in the same nucleus, demonstrates that the point mutation process is a very localized, submicroscopic event. Is point mutation a rapid or a gradual change? If it were typically a gradual change, or one which involved an instability of the gene for more than one cell generation, then 200 CHAPTER 23 point mutations would usually occur in clusters, even if within a cluster the same gene did not always mutate to the same allele. But many point mutants occur singly, and others, which appear in a cluster and seem to be identical, can usually be accounted for on the basis that a single cell containing the mu- tant gene divided a number of times before the tests to detect the mutant condition were made. While such data do not prove that point mutation is instantaneous, they indi- cate that it is usually completed within one cell generation and is, in this respect, more a quick than a gradual change. However, the number of point mutations obtained from X ray or ultraviolet ray treatments is reduced, if a posttreatment of certain types of visible light or of chemical substances is given im- mediately. Such posttreatments produce photo- or chemorecovery from point muta- tion. This proves that the point mutation process is often not completed for some minutes. It is only after the point mutation process is completed that the new genetic alternative is about as stable as the old. While the point mutants which arise at the present time are just about as stable as their parent genes, or other genes in the geno- type, this should not be taken to mean that all allelic and nonallelic genes have the same spontaneous mutation rate. A representa- tive sample of specific loci in Drosopliila gives an average of one point mutation at a given locus in each 200,000 germ cells tested. In mice, the per locus rate is about twice this, or one in 100,000. In man, scoring mutants that are detected when in heterozygous condi- tion, the per locus rate is one per 50,000 to 100,0000 germ cells per generation. Within each species, the different loci studied had about the same order of mutability. Never- theless, some genes are definitely more mu- table than others, and those that seem to be very mutable are called "mutable genes." The latter will be discussed in Chapter 25. The average spontaneous point mutation rate per genome has been estimated for Dro- sophi/a, mouse, and man. In Drosophila, one gamete in twenty (or one zygote in ten) con- tains a new detectable point mutant which arose in that generation. In mice, this fre- quency is about one in ten gametes, while in man this rate is about one in five gametes (or two in five zygotes). The point mutations which occur sponta- neously— that is, during the course of ob- servations made under natural conditions — bear no obvious relation to the environment, either with respect to the locus affected or to the type of alternative produced. However, modifications in the environment do influ- ence point mutation rate. For example, changes in temperature can change point mu- tation rate, each rise of 10° C, in the range of temperatures to which individuals are usually exposed, producing about a fivefold increase in mutation rate. This rate of increase is similar to, though somewhat higher than, what is obtained in ordinary chemical reac- tions with an increase in temperature. Vio- lent temperature changes in either direction produce an even greater effect upon point mutation rate. Actually, it is found that detrimental environmental conditions of al- most any kind cause an increase in point mutation rate. Certain physical and chemical agents which raise the mutation rate enormously are called mutagens. All high-energy radia- tions (see Chapter 21) are mutagenic as are many highly reactive chemical substances, in- cluding mustard gas and its derivatives, and also peroxides, epoxides, and carbamates. The point mutation rates obtained with muta- gens may be 150 times the spontaneous rate, and the loci affected and the types of mutant alternatives produced are not radically differ- ent from those which are involved in sponta- neous mutation. One can speak of a spec- trum of spontaneous point mutations, how- ever, in that certain loci are normally some- what more mutable than others. Ionizing Gene and Point Mutations 201 radiations produce a mutational spectrum much like the spontaneous one, as would seem reasonable from the fact that the radi- ant energy involved is more or less randomly distributed in the nucleus and generally en- hances chemical reactions of all kinds. How- ever, the point mutational spectrum is some- what different for different chemicals, and is different from that for natural or radiation mutation agents. This can be attributed to either the nonrandom penetration of these chemical substances into the nucleus, or the specific capacities that these have of com- bining with different nuclear chemicals, or both. Nevertheless, the frequency of point mutations, which increases linearly with the dose of ionizing radiation (although the rate is influenced by the amount of oxygen present), probably also increases linearly with the nuclear dose of many different chemical mutagens. So point mutations show no threshold dose with these mu- tagens, and the number of point mutations produced by a given total dose is constant, regardless of the rate of delivery. However, in the case of ultraviolet light, which is not a highly energetic radiation, the situation is otherwise. Here the individual quantum of energy has a probabiUty of inducing point mutation which is considerably less than 100 per cent. This means that several quanta can cooperate to produce mutation, so that the point mutation rate increases faster than linearly with dose, at least at low doses, and an attenuated dose is less mutagenic than a concentrated one. Point mutation is not restricted to the genes of any particular kind of cell. It oc- curs in males and females, in somatic tissues of all kinds, and in the diploid and haploid cells of the germ line. It has been found that perifertilization stages (later stages in game- togenesis and very early developmental stages) are relatively rich in spontaneous point mutations. It is not surprising that despite the very great differences in life span, there is not a greater difference in sponta- neous mutation rate between flies, mice, and men. For, if most germ line mutations occur in the perifertilization stages, these rates would be quite similar, since these different organisms are not very different in the length of time occupied by these stages. A further similarity exists among these species, in that there is not a very great difference in the num- ber of cell divisions required to go from a gamete of one generation to a gamete of the next. In fact, the differences in mutation rate for these organisms are approximately pro- portional to the differences in the number of germ cell divisions per generation. This brings to mind the following ques- tions: When, during the history of the gene, does the event of mutation occur? Can the old gene itself undergo mutation whether it is or is not in the process of synthesizing a new one? Can "mutations" occur during the course of synthesis of what is to be the new gene? The fact already mentioned, that point mutation rates in Drosop/iila, mouse, and man are proportional to the numbers of cell divisions that occur, suggests that some of these mutations occur at the time of synthesis of the new gene, although this does not specify whether it is the old or the new gene that becomes mutant. It has been found that the aging of spermatids and sperm of Dro- sophila increases their point mutation rate. Since these nondividing cells do not have their viability impaired when they are aneuploid, the increase in point mutations may be due to an effect upon the old gene, which is physiologically quiescent and prob- ably not actively synthesizing new genes. The occurrence of mutation in the old gene would mean that point mutational changes can occur while a gene is linearly attached to its genie neighbors. However, the higher mutation rate observed after cells are aged may also be explained as resulting from the accumulation, with time, of a mutagen which acts on the old or new gene once gene repli- 202 CHAPTER 23 cation is resumed. Finally, the possibility new gene is completed and attached to its still remains that changes can occur in the linear neighbors; these changes may be de- steps leading to gene synthesis, before the tected later as point mutants. SUMMARY AND CONCLUSIONS The mutational units in a genotype are, in order of size, the genome, fhe chromosome, chromosomal segments involving more than one gene, and the gene. Since a number of different alleles occur per gene, gene mutation may involve the entire gene, or one mutational site within the gene which has many alternatives, or many mutational sites which have one or more alternatives. It is possible that the genes operationally delimited by recombination and by mutation are not exactly equivalent materially. However, in the absence of critical evidence on this, we shall continue to assume that they are identical. Gene mutation is not limited in any way with regard to the effect it can have on synapsis. It is also in no way restricted by ploidy, type of cell or gene, but is limited with respect to the effect it can have on a gene's polarity. Tripolar genes are excluded, bipolarity being the usual, and unipolarity the less usual, alternative. Point mutations are the remainder of all mutations which are not known to involve inter- genic changes. Since point mutations include changes in single genes they may be em- ployed to determine the mutational characteristics of the gene. The frequency of point mutations increases linearly with the dose of high energy radiations, there being no effect of dose protraction, and no threshold dose below which the gene is safe from such change. Such mutations indicate that a given gene is relatively stable over many cell generations, changes in it being the result of very localized physico-chemical events which are completed in a matter of minutes, after which the new gene is similarly stable. Changes in the gene are enhanced or induced by temperature changes, aging, gene replication, and physical and chemical mutagens. It is possible that changes resulting in gene mutation can take place in the old gene, or in the new gene, or during the formation of the new gene. REFERENCES Alexander, P., "Radiation-Imitating Chemicals," Scient. Amer., 202, No. 1 :99-108, 1960- Muller, H. J., "Variation Due to Change in the Individual Gene," Amer. Nat., 56:32-50, 1922. Reprinted in Classic Papers in Genetics, Peters, J. A. (Ed.), Englewood CliflFs, N.J., Prentice-Hall, 1959, pp. 104-116. Muller, H. J., "Artificial Transmutation of the Gene," Science, 66:84-87, 1927. Reprinted in Classic Papers in Genetics, Peters, J. A. (Ed.), Englewood Cliffs, N.J., Prentice-Hall, 1959, pp. 149-155, and also in Great Experiments in Biology, Gabriel, M. L., and S. Fogel (Eds.), Englewood Cliffs, N.J., Prentice-Hall, 1955, pp. 260-266. QUESTIONS FOR DISCUSSION 23.1 . Is there a dose of X rays and/or of ultraviolet radiation which is safe, in that it cannot cause some point mutations? Explain. 23.2. Can we be sure that any given mutation involves a single gene change rather than an intergenic one? Explain. 23.3. Would we know of the existence of genes if all genes had the identical mutational capacity? Explain. Gene and Point Mutations 203 Lewis John Stadler {1896-1954) is noted for his studies on the nature of mutation and of t/ie gene {see p. xi). He and H. J. Muller discovered inde- pendently the mutagenic effect of X rays. {By permission of Genetics, Inc., vol. 41, p. 1, 1956.) 23.4. Would you expect the mutation rate to Polydactyly, P, from normal, p, to be greater among normal individuals in a pedigree for Polydactyly than it is among normals in general? Explain. How might you proceed to test this hypothesis? 23.5. Do the mutational properties discussed suggest any limitations with respect to the chemical composition of genes? Explain. 23.6. When a chromosome is broken, is the breakage point within a gene or between genes, or can both occur? Justify your answer. 23.7. Point mutations are sometimes called gene mutations. Do you think this is per- missible? Why? 23.8. In what way is the study of mutation dependent upon genes? In what way is the reverse true? 23.9. What is your opinion with regard to the validity of applying principles of point muta- tion directly to gene mutation? Chapter 24 POINT MUTANTS — THEIR DETECTION AND EFFECTS IN INDIVIDUALS P kOiNT mutants comprise the re- mainder of all genetically de- tected mutants for which no association with intergenic changes can be demonstrated. It is not only of historical importance, but also of current and future interest, to understand something about the genetic methods that are used for collect- ing point mutants. We shall consider two elegant procedures ^ using Drosophila melano- gaster, one for the detection of mutants that are recessive lethals, the other for "visible" mutations, at specific loci, which are viable when heterozygous. The technique for detecting recessive lethals is called "'Base'' (see Figure 24-1), and is designed to discover such mutants that arise in the germ line of the male, in X chro- mosome loci which are hemizygous, i.e., have no allele in the Y chromosome. The males used are wild-type, having all normal char- acteristics, including round, dull-red com- pound eyes. The females employed have X chromosomes homozygous for Bar eye (B), for apricot eye color (apr), and for a para- centric inversion (In) of almost the entire left arm {In sc'^^ sc^ whose right breakage point is designated sc'^^ and left 5c^) inside of which is a smaller inversion (InS). '"Base"' derives its name from Bar, apricot, scute in- version. Base females (or males) have nar- ^ These were invented by H. J. Muller. 204 row-Bar eyes of apricot color. The genotype of the Base female is written sc'"^^ B InS apr sc^/sc^'^^ B InS apr sc^. A wild-type male is mated to a Base female and the Fi daughters are obtained. These daughters are -\-/sc''^^ B InS apr sc^ and ap- pear heterozygous (wide) Bar (being other- wise wild-type). Since the very short right arm of the X is entirely heterochromatic, it is of no concern here. Because each Fi female is heterozygous for two paracentric inversions, any crossing over between the left arms of her X's pro- duces dicentric or acentric crossover strands which fail to enter the gametic nucleus (see Chapter 19). Accordingly, Fi females pro- duce eggs having an X that is, for our pur- poses, either completely maternal (5C'^^ B InS apr 5C^), or completely paternal (+). If this Fi daugh- ter mates with her Base brothers, half of the sons in the next generation (Fo) receive the -f maternal X, and half receive the Base ma- ternal X. So, if the progeny of a single Fi female are examined, it is a simple matter to recognize the presence of both types of sons, since it is usual to obtain more than forty sons per female. Note that each wild-type Fo son carries an identical copy of the X in the sperm that fertilized the egg which de- veloped as his mother (the Fi female). Even when the sperm used to form the Fi female carries an X-linked recessive lethal mutant, the Fi female will survive because she carries the + allele of it in her Base chromosome. However, each + F2 son will carry this mu- tant in hemizygous condition and will usually die before adulthood, so that no + sons ap- pear in Fo! It becomes clear, then, since an Fi female is formed by fertilization with a + X-carrying sperm, that the absence of + sons among her progeny is proof that the particular Pi sperm carried a recessive lethal mutant. Point Mutants — Detection and Effects 205 Such a lethal mutant must have occurred in the germ line after the fertilization that pro- duced the Pi male, for if it were present at fertilization, he would not have survived. It is unlikely that many of the lethals detected in sperm originate very early in development, for in this case a large portion of the somatic tissue would also carry the lethal and this would usually cause death before adulthood. Usually, the X-linked lethal present in sperm arises in a small portion of the germ line so that even when a few hundred sperm, of the several thousand sperm ejaculated, are tested, only one is found to carry a mutant. Occa- sionally, however, the mutation occurs early enough in the germ line so that several sperm tested from the same male carry what proves to be the same recessive lethal. When a thousand sperm from normal un- treated males are tested for X-linked reces- FIGURE 24-1. The breeding scheme used in the Base technique. \--x. , -Base ■i c Base Base >== I Base c/ Base 9 Base / I ^9 X Base Base (Individually) Base 9 9 Base ?c/ O (11 I n S^S O <2) (1) is absent if the F^ Base ehromosome contributed to the Pj L> contained a recessive lethal • (2) is absent if the f^ + chromosome contributed to the Pj O contained a recessive lethal T /\lK^ 206 CHAPTER 24 sive lethals by means of a thousand separate matings with Fi females, as described, ap- proximately two of these matings are found to yield no + sons, so that the recessive lethal mutation rate of 0.2% is a fairly typical one. For each dose of 1000 r of X rays to which the adult male is exposed, approxi- mately 3.1% more sperm are found to carry X-linked recessive lethals. The Base technique has certain disadvan- tages. When used as described, it detects only those recessive lethals which cause 100 per cent mortality before adulthood. Other recessive lethals that produce sterility or cause death of the adult before it can mate are not detected. No recessive lethals are de- tected unless they are hemizygous in the Fz male, as mentioned, and a considerable number are known to occur whose lethality is reduced by genes normally present in the Y chromosome. On the other hand, the advantages and ap- plications of the Base technique are numer- ous. A few can be mentioned here. The presence or absence of + males in Fo is easily and objectively determined. Since the reces- sive lethal which is detected in Fo is also car- ried by the heterozygous-Bar F2 females, this permits further study of the recessive lethal in that generation and subsequent ones. In such studies it can be discovered whether the lethals are associated with intergenic changes; those that are not are designated as recessive lethal point mutants. The Base technique can also be used to detect recessive lethals that occur in a Pi Base chromosome, the absence of Base males among the F2 prog- eny indicating such a mutation. Moreover, us- ing standardized environmental conditions, it becomes possible to detect hemizygous muta- tions which either lower the viability of the males without being lethal or raise their viability above normal. Most important is the additional possibility of studying the viability effects of recessive lethals when in heterozygous condition. Although the Base technique may be used also to detect X-linked mutants producing a visible morphological change when hemizy- gous, all those "visibles" which are also hemi- zygous lethals are missed. The ''Maxy"' technique (refer to Figure 24-2) overcomes this difficulty. The males used carry an X chromosome containing two medium sized paracentric inversions, In49 (having no ob- vious phenotypic effect) and B^'^ (having a dominant effect like Bar). The X also carries a minute paracentric inversion (of negligible phenotypic effect) associated with the reces- sive lethal IJl (located to the left of yellow body eolor, y), which is also present. This X also carries the recessive mutant oeelliless (oe) which removes the ocelli (simple eyes). The males are able to survive IJl because they carry the normal allele, IJ1+, as a mutant duplication in the longer arm of the Y chro- mosome. The genotype of the male may be written as: IJl In49 oc B"'/IJ1+.Y. One of the female's X's is the same as the one in the male. The second X carries IJl^ and a recessive lethal mutant / (located be- tween seiite bristles, se, and white eye, vv), for which the other X carries the normal allele, /+. This second X also carries the long in- version In 5c-^ sc^ (as in Base), plus reces- sive genes for yellow body color (j'), out- stretched wings and small eye (odsy), echinus, rough eyes (ec), forked bristles (/), singed bristles (sn), dusky wings (dy), cut wings (et), and recessive genes for the following eye colors: carnation (ear), garnet (g), vermilion (v), raspberry (ras), carmine {cm), ruby (rb), white (vv), and prune (pn). The arrangement of all the markers in this X is as follows: y I 5-c- ' car odsy f g dy v ras sn ct cm rb ec w pn sc^. Since, by convention, only mutants are in- dicated in the genetic formulae for these X's, you must realize that the 5'^^-containing X carries /+ as well as the normal alleles of all the other recessives in the second kind of X. Similarly, the X carrying the multiple reces- Poiut Mutants — Detection and Effects 207 sives also carries /J7+, though this is not indi- cated in the formula. Females of this stock do not produce gametes containing X chromosome cross- overs, since these are eliminated in the same manner as in the Basc'^ heterozygote. When females and males of this stock are mated together, only two kinds of male zygotes are produced. However, the half re- ceiving the /-containing X die, while the other half, which live, are genotypically exactly like their father. The female zygotes are also of two equally frequent types, the homozygotes for IJl dying, the ones surviving being identi- cal in genotype to their mother. This stock is self-perpetuating because it contains a bal- anced lethal system (see Chapter 20) in which the females are permanent heterozygotes and the males are of only one type. Note that if nondisjunction produces an XO zygote, this will die because Of the hemizygosity either of IJl or /. There are three possible types of nondisjunctional XXY females. One type which is homozygous for X's carrying IJl, lives, because of the />//+ on the Y, but it can- not breed because females that have oc in homozygous condition are sterile; the XXY type homozygous for / dies. The third type of XXY lives and breeds, but since it is het- erozygous for the two kinds of X's, the usefulness or continuity of the stock is not impaired. Phenotypically, males are ocelliless and bar-eyed, while females are slightly bar-eyed, but otherwise wild-type. You can readily see that mutations in the X of the male which involve any of the fifteen normal loci aflect- ing the eyes, body color, wings, or bristles, for which the female has recessive alleles, may be detected in their daughters. For ex- ample, if a sperm carries a mutation from y^ to j;, this will produce a yellow daughter when it fertilizes an egg carrying the multiple reces- sive X. This stock is, therefore, called "Maxy" because it was designed for the finding of "visible" mutations in the male's Ov o c 4- J_ ( 1 ( c Q. 00 E w w c + s > »^ Sr ! 1 o -t- 1 OQ c . T ^ 1 t FIGURE 24-2. The sex chromosomes of the males and females used hi the Maxy technique. X at specific loci, including y. The Maxy stock permits the detection of any mutation, spontaneous or induced, involving the nor- mal alleles of the fifteen recessives, provided the mutant does not produce the normal 208 CHAPTER 24 phenotype when heterozygous with the reces- sive allele, and provided it is not a dominant lethal. Once such mutants are obtained, they can be screened for point mutants. The study of recessive lethals on the X chromosome and on the autosomes shows that there are hundreds of loci whose point mutations may be recessively lethal. It should be noted that the recessive lethals de- tected by Base, and the visibles detected by Maxy, are not mutually exclusive types of mutants, for some Maxy-detected visibles are lethal when hemizygous, and about 10 per cent of Base-detected hemizygous lethals show some morphological effect when het- erozygous. It can be stated, in general, that any mutant in homo- or hemizygous condi- tion which is a "visible" will be found to pro- duce some change in viability, and, con- versely, that any mutant which affects vi- ability will be found to produce a "visible" effect, "visible" at least at the biochemical level. What, in general, is the nature of the pheno- typic effect of point mutants when homozy- gous or hemizygous? A mutant's biological activity is best described in terms of its effect upon reproductive potential, i.e., the capacity to produce surviving offspring. These terms include the mutant individual's capacity to reach the reproductive stage, its fertility and fecundity during this period, as well as the viability of its offspring until sexual ma- turity. We already know that each mutant has manifold effects due to a pedigree of causes (Chapter 10). It has been found that point mutants with small phenotypic effects are much more frequent than those with large effects. For instance, using the Base tech- nique, it is found that recessive mutants, which lower the viability of males without being lethal, are at least three to five times more frequent than those that are lethal (Figure 24-3). The vast majority of point mutants pro- duce a detrimental effect on the reproductive potential, those that are beneficial being ex- tremely rare. For example, the great ma- jority of mutations affecting a trait or organ cause its degeneration. This is understand- able in terms of the past evolutionary his- tory of a species. All the genotypes in a species have been subjected to selection for many generations, and those which produced the greatest reproductive potential were re- tained. Although point mutation at any locus is a rare event, many of the alterna- tives possible for each gene must have oc- curred at least several times in the past his- tory of the species. Of these alternatives only the more advantageous alleles were re- tained, and these are the ones found in pres- ent populations. So, when point mutation occurs today, it is likely to produce one of the genetic alternatives which had already occurred in the past and had been elimi- nated because it produced a lower biological fitness, that is, a lower reproductive poten- tial. It should be realized, moreover, that reproductive potential is the result of coordi- nated action of the whole genotype. The genotype may be likened to the machinery that makes modern automobiles, the en- vironment furnishing the raw materials for this process, with the automobile represent- ing the phenotype. Just as it is true for present genotypes, the machinery that manu- factures automobiles is complex and has had a long evolutionary development. The chance that a random local change in the present machinery will result in a better automobile is just as small as the chance that a newly occurring point mutation will in- crease reproductive potential. In what way does the phenotypic effect of a point mutant differ from that of its normal alternative? This can be studied by examin- ing the effect of increasing the relative num- ber of doses of the mutant present in the genotype. In Drosop/ii/a, for example, the normal fly has long bristles when the domi- nant gene Z?/j+ is present. There is a mutant Point Mutants — Detection and Ejfects 209 (OX ZH OliJ H"* ^ # ^ — >^ * ^ 3- >'^/ -' — 2- - 1- \lfry — 0 — n^ — ^ — r 1 1 1 1 1 1 -3.6 3.2 (/) CO III 2.8 C/5 (/) 2.4 O -1 2.0 Q UJ () 1.6 =5 O 1.2 z ^ 0.8 0.4 0 0 2 6 10 14 18 22 26 30 34 38 DOSE IN RADS(XIOO) FIGURE 24-3. Percentage of mutations, ±2 X standard error, recovered from Drosophila sperm exposed to different dosages of 18 mev electrons. The sex-linked recessive lethal frequencies (L) are joined by solid lines and are adjusted for the control rate; sex chromosome loss frequencies (S) are connected by broken lines and are corrected for the control rate; reciprocal translo- cation frequencies iT) between chromosomes II and III are connected by dot-dash lines. [From I. H. Herskowitz, H. J. Muller, J. S. Laughlin, Genetics, 44: 326, 1959.) 210 CHAPTER 24 Strain with shorter, thinner bristles due to the recessive allele bb {bobbed bristles). Now, bb has a locus that happens to be present both in the X and the Y chromosomes. You might suppose that the male, or female, homozygous for bb has bobbed bristles be- cause this allele results in thinning and short- ening the normal bristle. Since it is possible to obtain otherwise diploid XYY males and XXY females which carry three doses of bb, one would expect, according to this view, that the bristles formed would be still thinner and shorter. But, on the contrary,- in the presence of three doses of bb, the bristles are almost normal in size and shape. This demonstrates that bb functions in the same way as does bb^, but to a lesser degree. Mutants having a similar but lesser effect than the normal gene are called hypomorphs. Many point mutants are hypomorphs, since the addition of further doses of them causes the pheno- type to become more normal. Of the remainder of point mutants, most are amorphs, producing no phenotypic ef- fect, even when present in extra dose. White eye {w) in Drosophila is an example of this. Cases are also known of mutants which func- tion as neomorphs; these produce a new effect. Adding more doses of such a mutant causes more departure from normal, while adding more doses of the normal alternative has no effect. We can represent the relationship between the normal gene and its hypomorphic mutants diagrammatically, as shown in Figure 24-4.^ The vertical axis represents phenotypic ef- fect, the normal effect being indicated by +. The horizontal axis refers to the dosage of the normal gene or of a hypomorphic mu- tant. Notice that a single + gene by itself almost produces the full normal phenotypic effect (and often the difference between its effect and the normal effect is not readily detected). Two + genes reach the + pheno- ^ As shown by C. Stern. 3 Adapted from H. J. Muller. typic level. But, in the case of the hypo- morphic mutant, even three doses may not reach the phenotypic level produced by one + gene (recall the discussion of bb). Note also that genetic modifiers or environmental factors, which would shift the position on the horizontal axis and thereby shift the pheno- typic effect, have less and less influence as one proceeds from individuals carrying only one dose of mutant toward individuals carrying two + genes. You can see that natural selec- tion would favor alleles resulting in pheno- typic effects close to +, that is, near the curve's plateau, for this would insure pheno- typic stability. Any mutant which produced such a phenotypic effect would, in the course of time, become the normal gene in the pop- ulation and would automatically be domi- nant when heterozygous with a hypomorphic gene alternative. This model illustrates how the heterozygote with one + and one mutant gene has practically the same effect as the normal homozygote, and seems to be the best explanation for most cases of complete or almost complete dominance. This scheme also illustrates why so few mutants are bene- ficial, other things being equal, for the nor- mal gene alternative already produces an amount of phenotypic effect near optimum. In view of what was just discussed, you can understand that hypomorphic and amor- phic mutants are usually detrimental when pure (homo- or hemizygous). But you may wonder what their effects will be when heterozygous with the normal gene. If they are amorphs, the single + gene may fall short of producing the full phenotypic effect, and so such mutants would be ex- pected to be slightly detrimental when het- erozygous. Hypomorphs would be ex- pected to be less detrimental or not at all detrimental when heterozygous, at least with respect to the trait for which they are classi- fied as hypomorphic. But it should be re- called that each gene affects many different biochemical processes, and a mutant which 211 3M 1 DOSAGE OF GENES 2 + FIGURE 24-4. The relationship between dosage of normal and mutant genes and their phenotypic ejfect. is hypomorphic with regard to one trait may be amorphic with respect to another one. Thus, in Drosophila, the normal allele «/»/•+ results in dull-red eye color and also pigments the Malpighian tubules. One of its alleles, apr, results in a lighter eye color (being hypomorphic in this respect) but no color in the Malpighian tubules (being amorphic in this respect). Experience confirms expectation in this re- gard. It has been shown that most "reces- sive" lethal point mutants result in some detrimental effect on reproductive potential when heterozygous. Such mutants are not completely recessive, therefore, and it has been found, in Drosophila, that when hetero- zygous, they are cause of death before adulthood of about four per cent of indi- viduals. Mutants, which are detrimental but not lethal when pure, also usually show such a detrimental effect when heterozygous, this effect being somewhat less than that produced by heterozygous lethal point mu- tants. The principles of phenotypic action discussed here apply both to spontaneous and to induced point mutants. SUMMARY AND CONCLUSIONS The details of the Base and Maxy genetic schemes for the detection of recessive lethal and recessive visible mutants in Drosophila are described. Tiie study of point mutants of these types and others reveals that almost all are detrimental to the reproductive potential of individuals when pure (not hybrid ), and to a lesser extent when hybrid. Accordingly, most point mutants are not completely recessive to their normal genetic alternatives. Most normal genes fail to produce the full normal phenotypic effect in single dose, and most point mutants act on the phenotype in a hypomorphic or amorphic manner. 212 CHAPTER 24 REFERENCES MuUer, H. J., "A Semi-automatic Breeding System ('Maxy') for Finding Sex-linked Mutations at Specific 'Visible' Loci," Dro- sophila Info. Serv., 28:140-141, 1954. Muller, H. J., "The Nature of the Genetic Effects Produced by Radiation," in Radiation Biol- ogy, Hollaender, A. (Ed.), New York, McGraw-Hill, 1 : Chap. 7:351-473, 1954. Schalet, A., "A Study of Spontane- ous Visible Mutations in Dro- sophila Melanogaster," Proc. X Intern. Congr., Genetics, Mon- treal, 2:252 (Abstr.), 1958. See Supplement III. H. J. Muller, at Cold Spring Harbor, 1941. QUESTIONS FOR DISCUSSION 24.1. Are all of the mutants detected by the Base or Maxy techniques point mutants? Explain. 24.2. Suppose, in the Base technique, an F2 culture produced both expected types of daughters, but no sons at all. To what would you attribute this result? 24.3. How might you proceed to test whether a recessive lethal detected in the F^ of the Base technique was associated with an inversion or a reciprocal translocation? 24.4. A wild-type female produces 110 daughters but only 51 sons. How could you test whether this result was due to the presence, in heterozygous condition, of a recessive X-linked lethal? 24.5. How can you explain the phenotype of a rare female in the Maxy stock that pro- duces only the usual Maxy type of progeny, but which has both compound eyes distinctly lighter than normal? 24.6. Can the Maxy stock be used to detect the occurrence of visible mutants which arise in the female germ line? Explain. Can the Maxy stock be used to detect the frequency of recessive lethal mutations that arise in the male germ line? Explain. 24.7. Compare the relative suitability of man and Drosophila for the determination of mutation rates. 24.8. The genes in the X chromosomes are incompletely linked in the females of the Base stock, but completely linked in the females of the Maxy stock. Do you agree with this statement? Why? 24.9. Do you suppose that the general conclusions regarding the phenotypic effects of mutants in Drosophila also apply in the case of man? Why? 24.10. Reread the main part of this Chapter and write a Summary and Conclusions section of less than 200 words without consulting the one given. Compare the two sections critically and objectively. 24.11. What is the relation between the gene and the phenotypic effect by which it is de- tected? Chapter *25 THE GENETIC CONTROL OF MUTABILITY li N SEVERAL Chapters preceding this, we have become famiHar .with the characteristics of differ- ent units of mutation, ranging from the larg- est changes, genomic changes, to the smallest, gene changes. We have seen, moreover, that various externally applied environmental agents can produce mutations of all kinds. What is the nature of the role that the geno- type plays in mutability? A few moments of reflection will lead you to realize that in several respects the very nature of the geno- type influences its mutability. The fact that mitosis and meiosis occur in the precise way they do is evidence that the genotype normally prevents genomic and single whole chromosome changes in succes- sive generations of cells and individuals, re- spectively. There can be no doubt that these processes are under genie control — even if we cannot specify in each case, or in any particular case, the specific genes responsible for these orderly mechanisms for mutation- prevention. The synthesis of new genes must usually be so orderly as to prevent improper gene components from substituting for the proper ones, on the presumption that both types are present in the cell at the same time. The process of synapsis between homologous loci is adequately specific so that a chiasma typically occurs at precisely corresponding points of the two nonsister strands, and crossovers resulting in deficiencies and dupli- cations are avoided. (We have already men- tioned some evidence for the genetic control 213 of synapsis in the existence of coUochores, in Chapter 21, and in the case of asynaptic maize in Chapter 23.) It might be argued, however, that such ex- amples only serve to demonstrate that the processes mentioned reduce mutability as an inevitable consequence of their normal opera- tion. While it would be true in these cases that the present genotype appears to play a passive role, it must be admitted, since mitosis and meiosis are not intrinsic properties of genes, that during the course of evolution, the selection of genes to carry out these ac- tivities was an active process aimed at re- ducing mutability, or, in other words, aimed at maintaining genetic stability, while per- mitting replication and genetic recombina- tion via sexuality. While the genetic controls so far mentioned lead to a reduction in mutability, it should be recognized that the genotype also permits genetic changes to occur in controlled or regu- lated ways. This is demonstrated by the very process of sexuality, whose ploidy changes, from diploid to haploid and back again, must be under genetic control. Since mutational changes increase with mitotic activity (Chapter 23), and because mitosis and mitotic rate are known to be under genetic control (many cancer cells are mu- tants whose mitotic rate is increased), the genotype controls mutability in this way. We have already mentioned (Chapter 18) certain modifications of meiosis, doubtless also under genie control, which lead to ploidy changes in the next generation. Even within the somatic tissues of a multicellular organism, controlled genetic change is per- mitted in cells whose chromosomes become polyploid (as in human liver) or polytene (as in the Dipteran salivary gland). We have also already noted (Chapter 23) in Ascaris that changes from bipolarity to unipolarity occur in somatic tissues, as a result of which a number of small chromosomes are formed from a single large one. (The genotype sup- 214 CHAPTER 25 presses mutation in a polycentric chromo- some by suppressing the action of all but one centromere.) The frequency of nondisjunc- tion, which leads to aneusomy, has been shown to be dependent upon both the amount and distribution of heterochromatin, and also upon the types of chromosomal rearrangements present. So, insofar as the genotype regulates its heterochromatin and rearrangements, it is also regulating the in- cidence of nondisjunction. Similarly, the arrangement of meiotic nuclei in the oogenesis of Drosophila (Chapter 19) eliminates di- centrics produced by crossing over in para- centric inversion heterozygotes. Finally, the arrangement of the chromosomal material and the metabolic activity of the cell (amount of water and oxygen present, for example) are other ways in which mutability is regu- lated by the genotype itself. The preceding discussion has dealt largely with the prevention or regulated occurrence of intergenic changes. Is there other, specific evidence that the genotype regulates the oc- currence of point mutation? Compare the spontaneous point mutation rates of two lines of the same species of Drosophila, one living in a tropical and the other in a temper- ate climate. If the genotype was at the mercy of temperature, in nature, the tropical form would be expected to have a higher rate of spontaneous point mutation than the tem- perate form. However, when both lines are grown in the laboratory at the same tempera- ture, the tropical form has a lower mutation rate than the temperate one. This is good evidence that the tropical form has genetically suppressed (or the temperate form has geneti- cally enhanced) its mutational response to temperature. Accordingly, the two forms, in nature, probably show less difference in mu- tation rate than expected from the tempera- ture difference. Other strains of Drosophila melanogaster collected from different regions have different spontaneous point mutation rates. Some of this difference may be due to differences in mutability of the isoalleles (cf. p. 65) that are present and which result in the same wild-type appearance in each case; but part may be due also to a general control of mutability by the genotype. For some strains are known to contain mutator genes in whose presence the general point muta- tion rate is increased as much as tenfold. Of course, other alleles of mutator genes may therefore act as general suppressors of point mutability. Certain organisms (bacteria, for example) have mutants which make the indi- vidual generally less mutable when exposed to a given mutagen. Note also that the or- ganisms most advanced in evolution contain more chromosomal material per cell than do less advanced forms, and they have probably selected genotypes that reduce their sponta- neous mutation rate to avoid overmutation. Consider next certain results in maize. ^ The kernels of some plants are white, others are colored, while still others are white with colored speckles. At first, it appears as though we are dealing with a high rate of mutation of the "colorless" gene. It was found, however, that the white phenotype is the result of the presence of two genes adja- cent to or very near each other on the same chromosome. If these two loci become dis- sociated from each other, as when a particu- lar one is removed via a two-break deletion, for example, the mutant cell and all its daughter cells containing the remaining locus are colored. The locus removed is called Dissociation, Ds; this locus can be the cause of breakage in chromosome regions near it, and was shown to be a heterochromatic por- tion of the chromosome. If Ds is never dis- sociated from the adjacent locus, the kernel is white ; if it is dissociated during kernel forma- tion, the kernel has colored sectors or dots on a white background; if Ds is moved before the kernel forms, the kernel and later genera- tions of plants are completely colored. The mutation described here is the loss or removal ^ Based upon work of B. McClintock. The Genetic Control of Mutability 215 of Ds via breakage. This is not synonymous with the change in color, though the change in color is the phenotypic event which led to the detection and proof of the mutational event. The change in color is apparently a position effect (Chapter 22); the presence of Ds, next to the gene for color, suppresses color formation; its absence permits the gene for color to produce color. It was possible to prove that the very ca- pacity of Ds to cause breakages nearby is under genie control. These latter genes, since they activate Ds to break the chromo- some, thereby leading to the removal of Ds from its original position, are called Acti- vator, Ac, genes. Ac does not have to be on the same chromosome as Ds, and usually is not. FIGURE 25-1. TIxe effect o/ Activator on tlie action o/ Dissociation. (A) No Ac is present. T/ie /), the colored being about half lights (genetically like the light parent) and half mediums (like the light parent but lacking the transposed Mp) and a few reds (cases where Mp was transposed from P"" Mp, leaving P'' alone). How can the mechanism for transposing Mp FIGURE 25-5. Twin patch of mutant kernels, full red and light variegated, in a medium variegated pericarp ear. [Courtesy of R. A, Brink; photograph by The Calvin Com- pany reprinted by permission of McGraw- Hill Book, Co., Inc., from Study Guide and Workbook for Genetics by I. H. Hers- kowitz. Copyright, 1960.) away from P' Mp be visualized? The same kind of process that transposes Ds can be considered to apply here, also. This can be illustrated with Figure 25-7, where the medium parent cell has chromosomes (P'" Mp and P"") which are shown already divided, daughter strands still being con- nected at the centromere. A normal divis- ion would produce two daughter cells each carrying P^ Mp/P"", and each would give rise to medium sectors. But when mu- tation occurs, presumably involving two or more breaks, the Mp in one daughter strand may be transposed into a nonhomologous chromosome (hollow bar), and it is possible that the daughter cell which receives the transposed Mp will be the one carrying /''■ Mp, in which case the other daughter cell will carry only /"". Subsequent normal mitosis, of the cell containing P'" alone, will produce reds, and of the sister cell, lights, these cells becoming adjacent mutant patches in a medium background (see again Figure 25-5). Red X nonred {P'/P^ X P"'/P"') should produce about half nonred and about half red. We have already mentioned that it does. Reds do not have Mp adjacent to P^ Light X nonred {P" Mp/P"" plus transposed Mp X P"" P"") should produce half nonred. It does. If, in the last cross, transposed Mp is located in a nonhomologous chromosome, one quarter of Fi will be light and one quarter will be medium. But Mp can move from P'" Mp, yet still remain in the same chro- mosome but at a new position. Lights may therefore have their transposed Mp on chro- mosome 1. In this case, backcrossing will The Genetic Control of Mutability 219 produce more than one quarter of Fi that are lights and correspondingly fewer that are medium. Other properties of Mp have been dis- covered. Mp may become fixed at the P locus, so that a medium becomes a stable nonred form. Transposed Mp may occupy a variety of sites, two of which have already been mentioned (linked, and no longer hnked, to chromosome 1). In 57 of 87 cases, transposed Mp was still linked to chromo- some 1, having moved less than 50 crossover units from P. In the remaining cases Mp was transposed to any one of five different nonhomologous chromosomes. Of the 57 cases where transposed Mp was still linked to chromosome 1, 37 cases showed Mp within five crossover units of P, 10 were between /, PHENOTYPE GENOTYPE Medium Variegated r w P Mp / P Mutants Red r w P /P Light ^ r w Variegated P Mp / P + Transposed Mp / - FIGURE 25-6. New genetic liypothesis for pericarp variegation. \ \ 220 CHAPTER 52 FIGURE 25-7. Transposition of Modu- lator and the origin of twin sectors. DIVIDED PARENT DAUGHTER CHROMOSOMES CELLS (Medium Variegated Phenotype) CO-TWIN PHENOTYPES I Red I Light Variegated \ five and 15 units, and the remainder were farther away. Hence, Mp tends to move from the P locus by a short, rather than a long, jump. This suggests that contact be- tween old and new sites may be required for shifts and transpositions of Mp. Sometimes reds revert to variegated. In such cases, a transposed Mp is found to have been transposed near to P\ In fact, the fre- quency of such reversions from red to varie- gated can be studied by introducing into a chromosome containing P% a transposed Mp located various distances from P\ As shown in Figure 25-8, the closer transposed Mp is to P% the greater the frequency of reversions. In summary, then, medium mu- tates to red by loss of Mp from its position near the P"" locus. In this process, comple- mentary lights are produced which possess an extra Mp, transposed Mp. The medium type is reconstituted by the movement of a transposed Mp back near the P"" locus. Two additional points need to be made. The changes in phenotype involving reds, / The Genetic Control of Mutability 221 mediums, lights, and nonreds are not really figure 25-8. Effect of distance of Mp from mutations at the P locus. These changes P i^pon transposition rate of Mp to P. are the phenotypic consequences of muta- tions involving the transposition of Mp, and are in this respect like the changes conse- quent to the transposition of Ds. Transpo- sition of Mp to another locus may change the phenotype produced by the recipient locus. Thus, in a particular medium red carrying an allele on chromosome 9 that re- sulted in the starchy phenotype, a "mutation" to the waxy phenotype was observed. The ^».«~,^ waxy phenotype was unstable and frequently "mutated" back to starchy. Tests showed that Mp had been transposed to the starchy locus which then gave the waxy phenotype, and that the reversions to starchy were the ^__^- a result of Mp\ being transposed away from \ \ \ this locus. All the phenotypic changes con- sequent upon the movement of Ds and Mp '"^^^ strongly resemble position effects. > / >^ , 1. 1 PER CENT RECOMBINATION VARIEGATED SECTORS p _ ^ PER 1000 KERNELS 2.6 15 4.3 11 7.6 8 12.0 3 So far, we have not offered any evidence that the transposition of Mp is under genetic control. If Mp transposes away from P'' Mp 100 times in the absence of a trans- posed Mp, the presence of one transposed Mp reduces this frequency to about 60, while 42.0 0.2 the presence of two transposed M//s further reduces this value to about 5. Thus, the transposition of Mp from P'" Mp is con- trolled by the presence of transposed Mp\ \ Note that while breakability by Ds is regu- ^ lated by a different factor Ac, breakability W» " and its regulation are the consequence of a single kind of factor in the case of Mp. Factors like Ds and Mp are known to be 222 CHAPTER 25 fairly common in maize, and the phenotypic instability of various loci in other flowering plants, ferns, fungi, and bacteria may be due to similar factors. A genetic control of spontaneous "breakability" of chromosomes that resembles the Ac-Ds system has been found also in Drosophila melanogaster. We do not know to what extent transmissible changes are due, not to point mutations of the "mutable" and "less mutable" loci, whose phenotypic eff"ects we are following, but to the removal of some neighboring gene whose presence can cause a position ef- fect. However, not all point mutations can be such position eff'ects, of course, since dif- ferent genes must first arise by mutation in order to obtain position effects with them. Royal Alexander Brink in 1961. SUMMARY AND CONCLUSIONS The spontaneous occurrence of genomic and of single whole chromosome mutations is sup- pressed by the genotypic control of the processes of mitosis and meiosis. Structural rear- rangements in chromosomes are suppressed by the precision of synapsis. All these controls are possible only because genes are linearly arranged in chromosomes with sealed-off ends, such structural features themselves being primary properties of genes. Genetic change is genotypically regulated in certain cases involving the production of polyploid and polytene chromosomes, and of several monocentric chromosomes from a polycentric chromosome. Point mutation frequencies also are regulated genotypically. This is evidenced by the general control of mutation response to temperature changes or to mutagenic agents, by the occurrence of mutator genes, and of genes which produce breakages in chromosomes that lead to losses, shifts, and transpositions which may cause position effects, and by the occurrence of genes which regulate the operation of the genes causing the breakages. REFERENCES Brink, R. A., "Very Light Variegated Pericarp in Maize," Genetics, 39:724-740, 1954. McClintock, B., "The Origin and Behavior of Mutable Loci in Maize," Proc. Nat. Acad. Sci., U.S., 36:344-355, 1950. Reprinted in Classic Papers in Genetics, Peters, J. A. (Ed.), Englewood Cliffs, N.J., Prentice-Hall, 1959, pp. 199-209. McClintock, B., "Controlling Elements and the Gene," Cold Spr. Harb. Symp. Quant. Biol., 21:197-216, 1956. Peterson, P. A., "The Pale Green Mutable System in Maize," Genetics, 45:115-133, 1960. The Genetic Control of Mutability 223 Sandler, L., Hiraizumi, Y., and Sandler, I., "Meiotic Drive in Natural Populations of Dro- sophila Melanogaster. I. The Cytogenetic Basis of Segregation-Distortion," Ge- netics, 44:233-250, 1959. van Schaik, N. W., and Brink, R. A., "Transpositions of Modulator, a Modifier of the Variegated Pericarp Allele in Maize," Genetics, 44:725-738, 1959. QUESTIONS FOR DISCUSSION 25.1. How is the precision of the mitotic and meiotic processes related to the mutability of the genetic material? 25.2. Defend the statement that meiosis and mitosis are not intrinsic properties of genes. 25.3. If whole genome changes represent a class of mutation, should the changes in ploidy which occur in gametogenesis and fertilization be considered mutations? Why? 25.4. Does Dissociation itself provide evidence for the genetic control of mutability? Ex- plain. 25.5. What evidence can you present that variegation is not due to the effect of a single pair of genes, one of whose alleles is unstable? 25.6. Could you detect the transposition of a transposed Modulator to a locus near f"? Explain. 25.7. Why does a light variegated individual of maize, that has a transposed Modulator on the same chromosome as P"^ Mp, produce more than one quarter of F, offspring that are lights and less than this that are mediums, when this individual is backcrossed to nonred (/»" P")? 25.8. Could Modulatorlike genes be the cause of relatively rare "mutants" of amorphic, hypomorphic, or neomorphic types? What is the basts for your opinion? Chapter 26 THE GENE POOL IN CROSS-FERTILIZING POPULATIONS t: |he study of cross-fertilizing in- dividuals has led to our under- standing of the recombina- tional and mutational properties of genes. We have studied the nature of these genetic units and their phenotypic consequences in terms of the traits produced within individuals and their relatives. However, cross-fertiliz- ing individuals exist not only as individuals or as parts of families, but also as members of a general population. In such a general population each individual usually has an op- portunity to mate with numerous other indi- viduals. The gametes of all individuals mat- ing furnish a pool of genes, or gene pool, from which the genes of the next generation will be drawn. What we are concerned with, on this occasion, is the fate of a particular gene in the gene pool of successive genera- tions. Let us construct a gene pool and see what will happen to a gene in it in successive generations. Suppose, in the not too distant future, it is decided to colonize Mars with human beings from the Earth. Suppose that the popula- tion arriving there is sufficiently large, and that with respect to eye color genes, only the B (brown) allele and the completely reces- sive b (blue) allele are present, in such fre- quencies that the gene pool from the fathers and mothers is comprised of gametes that are 2B and M. On the presumption that the marriages which occur will be uninfluenced by eye color phenotype, what will be the genotypes of the Fi generation? What will be their phenotypes? Of what will the Fi gene pool consist? The answer to the first question may be obtained from Figure 26-1. As the result of the random union of gametes, .04, or 4%, of children will be SB, .32, or 32%, will be Bb, and .64, or 64%, bb. Phenotypically, the Fi population will be composed of 36% brown- and 64% blue-eyed people. (Since this eye color gene is located autosomally, the same proportions occur among boys and girls.) When, barring mutation, the Fi marry, what FEMALE GAMETES .2 B .8 b .04 BB .16 Bb .2 B Brown Brown MALE Eyes Eyes GAMETES .16 Bb .64 bb .8 b Brown Blue Eyes Eyes The F, Population .04 Brown (BB) + .32 Brown (Bb) + .64 Blue (bb) FIGURE 26-1. fi genotypes and tlie gene pool these produce. 224 The F Gene Pool .04 .16 .16 .64 The Gene Pool in Cross-Fertilizing Populations 225 will the gene pool be in their gametes? The 4% of BB individuals will furnish 4% of all gametes and these will be 5, and the 32% of Bb individuals will furnish 32% of all gametes of which half, or 16%,, will be B and half, or 16%, b. So, in the total gene pool there will be 20%, or .2, gametes with B. The b gametes comprise 80%, or .8, of the gene pool (16^o from the 32% Bb het- erozygotes and 64% from the 64% of bb individuals). Note that the gene pool of the Fi is identical to that of the Pi. It follows that in the F2 and all subsequent generations the same genotypic and phenotypic ratios will be found, because the frequencies of B and b in the gene pool will remain constant in every subsequent generation. What would be the consequence if, instead of starting the Martian colony with 80% b and 20% B, some other proportion had ob- tained? We can generalize the analysis by letting p equal the fraction of male and of female gametes in the population which car- ries B, and q equal the fraction of male and of female gametes which carries b. Natu- rally, for eggs, p + q = 1, as is true also for sperm. These sex cells will combine at ran- dom to produce the result shown in Figure 26-2. The resultant offspring population will be, then, p'^ BB + 2pq Bb + q- bb. The fraction of individuals who are brown-eyed will be p- + 2pq, while q- will be the blue- eyed fraction. The frequency of B and b among the gametes produced by the offspring population will be: 5 = p2 + pq = p(p + q) = p 6 = q- 4- pq = q(q + p) = q. SPERMS Thus the gene frequencies will remain the same as they were in the gametes of the pre- vious generation, and all future generations will have the same gene pool and the same relative frequencies of genotypes. The formula p- BB + 2pq Bb + q- bb describes the genotypic equilibrium produced by a static gene pool.^ It should be noted that this equilibrium principle is not dependent upon either the presence or the absence of dominance. Moreover, the B and b in the formula can represent any two alleles whose frequency in the gene pool is known, even if the sum of their frequencies is less than 1. If this equilibrium principle applied indefi- nitely, gene frequencies would remain un- changed, and the evolution of different genotypes (and accordingly of new pheno- types) would not occur. In the Martian model described, however, certain condi- tions had to be fulfilled in order to maintain genie equilibrium. One condition was met by barring mutation. \{ mutation is permitted to occur, to allelic states other than B and 6, it is clear that the frequency of these two alleles in the population will become reduced, upsetting the equilibrium. Moreover, the frequency of any allele will be changed if ^ This is called the Hardy-Weinbeii^ equilibrium prin- ciple (see references at the end of this Chapter). EGGS p B (Brown) q b (Blue) FIGURE 26-2. The types and fre- quencies of genotypes produced by a gene pool composed of p B and q b. pB (Brown) qb (Blue) p' BB pq Bb (Brown Eyes) (Brown Eyes) pq Bb q^ bb (Brown Eyes) (Blue Eyes) 226 CHAPTER 26 the mutation rates to and from it are different. In either or both events, the genetic equihb- rium will be shifted until a new one is attained. Thereafter, the new equilibrium will be main- tained until some new factor acts on mutation rate in a directional way. We have also presumed in our model that the reproductive potential is the same regard- less of what the genotype is with respect to eye color. But it is possible, under certain con- ditions, that persons with blue (or those with brown) eyes are preferred as mates, in which case the reproductive potential of an indi- vidual would not be independent of the alleles under consideration. Accordingly, if indi- viduals with a certain genetic endowment produce more surviving offspring than do those of a different genetic endowment, the genes which confer this higher biological fit- ness will tend to increase their frequency in the population, while those genes with lower fitness will tend to decrease. In this way, selection, operating on genotypes of different biological fitness, causes changes in gene fre- quencies, and shifts in the genotypic frequen- cies found at equilibrium. We have also presumed that the Martian population was a large one. When popu- lations are very large, oscillations that occur by chance in the number of children pro- duced by different genotypes do not matter for they do not change the gene pool. In small populations, however, such chance oscillations can change gene frequencies. Suppose, for example, the Martian popula- tion whose gene pool is M and 2B runs short of food, so that only one couple, de- termined by chance, can have children. The chance that this husband and wife will be blue-eyed is .64 X .64, or about .41, or 41%, which is the chance that the gene pool will drift at random in this particular manner, to produce the new gene frequencies of 1.0 for b and 0 for B. This random genetic drift can be illustrated also in a less extreme situation. If the Martian population is very large, and a certain family chances to produce a rela- tively large number of children for several generations, then the proportion of all indi- viduals having this family name will still be very small. But if the Martian population decreases while the reproductive rate of this family is unchanged, the proportion of all people with this surname will be increased. Finally, we have not mentioned the possi- bility that the Martian colony will have emi- grants or immigrants. If the emigrants have gene frequencies that differ from those in the population gene pool, the gene frequencies in the remaining population will be changed. If the immigrants have a different gene fre- quency from the natives and interbreed with them, this also will change the gene pool. In this way migration can shift gene equi- librium. We see then that a cross-fertilizing popula- tion will remain static, in genetic equilibrium, in the absence of mutation, selection, ran- dom genetic drift, and differential migration. The occurrence of one, another, or all of these will change the frequencies of genes in the gene pool and thereby shift the frequencies of genotypes at equilibrium. Different species possess different gene pools, and it is natural to presume that they are different species because of their different gene pools. In accordance with this view, since these four factors serve to change gene frequencies, they may be considered to be the principal causes of species formation. Insofar as the forma- tion of higher taxonomic categories is, like speciation, based upon change in gene pools, mutation (which supplies the raw materials), selection (which shapes these raw materials into the biologically fit genotypes of races and species), random genetic drift (which can produce rapid changes in gene frequency in small populations), and differential migra- tion (which can shift gene frequencies via interchange of individuals between popula- tions) are the principal causes of biological evolution. The Gene Pool in Cross-Fertilizing Populations 227 SUMMARY AND CONCLUSIONS The gametes which function to produce the next generation in cross-fertilizing populations comprise a gene pool. If the population (gene pool) size is very large, if mutation does not occur in any direction preferentially, if there is no differential selection for genotypes, and if migrants are genotypically just like the natives, the gene pool in successive generations will remain forever unchanged, as will also the relative frequencies of genotypes. However, if any of these conditions fails to obtain, there will be a shift in the nature of the gene pool, that is, gene frequencies will change, and so will the frequencies of different genotypes, until a new equilibrium is attained. It is suggested that not only species formation, but all of biological evolution is based upon changes in the gene pool. REFERENCES Dobzhansky, Th., Evolution, Genetics, and Man, New York, John Wiley & Sons, 1955. Dobzhansky, Th., Genetics and the Origin of Species, 3rd Ed., New York, Columbia Univer- sity Press, 1951. Hardy, G. H., "Mendelian Proportions in a Mixed Population," Science, 28:49-50, 1908. Reprinted in Classic Papers in Genetics, Peters, J. A. (Ed.), Englewood Cliffs, N.J., Prentice-Hall, 1959, pp. 60 62, and in Great Experiments in Biology, Gabriel, M. L., and S. Fogel (Eds.), Englewood Cliffs, N.J., Prentice-Hall, 1955, pp. 295-297. Li, C. C, Human Genetics, New York, McGraw-Hill, 1961. Theodosius Dobzhansky in 1957. Sewall Wright is noted for his research in phys- iological genetics and in tlie mathematics of popu- lation genetics. Photograph was taken in 1954. 228 CHAPTER 26 Rasmuson, M., Genetics on the Population Level, Stockholm, Sweden, Svenska Bokforlaget Bonniers; London, Heinemann, 1961. Weinberg, W., "Uber den Nachweiss des Vererbung beim Menschen," Jahresh. Verein f. vaterl. Naturk. in Wurttemberg, 64:368-382, 1908. Translated, in part, in Stern, C, "The Hardy-Weinberg Law," Science, 97:137-138, 1943. QUESTIONS FOR DISCUSSION 26.1. Does evolution have the same causes in populations reproducing only asexually, as in those reproducing sexually? Explain. 26.2. Suppose, in a population obeying the Hardy-Weinberg rule, mutation occurred for one generation, thereby changing the composition of the gene pool. How many additional generations would be required before a new genetic equilibrium would be established? Explain. 26.3. Discuss the statement: "The Hardy-Weinberg Law is the cornerstone of evolutionary genetics." 26.4. Assuming the Hardy-Weinberg principle obtains, what is the frequency of the gene R, if its only allele R' is homozygous in the following percentage of individuals in the population: 49%? 4%? 25^^70? 36%? 26.5. About 70% of Americans get a bitter taste from the drug phenyl thiocarbamide (PTC), and are called "tasters." The remaining 30% of the people get no bitter taste from PTC, and are "nontasters." All marriages between nontasters produce all nontaster offspring. All results support the view that a single pair of nonsex-linked genes determines the difference between tasters and nontasters, that dominance is complete between the only two kinds of alleles that occur, and that penetrance of the dominant allele is complete. a. Which of the two alleles is the dominant one? b. What proportion of all marriages between tasters and nontasters have no chance (barring mutation) of producing a nontaster child? c. What proportion of all marriages occurs between nontasters? tasters? 26.6. The proportion of A A individuals in a large crossbreeding population is .09. Assuming all genotypes with respect to this locus have the same reproductive potential, what proportion of the population should be heterozygous for Al 26.7. What do you suppose would happen to a population whose gene pool obeyed the Hardy-Weinberg rule for a very large number of generations? Why? 26.8. Can the same population obey the Hardy-Weinberg rule for one locus and not for another? Explain, Chapter 27 MUTATION AND SELECTION — NONRANDOM MATING AND HETEROSIS Ti Ihe composition of the gene pool is dependent upon several factors, including mutation and selection (Chapter 26). Selection acts at the phenotypic level to conserve in the population those genotypes which provide the greatest reproductive potential. Selection takes place at all stages in the life cycle of an individual. If a particular stage has been produced under the action of a haploid genotype, selection will favor the most adaptive phenotypes and thereby conserve the most adaptive hap- loid constitutions. If other stages in the life history involve the action of a diploid con- stitution, selection will favor the most adap- tive phenotypes and therefore the diploid genotypes they contain. Several implications of these statements need to be specified, namely, that selection acts not upon single genes, but upon genotypes, sometimes acting upon the phenotypes produced by single genomes, and at other times, in sexually re- producing organisms, acting upon the com- bined phenotypic effect of two genomes. It should be noted also that what is a relatively adaptive genotype at one stage of the life cycle may be relatively ill-adaptive at another stage, regardless of whether or not these stages have the same or different ploidies. It is, of course, the total adaptiveness of all these separate features which determines the over- all reproductive potential of an individual. Finally, it should be noted that in cross- fertilizing populations selection favors geno- 229 types which produce maximal fitness of the population as a whole. Because selection acts this way, it may be that some portion of the population may receive genotypes which are decidedly disadvantageous to the indi- viduals receiving them. If this is so, the same genetic components would be expected to be advantageous when present in other, more probable combinations. Since the human being is primarily of one ploidy, diploidy, it is upon his diploid- produced phenotype that selection principally operates. If we ask, "What is the fate of mutants in the gene pool?" our answer must include knowledge of the frequency with which the mutants arise and of their effects upon reproductive potential in a diploid genotype. The phenotypic effect of a mu- tant gene will depend, not only upon the nature of its allele, but also upon its relation- ship with the nonalleles in the genotype (Chapter 7). Let us consider, in human beings, the fate in the gene pool of mutants whose over-all phenotypic effect is dominant lethal, or dominant detrimental, or recessive lethal, or recessive detrimental, insofar as selection and mutation influence their fates. Dominant lethal mutations are lethal when heterozygous, and are eliminated from the gene pool in the same generation in which they arise. Accordingly, the biological fit- ness, adaptive value, or reproductive potential of such mutants is zero. If the normal homozygote {AiAx) is considered to have a selective disadvantage of 0, then the domi- nant lethal has complete selective disadvan- tage, its selection coefficient, s, being there- fore 1 . You can readily see that the mutation frequency or rate, u, to this dominant lethal condition must be equal to one half the fre- quency of affected individuals (AiAo), since each affected individual has one mutant and one normal gene. In the absence of special medical treatment, retinoblastoma, a kind of cancer of the eye, is an example of such a dominant lethal in man. 230 CHAPTER 27 Achondroplastic {chondroclyslrophic) dwarf- ism is characterized by normal head and trunk size, but shortened arms and legs, making for disproportionate dwarfism. This rare, fully penetrant (see p. 71) disease is due to the presence of a gene in heterozygous condition, which acts, therefore, as a domi- nant detrimental mutant. Such dwarfs {A\A-2) produce only 20 per cent as many children as are produced by normal people. This lower reproductive potential is expressed by assigning the AiA-i genotype a selection coefficient, s, of .8. The frequency of A^A-i in the population was found to be 10 dwarf babies in 94,075 births. This frequency must be due to the occurrence of dwarf children from normal parents, who carry new mutations to At, in addition to dwarf children, one of whose parents was dwarf. The gene frequency, p, o{ A-2 in the population must be 10 2(94,075), or .000053. From the incidence of dwarfs who were known to have normal parents, the GENOTYPE PHENOTYPE FREQUENCY AT EQUILIBRIUM A,Ai A, a, ajOj Normal Normal Dies P^ 2pq q2 u= Mutation rate from A, *" Oj q ="\l u/s Here s = 1, hence q = ^1] u= 10'^ = 0.000,01 Hence q ='^X .000,01 mutation rate, u, to A^ was determined to be .000042. If the value s = .8 is correct, p = u/s, or .000042 .8, or .0000525, which is in excellent agreement with the value of p observed. Note that for a dominant lethal p = u because s = 1. In the present case s is less than 1, so that p is larger than u. The fact that for dwarfism p is not very much larger than u illustrates the efficiency of natural selection in eliminating such mutants from the gene pool. The gene for juvenile amaurotic idiocy (a-i) has no apparent effect when heterozygous (Aiao), but causes homozygotes to die as children. It is, therefore, a recessive lethal mutant. Affected individuals (a-ya-i) are found with a frequency of 1/100,000, or .00001. What is the frequency of a-^ in the gene pool? As shown in Figure 27-1, the frequency at equilibrium of aofifi individuals is equal to q^. Accordingly, the frequency of 02 (q) must be equal to Vq^, or V.OOOOT, or about .003, while the frequency of ^1 must be 1 — .003, or .997. Note that the heterozygous car- riers are 600 times more frequent than af- flicted individuals. What is the mutation rate from Ai to (22? We have presumed that the gene pool is at equilibrium, that is, the rate at which ^2 enters the population by mu- tation must be equal to the rate at which it leaves the population in a2a2 homozygotes. Accordingly, u to ^2 must be .00001. The selection coefficient for normal individuals {AiAi and Aia2) is 0 and for fl2«2 is 1. We see, therefore, that at equilibrium the fre- quency in the gene pool of a recessive mutant 0.003 ACTUAL (P^) (2pq) (q') FREQUENCY AT cr>i III iRPil IM (0.997)' 2(0.997) (0.003) (.003)' FIGURE 27-1, Juvenile amaurotic idiocy. tVjUILlDKIUfVi 0.994 0.006 0.000,01 {See text for explanation.) Mutation and Selection — Mating and Heterosis 231 can be expressed as q = Vu/s, where s = 1 in the case of a recessive lethal. If the recessive mutant is detrimental without being lethal, s becomes less than 1 (but more than 0) and the frequency of the mutant in the gene pool becomes higher for the same muta- tion rate. Thus, if in the present example, s is Yi instead of 1 , q is twice as large. In deriving the types and frequencies of genotypes in a population at equilibrium, it was presumed that marriages were at random with respect to the genotypically determined trait under consideration. What happens if the different genotypes do not marry at random? Consider the disease phenylketo- nuria (Figure 27-2) which involves a type of feeblemindedness in individuals, homozygous for a recessive gene, who cannot properly metabolize the amino acid phenylalanine. The frequency in the gene pool of the normal gene (A) is .99, and of the abnormal gene (a) is .01. In the population at equilibrium, therefore, AA : Aa : aa individuals will have frequencies of 9801 10,000 : 198 10,000 : 1/10,000, respectively. Notice that Aa individuals are 198 times more frequent than aa, so that even if every aa did not reproduce, only one per cent of the a genes present in the gene pool would be eliminated each generation. This illustrates the inefficiency of selection against homozygotes for rare recessive genes, insofar as lowering the frequency of such genes is concerned. AA and Aa individuals apparently marry each other at random. FIGURE 27-2. Pedigree showing the occurrence oj phenylketonuria among the offspring oJ cousin marriages {denoted by thick marriage lines) However, it is also true that feebleminded people do not marry in the population at random. But this has little effect on the dis- tribution of genotypes in successive genera- tions, since aa people have so few of all the a genes present in the population. You can see that it is only marriages between Aa indi- viduals that are of consequence, since those are the major source of aa offspring. The example of phenylketonuria shows that, when a gene is rare and apparently completely recessive, nonrandom marriage has little influence upon its frequency, or the diploid genotypes in which it is found in the population. When the mutant is relatively frequent in the population, however, it is obvious that nonrandom marriages will raise the frequencies of certain genotypes and lower others. Moreover, if there are adap- tive differences for the different genotypes, the composition of the gene pool may be changed in a different direction, or at a dif- ferent rate, from what would be predicted for a population mating at random. Con- sider two ways in which mating may be non- random. The first involves the tendency of phenotypically similar individuals (disre- garding the sex differences) to mate, and is referred to as assortive mating. This kind of Od io DO-r-D £^K) cWSa iiiid^i 232 CHAPTER 27 breeding pattern is generally true in animals and in human beings, also. The genetic re- sult is the production of more homozygotes than would occur by chance matings. The second departure from random mating involves inbreeding, the tendency for mates to be more closely related in descent than they would be, were selection of mates made at random in the population. What is the effect of inbreeding when carried out for a single generation? We can estimate this by study- ing what happens to the genes that are hetero- zygous in the parent generation. There are various degrees of inbreeding, the closest form being self-fertilization, which occurs in many plants. In self-fertilization, the het- erozygote for a given pair of genes, Aa, pro- duces progeny one half of which are homozy- gotes, so that heterozygosity is reduced by one half with respect to this pair of heterozy- gous genes. The decrease in heterozygosity due to self-fertilization can be expressed in general, as follows: the chance that an off- spring will receive a particular allele in the male gamete is }2 and the chance it will re- ceive the same allele in the female gamete is y.. The chance the offspring will be a homo- zygote for that allele, therefore, is %. But there is an equal chance the offspring will become homozygous for the other allele, so that there is a total chance of 50% for homo- zygosis attributed to this type of inbreeding. If all members of the population self-fertilize, then in each successive generation, half of the genes that were heterozygous become homozygous. Suppose, in a portion of a population that mates at random, X% of the progeny are homozygotes. These come from matings between two heterozygotes, between two homozygotes for the same or for different alleles, and between a homozygote and a heterozygote. If the gene pool is at equilib- rium, the matings that tend to increase homozygosis are counterbalanced by others which decrease it, so that X% homozygosis remains constant generation after generation. Consider what happens in another portion of this population which happens to practice self-fertilization for one generation. Insofar as these self-fertilizing individuals are con- cerned, since they themselves already show X% homozygosis, their offspring will also have X% for this reason. But, if they show Z% heterozygosis, their offspring will have only }2Z% heterozygosis, and will show a total homozygosis of X + %Z%. In other words, each generation of self-fertilization makes half of all heterozygous genes homozy- gous, and the effect of self-fertilization, in a normally random mating population, is to increase the random mating rate of homozy- gosis by ^2 of the rate of heterozygosis. By how much is homozygosity increased in brother-sister (sib) matingsl The chance that a particular gene in the male sib's father is present in the male sib is )^, and the chance that the male sib's child will receive this is Yi. The chance for the occurrence of both events is Vi. The chance that the female sib re- ceives and transmits this same gene to her child is also M. The chance that the child of the sib mating will receive two of this same allele is M X M, or Ke, which is its chance of being homozygous for this gene. Since the child would have an equal chance to become a homozygote for the other allele in his grand- father and for each of the two alleles in his grandmother, this gives him 4 X Ke, or a 25% chance of homozygosis. In other words, sib matings will cause K of heterozygous genes to become homozygous. As discussed above, this chance of homozygosis is addi- tional to the chance of homozygosis which obtains for genes in the randomly mating portion of the population and which also obtains for sib matings. Matings between individuals who have one parent in common are called half-sib matings. In this case, the frequency that a given allele in the common parent will pass to the male half-sib is K, and the frequency Mutation and Selection — Mating and Heterosis 233 an offspring of this sib will receive this allele is Yi. The frequency of both events occurring is, therefore, %. The frequency is also Y^ for these events to occur through the female half- sib, so that the frequency of a given allele becoming homozygous from a half-sib mat- mg IS K X Ya, or Ke- Since the other allele in the common parent could, in this way, also become homozygous Ke of the time, the combined chance of homozygosity for half- sib matings is )i. So, Y?, of heterozygous genes become homozygous due to this type of inbreeding. The amount by which heterozygosity is re- duced because of inbreeding is called the inbreeding coefficient, F. In the case of cousin marriage, F is Yk- The values of F for more complicated pedigrees can also be worked out. We have seen that all forms of inbreeding increase homozygosity. Let us now calculate the consequence of cousin marriage upon the frequency of phenylketonuria. The fre- quency of heterozygotes per 10,000 people is 198 (see p. 231). Cousin marriage would re- duce heterozygosity by Y\6, or 12 individuals, of which six would be normal {AA) and six affected {aa). Since random mating would produce one affected individual per 10,000, cousin marriages would bring the total num- ber of affected homozygotes in this popula- tion to seven (six from inbreeding, plus one from random breeding). Accordingly, there is a sevenfold greater chance for phenylke- tonuric children from cousin marriages as from marriages between unrelated parents. An additional example can be given, of the increased risk of defect as a consequence of cousin marriages. In a Japanese population (Figure 27-3), congenital malformations, stillbirths, and infant deaths are 24 48 per cent higher in cousin marriages than when parents are unrelated. Since defects such as these are known to be due, in some cases, to recessive genes in homozygous condition, these data support the view that homozygosis resulting from inbreeding can produce detrimental results. The fact that inbreeding produces homozygosis, and that homozygosis can lead to the appearance of defects, does not automatically mean that inbreeding is disadvantageous under all cir- cumstances. For, while many individuals become homozygous for detrimental genes as a result of inbreeding, just as many be- come homozygous for the normal alleles. The success of self-fertilizing species is testi- mony to the advantage of homozygosity at least in these species. In populations that normally cross-ferti- lize, however, inbreeding usually results in a loss of vigor. This loss of vigor is directly connected to homozygosis. In what way is it theoretically possible to explain the adap- tive superiority of heterozygotes, which is usually known as heterosis or hybrid vigorl FIGURE 27-3. Increased risk of genetic defect witli cousin marriages. {Data from Hiroshima and Nagasaki.) Frequency from Increase in Frequency Per cent Unrelated Parents with Cousin Marriage Increase CONGENITAL MALFORMATION .011 .005 48 STILLBIRTHS .025 .006 24 INFANT DEATHS .023 .008 34 234 CHAPTER 27 Consider the three genotypic alternatives, AA, AA', A' A' relative to their phenotypic effects. If A is completely dominant to A' , and A' A' is less vigorous than AA and AA' , homozygosis resultant from breeding of AA' will clearly lead to detriment. This remains true even if A is incompletely dominant to A' or shows no dominance to it. In all these cases, the heterozygote is superior to one of the homozygotes. The second possibility remains, however, that the heterozygote, AA' , may be of greater adaptive value than either homozygote. Suppose, to illustrate this, that A is pleiotropic and has a relatively very adaptive effect with respect to trait M, but a relatively less adaptive effect with respect to trait N, while its mutant allele has the re- verse effect, namely, being relatively less adaptive for M, and relatively more adaptive for N. The heterozygote would, in the event of no dominance, be superior to either homo- zygote. Heterosis can be produced, there- fore, if the heterozygote is superior to either one homozygote or both homozygotes. The first type of heterotic effect can be demonstrated by crossing two pure lines homozygous for different detrimental reces- sives {A A bb CC dd EE X aa bb CC DD EE). The Fi {Aa bb CC Dd EE) will be uniform yet more vigorous (having four different normal alleles) than either parent (each of which had only three different normal alleles) because the dominant alleles hide the detrimental effects of the recessive alleles. The second type of heterotic effect can be illustrated in human beings. As mentioned in Chapter 10, homozygotes for the gene for sickle cell anemia {A' A') usually die from anemia before adolescence. AA individuals are of normal blood type, while AA' indi- viduals are either normal or have a slight anemia. In certain countries, the frequency of A' in the gene pool follows expectation for a recessive lethal gene. In other coun- tries, however, A' is more frequent than would be expected. How is this difference explained? It turns out that the heterozygote AA' is more resistant than the AA homozy- gote to certain kinds of malaria. Of course, in nonmalarial countries, A' confers no antimalarial advantage and so the fitness of the heterozygote (1 — s) is lower than that of the normal homozygote (1), while the A' A' individual has a fitness of 0. As expected, therefore, sickle cell anemia is rare or absent in most of the world where certain forms of malaria are absent. On the other hand, in certain malarial countries, even though heterozygotes may be slightly anemic, the advantage of being resistant to malaria produces a greater over- all fitness than does the AA genotype. Here the fitness of the heterozygote, AA' , is 1 and that of the normal homozygote, A A, is 1 — Si. Mutant homozygotes. A' A' , have a fitness of 1 — So, where S2 equals 1, since all A' A' die (even should A' A' be extremely resistant to malaria). What natural selection does in this case is to maintain both A and A' in the gene pool. A' having a frequency equal to — ^ — This fraction can be read "the ad- Si + Si vantage of A' (as shown by the advantage of AA' over AA) divided by the total disadvan- tage of A and A'."' You see, then, that when the heterozygote is more adaptive than either homozygote, showing heterosis in this way, natural selection will maintain a gene in the gene pool even though it is lethal when homozygous. It should be noted that while we have dis- cussed heterosis in terms of the phenotypic effects of the members of a pair of alleles, this does not mean that the unit of heterotic action is always a single pair of genes. Since we have already seen, in Chapters 7 and 8, that different pairs of genes interact in vari- ous ways in producing phenotypes, it would be no surprise to find that heterosis occurs as the result of the effect of certain combina- tions of nonalleles and alleles. It has been found, in D. pseudoohscura. 235 FIGURE 27-4. The variability of nurnial corn is being pointed out by Jamls h. Crow {Photographed in 1959 by Tlie Calvin Company.) that natural populations contain certain paracentric inversions. When laboratory populations are initiated, containing some individuals with the normal chromosome ar- rangement and others with a particular one of these inversions, in some cases, the popu- lation comes to contain only normal chromo- somes after a number of generations has passed. In such cases, the inversion chromo- some behaves like a detrimental gene which provides no advantage when heterozygous, and is ehminated from the gene pool. When other particular inversions are tested this way, however, an equilibrium is reached in which the normal and inverted chromosomes are both retained in the gene pool. In these cases, the inversion heterozygote is adaptively superior to either homozygote, showing heterosis, just as does the gene for sickling in malarial countries. It is not easy to de- cide the genetic basis for heterosis in such cases, however, since hybrid vigor could be due to the genes gained or lost at the time the inversion was initially produced, or it could be due to position effect, or to individual genes or groups of genes contained within the inverted region. (It should be recalled that individuals with paracentric inversions are not at a reproductive disadvantage in Drosophila. Should a heterotic system exist or develop in paracentric inversion heterozy- gotes, based upon the action of several genes contained within the inverted region, this heterotic arrangement would tend to remain intact in the heterozygote, because of the failure of single crossovers within the in- verted region to enter the haploid egg nucleus.) Finally, it would be appropriate to de- scribe briefly how hybrid vigor has been 236 CHAPTER 27 applied to agriculture, since it has been esti- mated that the use of hybrid corn alone has already enriched society by a billion dollars. You might ask: What is wrong with normal corn? The answer is that it is too variable in quality and vigor (Figure 27-4). The variability can be decreased by inbreeding, but unfortunately this also results in loss of vigor or other desirable traits. The way to overcome this impasse is first to obtain inbred lines, which are uniform because they are homozygous and which carry different desirable dominant genes (yet are also homo- zygous for different undesirable recessive genes). If different inbred lines are crossed, the Fi will be multiply heterozygous, uni- form, and more vigorous than either parental inbred line. Accordingly, hybrids can be made be- tween two selected inbred lines. But while these Fi plants are vigorous and uniform, they come from seeds produced on ears grown on one of the less vigorous inbred lines. For this reason such seeds are not suf- ficiently abundant to make it economical to use them in raising crops of corn. This difficulty is overcome in practice (Figure 27-5) by first making two different single cross hybrids by crossing four selected inbred lines two by two. Then the two hybrids are crossed. Seeds produced by this double cross are plentiful, since they are formed on a vigorous single cross hybrid plant, and can be sold inexpensively to farmers for planting. Breeding procedures resulting in hybrid vigor are applied to many plants and animals; heterosis is of great eco- nomic importance. SUMMARY AND CONCLUSIONS Two of the factors which decide the fate of a mutant in the gene pool are mutation and selection. For equal mutation rates, the lower the reproductive potential the mutant causes, the lower is the frequency of the mutant in the gene pool. Specific examples of rare mutants are described which lower reproductive fitness by being dominant lethal, dominant detri- mental, recessive lethal, and recessive detrimental. Nonrandom breeding, by assertive mating or inbreeding, increases the rate of homozy- gosis. The per-generation rate of reduction in heterozygosity due to inbreeding is Vi for self-fertilization, % for sib matings, % for half-sib matings, and Ke for cousin matings. Homozygosis in normally cross-fertilizing individuals leads to loss of vigor, while heterozy- gosis is accompanied by heterosis, or hybrid vigor. Heterosis is the phenotypic result of gene interaction, and occurs because the heterozygote either serves to mask different detrimental recessives which are homozygous in the parents, or is adaptively superior to both types of homozygote. Heterosis is of great economic im- portance. REFERENCES Allison, A. C, "Sickle Cells and Evolution," Scient. Amer., 195:87-94, 1956. Crow, J. P., Genetics Notes, 4th Ed., Minneapolis, Burgess, 1960. Dobzhansky, Th., Evolution, Genetics and Man, New York, John Wiley & Sons, 1955. Dobzhansky, Th., Genetics and the Origin of Species, 3rd Ed., New York, Columbia Uni- versity Press, 1951. Gowen, J. W. (Ed.), Heterosis, Ames, Iowa, Iowa State College Press, 1952. Mutation and Selection — Mating and Heterosis 237 INBRED A INBRED B INBRED C INBRED D FIGURE 27-5. The production of commercial liyhrid corn by the "double cross''^ breeding procedure. 238 CHAPTER 27 QUESTIONS FOR DISCUSSION 27.1. In each of the following cases, explain whether the rate with which a particular allele mutates is of primary importance in shifting its frequency in the population, when this gene: a. is a dominant lethal in early developmental stages. b. is a recessive lethal. c. expresses itself phenotypically only after the repro- ductive period of the individual. d. is very rare. e. occurs in small cross-fertilizing populations. 27.2. Can the adaptive value of the same gene differ: a. in haploids and diploids? b. in males and females? c. in two diploid cells of the same organism? Explain your decision in each case. 27.3. Other things being equal, what would happen to the frequency in the gene pool, of a dominant mutant whose selection coefficient changed from 1 to '4? What would happen in this respect if the mutant was completely recessive? 27.4. If persons carrying detrimental mutants fail to marry, these particular genes are re- moved from the gene pool. Under what conditions is the failure to marry likely to reduce appreciably the frequency of detrimental mutants in the gene pool? 27.5. Are inbreeding and assertive mating mutually exclusive departures from random mating {panmixis)! Explain. 27.6. Explain why the inbreeding coefficient, F, is Ke for cousin marriages. 27.7. Suppose the frequencies of A and a are .3 and .7, respectively, in a population obeying the Hardy-Weinberg rule and in which mating is at random. a. What per cent of the population is composed of homozygotes with respect to these genes? b. What would be your answer to a. after one generation in which hybrids could mate only with hybrids? c. What effect would the conditions in b. have upon the composition of the gene pool? 27.8. Discuss, from a genetic standpoint, the advantages and disadvantages of cousin marriages in man. 27.9. In Thailand, heterozygotes for a mutant gene that results in the formation of hemo- globin E are more frequent in the population than would be expected if the Hardy- Weinberg equilibrium obtained. How can you explain this? Chapter 28 MUTATIONAL LOADS AND THEIR CONSEQUENCES TO POPULATIONS FIGURE 28-1, Chromosomal complement of D. pseudoobscura. d: ROSOPHILA PSEUDOOBSCURA IS a fruit fly commonly found in northern Mexico and west- ern United States. When collected in the wild almost all individuals are alike pheno- typically, except for the sex differences, ap- pearing "wild-type" or normal. We cannot accept such a phenotypic uniformity as evi- dence for genotypic uniformity, however, since we already know of the possibility that a wild-type population may appear uniform yet contain concealed genetic variability in the form of isoalleles or recessive mutants having a point mutational origin. In fact, we have already indicated the existence in natural populations of Drosophila of isoalleles (Chapter 9), of pericentric inversions and re- ciprocal translocations (Chapter 21), and of paracentric inversions (Chapter 27). What we would like to have is an estimate of the total amount of genetic variability present in a natural population. This can be studied using D. pseudoobscura} D. pseudoobscura has five pairs of chromo- somes, composed of the usual X and Y sex chromosomes, three pairs of large rod- shaped autosomes (II, III, IV), and a dotlike pair of autosomes (V) (Figure 28-1), There are a number of laboratory strains of this species whose chromosomes are marked by various mutants of point and rearrangement type. It is possible, therefore, to perform a series of crosses between laboratory strains ^ Based upon work of Th. Dobzhansky and col- laborators. 239 and flies collected in the wild which yield information on the presence of mutants in the wild-type flies. In practice, autosomes II, III, and IV of wild-type flies were indi- vidually made homozygous to detect the presence of recessive mutants (cf. Figure 9- 4) that are lethal (causing death of all indi- viduals before adulthood), or semilethal (causing more than 90 and less than 100 per cent mortality before adulthood), or subvital (causing significantly less than normal but greater than 10 per cent survival to adult- hood), or sterile to females {female sterile) or sterile to males {male sterile). The results of such a study are summarized in Figure 28-2. About 25% of all auto- somes tested this way carried a recessive lethal or semilethal mutant. Recessive sub- vital mutants were found in about 40% of III chromosomes tested, and in more than 90% of tested II's and IV's, while mutants causing sterility were present in 4-14% of tested chromosomes. The natural popula- tion clearly carries a tremendous load of detrimental mutants. How is this load of mutants distributed in the fly population? Consider first one pair of the autosomes tested. Each member has a 25% chance of carrying a lethal or semilethal, and a 75% chance of being free of such a mutant. The chance that both members of a pair of chromosomes will carry a lethal or semilethal is (0.25)^, or 6.25%. You cannot tell from the data presented whether the lethals and semilethals found in 240 CHAPTER 28 FIGURE 28-2. Genetic load in natural populations of D. pseudoobscura. {After Til. Dobzhansky.) MUTANT TYPE PER CENT OF CHROMOSOMES II III IV Lethal or Semilethal 25 25 26 Subvital / 93 41 95 Female Sterile 11 ^ ^ 14 4 Male Sterile 12 a particular pair of autosomes are allelic (in which case nearly 6% of zygotes in nature would fail to become adults because of homo- zygosity for the mutants), or whether the mutants involve different loci (in which case 6.25% of zygotes would bedihybrid for linked mutants of this kind), or whether some com- bination of these two alternatives obtains. In any case, the chance that both members of a given chromosome pair would be free of le- thals or semilethals would be (0.75)'-, or 56%. What portion of the population would carry no lethal or semilethal on either mem- ber of autosomes II, III, and IV? This is calculated to be (0.75)^ X (0.75)^ X (0.75)^, or about 17%. If we consider the fact that the X and V chromosomes can also carry such mutants, the frequency of lethal- semilethal-free individuals in nature is still lower. When, now, the subvital mutants (which are an even more frequent class of mutant) and also sterility mutants are con- sidered, it becomes clear that very few, if any, flies in natural populations are free of a load of detrimental mutants. What is the mutant load in man? We have already discussed the fact that the vast majority of mutants are detrimental in homozygous condition (Chapter 24). Death rate data are available for a population in rural France in the last century which include fetal death, and all childhood and very early adult deaths. What we want to compare is Mutational Loads and Their Consequences 241 the death rate of offspring from unrelated parents with that found from cousin mar- riages.^ The death rate of progeny from un- related parents is .12, while it is .25 from cousin marriages. We shall not be con- cerned here with establishing the genetic or nongenetic cause of death in the normal outcrossed human population. It can be assumed, however, that the excess mortality of .13 (.25 — .12) has a genetic basis in the excess homozygosity which is consequent to cousin marriage. This is a reasonable as- sumption in the absence of any other known nongenetic factor which would tend to cause more or fewer offspring to die from marriages between cousins than from marriages be- tween unrelated parents. (These data would have a nongenetic bias, if, for example, it was a custom — which it is not — that all children from cousin marriages are purposely starved.) Apparently, then, 13% more offspring died because their parents were cousins than would have died normally. What is the total amount of recessive lethal effect present in the population in heterozygous condition? This can be calculated as follows. Recall (from Chapter 27) that, of all heterozygous genes, an extra ]u become homozygous in offspring of cousin marriages. Half of the Me, or }^2, must have become homozygous for the normal genes and half of }i6, or )i2, for their abnormal alleles. So, to estimate the total heterozygous content of mutants which would kill if they were homozygous, it is necessary to multiply .13 by 32. The result- ant value of about 4 represents a 400% chance that the ordinary individual carried in heterozygous condition a genetic load of detrimental mutants which would be lethal if homozygous. In other words, on the average, each person carried four lethal equivalents in heterozygous condition, or, four times the amount of detriment it would 2 Based upon an analysis of N. E. Morton, J. F. Crow, and H. J. Muller. take to kill if the genes involved were made homozygous. This analysis does not reveal how many genes are involved in the production of the four lethal equivalents. In some individuals these might be due to the presence in het- erozygous condition of 4 recessive lethals, or 8 mutants having 50% viability, or 16 mu- tants with 25% viability, etc., or any combi- nation of detrimental mutants whose total is four lethal equivalents. It should be realized that, since the last century, improve- ments in the environment (in housing, nutrition, and medical care) make it most hkely that the effect of these same mutants in present-day society would be expressed by somewhat less than four lethal equivalents. Similarly, in the light of such progress, the detrimental effects of these mutants in het- erozygous condition would be expected to be somewhat less at present than they were a century ago. Accordingly, in the last century, a hypothetical homozygous combi- nation which, because of variable penetrance and expressivity, produced no detectable ef- fect 25% of the time, detrimental effect (but not lethality prior to maturity) 15% of the time, and lethality before maturity 60% of the time, might at the present time have the respective values of 50%, 10%, 40%. In the earlier period this combination would have produced .6 of a lethal equivalent and at present .4. Notice also that the detriment which is not lethal before maturity would also be reduced, changing from 15% to 10%, or, speaking in terms of detrimental equiva- lents, what was .15 would now be .10. It is clear that the genes responsible for lethal equivalents and for detrimental equivalents must be the same, at least in part. It is equally clear that present-day man also carries a load of mutants. Some of those transmitted to him arose in his parents (probably two of each five zygotes carry a newly arisen mutant, as mentioned on p. 200), and others arose in his more remote an- 242 CHAPTER 28 cestry. It has been calculated^ that, on the average, each of us is heterozygous for what is probably a minimum of about eight mu- tant genes received in these ways. What happens to this load of mutants in successive generations? In order to predict, in a general way, the fate of the "usual" mutant in the popula- tion, it is necessary for us to determine the phenotypic effect of the "usual" mutant. Since the typical mutant is detrimental, at least to some degree, when homozygous, the homozygous condition tends to eliminate the mutant from the gene pool. But we have seen in Chapter 27 that there are two oppo- site effects possible for mutants when hetero- zygous— either the heterozygote is superior to both homozygot.'s (as is found for the gene for sickling in malarial countries), or the heterozygote is somewhat inferior to the nonmutant homozygote (as is true for most heterozygotes for point recessive lethals). In the former case, the heterotic effect would tend to increase the frequency of the mutant, so that both the normal and mutant variants would be retained in the population at equi- librium. Such a population, which normally retained in its gene pool more than one genie (or chromosomal) alternative, would, there- fore, exhibit balanced polymorphism. In the case where the heterozygote shows detriment, the heterozygous condition would increase the rate at which the mutant was eliminated from the gene pool. Experimental evidence in Drosophila '* and a statistical analysis of data for man ^ sup- port the view that the great majority of point mutants are detrimental when heterozygous. We shall, therefore, consider that the usual mutant is not heterotic when heterozygous, but is detrimental to a degree that is some- 3 By H. J. MuUer and by H. Slatis. "• Based upon work of H. J. Muller and coworkers, C. Stern and coworkers, J. F. Crow and coworkers, I. H. Herskowitz and R. C. Baumiller, and others. ^ Based upon an analysis by N. E. Morton. what less than it would be when homozygous. How is such a mutant gene eliminated from the population? It need not be ehminated by producing the death of an individual, although it is sometimes removed this way. A more general way to express the removal of a mutant gene from the gene pool is by genetic death, the failure to produce de- scendants carrying the mutant. Thus, all the genes in an individual, whether they be normal or mutant, suffer genetic death if that individual fails to produce children. Since mutants are stable, they are removed from the gene pool only by genetic death, or, rarely, by mutation of the mutant. A per- son carrying a dominant lethal like retino- blastoma suffers genetic death (as well as physical death). In this case, the mutant gene is eliminated from the population the generation in which it arises by mutation; it shows, therefore, only one generation of persistence. The dominant mutant for achon- droplasia produces an average reproductive potential of .2 as compared to normal and will persist for five generations, on the average, before suffering genetic death. This means that each generation, in a population maintaining approximately the same size in successive generations, the achondroplastic individual has an 80% chance of not trans- mitting the mutant. So, when this mutant arises, sometimes it will fail to be trans- mitted the very first generation, at other times it will suffer genetic death the fifth generation, and at still other times at the tenth. But, on the average, the mutant will persist five generations before being lost. You realize that even though genetic drift and migration can cause fluctuations in the frequency of the mutant, the principle of persistence will still obtain. Consider the fate in the population of a rare recessive lethal gene like that involved in producing juvenile amaurotic idiocy. Each time that homozygosis for this gene occurs, it results in genetic death, and two mutant Mutational Loads and Their Consequences 243 genes are eliminated from the gene pool. But consider the fate of the heterozygotes who are 600 times more frequent (Chapter 27), and carry 300 times as many of these genes as do the homozygotes. Since it is generally true that heterozygotes for a reces- sive lethal sufier genetic death about four per cent of the time (see p. 211), approxi- mately .04 X 600, or 24, heterozygous people would suffer genetic death, involving the re- moval of 48 genes, of which 24 would be the recessive lethal allele. Accordingly, 12 times as many of these particular recessive lethal genes suffer genetic death in the heterozy- gote than in the homozygote, even though the reduction in reproductive potential in a group of the former type of individuals is only Yiv, of what it is in a group of the latter type. It is apparent that the rarer a mutant is, the smaller will be the proportion of all genetic deaths it causes in homozygotes, and the larger the proportion that it causes in heterozygotes. For rare mutants, then, natural selection removes mutant genes primarily via the genetic death of heterozy- gotes, the small amount of detriment when heterozygous being more important from the population or gene pool standpoint than the greater detrimental effect when homozygous. However, each mutant is harmful to the population to the same degree, in terms of its effect on reproductive potential, in that each is eventually the cause of a genetic death, at which time it is removed from the gene pool. Thus, hypoploidy which acts as a dominant lethal persists only one genera- tion before it causes a genetic death. A point mutation which produces a reproductive disadvantage of only Ko% will persist, on the average, 1000 generations, at which time it will be the cause of genetic death. As a matter of fact, speaking not in terms of biological fitness, but in terms of the total amount of suffering to which a human popu- lation is subject, point mutants with the smallest heterozygous detriment are the most harmful type of mutant. Consider, on one hand, the gross chromosomal abnor- mality which kills in utero. This destroys a life early, so that the individual involved has not suffered very long timewise. In this case, also, the parents may suffer relatively httle, for such deaths may occur so early as to result in abortions which pass unnoticed. Consider, on the other hand, the effect of heterozygous point mutants in individuals who are past the reproductive age. These people already have or have not suffered genetic death, but continue, nevertheless, to be subjected to the previously produced, and newly produced, small phenotypic detriment of heterozygosity which adds to their aches, pains, and disease susceptibility. In this respect, then, the mutant with a smaller effect on reproductive potential causes more suffering than one with a larger effect, for the longer the persistence, the more damage that is done in postreproductive life. You might at first suppose that the amount of gene-caused human suffering can be re- duced through the practice of medicine. This is true, in terms of the individual being treated. For there is no doubt that the in- dividual who is diabetic for genetic reasons, and who is given insulin, is better off than he would be without this medicine. But re- member that this medicine does not cure the genetic defect. Moreover, the medicine, by increasing the diabetic's reproductive potential, serves to increase the persistence of the mutants involved, so that the genetic death which must eventually occur to remove the mutant is only postponed to a later generation, each additional generation re- quiring the same medication. It would be true that the total amount of human suffering also would be reduced, until the time of genetic extinction of the mutant, if in fact the medicine completely normalized the genetic defect. But to do this would require that the medicine replace the primary prod- 244 CHAPTER 28 uct(s) of the defective gene with that of the normal, in order to normaHze all of the pleiotropic effects of the mutant. However, insofar as most, if not all, current medicines act later than this in the pedigree of causes (Chapter 10), they serve to alleviate some detrimental effects, but not others, and in this way cause an increase in the totality of human suffering by increasing persistence. This situation will persist until genetics and medicine have advanced somewhat further than they have at present. In view of the foregoing you will agree that it is primarily the euploid or nearly euploid mutants which persist in the gene pool, and it must be these which are primarily responsible for changes in its composition during the course of evolution. By far the most common, and hence most important, class of mutants of this type, is the point mutant. You may have noticed, in Chapter 27, that it was only suggested that mutations provide the raw materials for biological evolution. Our hesitance in specifying that evolution is the natural outcome of changes in gene pools was based upon the observa- tion presented earlier that the great majority of mutants, including point mutants, are harmful in homozygous or hemizygous condi- tion. In the present Chapter and in Chap- ter 24, we have indicated that most mutants are also detrimental when heterozygous. Under these circumstances how can muta- tion provide the more adaptive genotypes so necessary for evolution to take place as a result of changes in the gene pool? This dilemma is resolved in the light of two facts. It is true, in a given genotype under a given set of environmental conditions, that the great majority of point mutants are detri- mental, and that perhaps only one point mutant in a thousand increases the repro- ductive potential of its carrier ever so slightly. Yet, provided the mutation rate is not too large and provided there is sufficient genetic recombination, these rare beneficial mutants offer the population the opportunity to be- come better adapted. The second point is that mutants which lower biological fitness under one set of environmental conditions may be more advantageous than the normal genes under different environmental cir- cumstances. So, for example, a mutant like vestigial wings in Drosophila is clearly inferior to its normal genetic alternative in an en- vironment where flight ability is advanta- geous, but this mutant might be advantageous for Drosophila living on a small island where flight is not only unnecessary but harmful, where insects that fly can be lost by being blown out to sea. As a second example of this, we can mention the fact that several decades ago the environment was DDT-free, and mutants which conferred immunity to DDT were doubtless less adaptive than the normal genetic alternatives present. But once DDT was introduced into the insect environment, such mutants, even if they were detrimental in other respects, provided a tremendous over-all reproductive advantage over their alternatives, so that they became established in the population as the new wild- type genes. Still other examples could be cited involving microorganisms, where mu- tants occur that confer resistance to anti- biotics; in an antibiotic-free environment these mutants would be less adaptive than the genes normally present. It becomes clear, then, that mutation pro- vides the opportunity for a population to become better adapted to its present set of environmental circumstances. It also pro- vides the raw materials needed to extend the population's range to different environments, which already exist in some other territory or which will arise through change in the environment of its present territory. A pop- ulation that is already very well adapted to its environment is appreciably harmed by the occurrence of mutation. But the en- vironments in different territories differ, and Mutational Loads and Their Consequences 245 the environment in a given territory will eventually change, so that a nonmutating population that is successful at one time will normally eventually face extinction in the future. Mutation, therefore, is the price paid by a population for future adaptiveness in the same or a different environment. We can now appreciate that mutation and selec- tion, together with genetic drift and migra- tion, represent the principal factors responsi- ble for the origin of more adaptive genotypes. We can also appreciate better the advan- tage that genetic recombination provides in speeding up the production of adaptive genotypes, and the importance of the genetic regulation of mutability. In view of what has already been presented, it is not at all difficult to predict the conse- quences of increasing the mutation rate in human beings. There can be no doubt that the exposure of human populations, to man- made penetrating radiations and certain reactive chemical substances, is increasing his mutation rate. Manmade, as well as spontaneous, mutations can occur in either the somatic line or the germ line. Somatic mutations are, of course, restricted to the per- son in which they occur. The earlier the mutation occurs in a person's life history, the larger will be the sector of somatic tissue to which the mutated cell gives rise. Suppose a mutagen causes mutation in a certain percentage of all cells. If these mu- tant cells are in an adult they will usually be surrounded by nonmutant ones, of the same tissue, whose action would usually suffice to produce a near-normal effect. A smaller number of cells would be mutated in an embryo than in an adult. However, the mutant embryonic cells could later give rise to whole tissues or organs which would be defective, and in which no compensatory action of normal tissue would be possible. Furthermore, insofar as many mutants af- fect the rate of cell division, the earlier in de- velopment they occur, the more abnormal will be the size of the structure to be pro- duced. It becomes understandable, then, even if developmental and postdevelopmental stages are equally mutable, that somatic mutations are more damaging to the indi- vidual, the earlier they occur in its develop- ment. Almost all somatic damage is done by newly arisen mutants in heterozygous condi- tion, since mutation involves loci which are usually nonmutant in the other genome. Although somatic mutants cannot be trans- mitted to the next generation, they can lower the reproductive potential of their car- riers, and in this way affect the gene pool for the next generation. Mutational damage to somatic cells de- pends upon whether or not the cell subse- quently divides. Certain highly differenti- ated cells in the human body, hke nerve cells, or the cells of the inner lining of the small in- testine, do not divide. It is ordinarily diffi- cult to detect mutations in such cells, since they have no progeny cells which can be classified as mutant and nonmutant. Such cells may be more or less mutable than cells which still retain the ability to divide, but in any case a variety of mutations can occur in them. These can include point mutations that inactivate genes or change the type of allele that is present, as well as structural rearrangements of all sizes. Nevertheless, the cell will remain euploid or nearly euploid because no cell division follows. Accord- ingly, phenotypic detriment to nondividing cells must be due almost entirely to point mutants in heterozygous condition and to position effect. (It is likely that position ef- fect occurs in human beings.) Although the functioning of nondividing cells may be considerably impaired for these reasons, so that they may behave as though they were aging prematurely, their sudden and imme- diate death due to mutation is probably very rare. The same mutations can occur in somatic 246 CHAPTER 28 cells that divide as occur in nondividing cells, but in this case, aneuploidy can result following nuclear division (recall the discus- sion of aneuploidy in Chapters 18 and 19). In dividing cells, most of the phenotypic damage is the result of aneuploidy, and most of this is the consequence of single break- ages that fail to restitute, at least for those mutation-inducing agents which are capable of breaking chromosomes. (It may be noted that all known agents that increase the point mutation rate also break chromosomes.) We should not consider the preceding dis- cussion of the somatic effects of mutation as a digression from the main aims of this Chap- ter, because such cells actually comprise a population which has been produced by asex- ual reproduction (cell division). Let us now consider, in a general way, the consequences of increasing the frequency of mutations in the human germ line. The earlier that mutation occurs in the germ line, the greater will be the portion of all germ cells produced which carry the mutant. Of course, the upper hmit of gametes carrying a particular induced mutant is usually 50 per cent. Consider the effect of exposing the gonads to manmade high-energy radiation (Figure 28-3). Before the exposure to manmade radiation, the load of mutants is presumably at equilibrium, the rate of origin of mutants equaling the rate of their loss via genetic death. Starting with the first generation to receive the additional radiation exposure, the mutant load increases each generation until a new equilibrium is reached. At that point the higher number GENETIC LOAD of genetic deaths will equal the higher num- ber of new mutants. If at some still later generation, the extra radiation exposure ceased, the mutational load would decrease gradually (because of variations in persist- ence) via genetic deaths, until the old equilib- rium were reached once again. It has become very important to learn in detail the genetic effects of high-energy radia- tion to which human populations are being subjected either purposely or circumstan- tially. In order to make the best evaluation, we would like to know, among other things, the precise way in which the energy of various radiations is distributed in tissue, the exact amount of gonadal exposure to radiations of different types, the kinds and frequencies of the different types of mutation each of these radiations would produce in the different stages of gametogenesis in males and in fe- males, the detriment of the induced mutants in hetero- and homozygous condition, the exact proportions of all mutants that are heterotic, and their amount of heterosis, as well as the persistence of these mutants. We certainly do not have, at present, as much information about any one of these factors as we would like to have, but available in- formation along these lines can already give us approximate answers (see references at the end of this Chapter). In the discussion following, it should be realized, therefore, that all figures used may be in error by as much as a factor of two or more. It has been a practice to discuss the germ line effect of radiation in terms of the amount FIGURE 28-3. Genetic load and exposure to radiation. K Radiation _ { Exposure \ GENERATIONS Mutational Loads and Their Consequences TAl of increase any particular exposure would produce in our spontaneous mutation rate. The general impression is held that we, as a species, are already adapted to the mutation rate ordinarily produced by nonmanmade mutagenic agents, and that if man causes this rate to double through his own activity, this will not pose a threat to his survival as a species! Accordingly, the question be- comes, how much manmade radiation would be needed to produce as many mutations as occur normally? A United Nations' report calculated that about 30 rads (roughly equal to 30 r) would be sufficient to double the human spontaneous mutation rate. This is called the doubling dose. It is reckoned that in a population of one million people (which is the approximate size of the population in the St. Louis area), 1 rad delivered to the gonads or sex organs of each person would produce between 100 and 4.000 mutants which would be transmitted to future gen- erations. This 1 rad of gonadal exposure for one generation would result in the birth of 100 to 4,000 people with new heterozygous mutants, the individuals showing the effects being spread out over the course of many generations. Only a part of the genetic deaths from these mutants would occur in the first generation, and these would not be evident when added to the number of genetic deaths consequent to spontaneous mutation. If the 1 rad gonadal exposure were repeated in every generation, eventually an equilib- rium would be established, in which in each generation there would be 100 to 4,000 people per million showing the effects of radiation- induced mutants in the form of genetic death. However, since the kinds of phenotypic effects produced by the radiation-induced mutants would be the same as those from mutants which occur normally, one would not be able to recognize the particular people who were hurt by the radiation. What part of our normal load of mutants comes from naturally occurring penetrating radiation? Since human beings receive about 5 rads in the course of a reproductive gen- eration, that is in 30 years, it is possible that as little as %q of )i of our mutations are normally radiation-induced. How much additional radiation are we ex- posed to in the course of medical treatment? It has been estimated that each person in the United States would receive, if medical use of radiation continued at its present level, a total dose to the sex cells of about 3 r per generation. Of course, while some people get no such amount of radiation, others get considerably more. But this average radia- tion dose to the germ cells from medical uses alone is 60% of the amount we receive spontaneously, and is raising our mutation rate about 10% above the spontaneous rate. With the increase, in the years to come, of the use of radiation for diagnosis and therapy, this amount of medical radiation might be- come greatly increased. Already, in a single year, radioactive materials were given in one million medical treatments. How many germ-line mutations are being produced by the radiation associated with fallout following atomic explosions? This is not an easy question to answer. For some radiation could reach the gonads from the fallout on the ground, and other radiation could come from what is breathed in, or from what is included with the food. In the case of fallout taken in with food, the distribution of particular radioactive sub- stances in the body will make a large differ- ence in the amount of radiation reaching the sex cells. In this respect, three most impor- tant radioactive substances in fallout are cesium-137, strontium-90, and carbon-14. Because of its distribution, cesium-137 is expected to produce more gonadal radiation from ingested fallout than does strontium-90. This is so because cesium is distributed through the tissues more or less evenly, in- cluding the gonads, while strontium is pref- erentially localized in bone. 248 CHAPTER 28 It should be noted that the period of time over which mutations are produced is dif- ferent for these different radioactive chemi- cals. For relatively short-lived radioactive substances like strontium and cesium, the induction of new mutations would be re- stricted almost entirely to a few generations following the explosions that produced them. This may be compared with the distribution in time of the mutations produced by car- bon-14. Carbon-14 has a half-life of 6,000 years. So, if the exposure were unchanged, even 200 generations from now there would be about half as many mutations produced, as there will be in the generation most mu- tated by the bombs already exploded. Ac- cordingly, because of its abundance and long half-life, carbon-14, before it decays to nitro- gen, has been calculated to deliver to the gonads 4 to 17 times as much radiation as cesium and strontium combined. This will produce proportionally more point muta- tions. In the United States National Academy of Sciences — National Research Council report of 1956 (see References), the gonadal dose expected from fallout, if weapons of the same type continued to be tested at the same rate, was given as about 0.1 rad in the next 30 years. On the basis of the United Nations report, this would cause approximately 10 to 400 mutations per million people. How much modification does this figure now need to bring it up to date? Several considera- tions need to be taken into account. One is carbon-14, whose long half-life was not taken into account in that report. Another is the changed rate of testing, which was such that, according to the United States Atomic Energy Commission, the amount of fallout- producing radioactive material in the strato- sphere was doubled by the numerous test explosions of nuclear weapons conducted by the U.S. and the U.S.S.R. in 1958 alone. Third, the unequal distribution of fallout in different parts of the world needs to be con- sidered. Fourth, since fallout is descending faster than expected, less decay has taken place in the stratosphere. Finally, changes in the quality of bombs tested and the sites of the tests must also be taken into account before accurate estimates of germ-line muta- tional damage due to fallout may be obtained. Each month brings more of the data re- quired to estimate the fallout risk to the germ cells. Apparently, the possible damage has been underestimated. For, whereas the International Commission on Radiological Protection recommended a maximum per- missible concentration of strontium-90 in food of 80 units in 1953, which was then adopted by various U.S. government agen- cies, in 1958 the Commission recommended this be lowered to 33 units, and the new value was recently employed as a guideline by the United States government. There are several genetic problems, in ad- dition to those already mentioned on page 246, which need to be solved, in order to esti- mate the effects of radiation upon future gen- erations. It is necessary to determine the relative mutability, for various types of mu- tation, of a dose given in a concentrated, as opposed to a protracted, manner. It is also necessary to learn as accurately as possible, the doubling dose for spermatogonia (about 40 r in the mouse) and oocytes (probably less than 40 r in mouse and man), for it is in these stages that the human germ cells used to pro- duce the next generation remain for the long- est period of time. It may be suggested that the largest number of germ-line mutations may occur in the oocytes. Spermatogonia are constantly dividing, during which time mutants producing a detrimental effect may be selected against so that a considerable portion are lost prior to gamete formation. However, there is no parallel situation in the germ line of the human female. The human female is born with all, or almost all, her future gametes already in the oocyte stage. No further mitotic cell multiplication takes Mutational Loads and Their Consequences 249 place in these cells, which remain relatively quiescent for decades before becoming ova. Not only is germinal selection against mu- tants via mitosis absent in women, but there is evidence suggesting that as oocytes age they become disproportionately sensitive to mutation (at least to nondisjunction that leads to the presence of extra whole chro- mosomes, polysemy, and probably to chro- mosome loss, also). While there is no doubt, in principle, that exposure to radiation has produced point mutants in the somatic and germ lines of man, this has not been easy to demonstrate in practice, for two main reasons. The first is that the point mutants expected are not qualitatively different from those which would occur spontaneously, and the second is that the quantitative effect, although large in total, is, in any one generation, small enough to be masked by the general varia- bility of human genotypes and environment. However, by means of the use of improved statistical methods, the evidence is becom- ing stronger and stronger that radiation has produced such genetic effects. On the other hand, there is clear proof that radiation can cause structural changes in human chromo- somes. With the recent perfection of cyto- logical methods for studying human chro- mosomes, and the evidence that aneusomy is a relatively frequent event in oocytes, it is Hkely that additional evidence will be forth- coming relative to the numbers and kinds of gross chromosomal mutations which differ- ent doses and kinds of radiation can induce in man. In conclusion two more points need to be made. In our discussion of the genetic ef- fects of low doses of radiation, we have recog- nized a danger which is not likely to be calamitous to the human gene pool. How- ever, the very high dosages which are possible in a nuclear war could be disastrous. For if 500 r is given the whole body in a short period of time, the chances are 50% that the person will die in a few months. If a person survived this period, his life expectancy would be reduced by some years, probably because of somatic mutations, and children conceived after exposure would be handicapped by many detrimental mutants. It is even possible, but not probable, that we could re- ceive enough radiation in a nuclear war to destroy the human species. Finally, it should be realized that we are being con- stantly exposed to manmade mutagenic chemicals. It is very probable that we are getting less germ-line mutagenic effect from chemicals than from radiation; on the other hand, however, it is possible that we are having more somatic mutants produced by chemical substances than by present radi- ation exposure. SUMMARY AND CONCLUSIONS Cross-fertilizing species carry a large load of mutants in heterozygous condition. The vast majority of these are detrimental when homozygous and, to a lesser extent, when heterozy- gous, although there are also some mutants that are heterotic. Other things being equal, all mutants are equally harmful in that all are eliminated from the population only via the genetic death of their carriers. For rare mutants, more detriment and more genetic deaths occur in heterozygotes than in homozygotes. Persistence of mutants in the population is inversely related to their effect upon reproductive potential. Mutants with the smallest detriment to reproductive potential cause the greatest total amount of human suffering. Mutation is the current price paid by a population for the possibility of having a greater reproductive potential in the same or a different environment in the future. So, despite the rarity, in a given environment, of mutants which increase reproductive potential, mutation provides the raw materials for evolution. 250 CHAPTER 28 Natural and manmade penetrating radiations are doubtless causing mutations in our somatic and germ cells, increasing our load of detrimental mutants. This exposure, though detrimental, is most likely no threat to man's survival as a species at present, although it could be in the future, if it became large enough. Further research is needed to assess accurately the magnitude of the effect of manmade radiations and chemical substances upon his mutation rate and well-being. REFERENCES Auerbach, C, Genetics in the Atomic Age, Fair Lawn, N.J., Essential Books, 1956. Background Material for the Development of Radiation Protection Standards, Report No. 1, Federal Radiation Council, U.S. Government Printing Office, Washington, D.C., 1960. Chu, E. H. Y., Giles, N. H., and Passano, K., "Types and Frequencies of Human Chromo- some Aberrations Induced by X-rays," Proc. Nat. Acad. Sci., U.S., 47:830-839, 1961. Crow, J. F., "Ionizing Radiation and Evolution," Scient. Amer., 201:138-160, 1959. Dobzhansky, Th., Evolution, Genetics, and Man, New York, John Wiley & Sons, 1955. Herskowitz, I. H., "Birth Defects and Chromosome Changes," Nuclear Information, 3 (No. 2):l-2, 4, 1960. Krieger, H., and Freire-Maia, N., "Estimate of the Load of Mutations in Homogeneous Populations from Data on Mixed Samples," Genetics, 46:1565-1566, 1961. Morton, N. E., "The Mutational Load Due to Detrimental Genes in Man," Amer. J. Human Genet., 12:348-364, 1960. MuUer, H. J., "Mutational Prophylaxis," Bull. N.Y. Acad. Med., 2nd Ser., 24:447-469, 1948. MuUer, H. J., "Radiation Damage to the Genetic Material," Amer. Scientist, 38:33-59, 126, 399-425, 1950. Report of the United Nations Scientific Committee on the Effects of Atomic Radiation, New York: General Assembly Official Records: 13th Session, Suppl. 17 (A/3838), Chaps. 5-6, Annexes G-I, 1958. Selected Materials on Radiation Protection Criteria and Standards: Their Basis and Use, Joint Committee on Atomic Energy, Congress of the United States, U.S. Govern- ment Printing Office, Washington, D.C., 1960. The Biological Effects of Atomic Radiation. Simvnary Reports, National Academy of Sciences — National Research Council, Washington, D.C., 1956 and 1960. (See Reports of the Genetics Committee.) QUESTIONS FOR DISCUSSION 28.1. Do you suppose that the mutations which occur in man serve a useful function? Why? 28.2. Compare the fate of a mutational load in asexually reproducing populations that are haploid, diploid, and autotetraploid. 28.3. Discuss the effect upon the gene pool of mutants restricted to the somatic line. 28.4. Can the gene that comprises part of a detrimental equivalent also comprise part of a lethal equivalent? Explain. 28.5. Give examples of polymorphism, and of balanced polymorphism in the genetics of man. Mutational Loads and Their Consequences 251 28.6. What is the relation between phenotypic detriment, genetic death, and genetic persistence? 28.7. Discuss the relative importance, to the individual and to the population, of point mutants and gross structural changes in chromosomes. 28.8. What is the difference, in terms of mutation, between a maximum permissible dose and a doubling dose of ionizing radiation? Is any dose of any radiation safe from a mutational standpoint? Explain. 28.9. Compare the genetic composition of the mutational load caused by fallout, medical uses of radiation, and atomic reactor accidents. 28.10. Do you believe it is essential that the general public become acquainted with the genetic etTects of radiation? Why? 28. 11. What are some of the beneficial uses of radiation? Are any of these based upon the genetic effects of the radiation? If so, give one or more examples. 28.12. One of the components of fallout is radioactive 1-131, which has a half-life of about a week. Discuss the genetic effects expected in the somatic and germ lines of persons exposed to fallout. Chapter 29 RACES AND THE ORIGIN OF SPECIES li 'n cross-fertilizing species, differ- ent individuals in a population are .heterozygous for different genes (see Chapter 28). This is true even though the gene pool is at equilibrium with the fac- tors which cause shifts in gene frequency — namely, mutation, selection, drift, and migra- tion. In other words, in reaching genetic equilibrium, all the members of cross- fertilizing populations do not eventually be- come homozygotes, nor do they all become heterozygotes. Such populations, therefore, do not become genetically either pure or uniform with the passage of time. While any given population is polymorphic for some genes, this does not mean that it is necessarily polymorphic with regard to a particular gene. So, for example, Indians in South America are all of O blood type, being homozygous (/ /) in this respect, but they have a polymorphic pool with respect to other genes. Moreover, an allele like P, for example, may be rare or absent in one pop- ulation, as is true in certain North American Indians, while it may be relatively frequent in the gene pool of another population, as in central Asia. Thus, populations located in different parts of the world may differ both in the types and frequencies of alleles which they carry in their gene pools. For many purposes it is desirable to identify a popula- tion with certain gene pool characteristics as a race. In certain studies the investigator may wish to define races only in terms of the distribu- 252 tion of the P gene for ABO blood type. It would then be perfectly reasonable and valid to define as different races, populations that do or do not contain /^ in their gene pool. On this classification, there would be only two races of man, the South American Indian (without P) and all other people (with P in their gene pool). In another study, however, it may be de- sirable to define races on the basis of the prevalence of / and P in the population. The distribution of these genes in the gene pool has been studied extensively in popula- tions all over the world. The results show that in western Europe, Iceland, Ireland, and parts of Spain, three fourths of the gene pool is /. However, as one proceeds east- ward from these regions, this frequency decreases. The opposite tendency is true for P. In fact, in a world map, P is most frequent in central Asia and some popu- lations in India, and becomes less and less frequent as one proceeds away from this center. Since the change in frequency of P is also a gradual one, it is not possible to draw lines on the map which would separate people into groups with sharply different gene frequencies. So, where these lines are drawn, and how many are drawn, are arbi- trary matters, as a consequence of which, more or fewer races will be defined. Not only are the genes for ABO blood groups useful in characterizing races, but so are other blood traits whose genetic basis is understood. It is actually valid, in defining races, to utilize differences in any trait so long as these are based upon genetic dif- ferences. So, for example, one can consider certain genetic differences in color of hair, eyes, and skin, and differences in stature and head shape, in delimiting races. Our knowl- edge of genetics should caution us, however, that the use of phenotypic criteria only may be quite unsatisfactory for classifying races. For the environment itself can cause pheno- typic differences (see Chapter 1), and the Races and the Origin of Species 253 same phenotypic result may be produced by different genotypes because of gene inter- action in dominance and epistasis (see Chapter 7). It should be reemphasized that the num- ber of races recognized is a matter of con- venience. For some purposes it may be ade- quate to separate mankind into only two races, while for others, as many as two hundred have been recognized. Most an- thropologists usually recognize about half a dozen basic races, or define about thirty, when finer details of some populations are to be considered. Regardless of the number of races defined, however, each is best characterized in terms of the genes it con- tains. Since the people in a population are either A, B, AB, or O in blood type, and there are no intermediates, there is no average genotype for ABO blood group. Nor is there an average genotype for any other polymorphic gene. Because there is no average genotype for a population, a race must be defined in terms of the relative fre- quency of alleles contained in its gene pool, and since there is no average genotype for a race, there is also no average phenotype. You see, therefore, the invalidity of trying to picture a typical (average) member of any race. A knowledge of the distribution of genes for ABO blood types in diff'erent popula- tions the world over provides important information to geneticists, anthropologists, and other scientists. To what can the dif- ferent distributions be attributed? Since people do not choose their marriage partners on the basis of their ABO blood type, and since there does not seem to be any pleio- tropic eff"ect which makes the possessor of one blood type sexually more attractive than another, we may conclude that mating is at random with respect to ABO genotype. There is some evidence, however, that in other respects not all ABO genotypes have the same biological fitness. It is possible that diff'erential mutation can also explain part of the diff"erences in distribution. But, the greatest shift in ABO gene frequencies in different populations in the past few thou- sand years has probably been the result of genetic drift and migration. In fact, the paths of past migrations have been traced, utilizing the gradual changes in the fre- quencies of ABO and other blood group genes in neighboring populations. It has already been mentioned (p. 235) that different paracentric inversions are found in natural populations of D. pseudoobscura. Even so, all of these flies found in nature are very similar phenotypically, even though they differ with respect to their chromosomal arrangements. This fly is common in the southwestern part of the United States (Figure 29-1), and different populations located there have been sampled to detect the relative frequency of these inversions.^ It is found that California populations are rich in the inversion types called Standard and Arrowhead. Eastward, in nearby Ari- zona and New Mexico, the populations con- tain very few Standard chromosomes and have the Arrowhead arrangement almost entirely. Finally, in still more easterly Texas, there are no Standard, some Arrow- head; most chromosomes are of Pikes Peak type. The shift in the frequency and type of in- versions in different geographic regions can- not be explained as the result of differential mutation, since the spontaneous rate at which inversions arise is extremely low. Moreover, there is no reason to believe that the gene flow among these populations has changed appreciably in the recent past, so that migration rates have probably had a relatively small influence upon genotypic frequencies; nor is there any reason to attribute to genetic drift a major role in causing the differences in inversion frequency * Based upon work of Th. Dobzhansky and col- laborators. 254 CHAPTER 29 FIGURE 29-1. Distribution of inversion types in D. pseudoobscura collected in the Southwestern United States. {After Th. Dobzhansky and C. Epling.) in the different areas. By a process of elimi- nation, therefore, we conclude that the pri- mary reason for these population differences lies in the different adaptive values which these different inversion types confer in dif- ferent territories. In support of this view are laboratory tests which demonstrate that, despite the lack of any obvious morphological effects, these inversions have such different physiological effects that each type is dif- ferent from the others in being more adapted to certain experimental environments than it is to others. It is reasonably certain, therefore, that in nature, too, these different gene arrangements are adaptive, the differ- ences among the three types of populations, which we may define as three different races, being the result of natural selection. Similar results have been obtained with three California races of the cinquefoil plant species, Potentilla glandulosa, which live at sea level, mid-elevation, and the alpine zone, respectively. The sea level race is killed when grown in the alpine environment. The alpine race grown at lower elevations proves less resistant to rust fungi than the lower ele- Races and the Origin of Species 255 vation races. By means of such experiments it was shown that each race is adapted to the conditions of its habitat. In different parts of the territory occupied by a species, there are different inorganic and organic (including organisms) environ- ments. It is clear, from what we have just described, that no single genotype would be equally well adapted to all the different en- vironments encountered within this territory. One way in which a cross-fertilizing species may, as a whole, attain maximal biological fitness is for it to remain genetically poly- morphic and to become differentiated into geographical populations or races which differ from each other genetically. In all of the examples discussed so far, the different races of cross-fertilizing species have occupied geographically separate terri- tories, and are said to be allopatric. Some- times, however, different races may be sympatric, that is, they may be found in the same territory. In the absence of geographi- cal separation, what factors operate to keep sympatric races from hybridizing to become one race? We can look for the answer to this from studies of the fate of races, origi- nally allopatric, which have become sym- patric. Man offers one example of this kind of change. Several thousand years ago, mankind was differentiated into a number of allopatric races. Since then, the develop- ment of civilization, and improved methods of travel, have made such races sympatric in part. But gene exchange in the now-sym- patric races may be prevented by social and economic forces, so that some of these races may retain their identity. Domesticated plants and animals offer another example of what may happen when allopatric races be- come sympatric. Consider the case in dogs. Many different breeds, or races, which were originally allopatric, may now all be found living in the same city. Yet these now-sympatric races do not exchange genes with sufficient frequency to form the single breed, or race, called mongrel, because their reproduction is controlled by man. It should be realized that, under other circumstances, allopatric races, which become sympatric, may form a single polymorphic race via cross- breeding. A species of cross-fertilizing organisms usually consists of a number of races adapted to the different environments in which they are found. All these races are kept in genetic continuity by interracial breeding and hybrid race types, so that the species, as a whole, has a single gene pool within which no portion is completely isolated from any other. How does one species differ from another? Let us restrict our attention to cross-fertilizing species. We shall require that two groups of organisms be genetically discontinuous from each other in order to be considered different species. (This means that each species has, in effect, a gene pool which is so isolated from the gene pool of another species, that neither the one nor the other can lose its identity via cross-breeding, or backcrossing subsequent to cross-breeding.) In addition, the gene pools of different species must be isolated from each other for genetic, and not merely environmental, reasons. New species have arisen during the course of past evolution, and since evolution con- tinues today, new species are forming at pres- ent also. By what mechanisms do new species of cross-fertilizing individuals arise? The mechanism considered most common is the production of two or more species from a single species. How could this come about? Suppose different portions of the same species become somewhat independent of each other reproductively, that is, they form different populations, and, while there is some interbreeding, most of the breeding is intrapopulation. In the course of time, the two populations may diverge genetically so that each is adapted to its own territory. We might now want to call these two popula- tions different races of the same species. 256 CHAPTER 29 These two races may continue to become more and more different in their gene pools because of mutation, natural selection of genotypes which further increase adaptive- ness, and genetic drift. As this differentia- tion process continues, the genes which make the two races adaptive in their own territories may, via their pleiotropic effects, make matings between the two races still less likely to occur, or may cause the hybrids of such matings to be less adaptive than the members of either parent race. The greater the degree of reproductive isolation, which is an accidental or incidental by-product of the different genotypes which are adaptive in the two territories of the two races, the greater would be the selective advantage of other mutants which further increase the reproductive isolation between the two races. If the two races continued to diverge geneti- cally in this way, they would eventually form separate and different gene pools, at which time they would have changed from two races of the same species to two different species. Note that speciation is an irreversible process, for once a gene pool has reached the species level, it can never lose its identity via cross- breeding with another species. In this generalized account of how specia- tion usually occurs, races have acted as incipient species. But, you must also recall that, under other circumstances, what are two races may also often become a single race. Thus, for example, while several thou- sand years ago different allopatric popula- tions of human beings were definitely dif- ferent races which might have formed dif- ferent species had the same conditions of life continued, some of these races have subse- quently merged into one race because of civilization and migration. What are the principal means by which races are known to become reproductively isolated from each other? The barriers lead- ing to complete reproductive isolation in- clude the following: 1. Geographical. Races may become sepa- rated by water, ice, mountains, wind, earthquakes, and volcanic activity. 2. Ecological. The habitat of the two races may be different or may become more different than it was originally, because of changes in temperature, humidity, sun- light, food, predators, and parasites. 3. Seasonal. In adapting to seasonal changes, one race may become fertile at a different time than the other, even if their territories overlap, or if the races are sympatric. 4. Sexual or ethological. The races may show preferences for intrarace mating. (In domesticated forms this preference may be decided upon by man.) 5. Morphological. The sex organs of the two races may be incompatible to various degrees. 6. Physiological. The sex cells of one race may fail to fertilize those of the other, so that the hybrid zygote is not formed at all, or is formed infrequently. 7. Hybrid inviability. Even when hybrid zygotes are formed, their development may be abnormal, so that they fail to complete development. 8. Hybrid sterility. Finally, even if hybrids complete development and even if they are hardy, they may be sterile. Environmental differences in geography, ecology, or season which act to separate races do not automatically produce geno- typic differences among them. They do furnish, however, the environmental varia- tions which provide a means of selecting genotypes which are adaptive, i.e., which have the greatest reproductive potential under the different conditions. Of course, mutation must occur to provide the raw ma- terials for this selection of more adapted genotypes, and since no single genotype is equally well adapted to all conditions, the races will come to contain different geno- Races and the Origin of Species 257 types. Completion of reproductive isolation may be accomplished by the remaining bar- riers listed. Reproductive isolation may be due genet- ically to either genie action or chromosomal behavior, or both. Thus, the many genes, by which the two incipient species differ, may produce seasonal, sexual, morphological, and physiological barriers, as well as hybrid in- viability. Although hybrid inviability is due to developmental disharmony consequent upon the presence of two genetically differ- ent genomes in each cell, hybrid sterility may be due, also, to an additional factor. The two races may have become quite different with respect to the arrangement of their genes, by means of structural changes within and between chromosomes, so that during meiosis, synapsis between the two different genomes is irregular. Failure of proper pairing will mean that the segregation proc- ess will be abnormal and the products of meiosis aneuploid. You should recall that aneuploidy in pollen is lethal, while aneu- ploid gametes in animals usually produce dominant lethality of the zygotes they form at fertilization. Are morphological differences a good indi- cation of species differences? It would seem likely that the more divergent two forms are morphologically, the more likely they are to differ physiologically, and also the more likely that these differences derive from very different gene pools which are isolated from each other. We would certainly expect, simply from a comparison of horse and mouse morphology, that these are different species. However, when the two groups being compared are more closely related in descent, it is found that morphology is not well correlated with reproductive isolation. Thus, for example, European cattle and the Tibetan yak are quite different in appearance and are usually placed in different genera. Yet these two can be crossed, and in Tibet, many cattle have yaklike traits. On the other hand, consider D. persimilis and D. pseudoobscura. These two species were once considered races of the same species, and are so similar morphologically that one can differentiate between their genitalia only if very careful measurements are made. Nevertheless, these two species have com- pletely isolated gene pools in nature, even where their territories overlap. Such mor- phologically similar species are called sibling species, and have originated from different races of a single species in relatively recent times. Sibling species have been found in other Drosop/iila, mosquitoes, and other in- sects, and have been found in plants, among the tarweeds of the aster family, and in the blue wild rye. D. pseudoobscura and D. persimilis illus- trate two other principles relative to species formation. Their study demonstrates that any particular reproductive barrier usually has a multigenic and/or a multichromosomal basis, and that any two species are separated not by one, but by a number of reproductive barriers. Each of the barriers involved is in- complete, but all together they result in complete reproductive isolation, so that there is no stream of genes between the two gene pools in nature. In the particular case of these two sibling species, the known barriers include the fact that pseudoobscura lives in drier and warmer habitats, and that females accept the mating advances of males of their own species more often than they do of males of the other. Pseudoobscura usually mates in the evening, persimilis in the morning; when interspecific hybrids are formed, they are relatively inviable, or, when viable, mostly sterile. In the formation of new species from races, the nature and origin of the reproductively isolating mechanisms involved shows that valid species do not originate by a single or simple mutation, but arise as the result of many different, independently occurring genetic changes. Moreover, we have seen 258 CHAPTER 29 that the genetic changes which lead to specia- tion are not accomphshed merely by the ac- cumulation of more mutants of the kinds which distinguish races. What is required for speciation are mutants with special ef- fects, effects which contribute to reproductive isolation. Populations usually must be physically separated while reproductive bar- riers are being built up, otherwise hybridiza- tion would break down these barriers. There is also experimental evidence, in support of the hypothesis described earlier, that natural selection, acting both directly and indirectly, will itself further the accumulation of genetic factors promoting reproductive isolation be- tween races. We have discussed, so far, how one species can give rise to two or more species, via races which serve as incipient species. Note that in defining a species, it was necessary to say that it had an isolated gene pool, that is, a PARENT SPECIES W n DIPLOID HYBRID gene pool closed to individuals of some other alternative condition (species). There is one possible type of species formation which would be unrecognized by this cri- terion, since the alternative state would no longer exist. It is conceivable, for example, that a species composed of a single popu- lation would gradually undergo numerous changes in its gene pool during the course of many generations. At the end of this time should we call the new population the same species, or should we give it a new species name? It is likely that, in some cases, had some of the members of the original popu- lation been miraculously preserved, the old form would be found reproductively isolated from the new. In this event we would be deahng with the formation of a new species whose origin was dependent upon the ex- tinction of an old one. The occurrence of this type of speciation will become a valid subject of study, once man learns how to preserve sample genotypes indefinitely. Not only may new cross-fertilizing species originate from a single species or its races, but many have arisen as the consequence of hybridization between two or more different species. What we are dealing with here are the consequences of interspecific liybricliza- tion. We already know that if interspecific hybrids are formed, they pose no threat to the isolation of the gene pools of their paren- tal species. The question we are asking is: Aneuploid Melotic Products AMPHIPLOID Euploid(n) Meiotic Products FIGURE 29-2. Interspecific hybridi- zation leading to new species forma- tion via amplnploidy. Races and the Origin of Species 259 By what mechanisms can interspecific hybrids form successful, sexually reproducing popu- lations that have their own closed gene pools? There are several methods by which inter- specific hybrids, of plants particularly, may be converted into stable intermediate types that are isolated from their parental species. The first method involves amphiploidy (Fig- ure 29-2). Suppose one species has 2n = 4 and an- other 2n = 6. The Fi hybrid between them will have five chromosomes. Even if the hybrid survives, it may be sterile because the chromosomes are all nonhomologous and none has a partner at meiosis. As a result, meiosis would proceed as if the organism were a haploid; the gametes usually pro- duced would not contain a single set of the five chromosomes, but would be aneuploid. If, however, the chromosomes in such an Fi hybrid are somehow doubled, artificially by the drug colchicine, or by some spontaneous agent, then the chromosome number in that individual or sector would be 2n = 10, or n = 5, and since each chromosome would now be paired, the chromosomes would behave normally in meiosis and produce euploid gametes. The progeny would be fertile, phenotypically more or less inter- mediate to, and isolated from, both parental species. It has been estimated that 20-25% of the present species of flowering plants have originated as interspecific hybrids whose chromosomes have become doubled in num- ber (being therefore "doubled hybrids" or amphiploids). Moreover, as many or more species have originated in this way in the past, and then later diverged to form diff'er- ent genera. Amphiploidy has been involved in the origin of cotton in the New World. In the present century, new species of goats- beard have arisen in nature via amphiploidy. Amphiploidy also can be produced artificially. Thus, it has been possible for man to cross radish (2n = 18) with cabbage (2n = 18) (Figure 29-3). This produces an Fi hybrid with 18 unpaired chromosomes at meiosis. If, however, the hybrid's chromosomes be- come doubled, an amphiploid is produced with 2n = 36 chromosomes (containing 9 pairs each from radish and cabbage). This amphiploid is fertile, genetically isolated from both radish and cabbage — and con- stitutes a new species. The breeding success of the amphiploid is greater, the more diff'erent are the chromo- somes of the two species which originally provided haploid genomes to the Fi hybrid. For if eachchromosome in Fi is diff'erent, then, after becoming doubled, each will have just one partner at meiosis and segregation will be normal. It is not surprising, therefore, that when the two species hybridizing are very similar chromosomally, an amphiploid of them produces trivalents and quadrivalents at meiosis, which lead to abnormal segrega- tion and sterility. While amphiploidy is not successful for hybrids between very similar species, there is a second way such interspecific hybrids may become stabilized as a new species. If the two hybridizing species are very similar chro- mosomally, they could have the same hap- loid number, and the Fi hybrid could have all these chromosomes synapse in pairs at meiosis. Segregation, independent segrega- tion, and crossing over could yield progeny of the hybrid whose recombinations may be- come stabilized in nature, yet isolated from either parental species. Consider certain species in the larkspur genus. Delphinium. D. gypsophilum is morphologically inter- mediate between D. recurvatum and D. hesperium. All three species have 2n = 16. It is possible to cross the "parent" species, recurvatum and hesperium, and obtain an Fi hybrid. If this Fi hybrid is crossed to gypsophilum, off'spring are obtained which are more regular and more fertile than those which would be produced by backcrossing this Fi hybrid to either parent species. 260 RADISH FIGURE 29-3 (Right). Seed pods of cabbage and radish, of their hybrid and aniphiploid. After G. D. Karpe- chenko.) AMPHIPLOID DIPLOID HYBRID FIGURE 29-4 {Below). Distribution o/ Delphinium species in California. Each species has a unique habitat. Similarly, crosses of gypsophilum with either of its parent species are not as regular or fertile as are those made between it and the hybrid of the parent species. This com- prises good evidence that gypsophilum arose as the hybrid between recurvatum and hesperium. Figure 29-4 shows the distribu- tion of these species in California. A third way in which interspecific hybrids can become stabilized as new species is by introgression. In this process, a new type arises after the interspecific hybrid back- crosses with one of the parental types. The backcross recombinant types favored by natural selection may contain some genetic components from both species. These re- combinants may be true breeding and may eventually become a new species. ^ D. GYPSOPHILUM D. RECURVATUM D. HESPERIUM SUMMARY AND CONCLUSIONS The races within a cross-fertilizing species are characterized by the content of their gene pools. Each of the different races is adapted to the territory in which it lives. The races involved may be sympatric or allopatric. Races become species by the accumulation of genetic differences whose end effect is genetic discontinuity — i.e., the formation of isolated Races and the Origin of Species 261 gene pools. The reproductive barriers separating any two gene pools are usually of several different types, each of which is incomplete, each of which has a polygenic and/or a poly- chromosomal basis, and need not be well correlated with morphological differences. It is generally recognized that most cross-fertilizing species arose from the further dif- ferentiation of races. It is possible, however, that a new species may arise also by the gradual change of one species, as a whole, into another species. Two (or more) species may give rise to a new one following interspecific hybridization. The interspecific hybrid may form a new species by means of amphiploidy, by selection of recombinants among its progeny, or by selection of individuals produced following intro- gression. REFERENCES Dobzhansky, Th., Genetics and the Origin of Species, 3rd Ed., New York, Columbia Uni- versity Press, 1951. Dobzhansky, Th., Evolution, Genetics, and Man, New York, John Wiley & Sons, 1955. Dodson, E. O., Evolution: Process and Product (Rev. Ed.), New York, Rinehart, 1960. Dunn, L. C, and Dobzhansky, Th., Heredity, Race, and Society, 3rd Ed., New York, New Amer. Lib. of World Lit., 1957. Merrill, D. J., Evolution and Genetics, New York, Holt, Rinehart and Winston, 1962. Stebbins, G. L., Variation and Evolution in Plants, New York, Columbia University Press, 1950. QUESTIONS FOR DISCUSSION 29.1. Discuss the validity of the concept of a pure race. 29.2. What presumptions need be made in order to use the frequencies of ABO blood types to trace the course of past migration? 29.3. Under what future circumstances would you expect the number of races of human beings to decrease? to increase? 29.4. Can the definition we have used for a species be applied to forms reproducing only asexually? Why? 29.5. Differentiate between genie sterility and chromosomal sterility. Invent an example of each type. 29.6. Discuss the hypothesis that a new species can result from the occurrence of a single mutational event. 29.7. Is geographical isolation a prerequisite for the formation of a new species? Explain. 29.8. What is the relative importance of mutation and genetic recombination in species formation? 29.9. Is a species a natural biological entity, or is it, like a race, defined to suit man's convenience? 29.10. Does the statement, "We are all members of the human race," make biological sense? Why? 29.11. Suppose intelligent life, phenotypically indistinguishable from man, arrived on earth. Would intermarriage with earth people be likely to produce fertile offspring? Why? 29.12. Invent circumstances under which the present single species of man would evolve into two or more species. Chapter 30 DEVELOPMENTAL GENETICS WHEN we are dealing with mu- tations other than point mutations, the genetic alter- natives can often be recognized cytologically by modifications produced in chromosomal appearance. In the case of point mutation, however, we are restricted to a study of the phenotypic consequences of the change in genotype. In fact, we may usually note the presence of a mutant (of any type) by its phenotypic effects, that is, by its effects on the characteristics of an individual. Since the characteristics determined by gene action are themselves not inherited, the question is, how are these traits produced? What happens between the time the zygote is formed and the time the trait appears? Development. We are interested, therefore, in the ways in which development is influenced by different genotypes, or in what we may call develop- mental genetics. You realize, of course, that regardless of the importance of a trait produced by a gene, we cannot learn anything about the role of the gene in the production of the trait, unless there is some genetic alternative which pro- duces a change phenotypically. We would never learn of the existence or of the role in development of a gene, if its presence, ab- sence, or alternative forms produced no difference in phenotype. Whenever two dif- ferent genetic constitutions produce two different effects on the phenotype, it should also be realized that what is recognized is not the total phenotypic effect of one genotype and how this effect has been changed by 262 another genotype; what is seen is only the change in development which has been made by the change in genotype. Let us invent an example that will illustrate both these points. Suppose a particular gene actually is responsible for the production of an entire protein composed of a chain of a hundred amino acids. So long as no genetic alternative is known which produces a change in this protein, there is no way of knowing whether this protein is the result of a specific gene's activity, or is entirely the result of the action of the total genotype together with the environment. But, suppose a mutant occurs which substitutes one amino acid in this protein for another, and that this pheno- typic change is detectable. From this result we could conclude only that the normal gene places one amino acid and the mutant gene another amino acid in this protein molecule. Note again that we would have learned not the total effect of the gene, but just the differ- ence between the phenotypic results of the two genetic alternatives. Under ordinary circumstances, when mu- tants present at fertilization in multicellular plant and animal forms are detected, it is because they produce some visible change in morphology. This is usually a macroscopic phenotypic change, identified a considerable time after the organism starts its development. Two questions arise in this connection. What is the genetic basis for the mutant involved? The answer to this can be obtained by utiliz- ing the principles and methods already dis- cussed in previous Chapters. How does the mutant change normal development to pro- duce the new morphological result? The answer to the latter question deals with learn- ing how phenotypes (of any type) come into being via gene action, and is the subject of phenogenetics, a study which is of broader scope than developmental genetics. Let us see what genetic and phenogenetic information can be obtained from studying one particular case. A novel phenotype Developmental Genetics 263 FIGURE 30-1. Normal ( riy,lu i and Creeper {left) roosters. {Courtesy ofL. C. Dunn; reprinted by permission of McGraw-Hill Book Co., Inc., from Study Guide and Workbook for Genetics by I. H. Herskowitz. Copyright, 1960.) appeared in the domestic fowl in which the legs were shortened so as to give the impres- sion that the bird was creeping. This ab- normal "Creeper" phenotype and the normal phenotype^ can be seen in the roosters at the left and right, respectively, of Figure 30-1. The genetic study of this phenotype gave the following results. Reciprocal crosses of Creeper by normal gave a 1 : 1 ratio of Creeper : normal chicks. Creepers crossed to Creepers gave, in the adult stage, 775 : 388 as Creeper : normal, a result which can be considered an excellent fit to a 2 : 1 ratio. It is reasonable to suppose, therefore, that Creeper is heterozygous for a single pair of segregating genes, in which the Creeper gene, Cp, is dominant to its normal allele, +. The 2 : 1 ratio is taken to indicate that the mutant homozygote Cp Cp is lethal (as is the case, see p. 65, for the mutant homozygote when yellow mice are crossed together). The view that Cp acts as a recessive lethal received support from a comparison of the survival rate of embryos from normal parents with that of embryos both of whose parents were Creeper. It was found that about 25% of ^ Studied by W. Landauer, by V. Hamburger, by D. Rudnick, and by L. C. Dunn. the embryos which would have survived in the former case died on or before the third day of incubation in the latter case. What is the developmental, phenogenetic connection between Cp Cp which acts as a recessive lethal, Cp + which produces Creeper, and + + which produces normal? Although Cp Cp usually dies at about the third day of incubatiorf, on rare occasions it may survive 19 days, or about until the time of hatching from the shell. Such a rare Creeper homozygote is shown at the left of Figure 30-2 (the comparable normal indi- vidual is at the right) and possesses the follow- ing syndrome of malformations: The eyes are split, are smaller than normal, and have no eyelids. The head is misshapen and the body is smaller. The skeleton is not ossified and, as seen on top of the black paper used as background in the Figure, only the digits of the limbs are well formed. A study of Cp + development shows, at seven days of incubation, that the leg buds are shorter than in normal embryos. This mor- phological manifestation of Cp action must be based upon events occurring earlier in development, for at 48 hours of incubation (Figure 30-3), a Cp + embryo (left) is smaller, 264 CHAPTER 30 FIGURE 30-2. Normal {right) and Creeper {left) homozygote at about N days oj development. (Courtesy of L. C. Dunn : reprinted by permission of McGraw-Hill Book Co., Inc., from Study Guide and Workbook for Genetics by I. H. Herskowitz. Copyright, 1960.) FIGURE 30-3. Normal {right) and Creeper {left) heterozygote embryos at about 48 hours of development. {Coiu-tesy ofL. C. Dunn; reprinted by permission of McGraw- Hill Book Co., Inc., from Study Guide and Workbook for Genetics by I. H. Hersko- witz. Copyright, 1960.) Developmental Genetics 265 less developed, and does not have the head flexure already present in a + + embryo (right). In fact, differences like this can be seen even 12 hours earlier, i.e., at 36 hours of incubation. In both the homozygote and heterozygote for Cp, the differentiation of cartilage has been interfered with. The Cp + individual has the disease called chondrodystrophy (or achondroplasia) (see p. 230), the Cp Cp indi- vidual has the cartilage disease called phoko- melia. Both diseases had been recognized more than a hundred years ago as occurring in human families; cases were found in which both parents were chondrodystrophic and some phokomelic children appeared, whose fingers protruded from the shoulders and whose toes came from a deficient hind limb. The condition observed in these latter individuals can be attributed to the presence of a mutant gene (like the Cp gene in fowl) in double dose, that is, when homozygous. It was already mentioned that at 36 hours of incubation, Cp + individuals are develop- ing more slowly than + + individuals. Be- ginning about that time, the tissue for the hind limb buds would normally be growing very rapidly, while other tissues were growing more slowly. Let us suppose that some of the effect of Cp in single and double dose is to cause a generalized slowing-down of growth. In this event, the structures most affected by the slowdown would be those growing most rapidly at the time. The observed effects of one or two Cp genes on the hind limbs and on the long bones of both fore and hind limbs follow expectation from a slowdown in growth rate which starts at about this particu- lar time in development. It should not be concluded, however, that the tissue for hind limb is completely passive to Cp action, and that its sole response is to slowdown in growth rate. It is possible, by means of transplantation experiments, to study the developmental fate of prospective hind limb tissue. If such tissue from a normal chick embryo is transplanted to a more for- ward position in another normal chick em- bryo, it grows out as a normal limb. If, how- ever, the prospective hind limb tissue is taken from a homozygous Creeper embryo and is transplanted to a more forward position in a normal chick embryo, it grows out as a Creeper type leg. This demonstrates that, even at a very early stage, before there is any hind limb as such, presumptive limb tissue from Creeper is already permanently deter- mined by the Creeper genotype to develop as Creeper limb. It also should not be presumed that all ab- normal tissues found in homozygous Creepers have been determined at an early stage in development, so that they possess only the Creeper alternative. It was mentioned that Cp Cp individuals have small, split eyes. The early eye anlage (imaginal disc) from a normal embryo can be transplanted to an abnormal position in a normal embryo. In this position it grows into an eye just hke that of homozygous Creeper. But an eye anlage from a Cp Cp embryo, transplanted to the eye-forming region of a normal embryo, grows into a normal eye. We may conclude, therefore, that the abnormal Creeper eye is due, not to some intrinsic differentiation fac- tor in eye tissue, but to some kind of abnor- mality in its surroundings. It may be sup- posed that in the Creeper homozygote the eye is probably undergoing a kind of starva- tion due to the bad circulation the genotype produces. This is supported by two lines of evidence. First, most prospective tissues of Cp Cp placed on a complete cuhure medium in vitro grow quite normally, although heart tissue grows less well than normal heart tissue does. Secondly, when limb rudiments from normal embryos are grown in vitro in a nutri- tionally dilute culture medium, they develop many of the characteristics of the Cp Cp Hmbs. The study of Creeper fowl demonstrates that the pleiotropic effects of this mutant 266 CHAPTER 30 found at the completion of development are due to gene-directed changes originating much earlier in development. In fact, we can infer, from the developmental fate of prospective limbs in Creeper embryos, that there are changes produced by a genotype which may precede any morphological changes. We can presume that what the Creeper gene does is to modify the physiology of the individual in such a way that general growth is slowed down, and the prospective fate of certain tissues is fixed, so that the morphological changes later noted are a direct consequence of these changes. The gene- caused physiological changes may be attrib- uted, in turn, to changes in cellular metabo- lism (which deals with the biochemical activi- ties associated with cells). We have already seen that genetically deter- mined metabolic changes taking place within certain cells (to produce an abnormal nutri- tional environment) may affect the function- ing of other cells (the differentiation of eye tissue). Let us consider two groups of studies with mice to see if we can learn more about the genetic control of effects produced exter- nal to the cell in which the gene acts. One group of investigations ^ involves a compara- tive study of normal and dwarf mice. These dwarf mice have all of their body parts re- duced in size to the same degree, so they are proportionally dwarfed, due to the presence of an apparently completely recessive gene in homozygous condition. During develop- ment both dwarf and normal mice grow equally fast, at first. Then, the dwarf sud- denly stops growing and never reaches sexual maturity. A microscopic study of the an- terior pituitary gland shows that the gland is very much smaller in the dwarf than it is in the normal mouse. Moreover, certain large cells, normally present, are absent in dwarf pituitaries, and it is these cells which appar- ently secrete growth hormone. That this is 2 Based upon work of G. D. Snell, of P. E. Smith and E, C. MacDowell, and of T. Francis. a case of genetically produced pituitary dwarfism is supported by the following type of experiment. Pairs of dwarf litter mates, about 30 days old, are used. Each day, for 30 days, one mouse of a pair is injected with extracts of pituitary glands from dwarf mice (Figure 30-4, B), while the other mouse is in- jected in a comparable way with extracts of pituitary glands from normal mice (Figure 30 4, A). During this period of treatment, the former mouse remains essentially dwarf, while the latter grows until it is virtually normal. Here, then, we are dealing with a chemical messenger, pituitary hormone, which regulates growth in general, and whose pres- ence is dependent upon a single pair of genes. The second group of studies is concerned with mouse tails. While the normal (+ +) mouse has a long tail, there is one strain in which a shortened tail (Brachyury, or Brachy) occurs.^ Brachy crossed to Brachy produces 73 Brachy : K normal offspring, suggesting that a gene Brachy (T) is dominant for short- tailness and recessive for lethality. Brachy mice should be, therefore, T-f. When the embryology was studied of offspring produced following the mating of Brachys with each other (r+ by r+), about 25% of the embryos were normal (+ +), about 50% showed tail degeneration at 1 1 days of devel- opment (r+), and about 25% of the em- bryos (T T) were monsters (Figure 30-5). These monsters had posterior limb buds mis- directed dorsally and zigzag neural tubes; moreover, they had no notochord. Since their whole posterior part was not developed, they could not form a placental connection and died between 10 and 11 days of develop- ment. Consider further the T T individual, whose somites in the posterior part of the body are grossly abnormal. It is known, from other embryological work, that proper somite for- mation is dependent upon the presence of 3 Based upon work of L. C. Dunn, P. Chesley, and D. Bennett. Developmental Genetics 267 FIGURE 30-4. Effect of injecting pituitary gland extracts into dwarf mice. {See text for explanation.) \ n 44 49 54 AGE IN DAYS 64 presumptive, that is, future, notochordal tissue. When normal presumptive notochord is present, the mesoderm surrounding it is induced to form cartilage and vertebral seg- ments. It would seem reasonable to at- tribute the failure of cartilage and vertebrae formation m T T individuals to the failure of presumptive notochord (which has lost the ability to develop into notochord) to induce the differentiation of mesoderm. Is this, in fact, the nature of the change, '\n T T indi- viduals, in the inductive relationship between these two adjacent tissues? This question can be studied using tissue cultures. A con- trol experiment shows that it is possible to explant presumptive notochordal tissue, from a normal individual, which has wrapped around it mesodermal tissue, from the same or another normal individual, and to obtain the development of cartilage and vertebral segments. Surprisingly enough, however, the mesoderm from normal embryos will develop into cartilage and vertebral segments when surrounding presumptive notochord from young T T embryos. Moreover, mesoderm from T r embryos does not form cartilage or vertebrae when surrounding presumptive notochord from normal embryos. We must conclude, then, contrary to expectation, that in the case of T T the normal inductive rela- tionship has been disturbed in an unexpected way, the mesoderm being no longer able to respond to the normal inductive stimuH of presumptive notochord. We have already seen how genetic change may influence or direct development of multi- cellular organisms by means of modifying (1) the relative growth rates of parts (Creeper Hmbs) and (2) the over-all growth rate (pitui- tary dwarfism). We have also found that genetic changes can act upon differentiation at a distance by means of (1) a general change 268 CHAPTER 30 BRACHY T BRACHY T+ 50% 25% I ^p-i^ 25% die 10^4 days MONSTER TT n days 16 days ^ birth BRACHY T+ NORMAL + + (Size Reduced) FIGURE 30-5. Brochyiiiy in the house mouse. {Courtesy ofL. C. Dunn ; re- printed hy permission of McGraw-Hill Book Co., Inc., from Study Guide and Workbook for Genetics by I. H. Herskowitz. Copyright, 1960.) in metabolism (starvation of the Creeper eye) or by means of (2) specific chemical messen- gers (mouse pituitary hormone). Tissues, being acted upon at a distance, can have their competence changed for genetic reasons (homozygous Creeper limbs). It was also found that adjacent tissues, which interact as induction systems, can have their diff'erentia- tion modified by changes in their response to inducing agents (nonresponsiveness of meso- derm to presumptive notochord in homo- zygous Brachy), and, although no example of this was described, probably, also, by changes in inducing capacity. It should be realized, however, even though differentiation during the development of higher multicellular or- ganisms involves intercellular interactions of all the types mentioned, that some cellular traits are produced solely through the intra- cellular action of the genotype. This is clear, for example, from the phenotypic effect of mutants induced during embryology which may be detrimental or lethal to the cells con- taining them, and from traits, as in Drosoph- ila, showing phenotypic mosaicism in direct correspondence with genotypic mosaicism. Developmental Genetics 269 SUMMARY AND CONCLUSIONS Phenogenetics, the study of how genetically determined phenotypes come into being, can be investigated for morphological traits. In this case, phenogenetics starts out as a study of the developmental genetics of morphology. Such studies reveal that the final morpho- logical outcome, which is usually a pleiotropic one, is based upon earlier morphological changes which are in turn preceded by physiological changes occurring still earlier in develop- ment. The developmental genetics of morphological features thus becomes explicable in terms of gene-caused physiological changes, or by means of the study of physiological genetics. The latter studies reveal that the physiological effect of the genotype is sometimes intra- cellular and sometimes intercellular. The action certain cells have on cells located elsewhere may involve a general or localized control of growth rates and differentiation. This action may occur nearby, via induction, or it may take place at a distance, by means of a general nutritive effect, or by hormones (or nerve impulse or muscular contraction). It is also found that the reacting tissue may have its competence to respond to these influences changed for genetic reasons. Comprehension of physiological genetics must, in turn, involve an understanding of how genes influence metabolism, and since metabolism involves the study of physical and chemical reactions, phenogenetics must ultimately be described in biophysical and biochemical terms. You may recall, from Chapter 10, that this was the path taken (morphology to physiology to biochemistry) in the phenogenetic study of the gene for sickle cell anemia. REFERENCES Beutler, E., Yeh, M., and Fairbanks, V. F., "The Normal Human Female as a Mosaic of X-Chromosome Activity; Studies Using the Gene for G-6-PD-Deficiency as a Marker," Proc. Nat. Acad. Sci., U.S., 48:9-16, 1962. Gluecksohn-Waelsch, 1951. S., "Physiological Genetics of the Mouse," Adv. in Genet., 4:2-49, Goldschmidt, R. B., Theoretical Ge- netics, Berkeley, University of California Press, 1955. Hadorn, E., Developmental Genetics and Lethal Factors, New York, John Wiley & Sons, 1961. Landauer, W., "On the Chemical Pro- duction of Developmental Ab- normalities," J. Cell Comp. Physiol., 43 (Suppl.):261-305, 1954. Waddington, C. H., Organizers and Genes, Cambridge University Press, 1947. Wright, S., "The Physiology of the Gene," Physiol. Rev., 41:487- 527, 1941. Richard Benedict Goldschmidt (1878-1958). (By permission of Ge- netics, Inc., vol. 45, p. 1, 1960.) 270 CHAPTER 30 QUESTIONS FOR DISCUSSION 30. 1 . In what way does the study of genes help us understand normal embryonic develop- ment? 30.2. If most somatic cells have the same genetic content, why do different cells differentiate in different ways? 30.3. In what ways can genes regulate embryonic development? 30.4. Do the studies of Creeper, of Brachy, or of pituitary dwarfism in mice offer any support for the view that most, if not all, genes have a single primary effect? 30.5. What is the relationship between phenogenetics, developmental genetics, physiological genetics, and biochemical genetics? 30.6. Discuss the comparative importance of genes that act earlier, as compared with those which act later, in development. 30.7. Do you suppose that all genes act at all times in all cells of the body? Why? 30.8. "This Chapter tells more about development than it does about genes." Do you agree? Why? Chapter *31 BIOCHEMICAL GENETICS (I) li "t was stated earlier (p. 16) that the nucleus is known to be very .active chemically. While this con- clusion can be justified by abundant evidence, let us consider one particular line of support at this time. It is possible ^ to remove the zygotic nucleus of a fertilized frog egg by microsurgery. The enucleated cell, so pro- duced, cannot perform normally the func- tions of maintenance, growth, cell division, and differentiation, and eventually undergoes degeneration because of the failure of normal metabolism — the normal chemical reactions necessary to carry on such functions. That the metabolic failure is attributable to the loss of the nucleus, rather than being an effect of the operation, can be proven by the fact that zygotes undergoing similar operations with- out being enucleated subsequently show normal behavior. Most important, more- over, is the fact that the same or a similar nucleus can be replaced in a second opera- tion, which is then followed by normal zygotic activity. We may conclude, therefore, that the nucleus is essential for normal metabo- Hsm, that is, for the cell's normal chemical activity, which has as its consequences cell maintenance, growth, multiplication, and differentiation. Let us make the simplest assumption, namely, that the nuclear components which are essential for normal metabolism are the genes in the chromosomes. Let us presume also, as the simplest explanation, that all of the features of metabolism which are unique ^ Based upon work of R. Briggs and J. T. King. 271 to cells are the direct or indirect consequence of genie action. On this basis, then, all as- pects of the phenotype having a genetic origin are founded upon the biochemical effects of genes. We would predict, because of the presence of numerous chemical substances in a cell, that one gene-initiated biochemical reaction would usually lead to others, which in turn would lead to still others, to form a kind of tree, whose successive branchings represent successive chemical reactions. Since all the branches would have been affected by the initial gene-caused biochemical change, one should find many different chemical, and/or physiological, and/or morphological consequences of it in the fully developed cell or individual. It would not be surprising to find, therefore, that a given genetic change has many different effects upon the phenotype, and that most, if not all, mutants have mani- fold or pleiotropic effects (see Chapters 10 and 30). It would also be expected, when these pleiotropic effects are traced back toward their origin, that the many different end effects would be found to be the conse- quence of fewer earlier-produced effects. Moreover, in tracing this pedigree of causes back toward its genie origin, we would also expect the more primary causes to be based upon metabolic changes, changes sometimes identifiable with modifications of particular chemical substances (such as hemoglobin in Chapter 10, or pituitary hormone in Chap- ter 30). With this orientation in mind, let us at- tempt a study of the biochemical basis of gene action, an area of investigation which we can label biochemical genetics. Where shall we look for information regarding the biochemical basis of gene action? It might be fruitful to study a trait like eye color, which itself is describable in terms of chemical sub- stances, pigments, for there might be, in this case, a relatively short series of steps to trace back before arriving at, or near, the primary gene-caused biochemical changes. 272 CHAPTER 31 You may recall (p. 53) that the dull-red eye color of wild-type Drosophila is due to the presence of both brown and red pigments. There are a large number of allelic and nonallelic point mutants that change eye color. Homozygotes for the mutant brown, bw, which are otherwise genetically wild-type, have brown eyes because they cannot make the red pigment. On the other hand, mutant individuals pure for vermilion, v, or for cinna- bar, en, cannot make the brown pigment, and so, even though otherwise genetically wild-type, have bright red eye color. Of course, flies pure for bw, and either v or en, have white eyes, since they cannot make either the red or the brown pigments. A series of investigations was initiated ^ to determine the effect that transplantation of prospective eye tissue has upon the type of eye color it later shows. The larva of Dro- 2 By G. W. Beadle and B. Ephrussi. FIGURE 31-1. Results of transplanting eye anlage between Drosophila larvae. sophila contains prospective compound eye tis- sue in the form of anhf^en, or imaginal discs. It became feasible to transplant an eye anlage from one larva into the body cavity of another and have development continue to comple- tion. When the adult emerged from the pupal case, its own eye color and, after being dug out, the eye color of the implant, could be scored. It was found that if an implant was genetically the same as the host, all eyes developed the expected color (Figure 31-1, E). Moreover, the eye anlage of most mutants, when transplanted into wild-type larvae, de- veloped the mutant eye color, even though the host eyes were wild-type. In these cases, therefore, the implant developed autono- mously, and the host provided nothing which modified the development of the mutant phenotype in this respect. There were two exceptions, however. If either v or en discs (from homozygotes for V or en) were transplanted into a wild-type host (B into A in the Figure), they developed into eyes with the dull-red color of the wild- type. In these cases, then, the wild-type host supplied something which the transplanted anlage needed to develop brown pigment. It was shown that what the wild-type host contributed was a diffusible substance. This substance itself was not brown pigment, but could be utilized metabolically by the implant to synthesize brown pigment. It is a reasonable presumption, therefore, that en and v are defective in the produc- tion of some substance which is present in wild-type, and which is essential for the subsequent formation of brown pigment. Three kinds of explanations are possible for the two exceptional transplantation results. First, en and v may be defective in the same way and fail to produce the same chemical precursor of brown pigment. If this is so, then reciprocal transplants of eye anlage between en and v larvae should produce only the mutant (bright red) eye color. But, it is also possible that the en and v mutants are Biochemical Genetics (/) 273 defective in different ways. In this event, a second explanation can be based upon the possibihty that the biochemical effects of the two different mutants are produced independ- ently of each other. This can be visualized in the following way. Suppose that brown pig- ment has as two of its precursors a v+ substance produced by the v+ gene and a c/i+ substance produced by the cn+ gene. If these sub- stances are produced independently of each other, then a v/v cn+jcn'^ larva should produce only cn+ substance, and a v+/v+ cn/cn larva only v+ substance. Reciprocal eye anlage transplants between these two larvae should produce wild-type eye color in the implants, if both precursors are diffusible, since which- ever of the two precursors is missing in the implant will be furnished by its host. Thirdly and finally, the two precursors of brown pigment may be different but related to, or dependent upon, each other in their production. The amount of relation or de- pendency between the two precursors might be of several kinds, and of almost any degree — ranging from almost complete independ- ence to complete dependency. Dependency would be complete and unambiguous if the formation of one precursor had to precede the formation of the other precursor. Sup- pose, to make this example specific, that the hypothesized cn+ substance is synthesized from the hypothesized v+ substance. This means that v+ substance is a precursor of cn+ substance. In this particular case, what should be the result of implanting a v anlage in a en host? The host (v+/v+ cn/cn) manu- factures v+ substance (which the implant, v/v cn^jcn'^ cannot make) ; the implant can convert this into cn+ substance which, in turn, can be converted to brown pigment. This pigment together with red would make the implant eye wild-type. What is the result expected from the reciprocal transplant of a en anlage in a v larva? Since the implanted en disc (v+/v+ cn/cn) lacks the ability to make cn+ substance, and does not receive this from the v host {v/vcn+/cn+), no brown pigment should be formed and the transplant should form a bright red eye color. When the reciprocal transplantations actu- ally were made (D into C, and C into D in Figure 31-1) the specific results last hypothe- sized were obtained; that is, a en disc in a v host remains cinnabar, while a v disc in a en host becomes wild-type. Such results also rule out the first two types of explanations presented. What we are presumably dealing with is a chain of chemical reactions (Figure 3 1-2 A) involving a minimum of four chemical substances: a precursor (1) which through the action of gene v+ is converted to v+ substance (2) which is converted by gene cn+ into cn+ substance (3), which is in turn converted to brown pigment (4). Note that v blocks the chemical pathway to brown pigment at an earlier stage than does en. The transplanta- tion results further support the concept that the chemical intermediates, formed prior to FIGURE 31-2. C/iain ofcfiemical reactions involved in the formation of brown eye pigment in Drosophila. A. Precursor -^ v^ Substance -^^ cn"^ Substance 4 Brown " Pigment Trypto- phan Kynurenine 3-Hydroxy- kynurenine Brown Pigment 274 CHAPTER 31 a blocked reaction, accumulate (as v+ sub- stance accumulates in a cinnabar larva), and can be used by a mutant that cannot form these intermediates (as does a v disc implanted in a en host). Subsequent work has confirmed these hypotheses and has elucidated the specific chemical nature of some of the substances involved; these are indicated in the appro- priate position in Figure 31-2B. It was found, for example, that the v+ substance is kynurenine, and that the cn+ substance is 3-hydroxykynurenine. Note that the v and en genes apparently block adjacent reactions in a sequence of reactions. You may ask next, by what mechanism do these mutant genes block these chemical reactions, or, con- versely, by what means do the normal genes v+ and c«+ accomplish the chemical conver- sions into kynurenine and 3-hydroxyky- nurenine, respectively. Kynurenine is converted to 3-hydroxyky- nurenine by the addition of a hydroxyl (OH) group. We can imagine two general ways that c«+ could accomplish this. One might involve the manufacture by c«+ of a unique, not otherwise produced, hydroxyl-containing substance capable of reacting with kynurenine to produce its chemical conversion. The other mechanism would presume that ky- nurenine and this hydroxyl-containing com- pound normally are both present in the same cell but react together in the desired way either not at all or with negligible frequency in the absence of cn+. In this case, the pres- ence of cr& would serve to expedite the chemi- cal reaction, so it occurs with the required frequency and speed. There are two unique features of cells known to be involved in expediting (or hin- dering) chemical reactions, features which are absent from nonliving mixtures of chemical substances. One of these involves the organi- zation of certain cell components into struc- tures {intracellular organelles) whose parts have specific physical relationships to each other. It is possible that different genotypes produce structural changes in the organelles so that a chemical reaction which could take place in one genotype is physically impossible in another genotype, because the reactants are spatially separated, and vice versa. The second unique feature for the regulation of metabolism in living material (protoplasm) is the occurrence of enzymes, organic catalysts which speed up chemical reactions between other chemical substances. It is also possible, then, that one genotype may have less, or none, of an enzyme produced by another genotype. Since many enzymes are known to be located in, or to comprise part of, organelles, it should be realized that a genetic change might result in a change in both struc- ture and enzyme simultaneously. It is also possible that the manufacture of a unique substance, which may be of a nonenzyme nature, might affect cell structure and/or enzyme formation. In fact, it is possible that a change in any one of these three kinds of effects could automatically involve the others. Can we eliminate any of these three ex- planations for gene action in the case of v+ and cn+? Even though kynurenine is diffus- ible between one cell and another, and there- fore within a cell also, we cannot eliminate a purely spatial change in the structure of some organelle as the primary effect of the en mu- tant. Moreover, it is also possible that en fails to manufacture some unique hydroxyl- containing substance to combine with ky- nurenine, or that it fails to produce the specific enzyme required to couple already present hydroxyl to kynurenine. Let us discuss next the biochemical genetics of certain traits in human beings, in the hope that we may be able to obtain more evidence concerning the mechanism whereby genes act to direct the chemical activities of protoplasm. There is a rare condition in man characterized by the fact that, beginning at birth, the urine, though normal in color when passed, soon Biochemical Genetics (/) 275 darkens on contact with air and turns from light to dark brown and finally to black. This characteristic persists throughout the life of the individual. Affected persons are not otherwise greatly inconvenienced, al- though in middle or old age there is a greater tendency to develop arthritis. Older affected individuals may also show a bluish discolora- tion of their nose, ears, and knuckles, so that cartilage, tendons, and even bones can de- velop this pigment. Family, pedigree, and population studies reveal (1) that normal parents may have affected children of either sex, (2) several siblings from normal parents may be affected, (3) affected children appear with a much higher incidence when their normal parents are related than when they are unrelated. In view of these results, and the fact that the blackening of the urine is expressed fully or not expressed at all, it can be concluded that affected individuals are homozygous for a single pair of autosomally linked genes. The blackening is known to be due to the oxidation of a substance present in urine called alcapton, or homogentisic acid, whose chemical description is 2,5-dihydroxyphenyl- acelic acid (cf. Figure 21-3). The disease is called alcaptonuria,^ and affected individuals are alcaptonurics. It should be noted also that several pedigrees have been found in which apparently the same phenotype is attributable to the action of a single dominant gene. It is not surprising that the same bio- chemical phenotype can be produced by two different genotypes, for we have already found this to be true in the failure of brown pigment formation in Drosophila. Since dominant alcaptonuria has not been studied extensively biochemically, we shall confine our attention henceforth to the recessive form of this disease. Alcaptonuria is clearly an inborn error of metabolism, and results in the daily excretion ^ The account following is based upon the work of A. E. Garrod and subsequent investigators. of several grams of alcapton. A study of the biochemistry of alcaptonurics shows that no other substance, among numerous others tested for, appears in abnormal quantities in the urine or blood, and that the reducing properties of the urine can be attributed en- tirely to the alcapton it contains. From these results it would seem that we have traced the pedigree of causes back to, or very close to, the primary effect of the gene. The question we may ask is whether the abnormal gene manufactures alcapton, or affects organelles, or modifies enzymes. If alcapton is a substance produced by the abnormal gene, it should be absent in homo- zygotes for the normal allele. Now, when alcaptonurics are fed five grams of alcapton, approximately this additional amount is excreted in the urine. But when normal individuals are fed the same quantity of al- capton, no alcapton is found in the urine. If, however, normal individuals are fed eight grams of alcapton, some alcapton is found in the urine. We can conclude from this that normal people have the abihty to metabolize alcapton to another form which does not become pigmented upon exposure to air, but that this ability has been lost, apparently completely, by alcaptonurics. So, the ab- normal gene does not produce its effect by forming alcapton as a unique substance. Apparently alcapton is a normal product of metabolism which does not accumulate in normal individuals because it is further metabolized rapidly, but which does accumu- late in alcaptonurics because of an organelle or enzymatic defect. Clear evidence has been obtained that it is an enzyme which has been changed in the abnormal genotype. For it has been found that the blood of alcaptonurics is deficient in an enzyme, normally present, which catalyzes the conversion of alcapton by oxidation to a noncolor-producing sub- stance, and this enzyme, homogentisate oxi- dase, is in fact missing in the liver of the al- captonuric. 276 CHAPTER 31 If alcapton is not produced by the gene for alcaptonuria, but is a normal metabolic inter- mediate, it may have chemical precursors. If such a chemical precursor is added to the diet of alcaptonurics, it should be converted to alcapton which should be excreted in increased amounts. When alcaptonurics are fed an excess of glucose, the amount of al- capton found is unchanged, indicating that glucose is not a precursor of alcapton. But, if p-OH phenylpyruvic acid, or if either of the amino acids tyrosine or phenylalanine is increased in the diet of alcaptonurics, their excretion of alcapton is increased almost correspondingly. Accordingly, it can be postulated that there is a series of chemical precursors of alcapton (Figure 31-3). In the scheme illustrated, phenylalanine is nor- FIGURE 31-3. Sequence of chemical substances involved in the formation and metab- olism of alcapton. 4 = homogentisate oxidase, 5 = isomerase, 6 = hydrolase. H-C II H-C %. I C-H C— H H-C-H I H— CNH2 COOH Phenylalanine H— C II H-C. H- ./ OH I C.v -H C- I H— CNH2 COOH Tyrosine OH I /^^ H-C C— H II I H-C. X— H C I H— C— H I COOH p-OH phenylpyruvic acid la HOOC • CH II H-C CO I CH2 CO H-C II H-C O II CH, I C— CH2 • COOH Fumaryiacetoacetic acid ^c II o o o H H I -C— COOH I H H-C II H-C OH I C, ^C^ I OH HH I — C— COOH I H HOOC-CH II HC-COOH Fumaric acid Maleylacetoacetic acid CH3 CO I CH2-COOH - Acetoacetic acid Alcapton (Homogentisic acid] CO2 -t- H2O Biochemical Genetics (/) 277 mally converted to tyrosine by the addition of an oxygen to the top carbon; tyrosine is normally converted to p-OH phenylpyruvic acid by replacing the amine (NH2) group by an oxygen; p-OH phenylpyruvic acid is converted, by still other chemical reactions, to alcapton. Alcapton is normally converted to acetoacetic acid, by a process which in- volves the splitting-open of the benzene ring; it is the first step in this conversion which fails in alcaptonurics. This hypothesized pathway from phenylalanine via alcapton to acetoacetic acid has been confirmed in sub- sequent work, as the result of which six en- zymatically catalyzed steps have been identi- fied. It should be realized, however, that tyro- sine, which is an essential component of pro- tein, can also partake in biochemical path- ways other than the one that leads to alcapton (Figure 31-3). It has been found, for ex- ample, that tyrosine is part of the pathway of chemical reactions leading to melanin formation. So, tyrosine, by a different chemi- cal pathway, is also a precursor of melanin. Albinism (lack or absence of melanin) could be caused genetically by the defective pro- duction of an enzyme necessary for the con- version of tyrosine to melanin. In another disease, which is due to a single rare recessive gene, affected individuals are feebleminded, or of lower than normal mental ability, and have other phenotypic changes, including light pigmentation. This pleiotro- pism has been directly correlated with the presence of phenylpyruvic acid, which is toxic, in the urine of affected individuals. It has been shown that the normal conversion of phenylalanine to tyrosine fails to occur in affected individuals, and instead, the amine in phenylalanine is replaced by an oxygen (thus forming a keto group), so that phenyl- pyruvic acid is produced (Figure 31-4). Diseased persons are therefore phenylpyruvics or phenylketonurics (see Chapter 27). The disease, phenylketonuria, can be partially / FIGURE 31-4. Formula for phenylpyruvic acid. H-C C-H II I H-C. ^C-H C I H— C— H I c=o COOH alleviated, or circumvented, if dietary phenyl- alanine is reduced to an amount that is enough for protein synthesis (for the presence of this amino acid is also essential in our proteins), but not enough so that any appreciable amount is converted to phenylpyruvic acid. Note that since tyrosine is also needed for human protein, this substance must also be included in sufficient quantity in the diet of phenylketonurics. Finally, it should be noted that a parahydroxylase enzyme that converts phenylalanine to tyrosine and is normally present in the liver has been found missing, or defective, in phenylketonurics. What is meant when an enzyme is said to be missing? This may mean either the total absence of the enzyme molecule, or else its presence in such a modified form that the molecule has lost the ability to perform its characteristic catalysis. These studies of metabolic defects have been of great service in identifying the places where genes act to direct metabolic processes. They also permit the determination of pre- cursors of a genetically defected step, and aid in the final elucidation of chains of biochemi- cal reactions, and of metabolic pathways. For example, substance X is proven a pre- cursor of substance Y, if mutant 1 cannot form Y but accumulates X, and if mutant 2 cannot form Y unless X is supplied (Figure 31-5). But biochemical genetics is of especial interest in another respect. We have seen, in the cases most thoroughly investigated, that 278 CHAPTER 31 FIGURE 31-5. Determination of precursors using mutant genes. A accumulates X but makes no Y. B makes no X but will make Y if X is supplied. C is the normal pathway. mutant 1 B mutant ? X added -^Y -^Y it has been possible to trace the pedigree of causes back to a point at which only one effect of the gene can be detected. This was true, for example, in the case of alcaptonu- ria. It is still possible (though improbable) that further study of this case would reveal that the gene for alcaptonuria, besides pro- ducing an effect upon the particular enzyme that metabohzes alcapton, has another effect which has no determinable relation to the enzyme effect. Such a finding would suggest that this gene acts upon the phenotype in more than one primary way. In most of the book up to this point, we have employed the phenotypic effects pro- duced by genetic differences to reveal the nature of the genetic material. Nevertheless, the properties of segments of the genetic material were described not in terms of pheno- typic effects, but in terms of recombination and mutation. In this Chapter and certain preceding ones (particularly those Chapters not marked by asterisks), we were concerned more with the nature and consequences of the phenotypic effects produced by the genetic material than we were with discovering the recombinational and mutational properties of this material. What have we learned about the way that the genetic material produces its phenotypic effects? We could cite abundant evidence that the genetic material does not act upon the phenotype as one single indivis- ible unit, but acts as though it is segmented into a number of units, or genes, each of which has a specific phenotypic effect. (Such units or genes for phenotypic effect were re- vealed because mutation made changes in genes, these different genes underwent re- combination, and served, in some way, to produce different phenotypic effects.) The simplest hypothesis is that the genetic unit of phenotypic effect is identical to the genetic unit of recombination, for in all our previous discussion the recombinational gene seemed to be identical with the phenotypi- cally functional gene. But, it must be em- phasized, because these two views of a gene are revealed through the use of different opera- tional procedures, that the gene as a unit of phenotypic effect, the functional gene, could be larger than, identical to, or smaller than the recombinational gene. (We have already noted, on p. 199, the possibility that the seg- ment of the genetic material defined by the recombinational gene may not be identical with the material affected when a single re- combinational gene undergoes mutation.) Our study of the genetic material revealed that it contains several different sized units of genetic recombination, including the genome, chromosome, and chromosome seg- ment. The term gene came to be used to refer to the smallest unit of the genetic ma- terial capable of undergoing recombination. The study of the genetic material also showed there were different sized units of mutation — the genome, chromosome, and chromo- some segment. It should now be apparent that we ought to embark upon a study to determine the smallest unit of the genetic material capable of producing a phenotypic Biochemical Genetics (I) 279 effect. This, too, can be called a gene. Now that we have genes which are to be defined by at least two different operations, it becomes inconvenient for the reader to have to decide from context, each time the term gene is used, whether the gene referred to is defined by recombination or by phenotypic effect. Accordingly, let us give these two genetic units specific names, even though we have no evidence so far that they differ in their material basis. We can define a recon as the smallest unit of the genetic material which is capable of recombination, and a cistron as the smallest unit of the genetic material capable of producing a phenotypic effect. When the less specific, nonoperational, term gene is used, henceforth, it will refer to the smallest unit of the genetic material as identified by these, and especially other, operations. Note that the recon, or cistron, is not de- fined in terms of properties which are beyond experimental test (see p. xi). For example, although the recon is part of the genetic ma- terial, we do not endow it with assumed or established properties of the total genetic material. Moreover, the recon is not the smallest unit of the genetic material capable of self-replication, of mutation, or of pheno- typic influence. Notice also that there is no restriction as to the lower limit for the size of the recon or the cistron. The study of biochemical genetics in the present Chapter (and also in Chapter 10) leads us to make the simplest hypothesis, namely, that a cistron produces only a single primary phenotypic effect, and that all its pleiotropic effects are consequent to this single activity. We can call this hypothesis a one cistron-one primary phenotypic effect relationship. If this hypothesis can be further tested and supported, we may be able to use the information gained in reverse, so to speak, to learn more about the size or scope of the genetic material required to produce a single primary effect. This would give us informa- tion regarding the nature of a cistron. But you should realize that the kind of informa- tion we obtain about the cistron will depend upon what we consider to be primary and what we identify as a phenotypic effect. SUMMARY AND CONCLUSIONS While the genes detected by recombination and by phenotypic effect are related to each other, their material basis need not be identical. The recon and cistron are defined, re- spectively, as the smallest unit of the genetic material capable of recombination, and of phenotypic effect. The biochemical activities necessary for the existence of protoplasm are controlled by the nucleus, presumably by the cistrons it contains. These chemical reactions occur in sequences that form often-branched metabolic pathways leading to the chemical, physical, physiological, developmental, and morphological aspects of the phenotype. As a conse- quence of this branching, most, if not all, cistrons have pleiotropic effects. The phenotypic differences produced by alternative cistrons can be traced by a pedigree of causes back toward the cistron. Such studies demonstrate that cistrons produce their effects at the metabolic level. It is reasoned that such action could involve the structural components of protoplasm (organelles) or particular chemical substances, including enzymes specifically, aUhough none of these effects need exclude any of the others. The study of inborn errors of metabolism in man demonstrates that cistrons control various steps in biochemical sequences, through their influence upon enzymes. At least in these particular cases, the effect on enzymes can be supposed to be the primary and only consequence of the action of a segment of the genetic material. In view of the experimental results, a one cistron-one primary effect relationship is hy- pothesized. 280 CHAPTER 31 REFERENCES Beam, A. G., "The Chemistry of Hereditary Disease," Scient. Amer., 195:126-136, 1956. Ephrussi, B., "Chemistry of 'Eye Color Hormones' of Drosophila," Quart. Rev. Biol., 17:327-338, 1942. Harris, H., Human Biochemical Genetics, Cambridge University Press, 310 pp., 1959. Hsia, D. Y.-Y., Inborn Errors of Metabolism, Chicago, The Year Book Publishers, 1959. Wagner, R. P., and Mitchell, H. K., Genetics and Metabolism, New Yoric, John Wiley & Sons, 1955. Harriet Ephrussi-Taylor {see p. 341), Boris Ephrussi, and Leo Szilard (see p. 336) at Cold Spring Harbor, N.Y. in 1951. {Courtesy of the Long Island Biological Association.) QUESTIONS FOR DISCUSSION 31.1. List five diseases in human beings that are caused by inborn errors of metabolism. 31.2. In what respect can an inborn error of metabolism be cured? 31.3. Do all mutations produce inborn errors of metabolism? Explain. 31.4. What evidence can you present that cistrons control different steps of a bio-synthetic sequence of reactions? 31.5. Has our study of recons and recon mutation been dependent upon the concept of a cistron? Explain. 31.6. Do you suppose that proof of the one cistron-one primary effect hypothesis would reveal anything regarding the chemical properties of a gene? Explain. 31.7. From which of these areas of investigation would you expect to obtain the most information regarding the cistron — morphology, physiology, biochemistry? Why? 31.8. Do you think that the concept of the cistron has any consequences for the practice of medicine? Explain. 31.9. In what way is our study of the cistron related to, or dependent upon, mutation ' and recons? 31.10. Discuss the evidence relative to the diffusibility of v+ substance and cn+ substance. Chapter *32 BIOCHEMICAL GENETICS (II) I "t has been hypothesized that the cistron has a single, primary effect .on the phenotype. This hypothe- sis is of value insofar as it provides motiva- tion for studies aimed at determining whether a given segment of genetic substance has, in fact, a single, primary action. We found, in the last Chapter, that investigations of specific genes, causing inborn errors of metabolism, gave support to this view. There is an addi- tional implication of the one cistron-one primary effect hypothesis. If we knew the nature of such a primary effect it should always prove to be the product of one cistron. It may be possible to subject this prediction to test also. In order to do so it will be neces- sary to decide which particular aspects of the phenotype are primary products of cistronic action. We have already discussed, in the previous Chapter, cases where the primary effect of a mutant cistron is upon the capacity of an enzyme to catalyze a particular reaction. Since different enzymes catalyze different reactions, each is said to show specificity of action. The mutants referred to resulted in a change in enzyme specificity. Let us pre- sume that the specificities of all enzymes found in protoplasm are the result of the pri- mary action of cistrons. If this is so, then it ought to be possible to study any particular enzyme and find that its specificity can be changed for genetic reasons. Let us proceed to study this hypothesis experimentally as a specific, if limited, test of the more general concept, one primary effect-one cistron. 281 We have already described the use of Neurosponi as material for studies of genetic recombination (beginning p. 121). Certain characteristics of Neurospora also make the organism very favorable material for bio- chemical studies. Neurospora can manufac- ture all of the components it needs for exist- ence and reproduction from a very simple, basic, food medium. This basic medium may consist solely of water, an array of inorganic salts, including sources of nitrogen, phos- phorus, sulfur, and various essential trace elements, a carbon and energy source such as cane sugar, and a single vitamin, biotin. From these raw materials it can synthesize some 20 different amino acids, all essential vitamins (but biotin), compounds like purines and pyrimidines, and everything else it needs for its total activity. According to the hy- pothesis under consideration, it should be possible to induce mutations in cistrons, which should then fail to correctly specify enzymes, so that various chemical syntheses should become blocked. Previous work has established that the last step in the synthesis of vitamin By, or thiamin, is normally accomplished by the enzymatic combination of a particular thiazole with a particular pyrimidine. If enzymes owe their specificities to cistrons, it should be possible to induce a mutation in the cistron that normally specifies this Bi-forming enzyme. If the mutant no longer specifies active Bi- forming enzyme, no Bi will be made. Then, since Bi is required for growth, the mutant mold will require Bi in its diet in order to grow. What is done ^ is to obtain haploid spores (produced asexually by the haploid organism grown from an ascospore) and treat them with a mutagenic agent (like X rays or ultra- violet light). The treated spores are then grown on the basic medium which has been supplemented with vitamin Bi. Under these circumstances the spores that grow will in- ^ Based upon work of G. W. Beadle and E. L. Tatum. 282 CHAPTER 32 elude those which can still make Bi them- selves, and those which can no longer make it, but which obtain Bi from the culture medium. Once the spores have grown sufficiently, a portion of each of the haploid growths can be placed on the basic, minimal medium, which contains no Bi. Those samples that continue to grow when transplanted to mini- mal medium are presumably nonmutant, in respect that they can perform all the biochemi- cal steps leading to Bi production; those samples that fail to grow presumably carry mutants involving failure to make Bi. Those failing to grow might be mutants which lack a precursor of Bi, a precursor whose presence as such is not essential for growth, but which is essential for subsequent Bi synthesis. Any mutant of this type can be eliminated from further consideration if it grows on minimal medium supplemented with the particular thiazole and pyrimidine which are the imme- diate precursors of vitamin Bi. (All other imaginable nutritional factors except Bi itself may also be added, but they would have no effect on the decision being made.) The residue of cultures, which grow on medium containing Bi but not on medium containing the immediate precursors of Bi, are clearly defective for the enzyme that catalyzes the last step in Bi synthesis. Each of the haploid strains remaining is then crossed to a haploid strain normal for Bi synthesis, and each diploid hybrid is studied separately as follows. After the hybrid under- goes meiosis (refer to pp. 121-124), a sac containing eight haploid ascospores is pro- duced. Each of the eight spores is removed and grown on Bi-supplemented minimal medium. If the haploid strain being tested is indeed Bi-deficient because of a mutant, a subsequent transplant of each of the eight haploid cultures to Bi-free minimal medium will produce exactly four that can grow and exactly four that cannot. Note that because all four products of meiosis are recovered and tested, each in duplicate, the 4 : 4 ratio is purely mechanical and is not subject to errors of sampling, as it would be if a random sample of spores were taken from a large pooled lot made up by mixing the spores from many sacs. When the experiment was per- formed, mutants were found. These had changed the specificity of the enzyme studied, as expected on our hypothesis. If, for a given mutant, a number of spore sacs are tested as described, it is also possible to map the location of the mutant relative to the centromere of the chromosome in which it is located. The way in which this is determined is illustrated in Figure 32-1, which shows only the single pair of chromo- somes involved. If, as shown in the left portion of the Figure, no chiasma occurs be- tween the loci of the mutant and the centro- mere, segregation of normal (+) and mutant {th) recons will occur at the first meiotic divi- sion, and, because the last two divisions in the ascus are tandem to the first, the eight ascospores will be in the relative order + + + + //2 ?/7 th th. If, however, a single chiasma does occur between the mutant and the centromere, as shown in the right portion of the Figure, segregation will occur in the second meiotic division, and the ascospores will be in the relative order + + th th + + th th. If a record is kept of the order of the spores in each ascus, then these two segregation arrangements can be identified when the spore genotypes are determined later. If 20% of all sacs showed second division segregation (two + spores alternating with two th spores), then 20% of the meiotic divisions had a chi- asma between the mutant and the centro- mere. This, you remember, means that the mutant is located 10 map units from the centromere. When a number of separately occurring point mutants, defective in the enzyme cata- lyzing the last step in Bi synthesis, were local- Biochemical Genetics {II) 283 TETRAD T + S 1^ > th ^ jk th FIRST MEIOTIC DIVISION v^ y SECOND MEIOTIC DIVISION /^ th S v^ J /^ ^ s \^^^ y /^ th ^ + 8 Spores + th MITOTIC th DIVISION + + th th 8 Spores + + + + th th th th FIGURE 32-1. Arrangement of spores in t/ie Neurospora ascus when segregation occurs at the first meiotic division {left) and at the second meiotic division {right), as determined by the absence and presence, respectively, of a chiasma between the segregating genes and the centromere. ized this way, they were all found to be on the same chromosome and appeared to be the same distance from the centromere. This suggests that the specificity of a particular enzyme is the result of the action of a particu- lar cistron. For the efficient detection of biochemical mutants in Neurospora, certain modifications are made in the procedure already outlined. Potentially mutant spores are grown on a medium supplemented with all substances which might conceivably be involved in bio- 284 CHAPTER 32 chemical mutation. Growing cultures are then transferred to basic (minimal) medium containing no additions, where failure to grow indicates a mutant culture which has lost the ability to synthesize some component added to the basic medium. The specific ability lost is determined by testing for growth in basic medium supplemented in turn with the individual enriching components of the complete medium. Techniques have been developed also to selectively eliminate non- mutant strains. Thus, spores given an oppor- tunity to grow on minimal medium can be subjected either to filtration, which separates the larger (growing) nonmutant cultures from the smaller (nongrowing) mutant ones, or to an antibiotic which kills actively growing cultures, but which has less or no effect on nongrowing ones. In this way, the sample later tested for mutants can be mutant- enriched. It is even possible to find mutants for unknown growth factors by supplement- ing the culture medium with extracts of nor- mal strains of Neurospora which contain various substances, both known and un- known, that are needed by the mold. The same mutants requiring unknown growth factors can then be used in the specific bio- assays needed for the isolation and identifica- tion of such substances. Such improvements in the techniques for detecting biochemical mutants in Neurospora expedited additional tests of the enzyme- cistron relationship. Two additional ex- amples will be described briefly. The first study deals with the final step in the synthesis of the amino acid tryptophan, which involves the catalyzed union of indol and the 3-carbon amino acid serine by the enzyme tryptophan synthetase. First, separately occurring trypto- phan-requiring point mutants were obtained; of these, the only ones tested were those that proved to be blocked in the final synthetic step and were lacking full tryptophan syn- thetase activity. Of 25 mutants qualifying, all proved to be located on the same chromo- some at about the same locus. The second example involved the final step in the syn- thesis of adenine which is catalyzed by the enzyme adenylosuccinase which splits succinic acid off adenylosuccinic acid to leave adenine. Of 137 independently occurring point muta- tions with little or no adenylosuccinase activity, again, all were on a particular chro- mosome at about the same locus. The cis- trons acting to specify tryptophan synthetase and adenylosuccinase are different, each hav- ing completely separate locations in the genome. These results, and similar ones for other enzymes in Neurospora, offer strong support for the hypothesis that the specificity of all enzymes is under cistronic control. More- over, different enzymes are specified by dif- ferent cistrons. The fact is observed that the addition of Bi, tryptophan, or adenine to the diet of mutants defective in the en- zymes directly responsible for their respec- tive syntheses makes the mold completely, or almost completely, normal. This is good evidence that the cistrons involved have only one function to perform, which is to deter- mine the specificity of one enzyme. If a cistron had more than one primary effect, surmounting one defect nutritionally would not be expected to produce normality or near- normality in all cases. From the results so far described, we might also be tempted to conclude that the total specificity of an en- zyme is the result of the primary action of a single cistron, because in all these cases the enzymatic defect was found to be due to a defect in one specific localized area of the genetic map. We can call this the one enzyme- one cistron hypothesis. All enzymes are protein, at least in part, and the specificity of an enzyme is known to be due to its protein content. Proteins are composed of amino acids (Figure 32-2) strung together in chains to form polypep- tides, so that the specificity of an enzyme must be attributed to the number and kinds Biochemical Genetics {II) 285 GLYCINE H H O I I II H— N — C— C— O— H I H ALANINE H H O I I II H— N H H I I VALINE H O ISOLEUCINE C — C — O — H H— N— C— C— O— H H — N i I H — C- I H I / n H — C — C— H I ^H H — C— H I H H H O I I II C — C--0— H I H— c— c; I H — C — H I H— C — H 1 H LEUCINE H H O I I II H — N — C— C— O— H I H — C — H I /H H — C — C^H I ^H H — C — H I H LYSINE H H O I I II H— N— C— C— O— H I H — C— H I H— C— H I H — C— H I H — C — H I H— N — H ARGININE H H O I I II H— N — C — C — O— H I H — C — H I H — C— H I H — C — H I N — H I C = NH I H — N — H HI5TIDINE H H O I I II H— N — C — C — O- I H— C— H I C=C— H I I H — N N \// C I H PROLINE H O I II ■H H — N — C— C— O- / \ H-C-H H-C-H \ / C / \ H H H- HYDROXYPROLINE H O I II •N— C — C— O— H / \ H-C-H H-C-H \ / C / \ H O — H SERINE THREONINE ASPARTIC ACID HHO HHO HHO I I II I I II I I II H— N— C— C— O— H H— N — C — C— O— H H— N— C— C— O— H I I I H— C — H H — C— O — H H— C — H I I I O — H H — C — H C=:0 I I H O— H GLUTAMIC ACID H H I I H— N — C I H — C — H I H— C — H I c = o I O — H O II C— O— H TYROSINE HHO I I II ■N— C — C— O— H I H — C— H I o CYSTEINE HHO I I II H — N — C— C — O- I H — C — H I S — H METHIONINE HHO I I II H— N— C — C— O— H I H — C— H I H — C— H I S I H — C— H I H CYSTINE H H I H — N O II -C— O— H ■H H C— O — H II H O TRYPTOPHAN HHO I I II H— N — C — C— O — H I -C — H I X o .H ;n— H PHENYLALANINE HHO I I II H— N — C — C— O — H I H— C— H I FIGURE 32-2. The twenty types of common amino acids. 286 CHAPTER 32 of amino acids it contains, their order in the polypeptide, the number of polypeptide chains, the way in which the parts of a poly- peptide chain are arranged relative to each other, and the way in which the different polypeptide chains in a protein are arranged relative to each other. It has been possible to study, in some de- tail, the enzyme tryptophan synthetase that is found in the bacterium Escherichia coli. This enzyme can be treated in vitro so that it dissociates into two proteins, i.e., two poly- peptide chains. By itself, neither of these chains has the usual enzymatic activity. But these two proteins can be reassociated, where- upon the normal enzymatic property is restored. Clearly, then, both portions need to be united in order to have this specific enzymatic action, so that part of the enzyme's specificity must be due to the joining of these two polypeptides. (You might suppose that this part of the specificity is due to the primary action of a single cistron.) But this must be only a portion of the total specificity of this enzyme. Another portion of it must reside in the nature of the polypeptide chains, which when joined make not just any enzyme, but tryptophan synthetase in particular. The fact that the two chains are so easily disso- ciable and reassociable indicates that these chains do not have a complex physical rela- tionship when joined together, and suggests that each chain might be specified independ- ently. In this case two distinct cistrons would be involved, each one specifying one poly- peptide chain. A number of bacterial mutants were ob- tained which lacked tryptophan synthetase activity.^ Some of these defected one poly- peptide chain and others defected the second. A genetic study showed that all the mutants defecting one chain were recombinationally separable from those defecting the other, although adjacent areas of the genetic map were involved. In this case, then, we have ^ Based upon the work of C. Yanofsky. the choice of considering the two adjacent areas either as a single cistron, or as two separate cistrons. Because the detailed specificity of this enzyme seems to depend upon what each of these two genetic areas does individually, we shall consider that two cistrons are involved. On this alternative, each cistron completely specifies a polypep- tide chain, and the union of the two chains comprising tryptophan synthetase may some- how be connected with the fact that the two cistrons involved are adjacent. How do these results, and our interpreta- tion of them, affect our general hypothesis of one primary effect-one cistron? The answer is that the general hypothesis is not at all affected. However, the specific hypothesis to test it, one enzyme-one cistron, should be made more comprehensive and be stated as one polypeptide-one cistron. This means that the total specificity of a polypeptide chain is determined by one cistron. According to the general hypothesis, the primary effect which a cistron has would be, at least in some cases, the complete specification of a polypeptide. Not all proteins are enzymes. If the hy- pothesis one polypeptide-one cistron is cor- rect, we should predict that each polypeptide chain in all proteins is completely specified by the primary and solitary action of a single cistron. Consider now certain results ob- tained by studying the protein hemoglobin.^ Human hemoglobin has a molecular weight of 66,700. In the horse, and probably in man, too, the molecule is spheroidal in shape, and its dimensions are 55 X 55 X 70 A (Angstrom units). It is composed of two half-molecules which are usually exact dupli- cates of each other. In each half-molecule there are two different polypeptide chains, called a and /3, each containing about 150 amino acids. The chains coil to form what are termed right-handed helices, and different ^ Based upon the work of V. M. Ingram, L. Pauling, H. A. Itano, H. Lehrmann, J. V. Neel, M. F. Perutz, and others. Biochemical Genetics (II) 287 chains are coiled about each other in a regular way. Each chain has an iron-containing heme group fitting into a pocket on the outer surface of the coil it makes. In the whole hemoglobin molecule, therefore, there are four heme groups, one for each of the two a and two (3 chains, and a total of about 600 amino acids. Henceforth, we shall be con- cerned with the protein, or globin, part of this molecule, since the heme groups are not involved in the variations to be considered. It has been possible to partially digest the globin portion of the molecule with trypsin, an enzyme which splits a polypeptide at every place where either of the amino acids lysine or arginine is present. This produces some 28 smaller polypeptides, or peptides, in dupli- cate (since there are two chains of each type), plus an undigested core that makes up about 25% of the globin. The 28 peptides can be separated from each other by their differential migration on filter paper when the digest containing them is subjected to an electrical field and various solvents. The result is that there are separate spots or "fingerprints" for each of the peptides on the filter paper (Figure 32-3). Each peptide (fingerprint) is given a different number, and can be further analyzed as to its amino acid content. Pep- tide 4, for example, normally contains eight amino acids in the following sequence: Val-His-Leu-Thr-Pro-G/w-Glu-Lys ' There is also evidence that the valine is an end amino acid in the /3 chain. This sequence is found in normal adult hemoglobin, called hemoglobin A. The core of globin can be digested with chymotrypsin and fingerprints obtained of its peptides. Persons heterozygous for the recon for sickling have the "sickle cell trait," which is readily detected when their red blood corpus- cles are exposed to an oxygen tension very much lower than normal, while persons "• Valine, histidine, leucine, threonine, proline, glu- tamic acid, glutamic acid, lysine. homozygous for this mutant have "sickle cell anemia," and their red cells will sickle even when the oxygen tension is not so drastically reduced. The hemoglobin of such people has been fingerprinted and analyzed as to amino acid content. The mutant homo- zygote has hemoglobin apparently identical with hemoglobin A, with the proven excep- tion that the sixth amino acid in peptide 4 has valine substituted for glutamic acid (the particular amino acid italicized in the pre- viously given sequence) (Figure 32-3). The heterozygote produces both this type of ab- normal hemoglobin, called hemoglobin S, and hemoglobin A. Previous study of the pleio- tropism of the recon for sickling (see Chapter 10) showed that all its phenotypic effects are traceable through a pedigree of causes to this single amino acid substitution in the /3 chain. This is excellent evidence that a change in the specification of a single polypeptide has been produced as a result of the change in the pri- mary action of a cistron whose nature had been changed previously by mutation. Another mutant is known that is located on the same chromosome as the recon for sickling and is probably an allele of it. This produces hemoglobin C which differs from hemoglobin A by replacing the same glutamic acid in the 0 chain, this time by lysine. Still another genetic change produces an- other hemoglobin, hemoglobin G. The amino acids in all the trypsin-produced peptides are the same as in hemoglobin A, except that the seventh one from the end in peptide 4 is glycine instead of glutamic acid. In this case, then, the amino acid sequence in peptide 4 is: Val-His-Leu-Thr-Pro-Glu-G/y-Lys Here, then, an amino acid in a different posi- tion is changed in the /3 chains. In hemoglobin E, a glutamic acid, normally found in pep- tide 26, is replaced by lysine, and it is likely that this is the only change in the whole mole- cule. Peptide 26 is also part of the ^ chain. While we have seen that there are mutants 288 CHAPTER 32 O) c::' .'-. O r^' 19%'' ^ c^o^^S ' 0 ^^ o oCJ _ + Hb-S FIGURE 32-3. The ^'fingerprints''' of hemoglobin obtained following trypsin treatment. (Courtesy of V. M. Ingram, from C. Baglioni, Biochim. Biophys. Acta, 48:392-396, 1961.) which cause a single amino acid located in different positions in the /3 chain to be re- placed by other single amino acids, the de- tailed genetic basis for the different mutants is not known as precisely, although in some cases the mutants are believed to be different alleles of the same recon. The available evidence is consistent with the view that all these mutants are at least on the same chro- mosome. Still other kinds of hemoglobin have been found in which the amino acid sequence in the a chain has been modified. This is true for hemoolobin I (in which a change occurs in peptide 23), and for hemoglobin '''Hopkins-2." A person homozygous for hemoglobin A has a molecule describable as af0-^ (see Fig- ure 32-4), one homozygous for the recon for sickling can be described a.t/3.2, and one ho- mozygous for the production of hemoglobin I Biochemical Genetics (II) 289 can be written a}^^. Hemoglobin H is pro- duced by persons homozygous for the recon for thalassemia (see p. 36) ; this type of hemo- globin has four (3 chains of A type (instead of two being a chains), so that it can be written j8f . Such homozygous persons also produce some hemoglobin A. It is also known that fetal hemoglobin, hemoglobin F, has two a chains like those in adult hemoglobin A; the other two chains are different from ^ and probably from a also, and are called y chains, so that hemoglobin F is a^J^- Finally, it is known that homozygotes for the sickling recon can make hemoglobin F which is ap- parently normal, a^y^, so that a change in the (3 chains has no effect on the y chains. The results with the recon for sickling prove that the synthesis of the nonenzymatic protein globin is cistron-directed in a primary way. What we would hke to decide is whether one or more cistrons are involved. There are several hnes of evidence which point to an independent specification of a and (3 chains: 1. Mutations that change the specifications of the (3 chain (producing hemoglobins S, C, E, G) produce no change in the a chain. 2. Mutations that change the a chain (pro- ducing hemoglobins I, Hopkins-2) pro- duce no change in the (3 chain. Further evidence consistent with the independ- ent specification of a and (3 chains comes from the study of individuals who possess both Hopkins-2 and S hemoglobins. Such individuals are known who have had one par- ent like themselves and the other of normal blood type (hemoglobin A). However, these individuals also have siblings who have Hopkins-2 but not S hemoglobin, and those who have the reverse. Accordingly, these Hopkins-2 + S persons cannot be mono- hybrid but must be dihybrid, for the abnormal hemoglobins occur both separately and to- gether in different siblings. Moreover, the number of sibHngs who must be recombinant is so large as to preclude the two mutant recons being linked very close together. We may write the genotype of these dihybrids as f^no-2 ,„A fjs f^\ Since the two recons are not very close together, they may be in different cistrons. It has been shown that the two a and the two (3 chains in a given globin molecule are identical, even in heterozygotes. If the Hopkins-2 + S individuals are dihybrid for recons in different cistrons, it would seem reasonable that the two a chains specified by ,„Ho-2 (i^j^^t ig^ a""""), or by m^'ia^), would be produced independently of the two ^ chains specified by n^([3'^), or by rt"^((S^). If so, then either product of the two different FIGURE 32-4. Normal hemoglobin and some ab- normal types due to mutation. TYPE Hgb A Hgb F Hgb S Hgb C Hgb G Hgb E Hgb I CHAINS A F ^t 3^ A E 2 ^2 Ho-2 A SPECIFIC NAME Aduir Normal Fetal Sickle cell Hgb Ho-2 oc 2 3 ^ Hgb H Hopkins No. 2 Thalassemia 290 CHAPTER 32 Qf-specifying cistrons might be found joined to either of the two different products of the j8-specifying cistrons. In this event, the di- hybrid under discussion would prove to have all four of the following types of globin: a?°--/3?, ar'^/3t, af^t a^f^t This has been found to be the case (see References at end of Chapter). The results, showing that sickle cell hetero- zygotes make normal 7 chains and defective (3 chains, suggest that three different cistrons are involved — one making a chains, one /3, and one 7. What working relationship might these three cistrons have? Subsequent to birth, the cistron that makes 7 chains normally has its action turned off, so to speak, and that for /3 chains is turned on. In the case of thalassemia major, the 7 cistron is turned off, and the /3 turned on, as in normal people, but the a cistron often fails to be turned on ; when the a cistron is turned on, hemoglobin A is produced, and when it is not, hemoglobin H (/St) is produced. (One might expect the t. major fetus to have 7^ and 0^7^ hemo- globin since the /3 chains are not being pro- duced. One would also expect that an adult with hemoglobin I, alfS^, would also pro- duce abnormal fetal hemoglobin of type All results support the view that each differ- ent polypeptide chain is specified by a single but different cistron. However, because of the rarity of heterozygotes, for mutants in different cistrons specifying hemoglobin, and the paucity of linkage data in man, it is difficult to obtain precise data on the relative positions of the different cistrons. For the same reasons, it is difficult to study the allel- ism of hemoglobin mutants affecting the same chain, which presumably involve defects in the same cistron. What kind of study would we like to be able to make in this respect? Heterozygotes for two mutants in the same cistron would usually have received one mu- tant from each parent. If the cistron was identical to, or smaller than, the recon, the genotype could be written — > the mutants would always segregate (barring mutation, including nondisjunction), and all gametes would receive either /Wi or W2 but never both or neither. This kind of result would prove the mutants were reconically allelic (see Chapter 22). If, however, the cistron was larger than a recon, being composed of a number of recons, the mutations involved would sometimes have been in two different recons of a cistron. In this case the heterozy- gote hypothesized would be reconically dihy- brid nil + and crossing over would, on rare occasions, give rise to gametes of mi m2 type and of + + type. (To obtain evidence for intracistron recombination one would prefer to test individuals who are heterozygous for both hemoglobins S and E, say, rather than those that are heterozygous for S and G. For the latter might involve adjacent recons be- tween which crossing over might be very rare, and one might fail to obtain the crossover and conclude incorrectly that these mutants were reconic alleles. One would certainly not choose heterozygotes for S and C hemo- globins for this purpose, for it is likely that these are, in fact, reconic alleles.) As indicated, the lack of appropriate num- bers of heterozygotes for mutants in the same cistron has thus far prevented us from making this test in man. This question can be investigated in Neurospora, however, which is a more suitable organism than is man for crossover studies. It was already mentioned that a large number of inde- pendently occurring point mutations, defect- ing adenylosuccinase, had been localized to the same region of a chromosome. We would like to know whether this enzyme contains only a single, very long polypeptide (which would be specified by one cistron), several polypeptides (which would be specified by several cistrons in the same general area of the chromosome), or many polypeptides. Biochemical Genetics (II) 291 Evidence has been obtained that this enzyme is dissociable into two portions. This be- havior is reminiscent of that of tryptophan synthetase, and suggests that two polypep- tides, and hence two cistrons, are involved in specifying this enzyme. It is possible, by crossing two mutant indi- viduals, to obtain progeny that are hybrid for separately arisen point mutants defecting adenylosuccinase. If these mutants are reconic alleles, and reverse mutations to adenine independence are recognized and dis- counted, the haploid ascospores produced by the hybrid will all still require adenine in the diet in order to grow. But, if the mutants involve different recons, then crossovers may be produced which have normal adenylo- succinase activity (see Figure 32-5). Large numbers of ascospores need to be screened to detect these recombinants, for these are expected to be rare. But this can be done simply by placing the ascospores on minimal medium and scorinii the number that form growing cultures. Of all tested spores, the percentage that grow, times two, equals the crossover percentage, or map dis- tance, between the defective recons. When 20 or so independently occurring point mu- tants were crossed together in various combi- nations, it was found that a small number did not show recombination, indicating that they are mutant in the same recon. But a large number of these mutants proved, by showing recombination, to involve different, non- allelic recons, and the map distances obtained between them were consistent with the hy- pothesis that they are located in a linear array. Moreover, their arrangement was found to be continuous with that of recons in different cistrons in the same chromosome. These results suggest that there are more recons involved in the specification of adenylosuccinase than there are cistrons. Accordingly, // is hypothesized that a cistron is composed of a mimber of linearly arranged recons. FIGURE 32-5. Normal {udenylosuccinase-specifying) cistron resulting from crossing over within two mutant (adenylosuccinase-defective) cistrons. TETRAD y Mutant Cistron 1 y Mutant Cistron 2 MEIOTIC PRODUCTS Mutant Cistron 1 Double Mutant Cistron Normal Cistron Mutant Cistron 2 292 CHAPTER 32 SUMMARY AND CONCLUSIONS The general hypothesis "one cistron-one primary effect" states that a cistron produces only one primary effect and that any primary effect is the result of the action of a single cistron. The specific hypothesis, one enzyme-one cistron, proposed as a test of the general hy- pothesis, was found to be essentially correct. However, its modification was necessitated by studies of the biochemical genetics of tryptophan synthetase and hemoglobin. These studies required that the specific hypothesis be made more general, so that it is stated as "one polypeptide-one cistron," and means that each polypeptide is specified completely by the primary action of a single cistron. It is concluded that one way a cistron can act in a primary way is to specify the amino acid content of a polypeptide. It is hypothesized that a cistron is composed of a number of linearly arranged recons. REFERENCES Beadle, G. W., and Tatum, E. L., "Genetic Control of Biochemical Reactions in Neuro- spora," Proc. Nat. Acad. Sci., U.S., 27:499-506, 1941; reprinted in Classic Papers in Genetics, Peters, J. A. (Ed.), Englewood Cliffs, N.J., Prentice-Hall, 1959, pp. 166-173. Harris, H., Human Biochemical Genetics, Cambridge University Press, 310 pp., 1959. Hsia, D. Y.-Y., Inborn Errors of Metabolism, Chicago, Year Book Publishers, 1959. Ingram, V. M., "How Do Genes Act?" Scient. Amer., 198:68-74, 1958. Itano, H. A., and Robinson, E. A., "Genetic Control of a- and /3-Chains of Hemoglobin," Proc. Nat. Acad. Sci., U.S., 46:1492-1501, 1960. Yanofsky, C, and Crawford, I. P., "The Effects of Deletions, Point Mutations, Reversions and Suppressor Mutations on the Two Components of the Tryptophan Synthetase of Escherichia CoH," Proc. Nat. Acad. Sci., U.S., 45:1016-1026, 1959; reprinted in Papers on Bacterial Genetics, Adelberg, E. A. (Ed.), Boston, Little, Brown, 1960, pp. 384-394. See Supplement IV and the first portion of Supplement V. QUESTIONS FOR DISCUSSION 32.1. What significance can you give to the fact that it is a glutamic acid in hemoglobin A which is replaced by another amino acid (valine, lysine, or glycine) in hemoglobins S, C, G, and E? 32.2. What are the disadvantages of human beings as material for the investigation of the cistron? 32.3. Can crossing over occur within a gene? Explain. 32.4. Is the hypothesis, one cistron-one primary function, equivalent to the hypothesis, one polypeptide-one cistron? Why? 32.5. What evidence can you give for rejecting the hypothesis that a cistron is equivalent to a single recon? 32.6. Would you expect that a chemical substance, specified in a primary way by a cistron, would be composed of linearly arranged parts? Why? 32.7. Can you apply the term cistron to the one or more recons that determine whether glutamic acid or lysine shall be located at a particular place in hemoglobin? Why? 32.8. What things do genes actually do? Has your answer any bearing upon the concept of a cistron? Explain. 32.9. Under what circumstances is it proper to use the term allele to describe cistronic alternatives rather than reconic alternatives? Chapter *33 CHEMICAL NATURE OF GENES WHILE many of the preceding Chapters dealt with defining the genetic material in terms of its capacity to recombine, to mutate, and to act by performing a single primary func- tion, no mention was made of the possibility of studying the chemical nature of the genetic material. Our attention is now drawn to the nature of the genetic material as revealed by studies utilizing the operation of chemi- cal analysis. Let us use the knowledge we already have to decide which of the cell's chemical components are, and which are not, suitable candidates for genetic material. Since we know that the nucleus contains genetic material in its chromosomes, any chemical substance which is located exclu- sively in the cytoplasm can be eliminated from consideration as the chemical basis for nuclear genetic material. In view of the fact that the properties which we know the genetic ma- terial possesses are complex, we should ex- pect the genetic substance to be of suitable complexity chemically. On this basis, we can eliminate from consideration all inor- ganic compounds (compounds not containing carbon), since such compounds do not pro- vide evidence of being suitably versatile in their chemical reactions. Elimination of in- organic compounds as genetic material, on this basis, is particularly reasonable with respect to those inorganic components which are present in greater abundance in the cyto- plasm than in the nucleus. We have already noted (p. 274) that the unique feature of protoplasm is the speed and 293 orderliness of its chemical activities. This we have attributed to the presence of pro- teins, in the form of enzymes and cellular structures. We have found, in Chapters 31 and 32, that the primary action of some cistrons is to specify the amino acids in a polypeptide chain. This would require that cistrons be capable of providing at least 20 different kinds of meanings or specifications (one for each of the 20 different kinds of amino acids found in protein), and suggests that intra- or intercistronic arrangement is somehow capable of manifesting the same degree of complexity as is exhibited by the polypeptide chain this arrangement specifies. It is entirely reasonable, therefore, to enter- tain the idea that the genetic material is itself protein, in which case it would clearly possess the correct amount of complexity. If the gene is protein in nature, we would expect to find protein in the chromosomes. Moreover, we might hope to find that the chromosomes contain a unique type of pro- tein, one not found in the cytoplasm. Chemi- cal analyses of nuclei and chromosomes fulfill these expectations in the form of the protein histone. Histone is a complex basic protein that is found only in chromosomes. How- ever, while it is found in the chromosomes of many organisms, it is not found in all chro- mosomes. Thus, for example, though it is present in the somatic nuclei of fish, it is replaced in the sperm of trout, salmon, sturgeon, and herring by a basic protein, protamine, of simpler composition. Assume protamine is genetic material. If the protamine in fish sperm is replaced by histone, in the somatic cells produced mitotically after fertilization, then the genetic specifications or information must be transferred from prota- mine to histone, which in turn is capable of acting as genetic material. In these organisms, then, the same genetic specifications would have to be carried in two chemical forms, protamine and histone. There is nothing in our previous knowledge to prevent us from 294 CHAPTER 33 FIGURE 33-1, Whole mount of a larval salivary gland of Drosophila. DMA stain is restricted to the nuclei. (Courtesy of J. Schultz.) accepting the view that the chemistry of the genetic substance might change in this way. We merely require, if there are alternative chemical compositions for the genetic ma- terial, that these be capable of performing a variety of activities in accordance with the principles already established. Nevertheless, the hypothesis that protamine and histone are both genetic material, in the same organism, is complicated, at least in the respect that it requires two chemical substances to account for a single genotype. It would be more satisfactory, because of its economy of as- sumptions, to discover a single nuclear chemi- cal which could serve as a candidate for the genetic material. There are other proteins found in chro- mosomes. Unfortunately, the quantity of these proteins changes according to the type and rate of metabolic activities performed by the cell. There is, therefore, no simple one- to-one relationship between their quantity and gene quantity. Accordingly, as in the protamine-histone case, additional hypoth- eses would be required in order to explain genetic behavior. We may conclude that de- spite the initial attractiveness of the hypoth- esis that the genetic material is proteinaceous, the types and amounts of nuclear protein ac- tually found do not offer any clear support for this view. There does remain another chemical sub- stance found in chromosomes, which is routinely absent in the cytoplasm (Figure 33-1). This is a type of nucleic acid called deoxyribonucleic acid ov DNA, which is found combined with basic proteins like protamine and histone, by means of a chemical linkage whose nature is not completely understood, to form deoxyribonucleoproteins. Before dis- cussing DNA as a candidate for being genetic material, let us first proceed to study the chemical composition of this material as it is found in chromosomes. Chemical Composition of DNA When DNA from chromosomes is analyzed chemically, it is found to contain organic ring compounds of which nitrogen is an integral part. The basic N-containing ring is six- membered like benzene, CeHe. Figure 33-2a shows the complete structural arrangement of benzene. Figure 33-2a' abbreviates this, by omitting the carbon atoms in the ring, and Figure 33-2a" also eliminates showing the hydrogen atoms attached to ring carbon atoms. The basic N-containing ring in DNA is called a pyrimidine. This has N substituted for the CH group at position 1, as well as at position 3, in benzene (Figure 33-2b). Chemical Nature of Genes 295 Figure 33-2b' and Figure 33-2b" show suc- cessive abbreviations of this formula, cor- responding to those used for benzene. The N found in DNA is also found in a derivative of the basic pyrimidine ring, called a purine, which is composed of a pyrimidine ring, minus the H atoms at positions 4 and 5, to which is joined an imidazole ring (5- membered), so that the carbons at these posi- tions are shared by both rings (as shown in Figure 33-2c, and the shorthand forms in c' and c")- Henceforth, the most abbreviated structural representation will be used for pyrimidines and purines. All pyriniidines and purines act chemically as bases. Figure 33 3 includes various types of py- rimidines, the names of those found in DNA being underlined. Note that all the deriva- tives of pyrimidine shown have an oxygen added at position 2 to replace the H which is H I H-cf^>C-H I II k H h^Vh H-L^H H a' BENZENE N H I C-H H- H H 1 N PYRIMIDINE H-C: I H sC— H c' PURINE FIGURE 33-2. Relations/lip between certain ring compounds. 296 CHAPTER 33 NH2 NH CYTOSINE (6-amino-2-oxypyrimidine) NHo 5-METHYL CYTOSINE (6-amino-2-oxy-5- methylpyrimidine) CH2OH 5-HYDROXYMETHYL CYTOSINE (6-amino-2-oxy-5-hydroxy- methylpyrimidine) URACIL (2,6-oxypyrimldine) THYMINE (2,6-oxy-5-methylpyrimidine) (5-methyl uracil) FIGURE 33-3. Pyrimidines. Names of pyrimidines found in DNA are underlined. relocated at position 3. This O is shown in the keto form (O = C^ , where R repre- \R sents an atom or group other than H). One of the two pyrimidine derivatives most com- monly found in DNA is cytosine. Cytosine differs from pyrimidine by having also an amino group (NH2) substituted for the H attached to the C at position 6. Accordingly, cytosine can also be called 6-amino-2-oxypy- rimidine. (Substitution, in cytosine, of CH3 for the H attached at position 5 produces 5-methyl cytosine, a DNA pyrimidine found in appreciable amounts in wheat germ, and in trace amounts in mammals, fish, and insects. Another pyrimidine, found only in the DNA of certain viruses that attack bacteria, has a hydroxymethyl group (CH2OH) replacing the H at position 5 of cytosine, and is therefore called 5-hydroxymethyl cytosine.) The other most frequently occurring py- rimidine in DNA is thymine. Thymine is unique in having a keto group replacing the H attached to the C at position 6, and has also replaced the H at position 5 by a methyl group. So thymine can be called 2,6-oxy- 5-methylpyrimidine. Note that the differ- ences between pyrimidines lie primarily in the variation in the groups present at the 5 and 6 positions in the ring. Figure 33 4 shows the structural formulae for various purines, the names of those found in DNA being underlined. One of the two purines which commonly occur in DNA is adenine. Adenine differs from the basic Chemical Nature of Genes 297 FIGURE 33-4. Purines. Names of purines found in DNA are underlined. ADENINE (6-aminopurine) 6-METHYLAMINOPURINE NH, 2-METHYL ADENINE (2-methyl-6-aminopurine) 6-DIMETHYLAMINOPURINE H— Nf^ NH H— N X CH3— N^^N H N 2-METHYLAMINO GUANINE NH2 "N""^N H 1 -METHYL GUANINE (2-amino-6-oxypurine) 29» CHAPTER 33 OH OH or OH OH OH D-RIBOSE OH 2'-DEOXY-D-RIBOSE FIGURE 33-5. Pentose sugars found in nucleic acids. formula of purine by having an NH2 group in place of H at position 6, so that this com- pound can be identified also as 6-amino- purine. (A purine similar to adenine, having a CH3 substitution on the NHo group at position 6, has been found in limited amounts in DNA, and is called, appropriately, 6- methylaminopurine .) The other purine most frequent in DNA is guanine (Figure 33-4). Guanine has an NH2 group at position 2 and an O in keto form at position 6, so it can be called also 2-amino-6- oxypurine. Note that differences among purines lie largely in the groups attached at the 2 and 6 positions of the double ring. D-ribose is a sugar (Figure 33-5a) contain- ing five carbons, being, therefore, a pentose sugar, of which four C are joined with an O to form a five-membered ring. Figure 33-5a' employs the convention, used hereafter, of not showing the carbons in the ring. DNA contains a pentose sugar modified from the D-ribose structure by the absence of an oxygen at position 2', so that this sugar is named 2'-deoxy-D-ribose, and is often called 2-deoxyribose, or deoxyribose (Figure 33-5b and b'). Each organic N-containing purine or py- rimidine base in DNA is normally joined to a deoxyribose sugar to form the combination called a deoxyriboside. The four main deoxyribosides in DNA are those for cytosine, called deoxycytidine, for thymine, called (deoxy) thymidine, for adenine, called deoxya- denosine, and for guanine, called deoxyguano- sine. The structure for these is shown in Figure 33-6. Note that the deoxyribose sugar always joins to these bases at its V posi- Chemical Nature of Genes 299 tion, the linkage being at position 3 of py- rimidines and at position 9 in the case of purines. In DNA, a phosphate group (PO4) is al- ways joined to a deoxyriboside forming a deoxyribonucleotide . The phosphate is at- tached either at position 3' or at 5' of the sugar, as shown in a generalized form in Figure 33-7. This is shown specifically for the deoxyribonucleotides containing the py- rimidine cytosine and the purine adenine in Figure 33-8. The deoxyriboside 5'-mono- phosphates of cytosine, thymine, adenine, and guanine are called, respectively, deoxycytidylic acid, thy mid y lie acid, deoxyadenylic acid, and deoxyguanylic acid. In summary, then, the basic unit of DNA is the deoxyribonucleotide which is composed of a phosphate joined to a deoxyriboside, which, in turn, is composed of a deoxyribose sugar joined to an organic base. These bases are either pyrimidines (most commonly cytosine and thymine) or purines (most commonly adenine and guanine). FIGURE 33-6. Common deoxyribosides. NH2 OH H Deoxycytidine CH. OH H Thymidine PYRIMIDINE DEOXYRIBOSIDES OH H Deoxyadenosine OH H Deoxyguanosine PURINE DEOXYRIBOSIDES 300 CHAPTER 33 FIGURE 33-7. Deoxyribonucleotides. OH Q Purine \ or ^\ /Pyrimidine h\h y3' \ O 1 H -o— p=o Purine or Pyrimidine Deoxyriboside 3'-monophosphate Deoxyriboside 5'-monophosphate Most of the DNA analyzed does not occur in single deoxyribonucleotide units, but is found to be composed of polydeoxyribonucle- otides, chains in which the individual deoxyri- bonucleotides comprise the links. The way these links are joined can be understood by examining the two deoxyriboside 5'-mono- phosphates at the right of Figure 33-8. These two compounds can become linked together if the topmost O of the bottom compound replaces the OH at position 3' of the sugar in the top compound. This reaction would be the equivalent of adding a phosphate to position 3' of the pentose in the top com- pound. The occurrence of such a reaction has already been mentioned, and is illustrated in the two molecules at the left of Figure 33-8. Since deoxyriboside 5'-monophosphates are capable of joining to each other by means of a phosphate linkage at 3', single unbranched chains of polydeoxyribonucleotides of great length are produced. Figure 33-9 shows a portion of such a chain. Note here that the polydeoxyribonucleotide is a hnear, un- branched, molecule, whose backbone is made up of sugar-phosphate linkages, and whose linearity is independent of the particular bases present at any point. This means that the structure of the chain is uninfluenced by the sequences of bases, which are therefore in indeterminate, or unspecified, array. Notice, moreover, that this polymer (a molecule com- posed of a number of identical units) of deoxyribonucleotides does not read the same in both directions. In the direction indicated by the arrows the sugar linkages to phosphates are 3'5', 3'5', etc., while in the opposite direction they read 5'3', 5'3', etc. Because of this, the polymerized DNA molecule is said to be polarized. There are two main methods of determining the amount of DNA present in the nucleus. One method is histochemical and employs whole tissues for the chemical extraction and measurement of DNA. In such work, one may perform the chemical analyses using masses of nuclei from which the surrounding protoplasm has been largely removed by special treatment. As a result of such studies, one can determine the average amount of DNA per nucleus. The second main method is a cytochemical one, by which the DNA content of individual nuclei, or chromosomes, or of chromosomal parts, is determined. This procedure makes use of the fact that DNA is the only substance in the cell which is stained when certain pro- cedures are followed. The Feidgen-Rossen- Chemical Nature of Genes 301 NH2 NHo Deoxycytidine 3'-monophosphate Deoxycytidine 5'-monophosphate or Deoxycytidylic acid NH, OH Deoxyadenosine 5'-monophosphate or Deoxyadenylic acid Deoxyadenosine 3'-monophosphate FIGURE 33-8. Specific deoxyrihonucleutides. beck technique stains DNA purple (see p. 19), while the methyl green method causes it to stain green. Not only are these stains, when applied properly, specific for DNA, but the amount of staining is directly proportional to the amount of DNA present. A given amount of stain retained in the nucleus will make a known quantitative change in the amount of different wave lengths of light it transmits, and this measurement can then be used to calculate the amount of DNA present. When, under the m/croscope, such a stained nucleus has different, appropriate, wave lengths of light in the visible spectrum sent through it, it is possible to determine, from changes in the density of its /7/?o/c»graphs, a measurement of its DNA content. From the portions of words italicized you can under- stand why this procedure is called micro- spectrophotometry. 302 CHAPTER 33 A different application of microspectro- photometry makes use of the fact that DNA is highly absorbent of ultraviolet light of wave lengths near 2600 A. When other substances, which absorb ultraviolet of these wave lengths, are removed, by enzymatic or other treatments, the quantity of DNA can be measured by its absorbence of these wave lengths. As one test of the validity of the FIGURE 33-9. Polydeoxyribonucleotide. 5'CH 5'CH * Pyrimidine or purine base of appropriate type (usually cyto- sine, thymine, adenine or gua- nine). absorbency, one can remove the DNA from the chromosome by the use of enzymes, deoxyribonucleases, or DNAases, which break up the long DNA chains so that the pieces can be washed out of the nuclei, leaving no DNA in the chromosome. Such treatment produces the expected loss of absorbency. Having digressed to study the chemical content and quantitative measurement of chromosomal DNA, let us list some results which bear upon a possible association of chromosomal DNA with the genetic material in the nucleus: 1. The amount of DNA increases during the metabolic stage until it is approximately double the amount present at the beginning of this stage. Mitosis partitions the DNA approximately equally among the two telo- phasic nuclei. Accordingly, all diploid nuclei of an individual have just about the same DNA content when first formed after mitosis. 2. The amount of DNA in a haploid gamete is approximately half that found in a newly formed diploid metabolic nucleus of the same individual. Fertilization, which restores the diploid chromosome condition, restores the DNA content characteristic of the diploid cell. 3. Cells which have extra sets of chromo- somes, being therefore polyploid, have a proportional increase in DNA content. 4. Different cells in a tissue, like those in the salivary gland of larval Drosophila, may show different degrees of polyteny in their chromosomes. The DNA content of these different nuclei is found to be proportional to the degree of polyteny. 5. The capacity of different wave lengths of ultraviolet light to induce mutations, in fungi, corn, Drosophila, and other organ- isms, is paralleled by the capacity of DNA to absorb these wave lengths. In other words, the mutational efficiency of ultra- violet light parallels the absorption curve of ultraviolet by DNA. Chemical Nature of Genes 303 6. Through the use of labeled atoms, tagged because they are radioactive or have an abnormal weight, it is found that parts of many cellular components are constantly being replaced. In these cases, then, there is an atomic turnover even though no addition is being made to the total amount of substance demonstrating the turnover. DNA is unusual in that it shows little, if any, turnover. 7. DNA is a long, linear, unbranched poly- mer, as would be expected if it represents a string of recons. Each deoxyribonucleo- tide is bipolar, in the respect that it typically can join only to two other deoxyribonucleotides via its 3' and 5' sugar linkages to phosphate, as would be expected if each deoxyribonucleotide was the equivalent of a nonterminal recon (cf. p. 197). We have discussed briefly the location of DNA, the amount and behavior of DNA be- fore, during, and at the conclusion of mitosis (1), meiosis and fertilization (2), the quantity of DNA in polyploid (3) and polytene (4) chromosomes, the relation of ultraviolet light mutability to DNA absorbence (5), and the constancy or stability with which DNA main- tains its integrity at the molecular level (6). In all these respects, the observations are consistent with the view that DNA either is the genetic material or is intimately associ- ated with the genetic material. Furthermore, the linear arrangement of recons has a parallel in the linear arrangement of deoxyribonucleo- tides in the DNA polymer (7). Chemical Composition of RNA Besides DNA, there is another type of nu- cleic acid found in the nucleus. This is called ribonucleic acid, or RNA. RNA is nor- mally found in combination with protein in the form of ribonucleoprotein. Because the RNA content of chromosomes varies within and among diploid cells of the same organ- ism, according to the metabolic activity of the cell, it can be concluded that RNA is un- likely to be the chemical basis of genes in typical (DNA-containing) chromosomes. Nevertheless, let us take this opportunity to discuss the chemical composition of RNA, noting in particular how it compares with DNA. Chromosomal RNA, like DNA, is a long, unbranched polymer of a basic unit called a ribonucleotide. The ribonucleotide is like the deoxyribonucleotide in being a combination of a base + sugar + phosphate; one way in which it differs is that the sugar is D-rihose (Figure 33-5). Another difference is found in the pyrimidine bases which it may contain. The two pyrimidines commonly found in RNA are cytosine (also common in DNA) and wrac/V (2.6-oxypyrimidine; not found in DNA). Uracil's structure is shown in Figure 33 3. The two purines commonly found in DNA, adenine and guanine, are also com- mon in ribonucleotides. In RNA, the base 4- sugar combination is called a riboside. Ribonucleotides are joined together by phosphates joined both at the 3' and 5' posi- tions of the sugar, just as in DNA, so that Figure 33-9 would equally well represent a polyribonucleotide if an O was added at each 2' position (making each sugar D-ribose), and if uracil was substituted for thymine as one of the bases usually included. It should be noted, finally, that RNA also absorbs ultra- violet light of 2600 A, but can be removed from the chromosome by treatment with ribonucleases, or RNAases. In summary, we can say that the chromo- somes contain two nucleic acids, DNA and RNA. These normally occur in combination with protein to form nucleoproteins (deoxy- ribonucleoprotein and ribonucleoprotein, re- spectively), in which these acids occur as polynucleotides (polydeoxyribonucleotides and polyribonucleotides, respectively), each of which is built of (mono-) nucleotides (deoxy- and ribonucleotides, respectively), composed of phosphates joined at 5' oinucleo- 304 CHAPTER 33 sides (deoxyribo- and ribosides, respectively), composed of a pentose sugar (2'-deoxy-D- ribose and D-ribose, respectively), joined to a pyrimidine (usually cytosine or thymine and cytosine or uracil, respectively) or to a purine (usually adenine or guanine). Some of this terminology is summarized in tabular form in Figure 33-10. Ahhough the RNA in chromosomes does not possess either the proper quantitative variation or constancy we would expect of ordinary chromosomal genes, it does possess the same linear organization as DNA, as would be expected for linearly arranged re- cons. Suffice it to say at this point that certain viruses (influenza, poliomyelitis and other encephalitic viruses, viruses like tobacco mosaic virus which attack plants, and even a virus attacking bacteria)' possess genetic properties, but do not contain DNA. These viruses are composed primarily of ribo- 1 See T. Loeb and N. D. Zinder (1961). nucleoprotein. Since DNA rather than pro- tein is favored as being the genetic chemical under typical chromosomal conditions, it is reasonable to entertain the view that it is RNA rather than the protein which is the chemical basis of genetic specification in these particular viruses. What we will attempt to do, in subsequent Chapters, is to present additional evidence that tests the view that DNA typically (and RNA in special cases) either is the genetic material or is intimately associated with it. Clearly we are seeking ultimately to deter- mine the chemical units of the genetic ma- terial which may correspond to the cistron and the recon, and the chemical basis for the mutation of single genes. In view of the like- lihood that the cistron contains more than a single recon, it should be realized that the chemical units, which correspond to the cistron and recon, are expected to be diff'erent, at least quantitatively. FIGURE 33-10. Terminology for nucleic acids and their components. NUCLEIC ACID COMMON PYRIMIDINE (PY) or PURINE (PU) BASE PENTOSE SUGAR NUCLEOSIDE (MONO-) NUCLEOTIDE with PO^at 5' 2'-deoxy-D-ribose deoxyriboside deoxyribonucleotide Cytosine PY Deoxycytidine Deoxycytidylic acid DNA Thymine PY Thymidine Thymidylic acid Adenine PU Deoxyadenosine Deoxyadenylic acid Guanine PU Deoxyguanosine Deoxyguanylic acid D - ribose riboside ribonucleotide Cytosine PY Cytidine 5'Cytidylic acid RNA Uracil PY Uridine 5'Uridylic acid Adenine PU Adenosine S' Adenylic acid Guanine PU Guanosine 5 Guanylic acid Chemical Nature of Genes 305 SUMMARY AND CONCLUSIONS When restricted to substances unique to the nucleus, the search for chemical substances which are genie, or intimately connected with the recombination, mutation, and function of genetic material, led to a consideration of protein as a possible candidate. The evidence available does not support protein as having such a primary role. In view of the localization of DNA, its quantity and distribution in mitosis, meiosis, fertiliza- tion, and polyploid and polytene chromosomes, the parallelism between DNA absorption and the mutability of ultraviolet light, the molecular integrity of DNA as revealed by turn- over studies, and its long, linear, unbranched arrangement, it is hypothesized that DNA is the genetic material in chromosomes or is at least intimately associated with the genetic material therein. It is also hypothesized that RNA may assume the genetic role of DNA in certain DNA-free viruses. Accordingly, some details of the chemical nature of RNA and DNA were presented. This Chapter initiates our attempt to discover the chemical units of the genetic material corresponding to the recon and cistron, and the chemical basis for single gene mutation. REFERENCES Chargaff, E., and Davidson, J. N. (Eds.), The Nucleic Acids, 2 Vols., New York, Academic Press, 1955. Loeb, T., and Zinder, N. D., "A Bacteriophage Containing RNA," Proc. Nat. Acad. Sci., U.S., 47:282-289, 1961. Potter, V. R., Nucleic Acid Outlines, Vol. 1, Minneapolis, Burgess Publ. Co., 1960. QUESTIONS FOR DISCUSSION 33.1. Do you think it is simpler to postulate that DNA rather than protein is genetic material? Why? 33.2. What is the chemical distinction between: a. a mononucleotide and a polynucleotide? b. a nucleotide and a nucleoside? c. a pyrimidine and a purine? d. a ribose and a deoxyribose sugar? 33.3. Draw the detailed chemical structure of a polyribonucleotide having the base se- quence adenine, uracil, guanine, cytosine. 33.4. Express thymine as a derivative of uracil. What part of the term deoxythymidine is superfluous? Why? 33.5. What evidence can you provide, from your own knowledge, to support the view that viruses possess genie properties? 33.6. How would you proceed to measure the absorbency of ultraviolet light by chro- mosomal DNA? chromosomal RNA? 33.7. Do you believe that the evidence so far presented provides conclusive proof that DNA is genetic material in chromosomes? Why? 33.8. What is your opinion of the hypothesis that DNA is the chemical basis for recons, but that protein is the chemical basis for cistrons? 33.9. Is DNA complex enough to serve as the chemical basis of cistrons? Explain. 33.10. Do you think the term chemon could be defined usefully? Justify your opinion. Chapter *34 ORGANIZATION, REPLICATION, AND TYPES OF DNA IN VIVO I "ndirect evidence, consistent with the view that DNA can serve as -the chemical basis of chromo- somal genetic material, was presented in the last Chapter. That Chapter described what may be called the primary structure oj DNA, as being a single, long, unbranched, polarized chain of nucleotides. In accord with the view that the DNA polymer is genetic, we would expect it to be linearly differentiated so that different portions of it could represent dif- ferent units of genetic material. This differ- entiation cannot be attributed to either the deoxyribose sugar or the phosphate, since one of each is present in every nucleotide. Differences in genetic information along the length of the DNA strand must be due, there- fore, to the bases present. Since species differ by numerous point mutations, we would ex- pect to find differences in the DNA's of dif- ferent species. Histochemical analyses have been made of the organic bases in DNA extracted from various species. Considering the total amount of the bases in an extract as 100%, Figure 34-1 gives the percentages of this found as adenine (A), thymine (T), guanine (G), and cytosine (C). Note that there is considerable variation in base content, ranging from organ- isms Relatively rich in A and T and poor in C and G (sea urchin), to those showing the reverse, where A and T are considerably less abundant than C and G (tubercle bacillus). These results demonstrate that the relative amounts of the four bases are different in 306 the DNA's from radically different species. Can these data tell us whether a shift in the sequence of bases also could produce differ- ences in the properties of the genetic material? That different orders of the same bases may also be involved, in specifying different genetic units, is suggested by the fact that the chicken, salmon, and locust, which must be very dif- ferent genetically, all have very similar base ratios. One might suggest as an alternative explanation that these species are molecular polyploids, differing only in the number of the same types of DNA molecules which they possess. This possibility can be eliminated from serious consideration in the light of our knowledge of the limited contribution which chromosomal polyploidy has made to evolu- tion, at least in the animal kingdom (Chapters 18,29). So long as histochemical analyses are made, of the total DNA of cells having a high DNA content, approximately the same base ratios would be expected to be obtained from dif- ferent members of a single species. This has proven true. Moreover, as expected, the same base ratios have been found in different normal and neoplastic tissues of the same and different human beings. Nevertheless, it is expected that a genome would contain many molecules of DNA which differ from each other in base sequence and content. The variation, found in different species, in the ratio A + T/G + C (which is about .4 for the tubercle bacillus and is about 1.8 in the sea urchin), is understandable in terms of our chemical knowledge, since the DNA strand imposes no limitation upon which base may be present, or how often it may appear along the length of the fiber. There is, how- ever, a remarkable equality in the amount of A and the amount of T in the DNA within a species, and also an equivalence in the amounts of G and C (refer to Figure 34-1). Since, in each species, A = T, and G = C, it is also apparent that A + G = T + C, or, in other words, the total number of DNA Organization, Replication, and Types of DNA in Vivo 307 ADENINE THYMINE GUANINE CYSTOSINE Man (sperm) 31.0 31.5 19.1 18.4 Chicken 28.8 29.2 20.5 21.5 Salmon 29.7 29.1 20.8 20.4 Locust 29.3 29.3 20.5 20.7 Sea urchin 32.8 32.1 17.7 17.7 Yeast 31.7 32.6 18.8 17.4 Tuberculosis bacillus 15.1 14.6 34.9 35.4 Escherichia coli 26.1 23.9 24.9 25.1 Vaccinia virus 29.5 29.9 20.6 20.3 E. coli bacteriophage Tj 32.6 32.6 18.2 16.6 FIGURE 34-1. Base composition of DNA from various ori^unisms. purines always equals the total number of DNA pyrimidines. While this regularity is common to all the chromosomal DNA's listed, there is nothing in the nature of the primary structure of DNA which would help explain it. However, the fact that the pri- mary structure of DNA is the same in all these organisms suggests that these regulari- ties may be connected with some additional, general, structural characteristic of chromo- somal DNA. An understanding of the basis for the A = T and G = C relationships may come from studies of an entirely different kind. It has been known for a long time that a beam of X rays is bent or refracted when it passes through material. If the material through which the rays pass is completely hetero- geneous in structure, no regularity is found in the way in which the emergent beam is refracted. But, if the material is composed of macromolecular units and/or molecular subunits, which are spatially arranged in a regular manner, then the emergent beam will form what is called an X-ray diffraction pattern. Moreover, a particular X-ray pattern can be used to identify units and subunits that are repeated at regular intervals of space. Thus, it has been found that each nucleotide in a DNA chain occupies a length of 3.4 A along the chain, and that this repetition is detectable by the characteristic X-ray diffrac- tion pattern it produces. X-ray diffraction patterns have been ob- tained for DNA from a variety of species. In some cases the DNA was not removed from the nucleus, in other cases it was re- moved from the nucleus and separated from protein as well. In all cases, provided the DNA was suitably hydrated, essentially the same patterns attributable to DNA were found (see Figure 34-2). A study of these 308 CHAPTER 34 FIGURE 34-2. X ray diffraction photographs of suitably hydrated fibers of DNA, showing the so-called B configuration. A. Pattern obtained f^' using the sodium salt of DNA. B. Pat- tern obtained using the lithium salt of DNA. This pattern permits a most thorough analysis of DNA. (Courtesy of Biophysics Research Unit, Medical Re- search Council, King's College, London.) ♦ ■ » , B FIGURE 34-3. The Watson-Crick dou- ble-stranded helix configuration of DNA. Organization, Replication, and Types of DNA in Vivo 309 common patterns showed, besides the 3.4 A repetition, other repeat units which would be explained only if DNA usually does not occur as a single strand. (On the other hand, X-ray diffraction studies show that RNA is usually single stranded.) Here, then, was a clear demonstration that there is a secondary structure to DNA which was hitherto un- expected, and there was every reason to believe this was the organization normally found in the chromosome. The simplest explanation consistent with the diffraction results was proposed by J. D. Watson and F. H. C. Crick (see the 1953a reference to them at the end of this Chapter). They hypothesized that DNA is normally two- stranded (see Figure 34-3). Each strand is a polynucleotide, and the two strands are coiled around each other in such a manner that they cannot be separated unless the ends are per- mitted to revolve. This kind of coiling is plectonemic (as is found in the strands of a rope) and can be contrasted with paranemic coiling in which two coils can be separated without their ends revolving (just as two bedsprings pushed together can be sepa- rated). The Watson-Crick model for the secondary organization of DNA macromolecules in- volves a double helix in which each strand is coiled right-handedly (i.e., clockwise). This is the same direction of coil as is found in the secondary structure of polypeptides (see p. 286). The model shows the pentose and phosphate backbone of each strand on the outside of the spiral, while the relatively flat bases which project into the center lie perpen- dicular to the long axis of the fiber. The backbone completes a turn each 34 A. Since each nucleotide occupies 3.4 A along the length of a strand, there are 10 nucleotides per complete turn and each nucleotide has a pitch of 36° relative to the long axis (so that 10 nucleotides complete the 360° required for a complete turn). The two helices are held together by chemi- cal bonds between bases on different strands. It has been found that the two strands can form a regular double helix, whose diam- eter is uniformly 20 A, only if the bases on different strands join in pairs, each of which is composed of one pyrimidine and one purine. Two pyrimidines together (being single rings) would be too short to bridge the gap between backbones, while two purines (being double rings) would take up too much space. Moreover, the pyrimidine-purine pair- ing must be either between C and G or be- tween T and A, for only in this way is the maximum number of stabilizing bondages between them produced. The type of stabiliz- ing bond holding the members of a base pair together is called a hydrogen bond or "//" bond."" The base pairs, with their H bonds shown as dotted lines, are diagrammed in Figure 34-4. (All these diagrams really should be at a 36° tilt from the horizontal.) The top half of the Figure shows the C-G (and G-C) arrangements. Note, in the C-G pair, that cytosine has been turned over (from left to right) relative to the way it was dia- grammed in Figure 33-3. Three H bonds are formed. Two occur between NH2 and O (the 6— NH2 of C with the 6— O of G; the 2—0 of C with the 2— NH, of G), and one occurs between the 1 — N of C and the 1 NH of G. The G;C pair is identical to C;G, as shown, except that, in this case, the base turned over is guanine. The bottom half of Figure 34-4 shows the other type of base pair (T:A or A:T, in which T and A, respectively, have been turned over relative to the way they were shown in Figures 33-3 and 33-4). In this pair only two H bonds are formed, one be- tween the 6 — O of T and the 6 — NH2 of A, and the other between the 1 — NH of T and the 1 — N of A. Although the H bond is a weak electrostatic bond (requiring only about 5 kcal of energy to break the H bond in N — H . . . O, whereas a regular C — C bond would require 50-100 kcal for breakage), there 310 CHAPTER 34 H \ N-H^.- O h^/ ^-^ \y_Ti Cytosine \ W Guanine H ..-^H-N / / -N H Guanine H Cytosine N-H^.. H H Thymine Adenine ^H— O^ ^"' H -N -N O H Thymine FIGURE 34-4. Base pairs formed between single DNA chains. are so many of them along a long double helix that the entire structure is fairly rigid and paracrystalline even when moderately hydrated. You will recall that the double helix con- figuration of DNA does not dictate the sequence of bases along the length of a chain. But you will also remember that the sizes of, and the H bonds in, the pyrimidines and purines did dictate that A in one chain can pair only with T in the other chain, and C with G, in order to form a double helix of constant diameter whose strands are held together by the maximum number of H bonds. Since A and T always go together, as do C and G, the equivalences A = T and C = G, found when DNA is analyzed chemically, become mean- ingful as being the direct consequence of the secondary structure of DNA. In fact, the chemical equivalences provide the first inde- pendent test of the Watson-Crick model, which was constructed initially on the basis of other considerations. Recall that in order to maximally H-bond a purine and a pyrimidine, it was necessary to represent one of the two as being turned over, so that the number I atoms of both face each other. This has an important conse- quence for the orientation of the two chains relative to each other, as is illustrated by means of Figure 34-5. The bases in the chain at the right all face the accustomed way, while those in the left chain are all turned over. In order that each base join to its sugar in the same way, the sugars must be arranged as shown. Notice, in proceeding downward from the top of the right chain, that the PO4 linkages to sugar read 3'5', 3'5', etc. But, when read the same way, the left chain is 5'3', 5'3', etc., so that the member chains in a double helix run in opposite directions, as indicated by the arrows. The X-ray diffraction results, which led to the double hehx hypothesis, do not tell us that all DNA in chromosomes is two- stranded, or that a double strand is never Organization, Replication, and Types of DNA in Vivo 311 FIGURE 34-5. The opposite direction oft/ie sugar-phospluite lin/DNA >DNA FIGURE 35-3. Growth of a DNA chain at its nucleoside end (left) and nucleotide end (right). Arrows show position of degradation by micrococcal DNAase plus splenic phosphodiesterase. 324 CHAPTER 35 can be seen in the diagram at the left of the Figure, P* should not occur in inorganic phosphates but should sometimes appear in other deoxyriboside 3'-monophosphates be- sides the one containing C. When the experi- ment was performed, the latter result was obtained. Not only was P* absent from in- organic phosphate, but it was found fre- quently in all four kinds of deoxyriboside 3'-monophosphates. An additional test of the view that the DNA chain grows at its 3' position is furnished by treatment of the limited product by a dif- ferent enzyme, snake venom diesterase. This enzyme digests DNA by breaking the bond between the phosphate and sugar at the 3' position, starting at the nucleoside end of the chain and proceeding toward the nucleotide end. In this way, the DNA is gradually digested into deoxyriboside 5'-phosphates as indicated by the arrows in Figure 35-4. When the limited product was treated this way, it was found, as expected, that almost all the radioactivity had been removed from the chain, even though only a very small portion of the DNA had been digested. Other results show clearly that the product of a limited re- action is DNA, which has one or very few deoxyribotides added to the nucleoside end of the chain. Still other evidence supports the view that the 3' point of lengthwise linkage is the same when net DNA is greatly increased as it is when the limited reaction occurs. The fact that lengthening of a DNA chain can occur in vitro, when any of the four com- mon deoxyribosides happen to be at the nucleoside terminus, is consistent with our knowledge of the nondependence of DNA pri- mary structure upon base sequence. Is there any other evidence that the DNA synthe- sized in vitro has the characteristics of DNA synthesized in vivol Let us summarize some of the physical properties of samples of DNA composed of 90% or more of the product synthesized in vitro. Such samples have physical characteristics that are similar to >DNA p.p. FIGURE 35-4. Degradation of DNA {arrows) by snake venom diesterase. that of DNA isolated from calf thymus, in- sofar as sedimentation rate and viscosity are concerned. From such characteristics a molecular weight of about 6 million was cal- culated, and it was inferred that the product was not usually single-stranded. In support of the latter inference was the finding that the macromolecular structure of the in vitro prod- uct was destroyed when heated for 10 minutes at 100°C, as expected if this treatment pro- duced single chains which collapsed to form compact, randomly coiled structures. Like thymus DNA, the enzymatic product shows Replication of DNA in Vitro 325 the same type of increase in ultraviolet absorption following digestion with pancre- atic DNAase. If the in vitro synthesis occurs in the same way as it does in vivo, one might expect that single-stranded DNA would serve as a better primer than does double-stranded DNA. Thus, we might expect the single-stranded DNA, isolated from the virus A' and A' -> A) and that bacteria can become transformed with respect to any chromosomal gene they possess. A' cells can be trans- formed to A" type which, in turn, provide large quantities of A"-DNA capable of trans- forming other A' cells to A". So, the DNA of transformed bacteria can be extracted in turn to provide greatly increased amounts of the same transforming principle. One trans- forming principle (A') can transform bacteria having any one of several alternative pheno- types (e.g., A or A")- If the A'-DNA, from bacteria transformed from A to A', is used to transform bacteria of a third genotype (A"), the only transformations produced are those involving the genes of the immediate donor (i.e., only A' and no A transformants are found). This demonstrates that trans- formations are not transmissible changes in- volving a simple addition of particular genetic material to the genotype, but entail the loss of host genetic material at the same time that the new genetic material is acquired. Thus, the genetic change in transformation is of a replacement type. How does transforming DNA accomplish this replacement? It is possible - to trace the fate of transforming DNA by labeling its phosphate groups with radioactive P^-. At various times after exposure to this labeled DNA, one portion of the treated bacteria is killed and analyzed with respect to the pres- ence of P^- in its DNA, while another portion is tested to determine whether it has been transformed. Only after bacteria have been exposed to the DNA extract for a suitable 2 Based upon work of L. S. Lerman and L. J. Tol- mach (1957). period of time is the labeled DNA found in the extract containing the host's chromo- somal DNA. Moreover, the frequency with which the host cell is transformed is directly proportional to the amount of labeled DNA so incorporated. The results mentioned in the last two para- graphs indicate that the transformer DNA actually enters the bacterium and replaces a segment of the host's chromosomal DNA,^ after which the newly introduced material replicates as a normal part of the chromo- some. Since it is DNA which alone carries the genetic information for transformation, we conclude that transformation provides direct and conclusive evidence that DNA is genetic material. Accordingly, chromosomal DNA contains the chemical units of the genetic material. All other transformation studies support this conclusion. We should now reexamine the assumption, made earlier in this Chapter, that transforma- tion involves mutation. The first results on transformation seemed to involve novel, rare changes in the genetic material, and were hence called mutations (see p. 137). We now know that transformation involves replace- ment of one segment of genetic material by another. No new type of genetic material appears; there is only a shuffling of already existent genes. Thus, no novel genes are pro- duced by transformation. Moreover, genetic transformation has been found not only in Pneumococcus, but in Hemophilis, Xanthom- onas. Salmonella, Bacillus, Neisseria, and Escherichia and other organisms as well. In the case of Neisseria, DNA is regularly liber- ated into the slime layer by autolyzing cells which are found in aging cultures. Such DNA is effective in transformation, as is the DNA obtained from penicillin-sensitive pneu- mococci lysed after treatment with penicillin. Not only is transformation widespread, but a given type may occur with frequencies as ^ See H. Ephrussi-Taylor (1951) for specific evidence for the latter observation. 342 CHAPTER 37 high as 25%. Such results demonstrate that transformation is not rare. In view of the possibility that transformation may be a routine process, it is best not to consider it as mutation. Accordingly, transformation, like segregation, independent segregation, crossing over, and fertilization, is probably best considered as a potential mechanism for normal genetic recombination. It is now believed that completion of the transformation process requires a series of discrete stages. 1. Cell competence. There are certain periods in cell division or in the growth of a culture during which transformation does not occur, whereas in other periods the cells are competent to react. 2. Binding of the transformer. When cells are in a competent stage, the transforming DNA is first transiently bound to the cell, and can be removed by several methods, in- cluding the action of added DNAase, before it is bound permanently. 3. Penetration of transformer. Perma- nently bound DNA is considered to have penetrated the recipient cell. If the trans- former DNA has been fragmented sonically, so that the DNA particles have a molecular weight of less than 4 X 10% these do not penetrate. Only high molecular weight DNA penetrates. Pneiimococcus contains about 6 million pairs of nucleotides per haploid nu- cleus, equivalent to about 200 molecules of about 10 X 10'' molecular weight. It has been found that about 60-4800 molecules of such a molecular weight penetrate. It is clear, therefore, that a large amount of DNA (equal to a considerable portion of the donor genome) succeeds in penetration. This, plus the fact that smaller pieces of DNA cannot penetrate, recalls the circumstances under which DNA uptake occurs in tissue culture (see p. 317). In such cases, DNA enters by phagocytosis, which occurs only when the DNA adheres to a suitably large particle. Perhaps, in bacteria also, a mechanism of penetration is involved in which the cell also takes an active part, and can do so only if the amount of DNA is sufficiently large. (It may be noted that the success of transforma- tion is inversely related to the thickness of a polysaccharide coat, which probably acts as some kind of barrier to binding or penetra- tion.) Whatever the specific mechanism for pene- tration may be, it is known that there are a finite number of sites on the bacterial surface which act as receptors for DNA. Since non- transforming DNA, such as DNA from a widely separated genus, can also penetrate readily, receptor sites can be saturated by nontransforming DNA, after which trans- forming DNA cannot penetrate. 4. Synapsis. Alternatives of the same trait (for example, resistance and sensitivity to streptomycin, or auxotrophy and prototro- phy for a particular nutrient) can be found in diff'erent species of bacteria. On the reason- able assumption that the same gene and its alternatives perform the same functions in different species, it ought to be possible to produce interspecific transformations. This has been accomplished, but, in any given case, interspecific transformation is usually less frequent than the intraspecific one. The fact that interspecific transformation takes place at all favors the idea that the trans- formed locus is normally part of the genotype of both species. The scarcity of interspecific transformations, therefore, is probably not due to the locus transformed; nor is it due to any failure of competence, or of binding or penetration of the foreign DNA. More- over, the transformation rate is actually lower, and is not an artifact due to a delay in pheno- typic expression which might occur in inter- specific, but not in intraspecific, transforma- tion. We are led, therefore, to hypothesize that the capacity to transform, which already- penetrated DNA has, is dependent upon the nature of the genes adjacent to those whose Bacteria : Recombination (I) 343 transformation is under test. These neighbor genes can be visualized as influencing trans- formation by their effect upon synapsis be- tween the transforming DNA and the cor- responding region of the host's genetic ma- terial. In intraspecific transformation, the loci adjacent to the transformed ones are homologous in transformer and host, so that synapsis between the two segments may occur properly, whereas in interspecific transforma- tion, these loci are likely to be nonhomolo- gous (or act to prevent synapsis), and, there- fore, may often fail to synapse. 5. Integration. Even if the hypothesized synapsis occurs properly between host and transformer DNA, some process has yet to occur by which the host gene, whose trans- formation is being followed, is lost from the chromosome, and the new locus becomes an integral part of it. Some understanding of the mechanism of this final stage in trans- formation may be gained from a study of the frequency of transformation. Diff'erent loci transform intraspecifically at diff'erent rates. Using genes that transform with suitably high frequencies, it has been possible to study the rate of double transformations, that is, the frequency with which bacteria are trans- formed with respect to two markers present in the donor DNA. In several cases (for example, penicillin- and streptomycin-re- sistance), the frequency of doubly trans- formed bacteria is somewhat less than the product of the frequencies for the single transformations. In these cases, this means that the transformer DNA carries the two loci either on separate particles, or else in widely separated positions on the same particle. On the other hand, the markers for streptomycin-resistance and mannitol-fer- mentation are transformed together with a frequency (.1%) which is about 17 times that expected from the product of the frequencies of the single transformations (.006%). This is taken to mean that these two genetic mark- ers are located on the same transforming particle, i.e., they are reasonably close to- gether in the same bacterial chromosome. How can we explain single and double transformations of closely linked loci? Be- cause of fragmentation during extraction, a given penetrating DNA particle may not always have the same composition relative to the two markers; it may sometimes carry only one of these, and at other times carry both markers. We can test the eff'ect, on single and double transformations, of reduc- ing the particle size of penetrating DNA. On the present hypothesis, we would expect, when particle size is reduced by DNAase or sonic treatment, that sometimes the par- ticles would be broken between the two markers, thereby reducing the relative fre- quency of the double transformation and increasing the relative frequencies of the single transformations. When the particle size is reduced, the over-all rate of transformation is lower, as expected. However, no change is found in the ratio of double to single trans- formations. This must mean that the two markers are so closely linked that they are separated only rarely when particles are frag- mented. Accordingly, this may be taken to mean that the penetrating particles must usually carry both markers, or neither, and that the failure to obtain 100% double trans- formations from the former type must be due to the fact that only a small portion of such a penetrant, synapsing particle is integrated. Integration of a portion of a synapsed particle may be visualized as occurring in two possible ways (Figure 37-1). One method could involve "breakage and exchange" of the kind that takes place in chromosomal rearrangement or crossing over. In this case (Figure 37-1 A), "breaks" would have to occur on each side of the marker to be intro- duced, so that a "2-strand-double crossover" (refer to p. 133) is produced. While double crossovers within a short distance would be expected to be extremely rare between two homologous chromosomes of higher organ- 344 CHAPTER 37 SEGMENT r CHROMOSOME a b c' c d' d e' e r f g h INTEGRATION COMPLETED REPLICATION DAUGHTER CHROMOSOME FIGURE 37-1. Postulated mechanisms for the incorporation of a segment of genetic information into a host chromosome. A. Breakage method; B. Copy- choice method. isms, this may occur under special circum- stances, and may be possible between the visibly less well-defined chromosome of bac- teria and the shorter synapsed segment of transformer DNA. The second method might involve what has been called copy-choice (Figure 37-1 B). In this procedure, a daughter chromosome is formed by the alternate use of the normal chromosome and the transformer DNA as a template, so that when completed it is exactly like the original chromosome except for a segment that was replicated using transformer DNA as a template. Although we cannot decide by which of these two mechanisms (or yet another) integration occurs, there is evidence that the process is fixed, so that it does or does not occur, within a single gen- eration, and it is likely that the transformed marker is replicated beginning with the second division cycle. It is clear that the portion of penetrant Bacteria: Recombination (/) 345 DNA which is not integrated is also not con- served as chromosomal gene material. If integration occurs by copy-choice then the transforming segment is not conserved either; whereas if integration occurs by crossing over the chromosomal segment replaced is not conserved. We have already mentioned that DNA from different sources and DNA particles of different sizes behave differently in various parts of the sequence leading to transforma- tion. Several studies have been made of DNA in vitro which may shed light on the transformation process in particular, and upon genes in general. When DNA in vitro is exposed to dilute concentrations of DNAase, the results '* indicate that single strands of the double helix are attacked first, and only later, when both strands have been attacked at reasonably proximal positions, is the molecule severed. Once severed, the smaller molecule penetrates poorly, you re- call. However, even if only the single strand has been attacked, transformation rate de- cHnes. This effect is attributed to the failure of penetrant molecules to transform, because the transforming locus, or a locus necessary for synapsis or integration, has been in- activated. We have already mentioned (refer to pp. 315, 325) that heating chromosomal DNA denatures it by causing strand separation. Under certain conditions, the single chain can fold so that a large number of comple- mentary base pairs are formed between bases at different levels of the single chain. After pneumococcal DNA has been heated for 10 minutes at 100° C, all chains are single and all H bonds are broken.^ If such a sample is cooled quickly under appropriate conditions, almost no double chains are formed, almost ■* Of L. S. Lerman and L. J. Tolmach. ^ The following account is based primarily on work reported by P. Doty, J. Marmur, J. Eigner, and G. Schildkraut (1960), and J. Marmur and D. Lane (1960). all chains being single, with half the molecular weight of the original DNA. This is called denatured DNA (cf. p. 315). On the other hand, if such a sample is cooled slowly, double strands are produced, which are united by complementary base pairing over most of their length. This is called renatured DNA. Denatured and renatured DNA differ in several properties. These include their appearance under the electron microscope (renatured DNA looks very much like native DNA while denatured DNA is irregularly coiled with clustered regions), their density (renatured and native DNA have similar and lighter densities than denatured DNA), and their ultraviolet absorbency (renatured and native DNA have similar but lower absorben- cies than denatured DNA). Several factors affect renaturation. Rena- turation is dependent upon the concentration of DNA in the slowly cooling mixture. When the concentration of single strands is high, so is the amount of renaturation, while slow cooling of single strands in low concentration does not produce any substantial recombina- tion of strands. A second factor, influencing union between strands, is the effect of salt concentration. The negatively charged phos- phate groups of single strands tend to prevent union with other strands. This can be over- come by adding KCl to the solution; this acts as a shield against the repulsion between phosphates. Accordingly, within a certain range, the more KCl that is present the greater is the amount of renaturation ob- tained by slow cooling of heated DNA. A third factor upon which strand recombination depends is the source of DNA. Assuming that the molecular weight of native DNA is approximately the same in all organisms, then a mammalian nucleus would have about a thousand times as many DNA molecules as a bacterial nucleus. Assume also, as is hkely to be true, that the DNA molecules within a genome all differ in base sequence. Then, for a given concentration of denatured DNA, 346 CHAPTER 37 there would be of the order of one thousand times fewer complementary chains present when the sample came from calf thymus than there would be in a sample that was obtained from Pneumococcus. It has been possible to show that when denatured DNA is heated to 80° C, a large amount of double strands is formed by the bacterial DNA but no detect- able amount is produced by the calf thymus DNA. Therefore, what is important in renaturation is the concentration of comple- mentary strands. Another physical-chemical change may oc- cur when DNA is heated in vitro. As already mentioned, native pneumococcal DNA has a molecular weight of about 10 million. When certain preparations of this native DNA are heated, the single chains obtained may have a molecular weight less than half this value. This can be explained, as the consequence of the presence of DNAase, as follows. Single chains are enzymatically severed by DNAase while still part of the double chain, as de- scribed previously, but the whole complex retains the double helix configuration. How- ever, once the complementary chains are separated by heat denaturation, the frag- ments of each single chain fall apart. It is also possible to make hybrid molecules by renaturing a mixture of N-14 and N-15 DNA from E. coli. These synthetic mole- cules can be identified by the position they assume in the ultracentrifuge tube (see dis- cussion beginning p. 312). Such hybrid mole- cules are also formed between single DNA strands from diff'erent species, but only if the species are closely related genetically (as would be suggested if they showed inter- specific transformation), and if they have similar base compositions. Because of the occurrence of genetic re- combination in bacteria via transformation, it is possible to test the biological activity of the various DNA's produced by strand sepa- ration and recombination. It is found, using Pneumococcus, that denatured DNA has very little transforming activity. (It is likely that this activity is due to the small amount of renaturation which could have taken place in the denatured solution.) On the other hand, the transforming ability of renatured DNA may be as much as 50% of that shown by an equivalent concentration of native DNA. Just as an increased concentration of DNA and a high ionic strength increase renatura- tion, so do they also increase transforming ability. The transforming ability of various chemi- cally hybrid molecules has been studied also. The pneumococci to be transformed were streptomycin-sensitive, and transformants were counted by the streptomycin-resistant colonies growing after these bacteria were plated on an agar medium containing strepto- mycin. The material to be tested for trans- forming ability was obtained as follows. A constant concentration of DNA, from strepto- mycin-resistant pneumococci, and a series of increasing concentrations of DNA, from streptomycin-sensitive pneumococci, were heat denatured together and then renatured together (by cooling slowly). The larger the amount of streptomycin-sensitive DNA in the mixture, the larger was the transformation rate to streptomycin resistance. This increase proves that the higher the concentration of streptomycin-sensitive DNA, the greater the number of renatured molecules capable of transformation. It should also be noted that the addition, to cooling, denatured, streptomycin-resistant DNA, of homologous DNA (DNA from the same species, as from sensitive Pneumococcus) in the native (double-stranded) state, showed no increase in transformation ability over the rate obtained in its absence. Also, if, during the slow cooling, denatured heterologous DNA (DNA from radically different species, as from Salmonella, Micrococcus, Strepto- coccus, or calf thymus) is added, even in large quantities, there was also no increase in transforming activity over the control rate. Bacteria: Recombination (/) 347 Was the increase in transformation rate, obtained in the presence of denatured, homol- ogous DNA, due to the formation of hybrid molecules or did the excess denatured strepto- mycin-sensitive DNA merely cause an in- crease in unions between pairs of streptomy- cin-resistant single strands? The latter pos- sibility cannot be the explanation, since, as mentioned previously, hybrid molecules can be formed between single strands even when these are derived from different (but related) species. It is clear, therefore, that hybrid molecules were produced, that these can trans- form, and that just as only one strand is re- quired for the replication of DNA, only one of the complementary strands is needed to carry all the information for cistronic action con- tained in the normal double helix. (We would expect that the viruses 0X174 and Si 3, cf. p. 311, also carry all their genetic information in a single DNA strand.) Three additional observations should be made. First, strand separation is accom- plished by heat in a matter of a few minutes or less. This is important since it suggests the direction further work may take in studying how this might occur with adequate rapidity in vivo. (It has been suggested that chain separation is normally produced enzymati- cally, through the activity of ravelase.) Strand recombination by slow cooling has as yet no known biological counterpart, al- though it might be a mechanism for genetic exchange between closely related DNA mole- cules. The second point is that the capacity to routinely separate and combine single strands should lead to a better understanding of transformation, in particular the mecha- nism of integration. Experiments along these lines and others would be greatly aided by the use of closely linked marker recons, one on each strand of the hybrid molecule. In fact, such studies have already been reported, in which double transformations have been obtained with a complex of strands containing different markers. Molecular hybrids may also be useful for comparing base sequences in closely related organisms even when genetic recombination between them cannot take place. The final point is that, in bacteria, the recon can be identified as the smallest unit of DNA capable of being integrated or re- placed in a host chromosome subjected to the transformation process. SUMMARY AND CONCLUSIONS Contrary to the simplifying assumption made on page 329, genetic recombination occurs in bacteria by means of genetic transformation. This process involves a sequence of events in which competent cells permanently bind transiently-bound, large molecular weight particles of DNA. Once the DNA particle has penetrated, it apparently undergoes a synapsis-like process with a corresponding segment of the bacterial chromosome. Trans- formation is completed when a small segment of the DNA particle becomes integrated by replacing a similar segment of the recipient chromosome. Transformation provides direct and conclusive evidence that chromosomal DNA is genetic material. In bacteria, the recon is the smallest unit of DNA involved in trans- formation. Strand separation and recombination in vitro produces denatured and renatured DNA, respectively. Renatured DNA can transform, even when a hybrid molecule contains one normal and one mutant chain. This proves that all the information for cistronic action can be carried in one of the two strands of double helix DNA. 348 CHAPTER 37 REFERENCES Avery, O. T., MacLeod, C. M., and McCarty, M., "Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types," J. Exp. Med., 79:137- 158, 1944; reprinted in Papers on Bacterial Genetics, Adelberg, E. A. (Ed.), Boston, Little, Brown, 1960, pp. 147-168, and in Classic Papers in Genetics, Peters, J. A. (Ed.), Englewood Cliffs, N.J., Prentice-Hall, 1959, pp. 173-192. Doty, P., Marmur, J., Eigner, J., and Schildkraut, C, "Strand Separation and Specific Recombination in Deoxyribonucleic Acids: Physical Chemical Studies," Proc. Nat. Acad. Sci., U.S., 46:461-476, 1960. Ephrussi-Taylor, H., "Genetic Aspects of Transformations of Pneumococci," Cold Spr. Harb. Sympos. Quant. Biol., 16:445-456, 1951. Herriot, R. M., "Formation of Heterozygotes by Annealing a Mixture of Transforming DNAs," Proc. Nat. Acad. Sci., U.S., 47:146-153, 1961. Hotchkiss, R. D., "Transfer of Penicillin Resistance in Pneumococci by the Desoxyribonu- cleate Derived from Resistant Cultures," Cold Spr. Harb. Sympos. Quant. Biol., 16:457-461, 1951; reprinted in Papers on Bacterial Genetics, Adelberg, E. A. (Ed.), Boston, Little, Brown, 1960, pp. 169-176. Lerman, L. S., and Tolmach, L. J., "Genetic Transformation. L Cellular Incorporation of DNA Accompanying Transformation in Pneuniococcus,'" Biochim. et Biophys. Acta, 26:68-82, 1957; reprinted in Papers on Bacterial Genetics, Adelberg, E. A. (Ed.), Boston, Little, Brown, 1960, pp. 177-191. Marmur, J., and Lane, D., "Strand Separation and Specific Recombination in Deoxyribo- nucleic Acids: Biological Studies," Proc. Nat. Acad. Sci., U.S., 46:453-461, 1960. QUESTIONS FOR DISCUSSION 37.1. On what basis is transformation classified as a mechanism for genetic recombination rather than as a mutation? Do you agree with this interpretation? Why? 37.2. In view of the fact that native and renatured DNA are lighter in density than de- natured DNA, was the experiment with denatured DNA, described on p. 315, performed correctly? Explain. 37.3. Devise an experiment to detect whether chain separation occurs during extensive /// vitro synthesis of DNA. 37.4. Do the transformation results obtained with chemically-hybrid DNA prove that only a single DNA strand is required for cistronic action? Explain. 37.5. Do the studies on transformation offer any clues as to the ploidy of Pneunwcoccusl Explain. 37.6. What kinds of problems would you study, if you had a feasible method of studying the fate of individual cells exposed to transforming DNA? 37.7. What do studies of genetic transformation reveal regarding the genetic nature of conserved and nonconserved chromosomal DNA? 37.8. Redraw Figure 37-1, showing hypothetical base sequences in double-stranded DNA. Has your drawing any bearing on your answer to question 37.6? Explain. 37.9. How can you explain the fact mentioned on p. 343 that the frequency of double trans- formations is sometimes somewhat less than the product of the frequencies of the single transformations? Chapter *38 BACTERIA: RECOMBINATION (II. Conjugation) I "t was found in Chapter 37 that genetic recombination may occur .in bacteria by means of genetic transformation. The transformation process has two unique features which had hitherto not been encountered. The first is that the donor DNA enters the recipient bacterium without the intervention of any other organ- ism, as was demonstrated by the infectivity of naked DNA. Accordingly, although trans- formation involves the genetic material of two different cells it is not a typical sexual process, since it does not depend upon contact between donor and recipient cell. The second unique feature is that the integration process leading to genetic recombination occurs in the pres- ence of a portion of the entire genome of the donor cell and the entire genome of the recipient, as a consequence of which only a small segment of the penetrant donor DNA replaces a small homologous segment of the recipient chromosome. It would seem to be a reasonable hypothesis that any homologous DNA may integrate, by the same mechanism involved in transforma- tion, provided it penetrates the cell. One can therefore institute a search for other means whereby DNA may be introduced into a recipient cell. The present Chapter deals with experiments designed to test whether or not DNA passes from one bacterium to another, when these are in contact. The first experiment ^ can be designed as ' Based upon work of J. Lederberg and E. L. Tatum (1946). 349 follows. A prototrophic strain (K12) of E. coli is treated with a mutagen (like X rays or ultraviolet light) to obtain single mutants requiring different nutritional supplements in order to grow. The mutagenic treatment is repeated on the single, and then on the double mutant auxotrophs to obtain finally two lines, each different from the other by three nutritional mutants, all six mutants having arisen independently. One triple mutant strain is auxotrophic for threonine (T"), leucine (L"), and thiamin (5r) while the other triple mutant is auxotrophic for biotin (B-), phenylalanine (Pa-), and cystine (C-). The genotypes of these two lines can be given, respectively, as TLBi B+Pa^C^ and T+L+B,+B~Pa C-. The pure fines are grown separately on complete liquid medium. Then about 10^ bacteria from one line are plated onto agar containing complete medium, to form a lawn of continuous colonies. In the case of the TLBy- line, three replica plates are made (see Figure 38-1), each one containing com- plete medium minus a different single nutrient (lacking T, L, and Bi, respectively). Oc- casionally, a replica has a clone growing on it, which can be shown to be due to mutation to prototrophy for the nutrient missing from the medium. However, such clonal growth is not found in the same corresponding posi- tion on all three replicas, or even on two replicas, with greater than chance frequency. The same results are obtained when the B Pa C line is tested on appropriate repli- cas. We conclude, therefore, that on rare occasions single mutants to prototrophy for one nutrient do occur, but that double or triple mutants do not occur with detectable frequency. Next, the same experiment is repeated, except that the two triple mutant strains are first mixed in the liquid medium before being plated on agar containing complete medium. In this case (Figure 38-2), six replicas are made with medium which is complete except in the 350 CHAPTER 38 following respects. Three lack B, Pa, and C besides T, or L, or Bi; the other three lack T, L, and Bi besides B, or Pa, or C. Individuals of the T~L~Bc strain cannot grow on the first three replicas mentioned because single re- quired nutrients are missing, and cannot grow in the last three because all three are missing. Individuals of the B,Pa~C~ strain cannot grow on the first three replicas because all three required nutrients are missing and cannot grow on the last three because one of these is absent. If the master plate contains a mutant to nutritional independence for one of the nutritionally dependent loci only one replica will show growth. For example, if on the master plate there was a r+ mutant among the individuals of the TL-Bc strain, a clone will grow only on the replica lacking B, Pa, C, and T. Actually, about 1 00 different positions on the master plate show growth on the replicas. This is a very much larger number than is detected in the six replicas that test spontaneous mutation rate when the two fines are plated separately. Some posi- tions show growth only on one of the six replicas. However, there are many positions that show growth on two replicas; these must have gained nutritional independence at two loci. Finally, there are also many posi- tions which grow on all six replicas, each representing the occurrence of complete prototrophs (T+L+Bi+B+Pa+C+). One can use the clones on the replicas, or return to the master plate, and demonstrate that these changes are transmissible and preadaptive. When tested, these clones prove to be pure, that is, the nutritional independence gained is not attributable to some kind of physical association between two or more different auxotrophs. The large number of clones growing on the replicas, plus the fact that many are complete prototrophs or are auxo- FIGURE 38-1. Use of replica-plating (shown diagrammatically) to detect spontaneous mutations in E. coli. Replica 1 detects one mutant to T+, replica 3 detects one mutant to Bi+, and replica 2' detects one mutant to Pa+. MASTER PLATE REPLICAS T L B, Medium Contains ^ o o o , -r -r -r ^ TLB, T L B, + - + + + - TLB, TLB, B Pa C ^ o o o Medium ^ B+p^+^H Contains ~ B Pa C B Pa C B Pa C Bacteria: Recombination {II) 351 FIGURE 38-2. Replica-plating (shown diagrammaticully) to detect genetic recombination in E. coli. A completely prototrophic recombinant is found at 12 o'' clock in all replicas. A recombinant for both Pa and C is found at 3 o'clock on replicas 5 and 6. Replica J has a clone growing at 9 o'clock which may be due either to recombination or to mutation to T+. T L B.B Pa C T L B, B Pa C MASTER PLATE T L B,B Pa C ^Medium Contair SIX REPLICAS T L B.B Pa C T L B,B Pa C T L B,B Pa C T L B, B Pa C T L B,B Pa C T L B,B Pa C 352 CHAPTER 38 trophs for only one nutrient, is proof that these results are not due to spontaneous mutation. They must, therefore, be attrib- uted to some type of genetic recombination. Could the genetic recombination observed be the result of transformation? This possi- bility is unlikely since almost all transforma- tions involve single loci. Recall that the frequency with which two loci are transformed in the same bacterium is much lower than that for single transformations, and is pre- sumably dependent upon the close proximity of two loci on the bacterial chromosome. Yet, in the present experiment, double re- combinants are common. In fact, certain recombinations for two loci occurred more frequently than did the recombination of these loci singly. Transformation would even less readily explain the large number of triple recombinants, the complete proto- trophs. Nevertheless, specific tests may be made to rule out transformation as an explanation. It is found that the number of prototrophs obtained by recombination is uninfluenced when DNAase is added to the mixing and plating media. No transforming activity is demonstrated when one culture is exposed to filtrates or autolysates of the other. Finally, a U-tube can be constructed with a filter of sintered glass separating the arms. Broth can be added and the two strains placed in differ- ent arms. The medium plus soluble sub- stances and small particles (including viruses) can be flushed back and forth through the filter. Yet no recombinants are found in platings made from either arm. We may conclude, therefore, that the genetic recombi- nation detected in E. coli is not due to trans- formation. It is also not dependent upon a virus. It can be shown, moreover, by plating a mixture of three lines of K12 diff"ering in the mutants which they carry, that the large number of different recombinants obtained can all be explained as the result of recombi- nation between any two lines; no individuals are obtained recombinant with respect to markers in all three lines. Apparently, this type of genetic recombination depends upon actual cell-to-cell contact between pairs of bacteria, and therefore involves a sexual proc- ess. The frequency of sexual recombination in the first experiment is only about one per million cells (100 sites of recombination per 100 million bacteria placed on the master plate). The rarity of the event makes it fruitless, at this point in the investigation, to search microscopically for evidences of bac- terial mating. (You should, however, recog- nize that the importance of a new phenom- enon should not be judged by the frequency with which it occurs in experiments that first detect it. Recall, for example, that the initial quantity of DNA first synthesized in vitro was infinitesimal in comparison with the amount synthesized in later work, and that the rate of transformation observed initially was very much smaller than the 10-25% rate which can be obtained today with modified techniques.) It should be noted that we have used a medium that selects certain recombinants for detection and not others. In the first experi- ment discussed, only recombinants possessing certain markers for nutritional independence were selected. These are called selective markers. Thus, while the prototroph r+L+ Bi+B^Pa^C^ was detectable, it was not pos- sible to test for the occurrence of the comple- mentary polyauxotroph T~L~BrBPa^C~. Since no test has been made for the multiple auxotroph one could doubt its occurrence. It is entirely reasonable that the immediate result of mating is a zygote which contains a combination of part or all of the genotypes of the two parental cells. Although we have assumed that integration could take place if DNA passed from one cell in contact with another, that is, between two bacteria in conjugation, we have so far no evidence that it does. In other words, the possibility re- Bacteria: Recombination {II) 353 --++r ++ s __++s ++ — r BMPTV, xBMPTV, F^ BMPTV, xBMPTV, Prototrophs B M P T Prototrophs B M P T 86% r V, 14% s V, 79% s V, 21% r V, FIGURE 38-3. Genetic recombinations, involving iinselected markers, obtained in reversed crosses. mains, at least with respect to the genes showing recombination, that the recombinant may be diploid. It is possible, however, to add markers that are not selected for or against when one is selecting for recombinant prototrophs. These are called unselected markers. E. coli is available in two genetic forms, one, Ki% is resistant to infection by the bacterial viruses Ti and Te while the other, Ki% is sensitive to infection by these viruses, which cause the bacteria to lyse. Using the auxotrophic mu- tants P~ (proline-requiring) and M^ (methio- nine-requiring), it is possible to make the cross - B'M-P+T+V/ X B+M^P-T-V,^ and select for the prototrophs B+M+P^T^. A number of prototrophs were obtained. These were then tested for sensitivity to virus Ti. Because Vi is an unselected marker both of its alternatives are testable. Eighty-six per cent of the prototrophs were typically resistant, Ff, and 14% typically sensitive, Vi' (Figure 38-3). Note that both of these alternatives are recombinant relative to some of the markers for prototrophy, and that the alternative present is typically expressed. A diploid Vi^'Vi^ would be either re- sistant, or sensitive, or intermediate in expres- sion. The fact that both typical sensitivity and typical resistance to virus occurs proves that some recombinants contain only one representative of this locus. It is therefore 2 See J. Lederberg (1947). reasonable to extrapolate from this finding and say that only one alternative is present of any gene in a recombinant, which is the same as saying that all the genes in a nuclear body of E. coli are normally haploid. This means that conjugation produces a tempo- rary (partial or complete) diploid condition which, following integration, results in hap- loid recombinants, just as is presumably the case in transformation. The haploid recom- binants may also be called segregants. When the reverse cross is made, V{ entering with the B^M+P-P- parent and Ki' with B M'P^T^, the prototrophs show approxi- mately the reversed percentages which are sensitive and resistant to Ti. In other words, the parent that provides P+T+ to the proto- troph also contributes the Vi locus which it contains about 80% of the time. The fact that there is an imbalance in the fre- quency of resistants and sensitives among prototrophs (i.e., their ratio is not 50% : 50%), which is reversed when the Vi markers are reversed in the parent cells, is clear evi- dence that Vi does not segregate (or integrate) independently of the markers with which it entered the zygote and which were subse- quently integrated to form the haploid proto- troph. Accordingly, Vi must be linked to P T, segregating from these loci (or failing to be integrated in the same segment with them) about 20% of the time. On a hnkage map, therefore, Vi is about 20 recombination 354 CHAPTER 38 (segregation or integration) units from P T. It is possible also to determine the linkage relationships between auxotrophic markers in the following way. The cross T+L+BrB' X T-LBi^B^ is made on complete medium, plated on complete medium, and replica- plated four times on complete medium minus T, or L, or Bi, or B. Prototrophic recombin- ants grow on all four replicas, single auxo- trophs grow on three, while double auxo- trophs grow on two of the replicas. Since prototrophs are found to be more frequent than either T+L-B,+B+ or T'L+Bi+B+, Tand L are linked. A further analysis of the results, for other markers and from other experiments, reveals that all the genetic markers tested in E. coli are linked to each other, and can be placed on a map arranged in a linear order according to their recombination (segregation or integration) distances. This means, in all likelihood, that E. coli has a single chromo- some. Can zygotes actually be demonstrated? Since it would be almost impossible to find these microscopically, we must look for them genetically. Following mating, certain clones behave as though they are mosaic for a num- ber of markers. When single cell isolates, made from such clones, are grown and tested, it is found ^ that their progeny may possess either of the parental genotypes of the origi- nal cross, or they may be recombinants be- tween the two. Clearly, the isolated cells were derived from more or less persistent heterozygotes, individuals that are diploid for various markers. Thus, the segregants within a clone offer unambiguous proof that they were derived from a true zygote. We conclude, therefore, that the zygote produced following bacterial conjugation usually has only a temporary existence which terminates, after recombination, in the production of haploid progeny. 3 By J. Lederberg and M. Zelle. SUMMARY AND CONCLUSIONS Genetic recombination occurs in Escherichia coli following the sexual process of conjugation. This organism normally has a haploid nuclear body, in which all tested genes belong to a single linear linkage group. REFERENCES Lederberg, J., "Gene Recombination and Linked Segregation in Escherichia Coli," Genetics, 32:505-525, 1947; reprinted in Papers on Bacterial Genetics, Adelberg, E. A. (Ed.), Boston, Little, Brown, 1960, pp. 247-267. Lederberg, J., "Bacterial Reproduction," Harvey Lect., 53:69-82, 1959. Lederberg, J., and Tatum, E. L., "Gene Recombination in Escherichia Coli," Nature, London, 158:558, 1946; reprinted in Classic Papers in Genetics, Peters, J. A. (Ed.), Englewood Cliffs, N.J., Prentice-Hall, 1959, pp. 192-194. QUESTIONS FOR DISCUSSION 38.1. Criticize an attempt to prove the occurrence of genetic recombination by conjugation, by simply mixing two bacterial strains which are singly auxotrophic for different nutrients, and subsequently testing for prototrophs. 38.2. Why is it futile to search cytologically for bacteria in the process of conjugation? 38.3. Differentiate between, and give an example of: a. A selective and an unselected marker. b. A singly auxotrophic and a prototrophic bacterium. Bacteria: Recombination {II) 355 38.4. In this Chapter, what is the evidence that the two auxotrophs which produced proto- trophic progeny did so because of conjugation rather than because of mutation, genetic transformation, or viral infection? 38.5. In what ways does genetic transformation differ from conjugation? 38.6. Invent suitable genotypes for parents, a zygote, and its clonal progeny, which would prove the existence of the zygote, from the genotypes of the members of the clone it produces. 38.7. Ignoring the centromere, draw all the different ways you can represent a chromosome whose recombination map is linear. 38.8. Do you think the Summary and Conclusions section for this Chapter is adequate? Why? 38.9. Do you suppose the discovery of sexuality in bacteria could have important implica- tions for the practice of medicine? Why? 38.10. What is meant by integration in genetics? Describe verbally the mechanisms by which it may occur. 38.11. List those features of crossing over which are difficult to explain on a copy-choice basis. 38.12. Discuss the genetic control of gene synthesis and gene degradation. 38.13. Criticize the statement, on p. 16, that reconic transmission can only occur between generations by means of a cellular bridge. Chapter *39 BACTERIA: RECOMBINATION (III. The Episome F) Ai RE THE members of a pair of conjugating bacteria equiva- -lent? In other words, does DNA from either one go into the other, so that both bacteria can act either as donor or recipient? Suppose two auxotrophically different, streptomycin-sensitive Hues can normally conjugate and give recombinant progeny. If both lines are exposed to strepto- mycin before, but not after being mixed, none of the pretreated individuals can divide, and since all eventually die, no recombinant clones are formed. When one of the two parental lines is given such a pretreatment with streptomycin, it is found ^ that no re- combinants are detected, whereas when the other parental line is pretreated prototrophic recombinants do occur. This demonstrates that the two parents are not equivalent. The former type of parent (giving no recombin- ants when pretreated) must always act as the DNA-receiving cell, which, after conjugation, normally becomes the zygote. So, when this parent is killed by streptomycin, it is impos- sible to obtain recombinant clones. The latter type of parent must always serve in conjugation as DNA donor, its death, after acting as donor, having no effect upon the zygote and subsequent recombination. The latter type which acts as genetic donor is called F+ (for "fertility"), whereas the former type, which acts as genetic recipient, is called F; these types serve, so to speak, male and female functions, respectively. In bacterial conjugation, therefore, the genetic transfer ' Following the work of W. Hayes (1953). 356 which takes place is a one-way process. The original wild-type strain of E. coli K12 is F+, and in the period during which one of the auxotrophic lines was being prepared (cf. pp. 349-351), an F~ variant must have arisen. F+ X F~ crosses are fertile; F~ X F~ crosses are sterile (show no recombination); F+ X F+ crosses are fertile only because F+ cells may on occasion spontaneously change to F~. If one F+ cell is placed in a culture of F~ cells, all the F~ cells are rapidly converted to F+ type! The rapidity of the change from F to F+ is such that the causative agent must multiply at least twice as fast as the typical cell (and, therefore, twice as fast as chromo- somal DNA), sex conversion occurring with an efiiciency about 10'^ times higher than that of recombination. Moreover, the new F+ cells transmit this trait to their progeny. We conclude, that in E. coli, F+ male sexuality is an infectious phenomenon due to a factor or particle which we can call EK Several properties are known regarding F^ It is transferred from male to female only upon contact, and it cannot be isolated as a cell-free particle retaining sex conversion potency (accordingly, it does not give evi- dence of being a typical virus). The matings that transfer F^ are more frequent but less stable than matings involving chromosomal transfer. Exposure of F+ individuals to the dye, acridine orange, inhibits the replication of F^ so that F+ cells are converted to F . However, this dye has no apparent effect on chromosomal genes. This fact, plus a divi- sion rate which is faster than that of the chromosome, is sufficient evidence for con- cluding that E^ is an extra-chromosomal particle. The F' particle modifies the cell harboring it, in several ways. Not only does F^ make a cell a potential male, but it has several effects upon the cell surface. F' must change the cell surface of a male, so that a male cell can recognize, and react with, a female cell which it contacts; it must be the cause of Bacteria: Recombination {III) 357 some kind of bridge-formation between male and female cell, over which F' is passed to the F~ cell; it must also be the cause of the formation of a receptor, at the surface of the cell, for a virus that attacks males only.^ Finally, the /ow/requency of recombination, Lfr, of chromosomal markers following the mating of F+ and F~ must also be a property attributable to F^ So far, we have found that E. coli has two mating types, F" and F+ (Lfr). Another mating type arose from F+ cells. This type produces a /?igh /requency of recombination of chromosomal genes, and is hence called Hfr. Since the fertility of Hfr cells is un- affected by pretreatment with streptomycin, Hfr cells are donors. Hfr can mate with F~ cells, and, with low fertility, with F+ (Lfr which have probably spontaneously reverted to F-). Crosses of Hfr X F" produce 100- 20,000 times as many recombinants as does the Lfr X F~ cross. Since the progeny of the Hfr X F^ are typically F~, and rarely Hfr, Hfr does not carry infective F^ particles. However, Hfr can revert to Lfr strains which show all the characteristics of F+, including infective F^ Since Hfr can only come from, and revert to, F+, it is concluded that F' must be retained in masked or bound form in Hfr strains, in which condition the presence of extrachromosomally located infective F' is prohibited. The occurrence of Hfr strains makes a cytological search for conjugating pairs more likely to be successful. This is found to be the case. Figure 39-1 is an electron micro- graph showing conjugation between an F^ cell and an Hfr cell. The Hfr cell has ultraviolet-killed bacterial virus particles (tadpole-shaped objects) adsorbed to its sur- face; the F" cell does not since it is genetical- ly resistant to this virus. The cytoplasmic bridge between the conjugants is obvious. When exconjugants of such visibly marked 2 See reference to T. Loeb and N. D. Zinder (1961) on p. 305. pairs of Hfr and F~ cells are isolated by micromanipulation and cultured, only the clones from the F" partner yield recombin- ants. We may note, in passing, that these findings conclude another demonstration, of numerous onesalready cited, of the mutual aid genetics and cytology have provided in the ad- vancement of both branches of investigation. Using Lfr strains, it is commonly found that most of the unselected markers in recombin- ant progeny are those derived from the F~ parent. This can be explained either by the transfer of the entire genome of the male into the female followed by the integration of only a portion of it, or by the transfer of only a portion of the male genome and its integra- tion in toto, or a combination of these two possibilities. Experiments can be designed to test one or more of these explanations.^ ^ The following discussion is based principally upon work of E. L. Wollman and F. Jacob (see references at end of this Chapter). FIGURE 39-1. Conjugation in E. coli. (Courtesy of T. F. Anderson.) 358 MINUTES RECOMBINANTS HAVING Hfr MARKERS 0 None 8 T ^V2 T, L 9 T, L, Az n T, L, Az, T, 18 T, L, Az, T, , Lac 25 T, L, Az, T, , Lac, Gal CHAPTER 39 FIGURE 39-2. Recombinants obtained following artificial interruption of con- jugation at various times after mixing F~ and Hfr strains. The Hfr strain has markers for T, L, Az, T\, Lac, Gal. {After W. Hayes.) Particular strains of Hfr and F", both marked with suitable genetic factors, are grown separately and then mixed in the proportion of 1 : 20, respectively, to assure rapid contact of all Hfr with F~ cells. At various intervals of time, up to 60 minutes after mixing, samples are withdrawn and subjected to a strong shearing force in a Waring Blendor. This serves as a very efficient means of separating bacteria in the act of conjugation, the treatment affecting neither the viability of the bacteria, nor their ability to undergo the process of recombina- tion, nor the expression of the various geno- types under test. Once separated, the bacteria are plated and scored for male markers which have integrated. It is found that 0 minutes after mixing no recombinants are obtained ; this is as expected, since at this time no male markers have yet been trans- ferred. After 50 minutes of conjugation almost all the male markers that are going to be transferred have done so. However, the time at which different male markers enter the female cell varies widely within this time interval. For example, T and L markers do not enter until after about nine minutes of conjugation, while the Gal marker (for galac- tose) requires about 25 minutes of conjuga- tion before it is transferred. T and L are known to be close together and widely sepa- rated from Gal in the recombination map of Lfr referred to earlier (p. 354). Accordingly, there is a definite relationship between time of transfer from Hfr to F~ and the location of the marker on the Hfr chromosome. If different portions of the Hfr chromo- some were entering the F~ cell at random times, the results mentioned would not be obtained. We conclude, therefore, that the Hfr chromosome is transferred in a prefer- ential order, one particular end of the DNA string usually entering the F~ cell first, since the loci that transfer do so in a regular linear procession (Figure 39-2). Other experiments reveal that energy is required for the transfer process and that the entrance rate is uniform from the first part of the chromosome to be transferred, O (representing the "origin"), up to and including the locus of Lac (for lactose). Whether or not they have received or lost a segment of an artificially ruptured chromo- some, both the female and male cells survive, Bacteria: Recombination {III) 359 since E. coli is normally multinucleate. In fact, such breakages are found to occur spon- taneously also. Because of spontaneous rupture, the spontaneous transfer of the Hfr chromosome is usually partial, so that a piece of variable size is injected into the recipient cell. The resultant zygote of an Hfr cross is, then, a partial diploid (as is true of a cell undergoing transformation), and can be called a merozygote, produced by a process of par- tial genetic exchange or meromixis. The last two new terms are applicable also in trans- formation. While the frequency of recombination is low for all chromosomal genes in an Lfr strain, it is relatively very high for markers in Hfr nearest O, decreases as the distance of the markers from O increases, and is only .01 -.001% for the markers most distal to O. It was mentioned that only rarely is an off- spring of Hfr X F" itself Hfr. This happens only in cases where there is evidence that the marker most distal to O has also been trans- ferred. This suggests that the locus re- sponsible for Hfr is located on the chromo- some. Moreover, no chromosomal locus is found to be transferred after the Hfr locus. We conclude, therefore, that Hfr is always located at the terminus of the chromosome which is in the process of being transferred. A fourth mating type is known,'* derived from F+ cultures, which produces a very //igh /requency of recombination, and is appropriately called Vhf. In Vhf strains (all are male), recombination rates for the mark- ers most distal to O occur with a frequency of 1-2%, a rate at least 100 times that found in Hfr strains. (Even so, less than about 1% of the progeny from mating Vhf and F are Vhf.) Three independently arisen Vhf strains are known. By means of artificial rupture experiments, the sequence of certain marker genes can be determined in each case. These sequences are shown in Figure 39-3, ^ See A. L. Taylor and E. A. Adelberg (1960). together with the frequency of recombinants per 100 Vhf cells in the mating mixture. What do these results show? The different Vhf strains demonstrate that the markers held in common are in the same sequence. However, the O point is in a differ- ent position in each case! Accordingly, so is the position of the Vhf locus at the end of the linkage map. This suggests the following hypotheses.^ The linkage group of E. coli is normally circular; the location of the Vhf- causing factor can occasionally change; be- fore chromosome transfer the linkage group is opened adjacent to the point of Vhf attach- ment, so that the Vhf locus is at the end oppo- ^ Following F. Jacob and E, L. Wollman. FIGURE 39-3. Recombination percentages for Vhf strains. O = point of origin; — = untested. A. Adelberg, 1960. {After A. L. Taylor and E. See References.) SELECTED MARKER his -I- gal + pro + met + mtl xyl + mal ade + org AB-3n 42 12 STRAIN AB-312 2.5 4 8 AB-313 4 22 — 3.7 C 25 3 49 2.8 26 43 1.5 40 32 C D 15 - — 6 _ _ 0.3 360 CHAPTER 39 site the O point. The results obtained from the study of Hfr strains ^ confirm all these assumptions, including the occasional reloca- tion of Hfr as a consequence of which a new O point and sequence of entry is determined. To what can we attribute the difference be- tween Vhf and Hfr strains? One simple explanation is that these differ in the rate with which they cause spontaneous chromo- some rupture during conjugation, Vhf having the lower rate. There is a remarkable similarity between a Vhf or Hfr locus in the chromosome and its capacity to produce breakages in nearby regions, on the one hand, and certain cases already described on pp. 214-222 in corn (Activator and Dissociation, and Modulator), or referred to on pp. 222 and 223 in Drosophila (Segregation-Distorter), on the other hand. Suffice it to say, at this point, that these cases may provide another example of what at first appears to be a wide variety of apparently different and unique phenomena and which later proves to be due to minor variations in the expression of a more general, common event. In the light of results with higher organisms, it is reasonable that the positions of spontaneous rupture are merely places where the already opened E. co// chromosome is subsequently broken by the action of Hfr, and to a lesser degree Vhf, these breakages being more probable the closer the region is to the Hfr or Vhf locus. It may be noted that the results are not contrary to the view that once the chromosome is broken, Hfr (or Vhf) ceases to cause additional breakages in the now-free, other segment. Since a frequently recombining male strain always has the same marker leading the others in transfer, we conclude that Hfr or Vhf can cause the ring chromosome to open at only one of the two regions immediately adjacent to it. The fact that the entry se- quence is different in different strains (the chromosome of AB-312 enters in the reverse * See A. L. Taylor and E. A. Adelberg (1961). direction from that of AB-311 or AB-313, as can be seen from Figures 39-3 and 39-4) may be due to an inversion of Hfr or of Vhf in moving from one chromosomal position to another. There is experimental evidence that the E. coli chromosome is composed of a single double-stranded DNA helix, although it is possible that there may be nonnucleic acid links between DNA molecules (each with a molecular weight of about 10 X 10^ and about 16,000 nucleotides long). (The ordi- nary chromosome of higher organisms may be composed of one or of as many as 16 double strands of DNA ; we do not yet know for certain how many DNA strands are pres- ent per chromatid.) Since the ring chromo- some of E. coli is opened where F attaches, you may wonder if these two new ends are able to join in restitution. These two open ends must usually remain unjoined, other- wise one would not observe, in Vhf lines, the selective marker nearest O transported and integrated from as many as 49% of donor males (Figure 39-3). The recombination frequencies observed after conjugation will depend, of course, upon the frequency of penetration of a marker plus the efficiency with which it becomes inte- grated. Interruption of mating experiments reveal the sequence of markers, regardless of the frequency (greater than 0) with which their integration occurs. Once the marker sequence is known, integration efficiency can be studied. For example, if matings are permitted to continue long enough so that just about all F~ cells are penetrated by the marker closest to O, the percentage of zygotes producing recombinants for that marker will indicate the efficiency of integration. Thus, if only about 20% of the recipient cells show integration of a marker near O, this locus has an integration efficiency of %. By essen- tially similar methods the integration effi- ciency for markers more distal to O can be determined. These are found to be of lower 361 FIGURE 39-4. Linear ''^chromosomes''' of Vlif strains. Arrows show direction of chromosome penetration during conjugation. {After A. L. Taylor and E. A. Adelberg, I960. See References.) ser/gly efficiency. Thus, the closer a gene is to O, the greater is its chance for integration. In this connection it may be fruitful to recall the observation (cf. p. 345) that when one strand of a double helix of donor DNA is broken via DNAase activity, incorporation of donor DNA into the recipient DNA frac- tion is unaffected but transformation rate is drastically reduced. Accordingly, the fact that the farther a locus is from O, the lower is its integration efficiency, may be a direct consequence of the action of an Hfr or Vhf locus which breaks one strand of double-hehx DNA and not the other. In this event, such loci could be producing effects not only on chromosome rupture (by severing both strands of the DNA double chain at the same level), but also on integration (by breaking only one chain of the two at any given level). In cases when penetration of a chromosome FIGURE 39-5. Genetic map of Escherichia coli in which distances are expressed in minutes; markers in parentheses are less precisely mapped. (After A. L. Taylor and E. A. Adelberg, I960. See references.) 362 CHAPTER 39 is stopped by Blendor treatment, we cannot decide whether the treatment breaks the chromosome, or the cytoplasmic bridge, or both. However, since conjugants, which have been visibly joined for two or more hours, may show only limited amounts of recombi- nation, it is likely that a chromosomal defect, and not bridge rupture, is responsible for spontaneously halting transfer and/or inte- gration. Taking into account the changes in rate of penetration and in integration efficiency, it is possible to construct, from interruption ex- periments using Vhf or Hfr males, a genetic map of E. coli markers whose relative dis- tances are expressed in minutes. This is shown in Figure 39-5. This map is essentially identical to the one derived from the relative frequencies of recombinants from Lfr X F^ crosses (p. 354). Let us now return to a consideration of F+ (Lfr) strains. One can perform an interrup- tion experiment to determine when the F^ particle in Lfr males is transferred in mating. It is found that F^ is first transferred about five minutes after mixing F+ and F . This is several minutes earlier than any marker on the chromosome is transferred. Moreover, there is no linkage of F^ with any marker on such a chromosomal segment. Accordingly, this is additional support for the extrachro- mosomal nature of F^ We have mentioned already that Hfr strains are always derived from F+ strains, as were the Vhf strains. We have also noted that Hfr strains may revert to F+. (So can Vhf.) We have taken this to mean that Hfr (and doubtless Vhf too) harbors a latent F^ particle. Since the fertility of Hfr is unaffected by exposure to acridine orange, the latent F' particle cannot be located extrachromo- somally, or it would disappear. Accordingly, the latent F^ particle in Hfr (or Vhf) must be located chromosomally. Since F^ is the only known factor essential for maleness, the locus we have assigned to the Hfr and Vhf genes must be the locus oj chromosomal FK Once F' enters the chromosome all remaining cyto- plasmic F' particles are normally lost. In the light of this information, what can we reason concerning what happens in F+ cells when, on rare occasions, they do transfer chromosomal material (so that, as a whole, the F+ clone gives a low frequency of recom- bination)? A number of hypotheses can be suggested, but we will consider a particular one. We can suppose that in order for chro- mosomal transfer to take place in F+ cells, an F' particle must attach to the chromosome, making it a typical Hfr chromosome. This can be tested as follows.^ After mixing suit- ably marked F+ and F^, replica plates are made to show the places where recombination has taken place. A search can then be made for Hfr strains among cells growing on the master plate. Although new Hfr strains occur rarely, they are obtained much more frequently from positions on the master plate where recombination has taken place than where it has not. Moreover, it is often found that the Hfr strain obtained produces a high frequency of recombination of the same marker whose recombination on the replica aided in the detection of the Hfr strain. In other words, it seems valid to believe that before an F"*" individual transfers chromo- somal material, it first changes to a particular Hfr (or Vhf) condition. This Hfr produces the recombination detected on the replica, while its clonal members on the master plate yield the same type of Hfr. This interpretation also receives support from other experiments. Some additional properties of F^ may now be listed. F^ is characterized by having a low affinity for the chromosome; it has no prefer- ential site of attachment, and once it attaches, the extrachromosomal multiplication of F^ ceases. A genetic variant is now known,^ F^, which has a high affinity for the chromo- some (so that it frequently produces an Hfr 7 Based upon work of F. Jacob and E. L. Wollman. 8 See E. A. Adelberg and S. N. Bums (1960). Bacteria: Recombination {III) 363 strain), and has a preferential site of attach- ment near Lac (lactose). However, the Hfr produced also carries F'-^ particles in the cyto- plasm. Because of its replicability and genetic variability, the male fertility factor of E. coli must be considered to be composed of genetic material. F is probably neither lytic nor otherwise rapidly lethal to F" cells. In view of this and the fact that it has a stable rela- tionship with the F+ cell, it may be considered a normal cellular component, when present. Since it can exist and reproduce extrachro- mosomally, F furnishes the first example so far presented of normal extrachromosomal genetic material. When F, as an autonomous extrachromosomal agent, is lost, either spon- taneously or after treatment with acridine orange, it represents genetic material that is not conserved for future generations. F also has the capacity to assume a regular locus on a chromosome. When integrated into the chromosome, it behaves as an ordinary chromosomal locus. In regular vegetative reproduction, chromosomal F is transmitted to all progeny, that is, it is con- served. In conjugation, however, chromo- somal F may not be conserved, since it is not transmitted to the zygote with appreciable frequency, except in the case of Vhf strains, and even when transmitted, it may fail to be integrated. To the genetic elements restricted to the chromosome, the only type of gene discussed in detail until this Chapter, we can now add the male fertility factor, F, which may be present or absent from the cell, and, when present, may be autonomous extrachromo- somally or integrated in the chromosome. To such genes which can participate in the cell facultatively, as extrachromosomal or as chromosomal elements, the name episome is given.'' 9 See F. Jacob and E. L. Wollman (1958). SUMMARY AND CONCLUSIONS In E. coli, there is only one female mating type, or genetic recipient (F") but several male mating types, or genetic donors (F+, Hfr, Vhf), Male mating type depends upon the pres- ence, location, and genotype of F. F is an episome. When present extrachromosomally, F is infective. When located chromosomally, the ring chromosome of E. coli is opened near the locus of F. The end opposite the F locus proceeds first, in the linear transfer of part or sometimes all of the opened chromosome into the recipient conjugant. REFERENCES Adelberg, E. A., and Burns, S. N., "Genetic Variation in the Sex Factor of Escherichia Coli," J. Bact., 79:321-330, 1960; reprinted in Papers on Bacterial Genetics, Adelberg, E. A. (Ed.), Boston, Little, Brown, 1960, pp 353-362. Hayes, W., "The Mechanism of Genetic Recombination in Escherichia coli,'' Cold Spr. Harb. Sympos. Quant. Biol., 18:75-93, 1953; reprinted in Papers on Bacterial Ge- netics, Adelberg, E. A. (Ed.), Boston, Little, Brown, 1960, pp. 268-299. Hayes, W., and Clowes, R. C. (Eds.), Microbial Genetics, Cambridge, Cambridge University Press, 300 pp., 1960. Jacob, F., and Wollman, E. L., "Episomes, Added Genetic Elements" (in French), C. P. Acad. Sci. (Paris), 247:154-156, 1958; translated and reprinted in Papers on Bacterial Genetics, Adelberg, E. A. (Ed.), Boston, Little, Brown, 1960, pp. 398-400. 364 CHAPTER 39 Jacob, F., and Wollman, E. L., "Genetic and Physical Determinations of Chromosomal Segments in Escherichia Coli," Sympos. Soc. Exp. Biol., 12:75-92, 1958; reprinted in Papers on Bacterial Genetics, Adelberg, E. A. (Ed ), Boston, Little, Brown, 1960, pp. 335-352. Jacob, F., and Wollman, E. L., Sexuality and the Genetics of Bacteria, 374 pp.. New York, Academic Press, 1961. Taylor, A. L., and Adelberg, E. A., "Linkage Analysis with Very High Frequency Males of Escherichia Coli," Genetics, 45:1233-1243, 1960. Taylor, A. L., and Adelberg, E. A., "Evidence for a Closed Linkage Group in Hfr Males oi Escherichia coli K-12," Biochem. Biophys. Res. Commun., 5:400-404, 1961. Wollman, E. L., Jacob, F., and Hayes, W., "Conjugation and Genetic Recombination in Escherichia coli K12," Cold Spr. Harb. Sympos. Quant. Biol., 21:141-162, 1956; reprinted in Papers on Bacterial Genetics, Adelberg, E. A. (Ed.), Boston, Little, Brown, 1960, pp. 300-334. r Elie L. Wollman {left) and Francois Jacob (right) in 1961. QUESTIONS FOR DISCUSSION 39.1. What events do you suppose occur between the time that donor DNA enters a female conjugant and the time of appearance of a segregant which is haploid for a segment of the donor DNA? 39.2. What is the evidence that F can integrate in a chromosome? That it can deintegrate? 39.3. What properties are attributable to F when this is integrated at a chromosomal locus? Bacteria: Recombination {III) 365 39.4. Does the occurrence of spontaneous ruptures of the donor bacterial chromosome interfere with mapping the linear order of genes via artificial interruptions of mating? Explain. 39.5. Assume, correctly, that the decay of P^' incorporated into DNA can break the E. coli chromosome, and that this decay is temperature-independent. Devise an experiment to determine the gene order in this bacterium. 39.6. What kinds of evidence can you present that per nucleus of £^. coli there is normally: a. A single "chromosome"? b. That this is a ring? 39.7. Give the genotypes of parents and recombinants, and the specific culture conditions you would employ in searching the progeny of F+ X F~ crosses for Hfr lines. 39.8. Would you, in the light of our knowledge of episomes, revise the discussion on p. 198 regarding recon polarity? Why? 39.9. What relationships exist between episomes and genetic recombination? Chapler *40 BACTERIA: RECOMBINATION (IV. Episomes and Nucleotide-Sharing) G ENETic recombination, by the .sexual process of conjugation, is known to occur in bacteria like Pseudomonas and Salmonella, as well as Escherichia. In Escherichia, male sexuality is attributed to the presence of an F particle, the particular male type being determined by the location and genotype of F. We have found that bacterial conjugation leads to new combinations of either or both the chromo- somal genes and the extrachromosomal, episomal, genes. Let us consider the sequence of events in- volved in the genetic recombination of the F particles themselves. What happens when an Hfr or Vhf strain reverts to F+ (Lfr)? The F particle which is integrated into the Hfr or Vhf chromosome is somehow deintegraled or liberated from it, enters the cytoplasm, repli- cates, and is infectious thereafter. Subse- quently, in some future generation, the F+ particle may reintegrate into a chromosome making it Hfr or Vhf. What would we expect to be the property of an F particle responsible for its integration into a chromosome? It is reasonable to require that the F particle be attracted to the chromosome. Could the cause of the attraction between F and chro- mosome be the same as that between a trans- forming segment of chromosome (or a piece of chromosome transmitted by conjugation) and the recipient chromosome? If so, the attraction aspect might be explained merely by supposing that the F particle is composed of DNA. But this assumption, alone, is not 366 sufficient to explain the integration of F, since in transformation the donor loci which inte- grate must be homologous to those replaced in the recipient cell (and this homology is probably also required for the integration of chromosome fragments introduced by con- jugation). We would have to suppose, in addition, that an integrating F particle con- tains a piece of DNA homologous to a seg- ment of DNA already present in the chromo- some. What might be the source of the ho- mologous segment F presumably contains? It might have been present "initially" or it might have been obtained on the last occasion that F deintegrated from the bacterial chro- mosome. If a free F particle sometimes carries a particular segment of chromosomal DNA, which it obtained at the time of deintegration, one should be able to find free F particles which have an affinity for a given chromo- some region when introduced into a normal F~ strain. This is indeed the case for the F- particle (see p. 363), which has a high affinity for a particular locus near Lac. On the other hand, the fact that F^ has a low affinity for the chromosome may mean that it has a smaller amount of chromosomal DNA at- tached to it than has F-. Using the F- particle as a particular ex- ample, it is possible to visualize several differ- ent genetic segments which might be liberated from the chromosome by deintegration. What may be liberated is the complete F- particle with, or without, normal chromo- somal material still attached, or a defective F- particle with, or without, normal chro- mosomal material still attached. Liberated, defective F- particles may or may not be capable of replication. (By being defective or incomplete, we mean that such particles would have lost their F characteristics, and would be undetected phenotypically. If, in fact, deintegration produces defective F- particles that cannot replicate, these particles would be lost at some future time, furnishing Bacteria: Recombination {IV) 367 an example of nonconserved chromosomal DNA.) What would be the reciprocal product if a defective F- particle is deintegrated? In that event, the chromosome would retain a por- tion of the F- genotype as a "memory." Thus, just as complete or incomplete F- might carry a piece of chromosome which serves as "memory," a chromosome may retain a part of the F- genotype as "memory." Should a complete F- with chromosomal memory infect a normal F" cell, it would be expected to inte- grate with relatively high frequency at a pref- erential position in the chromosome. And, reciprocally, should a chromosome with F- memory be exposed to complete F-, the epi- some also would be expected to integrate with relatively high frequency at a preferential locus. We have just expressed certain expecta- tions, regarding the frequency and locus of F integration, on the basis of certain assump- tions. Let us now see how experiments ^ (part of whose conclusions were already pre- sented in the last Chapter) bear upon these expectations. An F+ strain (carrying F^ extrachromosomally) gave rise to an Hfr strain, P4x, whose chromosome is transferred with the following orientation: O (origin or lead po'mt)-Pro-TL-Thi- . . . -Gal-Lac-SF (place of attachment of the sex factor). P4x X F" gives F~ progeny except for Lac recombin- ants which are in turn Hfr males, because of the close linkage of F' and Lac. A new strain, P4x-1, derived from P4x, has the following characteristics: (1) From in- terrupted conjugations it is established that P4x-1 is identical to P4x with respect to order and times of entry of chromosomal markers. Thus, for example, both transfer Pro at about six minutes, TL at about 20 minutes, and Lac last. However, the frequency of recombina- tion for Pro is reduced in P4x-1, being only ^ The preceding and following discussion is based largely upon the work of E. A. Adelberg and S. N. Burns (1960), and F. Jacob and E. A. Adelberg (1959). .3-. 5% of donor cells as compared to 4.8% of donor cells for P4x. (2) Moreover, in interrupted conjugation experiments with P4x-1 many of the recombinants for Pro or TL behave as males. It is also found that the male factor in P4x-1 is linked neither to these loci nor to any other chromosomal marker showing recombination, and that, like free F\ it enters the F^ cell about five minutes after conjugation begins. In brief, the results prove that the male sex factor in P4x-1 is located extrachromosomally. This cytoplasmic sex factor is called F- because it shows certain differences from F^ (cf. p. 362). Whereas F' can attach at any one of a number of different sites, giving Hfr and Vhf chromosomes which differ in O point position and in direction of transfer, F- attaches at a particular locus near Lac to give rise to an Hfr which always transfers its chro- mosomal loci in the same order and direction. The fact that P4x-1 transfers its chromo- some more frequently than does the typical F+ (F'-containing) male may mean that the F- particle has a chance for integrating at its re- stricted locus near Lac which is greater than the total chance that F^ has of integrating at any one of a number of different loci. On the other hand, P4x transfers more frequently than P4x-1. This suggests the possibility that F is already integrated in P4x, but is usually only paired with a homologous region in P4x-1, at which position it has the poten- tiality of being integrated. This suggested difference could explain the observation that P4x has no free F while P4x-1 has, repression of F reproduction cytoplasmically being a characteristic only of F when integrated into the chromosome. The experimental data are consistent with the view that, in P4x-1, inte- gration of the F- particle takes place only after conjugation is initiated. In those cases where F- is known to be transferred as an extrachromosomal particle to F~ cells, these cells are converted to males who transfer their chromosome relatively 368 CHAPTER 40 frequently with the same marker sequence found for P4x and P4x-1. This demonstrates that an ordinary F~ cell carries a chromosome which has, near Lac, a segment of DNA which is homologous to a segment carried by F^; that is, F- has a chromosomal segment serving as memory for pairing and integra- tion. It is possible, also, by treatment with acri- dine orange, to eliminate the extrachromo- somal F particles from the P4x-1 strain, con- verting it to F^. Such a strain will conjugate with males carrying either F^ or F^ extra- chromosomally. In both cases the F" strain can relatively frequently become a donor (male) which transfers its chromosome with the same orientation as does P4x and P4x-1. Clearly, then, the F~ chromosome derived from P4x-1 has retained near Lac a segment of F- as memory, the portion retained being one held in common by F' and F-. This experiment shows, moreover, that so far as the F portion of the particle is concerned, F' and F- are not detectably different. Ac- cordingly, we can think of F- as being an F^ particle with a longer, particular piece of chromosome attached. Since, thus far, the experimental results obtained support our expectations, let us continue our reasoning further. We have found evidence that F may carry chromo- somal DNA which is apparently still capable of replicating in its new location. Let us suppose also that this chromosomal segment is still capable of performing its normal func- tion. The fact that F^ carries no phenotypic effect expected of a normal chromosomal locus was presumed to mean that its chro- mosomal piece is short. Suppose a cistron usually contains hundreds or thousands of linearly arranged deoxyribotides. F' might carry, say, four chromosomal nucleotides as memory. In that case, the piece of chromo- some would contain only part of a cistron and could therefore produce only a portion of the product of the complete cistron. Since an incomplete primary gene product would not be unique, F' would be scored in practice as producing no gene product for this cistron. Yet the shortness of its chromosomal piece would mean that there would be many places where F' would find a similar deoxyribotide sequence when placed alongside the chromo- some in one direction or the other. This would explain the fact that F' can integrate at a number of loci, giving rise to Hfr or Vhf lines that transfer markers in opposite se- quences. The failure of F- to show a phenotypic effect for a normal chromosomal locus may be due either to the fact that its chromosomal piece, though longer than that of F', is still not long enough to include a cistron, or that it contains one or more, as yet unidentified, chromosomal cistrons. If the latter possibili- ties occur in fact, we should expect to find still different F' particles to which a known chro- mosomal marker is attached. This last ex- pectation can be tested experimentally. In studies of interrupted conjugation, using Lac'F~ cells and a Lac^ strain of Hfr where F^ is known to be integrated very close to Lac+, rare recombinants are obtained which have received Lac^ too early. Certain of these recombinants have the following prop- erties: 1 . They have received only F^ and Lac+. 2. They are unstable, and occasionally give rise to Lac~¥~ individuals. Hence the original recombinant was a merozygote carrying both Lac+ and Lac~ alleles. 3. When crossed to Lac~F~ cells they simul- taneously transfer F^ and Lac^ together with 50% or higher frequency. This transfer starts at a time soon after conju- gation begins, just as in the case of free F' (or F-), and is unlinked to other chro- mosomal markers. Thus, F'-Lac+ behaves as a free single unit. 4. The recombinant transfers its chromosome in the same sequence as, but with a lower Bacteria: Recombination {IV) 369 frequency (/io) than, the original Hfr hne. This is exactly what is found in compari- sons of P4x-1 with P4x. 5, The F^-Lac+ element can be transmitted in a series of successive conjugations, each recipient possessing the properties of the original recombinant. All these results are most simply explained by the deintegration, in the original Hfr strain, of an F^ particle carrying a chromo- somal piece bearing Lac+. The attached Lac+ piece is known, moreover, to contain three cistrons; these govern the synthesis of 13- galactosidase, j8-galactoside permease, and the repressor for this system, respectively. From subsequent integrations and deintegra- tions it is possible also to obtain F'-Lac^ particles, and other particles differing in Lac and F^ capacity. Finally, another Hfr, where F^ is integrated close to Pro, has been found to produce an ¥^-Pro particle having proper- ties analogous to those of the F^-Lac particle. Since F' can integrate at a variety of loci, we can extrapolate from such results that as a consequence of deintegration, any one of a variety of normal chromosomal loci may become a part of the genotype of cytoplasmic F^ When long enough, this memory piece of chromosome attached to F' will both repli- cate and produce its phenotypic effect. The evidence seems conclusive, then, that chro- mosomal DNA need not be located in the chromosome in order to replicate or function. We see, therefore, that F^ is involved in four types of genetic recombination. F^ makes possible recombination between a chromo- some segment of a donor and the chromo- some of a recipient; F^ can itself be added as a locus to the chromosome at a variety of positions, this being repressive of F^ located extrachromosomally; F^ can be removed from its chromosomal locus in toto (or in part) by deintegration; the deintegration that hberates F^ can include also neighboring cistrons. The last event produces recombina- tion of chromosomal genes between F^ and the chromosome. There is, therefore, a two-directional gene flow between the extra- chromosomal episome F and the chromo- some. So far, we have restricted our attention exclusively to the episome F in Escherichia coli. Evidence also has been obtained that colicines (certain substances - having the capacity to kill colon bacilli, i.e., having colicidal properties) are produced by a coli- cinogenic determinant which acts as an epi- some in E. coli. The colicinogenic determi- nant can leave its chromosomal locus, multi- ply autonomously extrachromosomally, and later integrate into the chromosome.^ When extrachromosomal, the free episome can mi- grate actively, as F does, arriving in the F~ cell as early as 2}^ minutes after conjugation is initiated. Do episomes occur in organisms other than bacteria? It has already been suggested (re- fer to p. 360) that certain genetic elements in the chromosomes of corn and of Drosophila possess characteristics similar to those of episomes.'' In this connection let us also consider the relationship between the centro- mere and centrosome. The centrosome is an organelle often found at each pole of a spindle, particularly in animal cells, and a granular structure called the centriole is some- times seen within it. Similar granules can sometimes be seen within the centromere (Figure 40-1). The granules in both the centromere and centrosome stain the same (both doubtless containing DNA), and so does the material surrounding these granules. In the hving cell, also, centromere and centrosome have a similar appearance. (The granules within the centromere are thicken- ings of the DNA thread which passes through the centromere and which is continuous with ' At least two colicines are lipocarbohydrate-protein complexes. 3 See F. Jacob and E. L. Wollman (1958). ■• See F. Jacob and E. L. Wollman (1961), p. 364. 370 CHAPTER 40 B FIGURE 40-1. The centromere and its granules in corn. {Courtesy of A. Lima de Faria, ''Com- pound Structure of the Kinetochore in Maize," J. Hered., 49:299, 1958.) the DNA thread in the chromosome arms.) The morphological similarity of centromere and centrosome is paralleled by their simi- larity in behavior. Centromeres are some- times attracted to each other and to the cen- trosomes. At anaphase, the centromeres migrate toward the centrosomes. The centro- some also has the capacity for movement, as demonstrated by its preanaphase movement and its behavior after a sperm has penetrated an egg. From these facts some sort of kinship is suggested ^ between centrosome and centro- mere. This view is strikingly supported by an additional piece of evidence. It has been found,*' during the meiosis of certain mollusc sperms, that certain chromosomes degener- ate, as a result of which free, "naked" centromeres are produced. These now-free centromeres form a group with the centro- ' Originally by C. D. Darlington, by F. Schrader, and by others. ^ By A. W. Pollister and P. F. Pollister. some, and thereafter duplicate centrosomal behavior and appearance exactly. In effect, then, the freed centromeres become extra centrosomes. The preceding evidences sug- gest the hypothesis that the centromere and centrosome represent alternative states of a presently or previously existing episome. The change from one episomal state to the other would probably be influenced by the presence of a nuclear membrane, by various other genetic factors present in highly organized cells, as well as by mutations which have occurred in the episome when in its alternative states. It is known that at the base of each cilium or flagellum is a granular body, the kineto- some, which is responsible for the motion of the organelle. There is excellent evidence that these kinetosomes are homologous to centrioles (or centrosomes). This suggests that kinetosomes are also episomes or episo- mal derivatives. It has been suggested ' that the episome F functions like a centromere. Is it possible that the movements attributed to F and the centromere and centrosome have the same basis as the flagellar and ciliary movement produced by kinetosomes? Let us speculate with regard to certain matters which may not, at first, seem to have any possible relation to episomes.^ Heterochromatin (cf. p. 181) behaves cyto- logically and physiologically (phenotypically) less specifically than does euchromatin. Moreover, because portions of heterochro- matin can be added or subtracted from the genotype without causing great phenotypic modification, it is reasonable to believe that the cistronic product of heterochromatin is not only unspecific, but is duplicated in suc- cessive heterochromatic segments. In view of the likelihood that the cistrons of hetero- chromatin produce an unspecific product, it is hypothesized that each has a short nucleo- ' Originally by R. C. Baumiller. « See I. H. Herskowitz (1961). The reading of the remainder of this Chapter is optional. Bacteria: Recombination (IV) 371 tide sequence, since the complexity, and hence the specificity, of a cistron's product would be expected to be directly correlated with the number of nucleotides in the cistron. It can also be hypothesized that most, if not all, of the heterochromatin in a cell is composed of a monotonous repetition of the same nucleotide pair, usually A-T. This view receives support from the following observations: (1) The sequence of A, T, C, G on a single strand of DNA is far from random; at least 70% of the bases are dis- tributed so that three or more pyrimidines (and hence purines, too) occur in successive nucleotides; sequences of five successive T's have been identified; 4.9% of a tested DNA contains sequences of eight or more pyrimi- dines in succession; (2) a polymer of A-T alone may make up as much as 30% of cer- tain crab sperm DNA; (3) the amount of thymine is increased in Drosophila cells carry- ing an extra, almost entirely helerochromatic, Y chromosome; (4) 5-bromo deoxyuridine, which is known to replace thymine in DNA, causes a high frequency of breaks near or in heterochromatic regions. Suppose, to exemplify these suggestions, that all nucleotides in a particular stretch of heterochromatin have the same formula. A, and that three in succession (AAA) comprise a cistron. (We can ignore, at this time, the complementary chain composed of T's.) Since the number of successive A's is expected to be considerable, it is reasonable that a particular A may be used cistronically, some- times with two A's to its right, and other times with two A's to its left. In other words, in heterochromatin, a nucleotide can be shared by adjacent cistrons. On the other hand, a typical, highly specific cistron in euchromatin would be expected to be com- posed of several hundred or a thousand linearly arranged nucleotides. Suppose a particular euchromatic cistron terminates in -TAA. If a break occurs between the -TA and A, and another occurs almost anywhere in the heterochromatin, and cross-union of broken ends occurs, the euchromatic cistron will have its proper nucleotide sequence com- pleted. But, since any three successive A's can serve as template for making hetero- chromatic product, there could be nucJeotide- sharing between the now-adjacent euchro- matic and heterochromatic cistrons. In this case, whenever the heterochromatic cistron was used to make product, no complete prod- uct would be formed from the euchromatic cistron. Thus, so long as the adjacent heterochromatic cistron made its product, the euchromatic cistron would be functionally suppressed, and would be scored as an amorph (see p. 210). If conditions changed, so that the euchromatic cistron was able to function as usual, the normal euchromatic phenotype would result. If the heterochro- matic and euchromatic cistrons were func- tional alternately, no detectable phenotypic effect would be expected with regard to the heterochromatic product, but phenotypic mosaicism could result because of the inter- mittently produced euchromatic product. In these ways, suppressed or variegated pheno- typic effects, which are known to be due to the placement of heterochromatin near eu- chromatin, may be explained. Such position effects (cf. Chapters 22 and 25) are frequent following structural changes in chromosomes of Drosophila. Some of these cases of pheno- typic suppression involve genetic elements like Segregation-Distorter in Drosophila and Dissociation in corn, which are associated with heterochromatin, can also cause break- age, and can change their location in the genome. Since such factors resemble epi- somes, it may not be too far-fetched to study these and known episomes for the occurrence and possible consequences (phenotypic sup- pression, organelle movement, and chromo- somal breakage) of nucleotide-sharing. Many of the ideas relative to homologous organelles, nucleotide-sharing, and suppres- sive and variegated position effects discussed 372 CHAPTER 40 in the last portion of this Chapter are highly speculation is identified as such, no perma- speculative. (Speculation in science is per- nent scientific damage ensues.) In the mitted, however, if it conforms to the facts Chapters that follow you should keep these known at the time, and if it leads to expecta- ideas in mind, and search for more informa- tions subject to test in feasible experiments. tion to test the correctness of part or all of When these rules are followed, and the the hypotheses presented. SUMMARY AND CONCLUSIONS The free episome F may carry chromosomal markers, and chromosomes may carry only a part of F. This results in a two-directional flow of genetic material between extrachromoso- mal F and the chromosome, which represents a new type of genetic recombination. The chromosomal markers carried by free F are still capable of replication and of pheno- typic function, having retained these genie characteristics though removed from the chro- mosome. Colicines are the product of an episome. Episomes may exist in organisms more complex than bacteria. The characteristics of centrosomes, kinetosomes, and centromeres suggest that these show a present or past episomal relationship with each other. Elements like Modulator and Dissociation have some of the properties of episomes. Speculation leads to the tentative concept that nucleotides may be shared by adjacent cistrons. This might lead to suppressed or variegated phenotypic effects, chromosomal breakage, and movement of cell organelles. The study of episomes and of episomal-like factors may provide a test of the occurrence and consequences of nucleotide-sharing. REFERENCES Adelberg, E. A., and Burns, S. N., "Genetic Variation in the Sex Factor of Escherichia Coli," J. Bact., 79:321-330, 1960; reprinted in Papers on Bacterial Genetics, Adelberg, E. A. (Ed.), Boston, Little, Brown, 1960, pp. 353-362. Jacob, F., and Wollman, E. L., "Episomes, Added Genetic Elements" (in French), C. R. Acad. Sci. (Paris), 247:154-156, 1958; translated and reprinted in Papers on Bacterial Genetics, Adelberg, E. A. (Ed.), Boston, Little, Brown, 1960, pp. 398-400. Herskowitz, L H., "The Hypothesis of Nucleotide Sharing by Adjacent Functional Units of DNA," (Abstr.) Genetics, 46:870, 1961. PoUister, A. W., and Pollister, P. F., "The Relation Between Centriole and Centromere in Atypical Spermatogenesis of Viviparid Snails," Ann. N.Y. Acad. Sci., 45:1-48, 1943. Spencer, J. H., and ChargafF, E., "Pyrimidine Nucleotide Sequences in Deoxyribonucleic Acids," Biochim. Biophys. Acta, 51:209-211, 1961. QUESTIONS FOR DISCUSSION 40.1. Answer question 39.9 on page 365. 40.2. Do all matings transfer F particles of one genotype or another? 40.3. Discuss the relationship between the transmission of free F particles and of a segment of the male chromosome. 40.4. Discuss the reality of a bacterial "chromosome" and its linear arrangement. 40.5. By what series of events can you explain the origin of strain P4x-1 from P4x? 40.6. Specify a particular Hfr strain oi E. coli and tell how you would proceed to obtain an F-Pro (proline) particle. Bacteria: Recombination (IV) yj3 40.7. What characteristics of Dissociation and Modulator resemble those of the episome F? 40.8. By what mechanisms for shuffling genes might nucleotide-sharing be initiated? 40.9. Discuss the possible role of nucleotide-sharing in integration, and in deintegrated episomes. 40.10. What do you think of the hypothesis that most, if not all, episomes or episomal derivatives are nucleotide-sharing, and that nucleotide-sharing is associated with motility? 40.11. How do you suppose episomes originate? 40.12. Have integrated episomes and episomal derivatives the general capacity to break chromosomes? Explain. Could nucleotide-sharing be associated with such a capacity? Explain. 40.13. Devise nucleotide sequences which would explain the differences between Bar, Ultrabar, and wild-type eye shape in Drosophila. 40.14. Devise nucleotide sequences which could be used to explain cis-trans position effects. List the assumptions you have made in such an explanation. Chapter *41 BACTERIA: RECOMBINATION (V. Transduction) Ti Ihe four Chapters preceding this have dealt with the shuf- fling of genetic material in bac- teria. In the cases discussed, the new genetic combinations typically involved the reloca- tion of only a portion of a bacterial genome. While, in the case of conjugation, a segment of chromosomal DNA passes from donor to recipient bacterium through a cytoplasmic bridge formed by the activity of an F particle, no organismal assistance is necessary for the entrance of donor DNA in all of the cases of transformation previously discussed. It has been shown in the last two Chapters that the F particle itself undergoes recombination, not only because it is infectious, but because it can enter and leave the bacterial chromo- some, i.e., because it is an episome. More- over, because of the episomal cycle, F may acquire chromosomal memory and the chro- mosome may acquire F memory. Such new nucleotide combinations in turn foster further genetic recombinations which result in a flow of cistrons between F and chromosome. It has been reasoned earlier (p. 349) that any homologous segment of DNA introduced into a bacterium has the potentiality of pair- ing with, and integrating into, a bacterial chromosome. Have we exhausted the mecha- nisms for introducing homologous DNA into bacteria? Bacteriophages or phages ^ are viruses that have the capacity to destroy bacteria. After these fasten onto the bacteri- um, all or part of the phage that is external ^ The Greek letter 0, phi, is used to denote phage. 374 to the bacterium can be shaken off" by the shearing action of a Blendor. Nevertheless, the course of the infection is unchanged by such treatment, that is, the virus still produces its characteristic eff"ects on the bacterium. This can be taken to mean that the part of the virus essential for these eff^ects actually enters the bacterial cytoplasm, and that what remains attached to the outside of the bacterium is dispensable in this regard. In view of this behavior by phage, two new ways for the entry of homologous DNA into a phage-infected cell can be envisaged. First, the virus might carry externally, adsorbed to its outer surface, a segment of DNA derived from its previous bacterial host. This piece may penetrate the new host at the same time that the essential part of the phage enters, the latter action providing a place of entry for the bacterial DNA. The second mode of DNA entrance in- volves the internal contents of the phage which enter the bacterium. The part of the virus which enters the bacterium may contain DNA which possesses a nucleotide sequence also found in the bacterial chromosome. There are two possible origins for such homol- ogous DNA. It might be (1) a segment of DNA, not normally a part of the virus, which originated in the previous host chromosome (in which case the phage may or may not be defective in its own DNA or RNA content), or (2) a segment of DNA that is normally found as a (continuous) part of the viral DNA. With this introduction, let us examine the results of a series of experiments - employing the mouse typhoid organism, Sahnonella typhimiirium. This bacterium is a close relative of E. coli, and also can be cultured on a simple medium. A number of auxotrophic strains of Sahnonella have been obtained, in- cluding one that requires methionine {M'T+) and another that requires threonine (M+T~). ^ The following discussion is based upon the work of N. D. Zinder and J. Lederberg (1952). Bacteria: Recombination {V) yis When these two strains were mixed and then plated on medium lacking methionine and threonine, prototrophic colonies ap- peared in such large numbers that they could be explained entirely, or almost entire- ly, as being the result of genetic recombina- tion, and not of mutation. Prototrophs also were obtained, if a liquid culture of the M+T' strain was centrifuged (to remove most of the bacteria), the supernate heated for 20 to 30 minutes (to kill any remaining bacteria), and the supernate added to the M T+ strain. This demonstrates that no living M+T^ donor cells are required in order to furnish the M+ factor for the estabhshment of prototrophy. So, this is clearly not a case of genetic recombina- tion involving conjugation. Moreover, the filtrate retained its full M+ capacity after treatment with DNAase. Accordingly, this is not a case of genetic recombination via trans- formation. Since the M+ factor could pass through filters that held back bacteria but not viruses, it can be called a filterable agent (FA). It was also found that the M+T- strain harbored a phage. This virus, P22, is said to be nonvirident or temperate, for only occasion- ally does it become virulent, at which time it replicates itself and then lyses or bursts the host cell to liberate as many as several hun- dred progeny phage. Accordingly, temperate phage does not cause conspicuous lysis. Since the M+T~ strain of Salmonella carries P22 as a temperate virus and is potentially capable of having its cells lysed, it is said to be a lyso- genic strain. Lysogeny also confers on the bacterium immunity to infection by identical or homologous phage. The MT^ strain happens normally to lack P22, that is, it is a nonlysogenic or sensitive strain. When a sensitive strain is infected with temperate phage, a certain fraction of the cells lyse and liberate phage, but another fraction survives, becomes lysogenic, and gives rise to lysogenic progeny. Lysogenic bacteria can be lysed artificially and tested for phage. None is detected. Apparently, in cells that become lysogenic, the phage is converted to a new form, called prophage, which reproduces at the same rate as the host. When a lysogenic cell is to be lysed by viral action, prophage normally first rapidly replicates a number of times to produce infective phage which is liberated at the time of lysis. A natural question to ask now is: What is the relationship between the filterable agent M+ and the phage P22? The following facts were determined experimentally. Both FA A/+ and phage P22 (1) were unafi'ected by RNAase and DNAase, (2) showed the same inactivation pattern with temperature changes, (3) had the same susceptibility to an antiserum that blocks the attachment of phage to the bacterium, (4) attached to susceptible cells simultaneously, (5) had the same size and mass as determined by filtration and sedimentation tests, (6) appeared in the medium at the same time and in a constant ratio, and (7) retained this constant ratio even though various purification and concen- tration procedures were applied. It is evident from these results, therefore, that FA M+ is phage-bound. Since the genetic material of Salmonella is known to be composed of DNA, so is the genetic factor M+. Moreover, since fact (1) precludes the attachment of the M+ genetic factor to the outer surface of the phage particle, the M+ gene must be located in the interior of the virus. Genetic transduction is the term we shall use to define the process of genetic recombination which is made possible when homologous DNA, contained within a virus particle, is sent into a recipient cell. Is there any limitation to the genetic ma- terial which can be transduced by P22? P22 can be grown on bacteria which are geneti- cally marked M+T+X+Y~Z-, and the crop of phage produced, following this infection, can be harvested. Part of the harvested phage is then tested on suitable indicator strains (M~, T-, X-, Y-, Z-) one at a time. The results show transduction of M+, of T+, and of A'+j 376 CHAPTER 41 but not of Y+ or Z+. Another part of the harvested phage is not tested yet but grown in- stead on another genetically marked strain, for example, M+TX^ Y+Z . The new crop of phage produced is harvested and then tested on the indicator strains already mentioned. It is found now that the new crop has lost T+ transducing ability but has gained Y+ trans- ducing capacity. These results demonstrate that a phage filtrate has a range of markers for transduction which is exactly the range of the markers present in the bacteria on which the phage was last grown. In other words, the phage is passive with respect to the con- tent of genes it transduces, and retains no transducing memory from hosts previous to the last. Since additional tests demonstrate that practically any locus in Salmonella is transduceable by P22, we may call this a case of generalized transduction. In generalized transduction a particular marker is trans- duced once for about each lO*' infecting phage particles. Although almost any chromosomal marker is transduceable by P22, what is the length or scope of the transduced DNA? P22 can be grown on M+T+X+, harvested, and then grown on M-J-X-. The latter bacteria are replica- plated on different media, of which one selects for A/+ recombinants, another selects for r+, and a third selects for X+. When the M+ clones are further typed they are still T-X-. Similarly, T+ clones are still M X^ and X+ clones are still M~T~. This demon- strates that usually a single bacterial marker is transduced. In this respect, then, trans- duction is similar to transformation but is different from conjugation, where, especially in Vhf strains, large blocks of genes may be transmitted and integrated. Several examples are known in Salmonella, however, in which several genetic markers are transduced together, in what may be called linked transduction or cotransduction. It was established, in other work, that the biological synthesis of the amino acid tryptophan is part of a sequence of genetically determined reactions that proceeds from anthranilic acid to indol to tryptophan. Cotransductions were found ^ of genes controlling different steps of this biosynthetic sequence, indicating that these genes are closely linked to each other. Histidine biosynthesis in Salmonella in- volves at least eight loci, of which four pro- duce identifiable effects on the sequence of chemical reactions involved. Linked trans- ductions have been found between two or more of these loci."* In fact, using the relative frequencies of different cotransductions and other evidences, it was possible to prove that all eight loci are continuous with each other and are arranged linearly (see Figure 46-1, page 422). The close linkage of cistrons controUing different parts of a biosynthetic sequence is not a universal phenomenon, however. But, when such close linkage does occur, it may be adaptive, in that a single mechanism may suffice to turn off or on the whole series of enzymatic reactions. (In this regard it may be suggested that nucleotide- sharing could provide a mechanism for turn- ing chemical sequences on or off, depending upon which of the overlapping cistrons was functional on different occasions.) Cotrans- duction of closely linked markers is known to occur ^ also in E. coli infected with phage PI. When, in a generalized transduction experi- ment, a prototroph is obtained by transducing an auxotroph, the new prototroph is stable and produces clones phenotypically identical to typical prototrophs. We may call this complete transduction. In this case, the intro- duced prototrophic gene must have inte- grated into the Salmonella chromosome in place of the recipient's auxotrophic gene. It was noticed, however, that in addition to the large colonies formed, each of which repre- sented a complete transduction, there were ^ By M. Demerec and coworkers. '' By M. Demerec, P. E. Hartman, and coworkers. ^ From the work of E. Lennox. Bacteria: Recombination {V) 211 FIGURE 41-1. Large and minute colonies of Salmonella, representing complete and abortive transductions, respectively. {Courtesy of P. E. Hart man.) about ten times as many minute colonies (see Figure 41-1). These minute colonies did not appear in platings of auxotrophic mutants on deficient medium. They were also not attributable to any interaction between auxo- trophs and colonies of normal or transduced prototrophs located elsewhere on the plate. It was possible to show, in a variety of cases and by various methods,*' that each minute colony contained but a single genetically prototrophic cell. Minute colony formation was demonstrated to have the following origin. The initial cell of the minute colony received through phage infection the segment of DNA containing the gene for prototrophy under test. However, this gene (1) failed to be integrated, (2) failed to replicate, (3) but retained its functional capacity to produce a phenotypic effect. As a consequence, a partial hybrid or heterogenote was produced in which the injected cistron for prototrophy was functional. Because prototrophic cis- "• By B. A. D. Stocker, J. Lederberg, N. D. Zinder, and by H. Ozeki (1956). tronic product was made, the cell was able to grow and then divide. However, only one of the first two daughter cells received the extra chromosomal segment, or exogenote. The daughter cell without the exogenote was able to grow and divide only until the prototrophic cistronic product became too scarce; on the other hand, the heterogenotic daughter cell could continue to grow and divide, in turn producing only one heterogenotic daughter cell. In this way, a minute colony is produced which contains a single genetically proto- trophic cell. This consequence, of the failure of complete transduction, is called abortive transduction. Hypothetically, there are two possible fates for the exogenote in an abortive transduction. Eventually, the exogenote might either be lost (by mutation) or it might undergo inte- gration to result in a complete transduction. The latter alternative has, so far, not been found to occur. Regardless of its ultimate fate, we can agree that the exogenote is cistronic in nature. Note that the exogenote is still considered to be a segment of genetic material, even though it does not self-rep- licate. But, remember, self-replication was an assumed characteristic of the total genetic material, and that no such capacity was re- quired of a cistron when this was defined. In most transduction studies, an excess of phage is used. In such experiments trans- duced cells are always found to have simul- taneously become lysogenic. This means that the cell being transduced has received not only the exogenote segment but an apparently complete genome of a phage as well, the former resulting in genetic recombination, the latter in immunity and eventual lysis of some progeny (lysogeny). Is the phage particle whose contents enter the host cell and make it lysogenic, the same particle which introduces the exogenote? This need not be so, since the use of high concentrations or multiplicities of infecting phage means that the host could have been penetrated by the 378 CHAPTER 41 contents of two or more phage particles, one particle furnishing the exogenote and another causing lysogeny. Using different concentra- tions of phage, and low multiplicities es- pecially, it was possible to prove that only one phage particle is needed per transduction. It was shown,^ moreover, that a single phage which attacks a susceptible bacterium can produce only one of three mutually exclusive effects on its host, namely, death (by lysis some 60 minutes later), lysogeny, or trans- duction. A phage that produces generalized, complete or abortive, transduction is, there- fore, defective with regard to its phage genotype and is subsequently unable to repli- cate, at least when it is the only phage of that type infecting the cell. What has happened is that a small chromosome segment of the last host bacterium must have replaced a particular segment of the phage genome whose presence is necessary for the subse- quent lysis or lysogenization of a new host. We had already anticipated this possibility (p. 374). We cannot tell in the present case, however, whether the DNA of the exogenote is separate from, or attached to, the defective phage genome. E. coli strain K-12 is normally lysogenic for the temperate phage lambda (X), to which it is relatively insensitive. A mutant strain of E. coli was found which is sensitive to lambda, that is, which is nonlysogenic. Since the nonlysogenic strain exposed to lambda is often lysed, it can be used to test for the presence of lambda in a filtrate. Sometimes, the sensitive strain becomes lysogenized. This permits the study of the transducing capacity of lambda by using a lysogenic strain with different genetic markers from those of the sensitive, nonlysogenic, strain. The strains and procedure, then, are comp- arable to those used to study transduction by P22 in Salmonella. Lambda to be tested for transducing ability can be collected from the filtrate of the lysogenic strain. Lambda can ^ By J. N. Adams and S. E. Luria. also be harvested, in greater quantity, a few hours following a short treatment of lyso- genics with uUraviolet light. Such UV- induction causes prophage to replicate prog- eny phage which lyse the cell. When the transduceability of various markers is tested, it is found that only a very limited range of markers can be carried by lambda. These are restricted to a cluster of loci. Gal, con- troUing galactose fermentation, loci which, from conjugation studies, are known to be very closely linked to each other. Lambda is therefore capable only of restricted transduc- tion. Lysis, and the consequent liberation of infective phage, can be induced by ultra- violet light, as mentioned. It is also found that when lysogenic Hfr conjugate with sensi- tive F~, a number of zygotes are induced to lyse and liberate infective phage. This method of inducing prophage to replicate and liberate infective phage by lysis, initiated by conjugation, is called zygotic induction. It is found, moreover, that zygotic induction oc- curs, with a given Hfr strain, only if about 26 minutes of conjugation takes place before it is interrupted. This suggests that the chromosome has a locus, Lp (or /;-), with which prophage is physically associated. In a nonlysogenic cell there is no prophage attached to or associated with the Lp site, whereas in a lysogenic cell there is. More- over, in crosses between nonlysogenic Hfr {Lp without prophage) and lysogenic F~ cells {Lp with prophage), there is no zygotic induc- tion, and the nonlysogenic trait {Lp without prophage) is transferred and segregates (cf. p. 353) among recombinants just as any other genetic marker. From these results and others, it is found that Lp, the locus for lambda prophage maintenance, is closely linked to the Gal loci which lambda may subsequently transduce (see Figure 39-4, page 361, where the locus under discussion is given as X). The original lambda-containing lysogenic Bacteria: Recombination (K) 379 bacterium is stable and haploid with respect to the Gal "locus" and produces per 100 thousand lambda only about one Ga/-carrying lambda. Such progeny phage are said to be capable of producing a low frequency of transduction (LFT). About two thirds of the transduced cells from LFT phage form clones that are unstable with respect to Gal, i.e., that segregate out the Gal genotype of the recipient cell. In other words, a Gal bac- terium transduced with lambda carrying Gal+ is usually an unstable heterogenote, being diploid and heterozygous for the Gal locus, occasionally segregating Gal~ progeny. (It should be noted that the merozygote pro- duced by lambda transduction differs from the merozygote produced by P22 in an abor- tive transduction. In the latter case the transduced segment is incapable of replica- tion, whereas in the former case the trans- duced segment can replicate, so that clones of merozygotes may be produced.) When infective lambda is induced from a lysogenic individual merozygotic for Gah the transduc- ing lysate may contain 100 times as many phage carrying a Gal locus as does the lysate of haploids. Such a crop of phage is capable of what may be called a high frequency of transduction (HFT). (There is a second difference between generalized and restricted transduction. In generalized transduction, transducing phage may be obtained from the lysate of nonlysogenic cells infected with free phage, whereas this is not so in the case of restricted transduction. Thus, in the case of restricted transduction, transducing phage are released only from lysogenic [haploid, or merozygotic] bacteria.)^ From the results obtained by employing different multiplicities and combinations of transducing lambda (harvested from lysogenic cells) and nontransducing lambda (harvested soon after nonlysogenic cells are infected), it * The preceding account is based largely upon the work of J. Lederberg, E. M. Lederberg, and M. L. Morse, and of E. L. Wollman and F. Jacob. has been possible to prove,'' just as is true for generalized transduction, that transducing lambda is defective for a portion of the lambda genome. What happens is that the Gal loci being transduced probably replace a segment of the lambda genome. The transducing defective lambda particle (called Wg) has retained certain phage properties and lost others. Thus, when only a single GcrZ-transducing particle is involved in an infection, the particle is still capable of eventually killing and lysing the cell. But it has lost the ability to replicate and produce infective phage progeny, so that the prophage it forms must be defective. Also, such a particle can only rarely lysogenize its host. This means the host is only rarely immune to further infection with lambda. Accordingly, a cell infected by such a defective lambda is still subject to infection by nontransducing phage, whose additional presence (1) makes the host lysogenic and (2) contributes a function which permits the defective prophage to multiply. At the time of lysis of such a doubly infected cell, infective phages of both nontransducing and transducing ability are liberated. This situation is similar to that already described in Salmonella which cannot be lysed or lysogenized if infected by a single transducing particle but which can demonstrate either of these characteristics if the host is also infected with one or more normal, nontransducing P22 phage particles. Transformation is not known to occur in E. coli, probably due to some difficulty in DNA penetration. One would predict, how- ever, if the DNA of a defective lambda were isolated and somehow introduced into a cell, that it would sometimes behave as a trans- forming principle with respect to Gal loci. Even if naked DNA does not penetrate E. coli by itself, it might be hoped that it would adhere to the outside of a nontransducing phage, and enter the host at the time the 5 By W. Arber, G. Kellenberger, and J. Weigle(I957), and by A. Campbell. 380 CHAPTER 41 infecting phage contents do, in accord with the expectation mentioned on page 374. Indeed, this is known to occur; ^'^ that is, using nontransducing lambda as a carrier or "help- er," DNA isolated from defective transduc- ing phage is capable of Gal transformation. You should have noticed, in the discussion of lambda, that such a temperate phage has two alternative pathways of action open to it upon infecting a sensitive bacterium. Either it enters the cytoplasm where it replicates faster than the chromosome, or it integrates with the chromosome where, as prophage, it resides associated with the Lp locus and is synchronously replicated as a regular chro- mosomal marker. Accordingly, lambda, and probably most, if not all, other temperate phages are episomes. What is the difference between temperate phages capable of generalized and restricted ^" From the work of A. D. Kaiser and D. S. Hogness (1960). transduction? Extrapolating our reasoning regarding the episome F, it can be suggested that the nucleotide sequence held in common between prophage and chromosome is shorter for generalized transducing phages than it is for restrictive transducing phages, the former having a greater number of, and hence less specific, places of attachment as prophage than do the latter. Several experiments sup- port the view that, at least sometimes, pro- phage may make the host cell immune to further infection by homologous phage, not by preventing the penetration of the DNA, but by preventing its replication. This action parallels the suppression of free-F replication by integrated F. Transduction by temperate phages has been found to occur also in Pseudomonas, Vibrio, Staphylococcus, and Proteus. It would not be surprising to find that transduction can occur in a wide variety of cells, including human cells. SUMMARY AND CONCLUSIONS Genetic recombination of loci characteristically identified with the bacterial chromosome can be mediated by temperate bacteriophages in the processes of genetic transduction and genetic transformation. A transducing phage is defective in its own genome. The deficient portion is probably replaced by a small segment of DNA acquired at the time the prophage deintegrated from its last host. The transduced segment may be derived from a wide variety of regions of the bacterial chromosome, as in generalized transduction, or from a narrowly limited region, as in restricted transduction. The DNA segment transduced may, by integration, replace a chromosomal marker of the host (as in complete transduction), or it may produce a merozy- gote, in which case the exogenote is still capable of acting cistronically, whether it can replicate (as can Gal exogenotes in E. coli) or cannot (as in abortive transduction in Sal- monella). Most, if not all, temperate phages are episomes, which when attached to the chromosome have some characteristics resembling those of attached F. REFERENCES Arber, W., Kellenberger, G., and Weigle, J., "The Defectiveness of Lambda-Transducing Phage" (in French), Schweiz. Zeitschr. Allgemeine Path, und Bact., 20:659-665, 1957; translated and reprinted in Papers on Bacterial Genetics, Adelberg, E. A. (Ed.), Boston, Little, Brown, 1960, pp. 224-229. Jacob, F., and WoUman, E. L., "Spontaneous Induction of the Development of Bacterio- phage X in Genetic Recombination of Escherichia Coli K12" (in French), C. R. Acad. Sci., Paris, 239:317-319, 1954; translated and reprinted in Papers on Bacterial Viruses, Stent, G. S. (Ed.), Boston, Little, Brown, 1960, pp. 336-338. Bacteria: Recombination (F) 381 Jacob, F., and WoUman, E. L., "Genetic Aspects of Lysogeny," pp. 468-500 in A Symposium on the Chemical Basis of Heredity, McElroy, W. D., and Glass, B. (Eds.), Baltimore, The Johns Hopkins Press, 1957. Kaiser, A. D., and Hogness, D. S., "The Transformation of Escherichia Coli with Deoxy- ribonucleic Acid Isolated from Bacteriophage Xdg," J. Mol. Biol., 2:392-415, 1960. Morse, M. L„ Lederberg, E. M., and Lederberg, J., "Transduction in Escherichia Coli K-12," Genetics, 41:142-156, 1956; reprinted in Papers on Bacterial Genetics, Adelberg, E. A. (Ed.), Boston, Little, Brown, 1960, pp. 209-223. Ozeki, H., "Abortive Transduction in Purine-Requiring Mutants of Salmonella Typhi- murium," Carnegie Inst. Wash. Publ. 612, Genetic Studies with Bacteria, 97-106, 1956; reprinted in Papers on Bacterial Genetics, Adelberg, E. A. (Ed.), Boston, Little, Brown, 1960, pp. 230-238. WoUman, E. L., and Jacob, P., "Lysogeny and Genetic Recombination in Escherichia Coli K12" (in French), C. R. Acad. Sci., Paris, 239:455-456, 1954; trans- lated and reprinted in Papers on Bacterial Viruses, Stent, G. S. (Ed.), Boston, Little, Brown, 1960, pp. 334-335. Zinder, N. D., "'Transduction' in Bacteria," Scient. Amer., 199:38-43, 1958. Zinder, N. D., and Lederberg, J., "Genetic Exchange in Salmonella," J. Bact., 64:679-699, 1952. QUESTIONS FOR DISCUSSION 41.1. How would you define the term provirus? How do the terms merozygote and heterogenote differ? How would you define a homogenotel 41.2. What characteristics are conferred upon a host cell infected by a nontransducing temperate phage which (1) becomes a prophage? (2) does not become a prophage? 41.3. How would you proceed to prove that there is only one exogenote in a microcolony of Salmonella produced following abortive transduction? 41.4. Discuss the statement: "Temperate phage has chromosomal memory, and the chromosome has temperate phage memory." 41.5. F particles are known which carry the prophage of X as "memory." How could you prove the existence of such a particle? 41.6. Describe the procedure and genotypes you might use in demonstrating that E. coli can undergo genetic transformation with respect to Gal. 41.7. Is there any reason for believing that the close linkage of genes with related effects might be more advantageous in microorganisms than in higher organisms? Explain. 41.8. List the different ways that the Gal locus in E. coli may undergo recombination. 41.9. Would you say that temperate phages are good or bad for bacteria? Explain. Give an example of this process Norton D. Zinder, about 1954. 41.10. How would you define sex-duction or F-ductionI in E. coli. 41.11. Is a cell which has presumably stopped undergoing mutation, genetic recombination, and self-replication of its DNA still considered to contain genetic material? Explain. Chapter *42 VIRUSES: RECOMBINATION IN BACTERIOPHAGE (I) 1 "t has been already stated that the genetic material of bacterial chro- .mosomes is DNA. It has been inferred (p. 366) that the episome F is also composed of DNA, and proof of this has been obtained. The possibility may not be ex- cluded at the present time, however, that some episome other than F may have RNA for its unique genetic material, for this substance, too, could provide nucleotide sequences suit- able for pairing with chromosomal segments of DNA. It is even possible to imagine that RNA might become integrated into a DNA chromosome.^ (RNA episomal particles that deintegrate from the chromosome and retain a DNA segment as chromosomal memory could subsequently readily reintegrate.) Since we concluded near the close of the last Chapter that most, if not all, temperate phages are episomes, it would seem desirable at this point to discuss the morphology, chemical composition, and some details of the behavior of bacteriophages. Different phages have different morpholo- gies. Consider the structure of phages of the T-even group (T2, T4, T6), since these have been studied in some detail. ^ Such phages are tadpole-shaped, 0.1-0.2/i long, or about a tenth the bacterial diameter (see Figure 42-1). The head has the form of a bipyrami- dal hexagonal prism, while the tail is cylindri- ^ See reference to J. S. Krakow, H. O. Kammen, and E. S. Canellakis at end of Chapter. 2 See reference to S. Brenner et at. (1959) at end of Chapter, 382 cal and is the structure used for attaching the phage to the host cell. The membrane sur- rounding the head is composed of a large number of repeated subunits each having a molecular weight of about 80,000. The tail consists of an outer sheath which is composed of spirally arranged subunits which form a hollow cylinder. The sheath can contract in length as a consequence of which its diameter is increased but its volume is approximately unchanged. The sheath is composed of about 200 subunits, each of approximately 50,000 molecular weight. Beneath the sheath is the core of the tail. The core is a hollow cylinder with a central hole about 25 A in diameter. At the distal end of the core is a hexagonal plate to which six tail fibers are attached. Each tail fiber has a kink in the middle and seems to contain subunits with a molecular weight not less than 100,000. The head membrane, sheath and tail fibers are com- posed of proteins, each of whose digestion with trypsin gives a unique fingerprint. Therefore, each of these proteins is different. The core also is composed of protein. A serologically distinct protein, comprising 4-6% of the total protein, is found in the interior of the phage particle; polyamines, putrescme, spermadine, lysozyme, and a minor polypeptide are also reported in the phage interior. In addition to the components already mentioned, the phage interior contains DNA whose volume is approximately equivalent to that of the total protein. This DNA is com- posed of a double helix about 200,000 nucleo- tides long. Since such a polynucleotide would be about 68/x long, it is clear that the DNA inside the phage must be highly coiled upon itself. No RNA is reported to be contained in these phages. However, not all phages contain DNA. At least one phage is known to contain RNA and no DNA. More- over, the physical and chemical complexity of the T-even phages is not typical of all viruses. Certain plant viruses, like tobacco 700 A 383 FIGURE 42-1. Diagrammatic representation of the struc- tures observed in intact and triggered T-even phages of E. coli. HEAD >TAIL mosaic virus and turnip yellow mosaic virus, are relatively simple structures. We are now in a position to examine the evidence concerning the chemical identity of the genetic material in typical DNA-contain- ing phages. Since DNA contains no S and T2 phage protein contains no P, the DNA in one sample of phage can be labeled by feeding the E. coil host cells radioactive P^% while the protein in another sample of phage can be labeled by feeding the host cells radioactive S^^ The two samples of radioactive phage are then permitted to infect nonlabeled cells. ^ ^ This account follows the work of A. D. Hershey and M. Chase (1952). It is found that all of the P^'- (hence all of the DNA) enters the bacterium while all but about 3% of the S'*^ (hence almost all the protein) remains outside. As mentioned in the last Chapter (p. 374), protein portions of a phage can be shaken off the host cell by Blendor treatment without affecting the normal outcome of infection (transduction, lysis, lysogenation). This result is consistent with the view that it is DNA which is the carrier of phage genetic information. It has already been mentioned (p. 379) that naked DNA does not penetrate normal E. coli. It is possible to remove the cell wall of E. coli by suitable culture conditions to pro- 384 CHAPTER 42 duce what is called a protoplast. When protoplasts are exposed to almost naked DNA from phage T2, complete and typical T2 progeny phage are later released by lysis."* This result strongly suggests that it is the phage DNA which contains all the genetic information for the production of progeny phage. By various methods (treatment with phenol or CaClo), it is possible to remove all the protein coat from phage, leaving naked phage DNA. When protoplasts of E. coH are treated with naked, single-stranded DNA of phage X\1A progeny are produced, complete with the protein envelope characteristic of this phage. ^ (Note that there are only about 5,000 deoxyribotides per 0X174 particle.) Accordingly, the genetic material in DNA-containing phage is solely DNA. Following the tail-first attachment of a phage to a host cell, the course of events leading to lysis can be summarized as follows (Figure 42-2). All the DNA and a small amount of protein are injected into the host, possibly assisted by the contraction of the spiral sheath protein. Then follows an eclipse period during which no infective phage can be demonstrated in the recently infected bacterium (Figure 42-2, B-D), even if this is artificially lysed. In this period, the infected cell is said to carry vegetative phage. During the eclipse period the phage DNA is repli- cated to produce a pool of DNA units. From time to time, this pool of DNA is sampled and a fraction of it undergoes con- "* As shown by J. Spizizen and by D. Fraser, H. Mahler, A. Shug, and C. Thomas, Jr. ' By G. G. Guthrie and R. L. Sinsheimer. densation into phage genomes which are surrounded by a new skin (head and tail), formed from a cycle of protein synthesis and organization (Figure 42-2D). The produc- tion of the first infective phage signals the end of the eclipse phase (Figure 42-2E). (If bacteria are prematurely lysed toward the end of the eclipse phase, one can find empty phage heads in the lysates.) About 20-40 minutes after infection, the bacteria produce endolysins which lyse the cell wall and liberate infective phage into the medium. This completes the lytic cycle of a bacterio- phage. This is the only cycle possible for virulent phages such as those of the T series attacking E. coli. This is also one of the two cycles possible for temperate phage, which has the other alternative upon entering a bacterium of establishing a relationship with the bacterial chromosome as a prophage and making the bacterium lysogenic. Of course, occasionally, the prophage dissociates from the chromosome, becoming vegetative phage whose replication produces infective phage released at lysis. Methods for detecting the presence and amount of virulent phage are based upon the capacity of the phage to lyse sensitive bacteria grown either in liquid or solid medium. In the former medium, the assay is made by determining the time required for complete lysis of a liquid culture of sensitive bacteria; this is denoted by the clearing of the originally turbid culture. In the latter medium, the surface of an agar-containing plate is heavily seeded with sensitive bacteria whose clones grow together to form a continuous and somewhat opaque lawn. If a few virulent FIGURE 42-2. (opposite). Electron micrographs of growth ofT2 virus inside the E. coli host cell. A. Bacillus before infection. B. Four minutes after infection. C. Ten minutes after infection. The thin section photographed includes the protein coat of T2 which can be seen attached to the bacterial surface. D. Twelve minutes after infection. New virus particles are starting to condense. E. Thirty minutes after infection. More than 50 T2 particles are completely formed and the host is about ready to lyse. (Courtesy of E. Kellenberger. Reprinted from the Scientific American, 204:100, 1961.) Viruses: Recombination in Bacteriophage (f) 385 !>*- E 386 CHAPTER 42 phage particles are added on top of such a bacterial lawn, each particle will enter a different bacterium, lyse it, and release up to several hundred daughter particles. These will proceed to attack bacteria near the original burst, and subsequently cause them to lyse. When repeated, this cycle will result in a progressively increasing zone of lysis which appears as a clearing or plaque in the bacterial lawn. Each plaque will represent a phage colony derived from one ancestral particle. The type of plaque formed depends upon the medium, host, and phage. When, how- ever, all other factors are controlled, it is found that genetically different virulent phages may produce characteristically differ- ent plaques. Differences in plaques may in- volve size, the presence or absence of a halo, the nature of the edges, and color differences on colored agar. One can, therefore, study the inheritance of phage mutants affecting plaque type. Genetically different phages also differ in the hosts which they are able to infect, and mutants occur in phage which change the range of hosts which may be attacked. Accordingly, the inheritance of phage mutants affecting host range may also be studied. What kinds of results are ob- tained when both types of mutants are in- volved simultaneously? It is possible to obtain a strain of virulent T phage which is mutant both for host range, h, and plaque type, r. When sensitive bac- teria are singly infected, the mutation rates to the wild-type alleles (/?+ or r+) can be deter- mined. It is also possible, using wild-type phages, to determine the mutation rates to the two kinds of mutant alleles. The sensi- tive bacterial strain also may be exposed to a high concentration of a mixture of the doubly mutant (// r) and wild-type (//+ /•+) phages, as a result of which some doubly infected cells occur that carry both phage types. When the daughter phages from such exposures are harvested and tested, it is found that not only the parental types occur (/? r and /?+ /•+) but that recombinational types (/?+ r and h /•+) occur in such high frequency as to exclude a mutational explanation for most of them (Figure 42-3). Accordingly, such experi- ments ^ prove that genetic recombination occurs between phage particles in a multiply infected cell. Using a variety of mutants, and on the basis of the relative frequencies with which different recombinants appear in phage released from multiply infected cells (this ^ Following the work of M. Delbruck and W. T. Bailey, and of A. D. Hershey and R. Rotman. FIGURE 42-3. Plaques produced by parental and recombinant phage types. Progeny phage of a cross between h r+ andh^ r were tested on a mixture of suitable indicator bacteria. The small clear and the large turbid plaques are made by the parental types of phage progeny (h r^ and h+ r, respectively). The large clear and the small turbid plaques are pro- duced by the recombinant types of progeny (h r and h+ r+, respectively). {Courtesy of A. D. Hershey.) Viruses: Recombination in Bacteriophage (I) 387 procedure being the equivalent of studying the resuhs of "crossing" genetically different virulent phages), it is possible to construct a genetic map of phage in which the mutant loci are arranged in linear order. Plaque mutant r is rapidly lysing and pro- duces a larger plaque with sharper margins than is produced by the wild-type allele /•+. Mixed infections with r and /-+ phages usually yield progeny phages that produce plaques of one or the other type. However, two per cent of the plaques observed are mottled, that is, are plaques which appear partly r and partly r+. When the phages in mottled plaques are tested, both (and only) r and r+ types are found. Accordingly, such mottled plaques cannot be explained as being derived from single haploid recombinant progeny. These mottled plaques can be shown not to be due to some action by clumps of phage particles. Finally, such plaques cannot be explained as being due to mutations in unstable r mutants, for such unstable r phages are known to pro- duce sectored, rather than mottled, plaques, whose phage contents when tested either yield sectored plaques again or r plaques, but none of r+ type. From these results and others, it is proved ^ that the two per cent of phage that produce mottled plaques are heterozygotes for a short region including the r locus. It is also found that such hetero- zygotes are recombinant for genetic markers on opposite sides of the short heterozygotic region.^ This suggests that, in phage, the processes leading to heterozygosis are the same as those leading to recombination. One of the ways that heterozygosis and genetic recombination in phage can be visual- ized to occur is as follows. In a mixed infec- tion, the DNA's of different vegetative phage particles repeatedly pair and unpair until the host lyses. It is during these "matings" that replication of new vegetative phage occurs. Suppose replication proceeds by a copy- 7 A. D. Hershey and M. Chase (1951). ^ As indicated by C. Levinthal. choice mechanism, and the two mating strands are genetically different at two closely linked points. If new phage DNA was formed by using the DNA of only one vege- tative phage as template, the progeny phage receiving this would, of course, be non- recombinant. If, however, as diagrammed in Figure 42-4, one vegetative phage is used as a template to make a partial replica that extends through one marked locus («+) and the other mate is used as a template to make a partial replica that extends through the other marked locus (/)+), synthesis may con- tinue so that both partial replicas contain the short segment between the markers. A progeny phage which receives such over- lapping partial replicas would be recombin- ant for the markers we are following and diploid for the segment between them. It is possible that the ends of the partial replicas may join to produce a single strand with a tandem duplication. Had the parent phages differed in the genie alternatives (xi vs. X2) present in the segment which became diploid, the recombinant phage would also be partially heterozygous. Of course, should partial replicas overlap in some other region, the progeny phage containing the overlap would not be recombinant for the markers we have been following, and recombination would be identified only if this new region had been suitably marked genetically. It is known that a single phage particle can be heterozygous for several loci, provided these are located far enough apart. In fact, it is unlikely, contrary to the possibility already mentioned, that any phage is completely haploid. Con- sidered in this way, phage recombination is a consequence of the occurrence of copy-choice in the formation of partial DNA replicas. This explanation receives support '^ from the fact that the observed frequency of hetero- zygotes is great enough to explain the ob- served frequency of recombination. The preceding explanation of the mecha- ' From work of C. Levinthal. CHAPTER 42 FIGURE 42-4. The hypothesized synthesis of over- lapping partial replicas. The phage cross is a+(xi)b- X a-(x2)b+. (x,) b _ (x,) ^ (x, ■ b^ b- (xJ (xJ (x,) PARTIALLY DIPLOID RECOMBINANT MATING PARTIAL REPLICATION nism of phage recombination is based largely on experiments employing virulent T phages. In other experiments, ^° crosses are made, in unlabeled host cells, between different mu- tants of the temperate phage, lambda, of which one is unlabeled and the other is labeled with the isotopes C^^ and N'\ Density-gradient ultracentrifugation is then used to determine the distribution of labeled parental DNA among both parental and recombinant genotypes. It is found, from this and other experiments, that discrete amounts of the original parental DNA appear in the recombinant phages. The simplest model accounting for the results is that recombinants are formed following breaks in the parental DNA strands. Such results do not support, but do not exclude, the usual form of the hypothesis of phage recombina- tion by a copy-choice mechanism, according to which the recombinant phage DNA should be made of entirely new, unlabeled, material. 1° M. Meselson and J. J. Weigle (1961), and G. Kel- lenberger, M. L. Zichichi, and J. J. Weigle (1961). (x,) b . PARENTAL A. D. Hershey, about 1960. Viruses: Recombination in Bacteriophage (/) 389 SUMMARY AND CONCLUSIONS T-even phages are virulent in E. coli. Their morphology and lytic cycle are discussed, their genetic material identified chemically as DNA. Following multiple infection with phages carrying different genetic markers, genetic recombinants are found among the progeny. From the results of such phage crosses a recombination map can be constructed in which the genes are arranged linearly. Recombinant phages are often also diploid for a short region between the recombinant markers. Both phage recombination and partial diploidy may sometimes be the consequence of a copy-choice mechanism of DNA replication during which there has been a switching of the templates utilized in the making of partial replicas. Sometimes, if not always, phage recombination involves parental strands which have broken. REFERENCES Brenner, S., Streisinger, G., Home, R. W., Champe, S. P., Barnett, L., Benzer, S., and Rees, M. W., "Structural Components of Bacteriophage," J. Mol. Biol., 1:281-282, 1959. Hershey, A. D., and Chase, M., "Genetic Recombination and Heterozygosis in Bacterio- phage," Cold Spr. Harb. Sympos. Quant. Biol., 16:471-479, 1951; reprinted in Papers on Bacterial Viruses, Stent, G. S. (Ed.), Boston, Little, Brown, 1960, pp. 179-192. Hershey, A. D., and Chase, M., "Independent Functions of Viral Protein and Nucleic Acid in Growth of Bacteriophage," J. Gen. Physiol, 36:39-54, 1952; reprinted in Papers on Bacterial Viruses, Stent, G. S. (Ed.), Boston, Little, Brown, 1960, pp. 87-104. Kellenberger, G., Zichichi, M. L., and Weigle, J. J., "Exchange of DNA in the Recombi- nation of Bacteriophage X," Proc. Nat. Acad. Sci., U.S , 47:869-878, 1961. Krakow, J. S., Kammen, H. O., and Canellakis, E. S., "The Incorporation of Ribonucleo- tides into Terminal Positions of Deoxyribonucleic Acid," Biochim. et Biophys. Acta, 53:52-64, 1961. Meselson, M., and Weigle, J. J., "Chromosome Breakage Accompanying Genetic Recombi- nation in Bacteriophage," Proc. Nat. Acad. Sci., U.S., 47:857-868, 1961. QUESTIONS FOR DISCUSSION 42.1. Is the hole in the tail of T-even phages large enough for the passage of one or of two double helices of DNA? Explain. In what respect does your answer bear on the manner of entry of phage DNA into a bacterial host? 42.2. What are the advantages of studying phages using bacteria growing on solid, rather than in liquid, culture medium? 42.3. Do you think it would be feasible to study the genetic basis for different morphological or for different protein components of a phage? Explain. 42.4. If a phage is virulent in one strain of bacteria and not in another, is it temperate for the latter? Explain. 42.5. What is meant by a phage cross? Describe how you would know that you made one. 42.6. Are the genes which show recombination always diploid in a recombinant phage? 42.7. Does the fact that complete progeny phages are liberated after naked DNA from 0X174 infects a protoplast mean that all the information for making 0X174 DNA and 0X174 protein is contained in the phage's DNA? Explain. 42.8. Are the hypotheses of phage recombination by breakage and by copy-choice mutually exclusive? Explain. Chapter *43 VIRUSES: RECOMBINATION IN BACTERIOPHAGE (II) a: LTHOUGH most of the work discussed in the last Chapter utilized virulent phages, the concluding portion mentioned that genetic recombination also occurs between temperate phages. It was mentioned specifically that multiple infection of sensitive cells by different mutants of lambda is followed by the occur- rence of genetically recombinant phages among the progeny. From the frequencies of such recombinants it is possible to arrange the mutants in a single linear linkage map, just as was stated can be done for different mu- tants in the virulent T phages. The difference between a virulent and a temperate phage lies in the capacity which the latter has to lysogenize its host. Is lysogeny itself dependent upon the phage genotype? When temperate phages infect sensitive bacteria, the plaques produced have a turbid region in their center due to the growth there of bacteria which were ly- sogenized, not lysed. In such a temperate strain, mutants can occur whose capacity for lysogenization is decreased or lost (in the latter case the phage becomes a virulent one), and are detectable because they form less turbid or clear plaques, respectively. This proves that lysogenization is based upon a part of the phage genotype which is expressed in the phenotype of its host. Moreover, "matings," between phages carrying different lysogenization mutants and other markers, show that these lysogeny loci are a regular part of the phage genetic map. 390 While certain mutations affecting lysogeny seem to affect the stability of prophage in the course of bacterial multiplication, the most important ones seem to affect the very process by which phage is converted to prophage. Mutants of the latter type are called c {clear- ing) mutants and these occur in a cluster of loci located within the genetic map of the phage. In lambda, there are three groups of c mutants arranged linearly in segments called Cs, Ci, and Co (Figure 43-1). Mutants in the Ci segment no longer have any measurable capacity to lysogenize, whereas those on either side (being in Cs or C2) have their ability for lysogenization reduced to about .1 to .01 of that of wild-type lambda. It has been possible to isolate more than a dozen temperate phages in E. coli, of which some show ultraviolet and zygotic induction and others do not. All seven of the viruses which give rise to inducible prophages were found to be associated with the chromosome at different loci (Lp or ly), all of which are located in a linear order close to and on the same side of Gal (Figure 43-2). These phages are all different, in the respect that a host lysogenic for one of them is immune to subsequent infection by the same phage, but is not immune to subsequent infection by any of the others. When multiple infections are made, to cross lambda with any one of the other six phages, some genetic recombinations are found in the progeny phage in each case. However, the markers capable of recombina- tion differ, and this is dependent upon the type of phage with which lambda is crossed. This demonstrates that these six phages differ in the number of loci they contain which are identical or homologous to loci in lambda. The results of crossing lambda and phage 0434 are particularly instructive. ^ COI H \ H^=ttf ^v^ Cj J L C| C2 im FIGURE 43-1. Diagrammatic representation of the linkage group of the temperate bacteriophage X. The upper diagram shows the linear arrangement of various markers for host range or plaque size or type. The d symbols refer to specific de- fective mutants. The c region is marked by a thicker line and is shown enlarged in the lower diagram. It is composed of three sub-regions, c., Ci, C2. Im refers to the segment controlling immunity. {After F. Jacob and J. Monod.) completely lambda except for the Ci region of the lambda genetic map. Such a hybrid 434 still behaves as 434 in the following respects. It still occupies only the 434 position in the E. coli map, and makes the cell immune only to subsequent infection by 434 or hybrid 434. We may conclude from this that the Ci region of lambda is the only portion of the lambda genotype concerned with selecting its par- ticular prophage site on the chromosome, and the only essential portion determining im- munity to infection by homologous phage. Let us restate these results in terms of lambda prophage. Since the characteristics of pro- phage include its association with a particular chromosomal site and with the immunity of its host, it is concluded that both these traits of prophage are intimately associated only with the Ci portion of the phage genome. We shall also call Ci the "prophage region" of other phage genomes. Whereas different Ci mutants of lambda can undergo recombination, there is no re- combination between the Ci's of lambda and 434, indicating a genetic dissimilarity between them. It becomes clear that the presence of a given Ci region inhibits the vegetative multi- plication of superinfecting particles possessing the same Ci regions. This immunity is found to be due to the occurrence of a repressor substance, in the cytoplasm, whose formation and specificity appear to be determined by the Ci region. 1 1 The preceding discussion is based primarily upon work of F. Jacob and E. L. Wollman (1957), of A. D. Kaiser and F. Jacob (1957), and of F. Jacob and A. Campbell. FIGURE 43-2. Part of the E. coli linkage map showing the location of certain inducible prophages. Lac, Galb 82 X 434 --I 1 \ 1 h- 424 381 21 1466 H 1 h+— • 392 CHAPTER 43 Prophage has another characteristic, namely, the capacity to produce infective phage. It should be noted that while the chromosomal locus and immunity properties of prophage are explicable on the basis of a small portion of the phage's total DNA content, the Ci region, this does not neces- sarily mean that this region alone includes all the genetic information or specifications needed for prophage to become vegetative phage, and therefore infective phage. The preceding discussion may suggest to you that not all of the genetic material present in a phage is essential for the existence of phage as an organism. This possibility should not be surprising, since we are already acquainted, in other organisms, with homo- zygous deficiencies (of euchromatic or hetero- chromatic regions) which still permit viability of the organism (even though such a change may be more or less adaptive). In these cases, then, there is genetic material present which is nonessential for the formation of the organism. Although we have seen that in order to have a phage organism at least the prophage loci in the Ci region are essential, we do not have any way, at present, of determining how many additional loci are essential. Let us assume, contrary to the expectation just mentioned, that all genes present in phage are essential for phage existence. If so, one would expect to find that progeny phage contain the same genetic material as the parent phage. Or, to put this expectation another way, the very DNA molecules pres- ent in the parental phage particle should be present in one or more progeny phage. This can be tested in the following way. The DNA of phage is labeled with P^-', by harvesting the phage that lyse bacteria growing in medium whose sole P source is P^'-. The labeled phages are then permitted to infect unlabeled bac- teria, and the total amount of label included in the harvested progeny is compared with the total amount present in the parent phage. It is found ^ that only about 40% of the parentally labeled DNA is included in the progeny, that is, 60% of the specific DNA nucleotides (P atoms) present in the parents are not found in the offspring. Thus, 60% of the DNA of phage is nonconserved. There are several explanations possible for this. One possibility is that all or almost all of the genetic material of phage is essential but that replication and the formation of progeny phage is inefficient, so that not all the parental DNA is retained, 60% of it being replaced by unlabeled daughter DNA genetic material of exactly the same type. The presence of a mixture of labeled and unlabeled DNA in the same particle indicates the occurrence of genetic recombination (see p. 388). Even though the parental phage DNA is sometimes broken to produce genetic recombinant prog- eny, there is very clear evidence^ that the DNA of phage T2 normally consists of a single unbroken molecule whose molecular weight is 130-160 X 10«. In discussing the advantages of bacteria as material for genetic studies (p. 330), it was noted that very large bacterial populations are readily manipulated experimentally. This makes it feasible to discover and study rare events of mutation and genetic recombination. Phage also has this particular advantage. Consider how this advantage may be put to use in the investigation of a particular kind of phage mutant,"* whose study may reveal the characteristics of the fine structure of the genetic material. We have already mentioned (on p. 387) that wild -type (r+) T-even phages produce small, rough-edged plaques when plated on E. coli, whereas rapid lysis (r) mutants pro- 2 By J. D. Watson and O. Maal0e, by C. Levinthal, and by others. 3 From I. Rubenstein, C. A. Thomas, Jr., and A. D. Hershey (1961), and P. F. Davidson, D. Freifelder, R. Hede, and C. Levinthal (1961). "• The discussion following is based largely upon the work of S. Benzer (1955, 1957). Viruses: Recombination in Bacteriophage {II) 393 GENOTYPE PLAQUES FORMED ON HOST STRAIN B K rl or rlll Mutants r r rll Mutants r None FIGURE 43-3. Be/mvior of r mutants of T-even phages in t/ie B and K strains ofE. coli. duce large, sharp-edged plaques. When the r mutants are mapped they are found to occupy three distinct regions of the phage genetic map, rl, rll, and rlll. The r mutants in all three regions can be detected and harvested using E. coli strain B. However, the mutants in the rll region are unique in having an additional attribute, namely, that they cannot form plaques when their host is strain K of E. coli (which is lysogenic for lambda) although the rl and rlll mutants and r+ phages can do so (Figure 43-3). Thus, mutants in the rll region show a restriction in host range as compared with r+ or r mutants in other regions. We shall restrict our atten- tion henceforth to the mutants of the rll group in phage T4, The host range restriction of rll mutants is useful not only for their identification, but for the study of rates of mutation and of genetic recombination. After identifying a mutant as being in the rll region, its mutation rate to /•+ can be determined readily by plat- ings on strain K, since only r+ mutants will form plaques there. From a large number of rll mutants, those which were stable and had a low mutation frequency (sometimes as low as 1 per 10** phages) were selected for further study. Using high multiplicities of phage infection, the results with these showed that mutants in the rll region fall into one of two groups. If a bacterium is infected by one mutant from each of the two groups, both viruses can reproduce and lyse the cell. (In this case almost all the multiply infected K bacteria on a plate would lyse, clearing the entire plate in about half an hour.) This behavior can be explained by considering that the rll region is composed of two sub- regions, A and B. Each subregion in normal phage independently forms a phenotypic product, the products of both subregions being required to cause the r+ phenotype. So, a mutant defective only in the A subregion still makes proper B product, and vice versa. In a bacterium multiply infected with one phage mutant in A and another mutant in B, FIGURE 43-4. T/ie occurrence or non-occurrence of complementation between different rll mutants. X y r X r FUNCTIONAL COMPLEMENTATION r'^X r^ NO COMPLEMENTATION 394 CHAPTER 43 the two mutants can act phenotypically in a complementary manner, or show comple- mentation, to produce the r+ phenotype (Fig- ure 43-4). On the other hand, if muUiple infection occurs with two different mutants both located in subregion A (or B), they will not be capable of phenotypic cooperation to produce the r+ phenotype, since both phages produce defective or no A (or B) product. While the entire plate does not clear in about a half hour in this case, one may observe the occasional incidence of plaques in different regions of the plate. Sometimes the fre- quency of these plaques is no greater than one would expect to find due to the rate of spontaneous mutation of the two mutants. This would indicate the nonoccurrence of /•+ genetic recombinants, which failed to ap- pear either because the two mutants are lo- cated too close to each other on the genetic map and/or because both have a common nucleotide defect. Other times the frequency of plaques is so clearly larger than that expected from mutation that the excess can be attributed to recombination between the two A (or B) mutants which results in progeny phages whose rll region is normal (and pro- duce the r+ phenotype) or is doubly mutant (and are undetected). Two mutants in subregion A, rl and r2, may fail to show recombination with each other. However, one of these, say rl, shows recombination with mutant r3, while mutant r2 does not. Such results can sometimes be shown to be due to the fact that mutant r2 is deficient not only in its own locus but for all or part of rl and r3 also. Accordingly, the mutant order must be 123 (or 321) in such cases. Other mutants behave as point mu- tants, giving no evidence of deficiency. Of more than 1500 spontaneously occurring rll mutants typed, about 300 were different, that is, each was separable from all the others by recombination (Figure 43-5). Using over- lapping deficiencies and mutants showing no evidence of being deficiencies, it is possible to arrange all the r loci in the A and B sub- regions in a single linear sequence, the re- combination rates between two mutants being constant in different tests. Thus, even in its fine structure, the genetic recombination map of bacteriophage is linear. The great efficiency in detecting r+ mutants by the plating of rll mutants on strain K has already been impHed by the statement that mutation rates as low as 1 in 10* can be detected. This method also has approxi- mately the same efficiency for detecting re- combinations. The smallest reproducible frequency of recombination, 0.02%, was found between the mutants /•240 and r359. Of numerous other mutants tested, all gave either no recombination or a higher per- centage of recombination. Note that the methods used can detect recombination fre- quencies that are a hundred or even more times less frequent than 0.02%. Even so, none were found, as if 0.02% is close to the lower limit of recombination. To what use may we put this observation? Since the genetic material of phage T4 is DNA, a lower limit for recombination frequency may give us an idea to what degree DNA is finitely divisible for purposes of recombination. Can we translate genetic recombination distance into DNA nucleotide distance? Or, in other words, can we set any limits in terms of nucleotides to the size of the recon in phage T4? In order to attempt to estimate the nucleo- tide scope of a recon we shall have to make the following primary assumptions: (1) the probability for genetic recombination is con- stant per molecular distance throughout the phage genetic map, and (2) phage DNA is present as a single copy in the form of a single double-stranded Watson-Crick hehx. With reference to (1) it is further supposed that the genetic markers studied are repre- sentative of all loci present and that the total length of the genetic map is accurately Viruses : Recombination in Bacteriophage {II) 395 estimated by the summation of a number of small distances. On these bases, then, the total genetic map of phage T4 can be calcu- lated to be approximately 800 recombination units long, i.e., shows 800% recombination with respect to its total genetic content. (Recall that a recombination map based upon crossing over can be longer than 100 re- combination [in this case, crossover] units.) With reference to the primary assumption (2) it may be recalled that there are about 400,000 nucleotides per phage, represented by a double helix of 200,000 linearly arranged nucleotides. The fraction 800% recombinations/200,000 nucleotides equals 0.004%, and expresses the percentage of recombinations which occurs per linear nucleotide. On the reasonable assumption that recombination cannot take place within a nucleotide, there would be 199,999 points between nucleotides where exchange may occur if the phage chromosome is a rod, and 200,000 such points if the phage chromosome is a ring. Expressed in this particular way, we can say that if two r mutants each differ from their wild-type form by only a single nucleotide, and if the two nucleotides changed are adjacent in /•+, then recombinants (r+ and double mutant) would be expected to occur among 0.004% of the progeny obtained by crossing the mutants. Suppose that the lowest recombination frequency observed, 0.02%, is actually the minimal rate. This means that, on the aver- age, only every fifth (.02/.004) internucleotide point is capable of undergoing recombination, so that the recon is estimated to be as small as five nucleotides in length. Because tests fail to give evidence that phage nucleotide sequence is interrupted by non-nucleotide material, it would seem reasonable that re- combination could occur at any internucleo- tide position. Accordingly, in view of the fact that the observed value of 0.02% is a maximal value for the least amount of re- combination, and in view of the uncertainties which exist with regard to the length of the genetic map and the number of nucleotides in the phage genome, we can entertain the working hypothesis that one recon equals one nucleotide. ^ This is consistent with our expec- tation regarding the polarity of a recon (see p. 303). On this hypothesis, it should be clear that the term allele is properly restricted to the alternatives that occur for a particular recon. In this way we are describing reconic alleles, within which there can be no recombi- nation or crossing over (see Chapter 22 and p. 290 for previous usage of the term allele in this sense). Finally, let us consider the functional characteristics of the rll region. So long as we are considering the production of the r and r+ phenotypes, the rll region has a single function. In this respect the whole region behaves as a single cistron. But the rll region is composed of two subregions, A and B, a mutant in one subregion being able to functionally complement a mutant in the other. Such complementation suggests that A and B are independent at this level of functioning, and therefore each may be con- sidered to be a separate cistron having a dif- ferent function. The whole rll region contains about 1000 linearly arranged nu- cleotides, each subregion containing hundreds of nucleotides. We do not know, but A and B may order the amino acid sequence in dif- ferent polypeptide chains, both of which must be correctly specified in order to produce the r+ phenotype. While the specification of a polypeptide chain is accepted as one kind of primary effect possible for a cistron, it should be noted again (refer to Chapter 32) that the primary effect of a cistron may not always be to specify a polypeptide. Substances other than polypeptides may be specified by the primary action of a cistron; these substances might conceivably be simpler than a polypep- tide, for example single amino acids. In this ^ Support for this view is found in work by D. R. Helinski and C. Yanofsky (1962). 396 CHAPTER 43 A ciSTRON ^t<><>0+0<><><><><><> -^^ AP HB F P 635 O 5N ItfeS G 1310 585 fclfe C 1563 0 ^i^ 612 6 866(3 uvioo C) 126 80 70 7^ AP 181 Ifa ^>-o -o -o -o -ooooo — ooo — o- -o 315 661 uv 1777 351 240 NT Wl C 3S1 279 5311316 1702 G SD 5")6 131 l3'?4 HS 271 IS5 ^ oS5 NT J JIS 470 1«1 181 "fl 121 106 51! 3JI -QOOO -O -O B6 B5 BHba <)<>o-o-o-o^^^cK>- Blalb Viruses: Recombination in Bacteriophage (//) 397 -0-0-00-CK>CK>0- > -Po -oooooc H O AP P BC fc07 P H Ll/ -0<>0<)OhD<>CK>0-<> i -O OOOCKD-OOOOOOO- -OOOO -O- <>^- 1518 NT -ooooo- -o- -o-oo-o- oooocHxyAr -00-0-0-000^^, P UV ill J H 547 5D G 1883 -rtS H 15l3 H25 SOT b03 W70 U\l K F UV EH UV 1130 733 H47 ^^ W 1 J55 JOl Ifc JIH JO 94 I Dig MPU 101 -Ok -O OOK>OK>OK>0- -O^O- -CKKX //uv; H F 745 %0 NB EM l^ J 1445 1345 573 P J2\5 103 13JJ UV H04 3331 AP EM H ?ti SD B i35 J7 IIU 50 54g 148 5 132 76 tH 51 61 72 -OO- OOOO B CISTRON ICH 447 J44 UV 3cO AOOOK> -OOOOOOOO- OOO OO- -^^6""' BMbl BMal BHal B3 -?0- -?- -BOHD- o- o- O EM O DAP AP JOib SO EM 337 "^ NT 35 S3 i3 87 318 Blal Bib BlOal eiOal BlOb 398 CHAPTER 43 case we would also be correct to call a cistron the much shorter linear sequence of nucleo- tides required to specify a single amino acid. (Recall that it has been suggested on p. 370 that heterochromatin is composed of short cistrons that specify, not polypeptides, but a much simpler kind of chemical product.) It will be necessary, therefore, to realize that although a cistron is recognized as a linear sequence of nucleotides which produces a single primary phenotypic effect, the particu- lar primary function under consideration will determine the complexity of the product and therefore the length of the cistron involved. Seymour Benzer, in 1961. SUMMARY AND CONCLUSIONS Genetic recombination can occur between temperate phages of different, but related, types, but not between their prophage regions. The prophage stage of a phage is (1) associated with a unique chromosomal site, (2) the cause of immunity, (3) essential for the subsequent production of infective phage. The first two properties of prophage are identified with the Ci region of the phage genetic map. However, this region does not necessarily contain all the genetic material necessary for the production of complete infective phage. Not all of the parental phage DNA is conserved in subsequent phage progeny. The genetic fine structure of the rll region of 0T4 is revealed by studies of mutation, complementation, and genetic recombination. The data from this study and others suggest the hypothesis that one recon equals one nucleotide. REFERENCES Benzer, S., "Fine Structure of a Genetic Region in Bacteriophage," Proc. Nat. Acad. Sci., U.S., 41:344-354, 1955; reprinted in Papers on Bacterial Viruses, Stent, G. S. (Ed.), Boston, Little, Brown, 1960, pp. 209-219. Benzer, S., "The Elementary Units of Heredity," pp. 70-93 in A Symposium on the Cliemical Basis of Heredity, McElroy, W. D., and Glass, B. (Eds.), Baltimore, Johns Hopkins Press, 1957. Benzer, S., "On the Topography of the Genetic Fine Structure," Proc. Nat. Acad. Sci., U.S., 47:403-415, 1961. Benzer, S., "The Fine Structure of the Gene," Scient. Amer., 206 (No. l):70-84, 1962. Davidson, P. F., Freifelder, D., Hede, R., and Levinthal, C, "The Structural Unity of the DNA of T2 Bacteriophage," Proc. Nat. Acad. Sci., U.S., 47:1123-1129, 1961. Viruses: Recombination in Bacteriophage {II) 399 Helinski, D. R., and Yanofsky, C, "Correspondence between Genetic Data and the Position of Amino Acid Alteration in a Protein," Proc. Nat. Acad. Sci., U.S., 48:173-183, 1962. Jacob, F., and Monod, J., "Genetic Regulatory Mechanisms in the Synthesis of Proteins," J. Mol. Biol., 3:318-356, 1961. Jacob, F., and Wollman, E. L., "Genetic Aspects of Lysogeny," pp. 468-500 in A Symposium on t/ie C/iemical Basis of Heredity, McElroy, W. D., and Glass, B. (Eds.), Baltimore, Johns Hopkins Press, 1957. Jacob, F., and Wollman, E. L., "Viruses and Genes," Scient. Amer., 204 (No. 6):92-107, 1961. Kaiser, A. D., and Jacob, F., "Recombination Between Related Temperate Bacteriophages and the Genetic Control of Immunity and Prophage Localization," Virology, 4:509- 521, 1957; reprinted in Papers on Bacterial Viruses, Stent, G. S. (Ed.), Boston, Little, Brown, 1960, pp. 353-365 Rubenstein, L, Thomas, C. A., Jr., and Hershey, A. D., "The Molecular Weights of 2T Bacteriophage DNA and its First and Second Breakage Products," Proc. Nat. Acad. Sci., U.S., 47:1113-1122, 1961. Watson, J. D., and Maalde, O., "Nucleic Acid Transfer from Parental to Progeny Bac- teriophage," Biochim. et Biophys. Acta, 10:432-442, 1953; reprinted in Papers on Bacterial Viruses, Stent, G. S. (Ed.), Boston, Little, Brown, 1960, pp. 105-115. See last portion of Supplement V. QUESTIONS FOR DISCUSSION 43.1. In what respects is 0X similar to and different from an F particle? 43.2. A temperate phage is known which is capable of transducing any known chromosomal marker in E. coli. Would you expect to be able to locate the chromosomal site for its prophage? Explain. 43.3. Does the finding that a single phage particle may transduce a bacterial fragment carrying not only a bacterial marker but two linked prophages have any bearing upon the essentiality of the entire phage genome for infection and/or the production of phage progeny? Explain. 43.4. How can you distinguish a mutant in the rll region from one in the rl or rlll region? 43.5. Describe how the trans test is used to show functional complementation between two mutants in phage. 43.6. What would you expect to be the near-maximum number of nucleotides transduceable by a phage which is still capable of phage activity? On what is your opinion based? 43.7. How would the estimated nucleotide length of a recon be affected if 80% of phage DNA was actually conserved? If the average protein-specifying cistron was 2000 nucleotides long, how many different proteins could be specified by T4? by 0X174? 43.8. What do you consider to be the most remarkable feature of 0X174? 43.9. Mutants which show functional complementation in the pan-2 region of Neurospora can be arranged in the same linear order by complementation and by genetic recombi- nation. Is it necessarily true that both maps also will be identical for other regions? Explain. 43.10. What is a cistron? How is your answer related to its length in nucleotides? 43.11. What have you learned in the present Chapter regarding the chemical nature of different genetic units? Chapter *44 VIRUSES: BACTERIAL, ANIMAL, AND PLANT JLmc "n discussing the genetics of the rll region of the T4 phage genetic lap, it was mentioned (p. 394) that the more than 1500 spontaneously oc- curring mutants tested could be explained as involving changes in one or more of about 300 different sites in the rll region. This means that some sites of mutation must have been involved more than once. In fact, the number of times different sites were involved in mutation is quite variable. In terms of DNA this must mean that certain nucleo- tides, singly or in groups, are much more Hkely to undergo spontaneous mutation than others, there being, so to speak, mutational '''hot spots" within the rll region. Since recombination studies permit the analysis of the rll region at the level of the nucleotide, the DNA of T4 may serve as material for studies on the nature of mutation which may lead to a clearer definition of mutation in chemical terms. It should be noted at this point that even-number T phages (T2, T4, T6) have 5-hydroxymethyl cytosine (Figure 33-2, and p. 296) instead of cytosine in their DNA. In all other respects, the DNA is typical. It was already noted, on page 325, that 5-bromo uracil (Figure 35-5) can substitute for thymine, and only thymine, in the synthesis of DNA in vitro. What would be the mutational consequence of in- corporating 5-bromo uracil into T4 DNA? ^ 1 The discussion following is based largely upon the work of S. Benzer and E. Freese (1958) and subse- quent work by E. Freese and coworkers. 400 Addition of 5-bromo uracil to the normal cul- ture medium in which T4-infected cells are growing would not necessarily result in the in- corporation of this base analog in T4 DNA, since thymine could be synthesized by the bacterium and it, rather than the analog, might be utilized preferentially or exclusively in the synthesis of phage DNA. In order to assure that no thymine is synthesized or available as raw material for DNA synthesis, sulfanilamide is added to the culture medium. This drug, which by itself does not appreci- ably increase the mutation rate, inhibits nearly all syntheses leading to the addition of methyl or hydroxymethyl groups to com- pounds. Accordingly, the medium is sup- plemented with a variety of essential chemical substances already containing methyl and hydroxymethyl groups but not with the deoxyribotides of thymine or of 5-hydroxy- methyl cytosine. (The latter deoxyribotide is omitted to prevent its possible conversion to an analog of thymine which might be incorporated in preference to the 5-bromo uracil.) In this way, the bacterium can prop- erly function as a phage host, but cannot, for example, produce thymine (5-methyl uracil) by methylating the uracil which is present in abundance in the cytoplasm and its RNA, Under these conditions, then, 5-bromo uracil will be used as a substitute for thymine in DNA synthesis. It should be noted, however, that the base analog may also be falsely incorporated in place of 5-hydroxymethyl cytosine, or act in other ways, in producing a mutagenic effect. Under these experimental conditions, it is found that 5-bromo uracil is highly muta- genic in the rll region. A comparison of 5-bromo uracil-induced and spontaneously occurring rll mutants reveals that the induced mutants also occur in clusters on the genetic map, but that the hot spots are in different positions. Moreover, contrary to the spon- taneous mutants, very few of those induced are of gross (internucleotide) type, and almost Viruses: Bacterial, Animal, and Plant 401 all are subsequently capable of reverse muta- tion to, or near, the r+ phenotype. Although the mutational spectra (see p. 200) for 5-bromo uracil, for other chemical mutagens, and for spontaneous mutants are all different at the nucleotide level, we cannot specify, with any certainty, the exact chemical basis for the induced mutations. This is so because there are a number of possible metabolic paths through which the mutagen may be producing its effect. It is clear that the chemical basis for mutagenic action is best studied when the pathway between muta- gen and gene is the shortest possible.^ In this connection, just as it is preferable to expose sperm rather than any other cell of that organism to a chemical mutagen, clearly it is more desirable to treat phage or trans- forming DNA directly, rather than indirectly, via its host. What is the molecular basis of mutationl Since the genetic material is a linear array of nucleotides, consider how mutation might involve single whole nucleotides. Loss or gain of a single whole nucleotide might be expected to result from breaking the nucleo- tide string backbone at two or more places, followed by deletion of a whole nucleotide or its insertion in a new position. This might occur especially frequently after exposure to a physical mutagen which ionizes, and would involve, at least occasionally, only the already formed "old" gene material. However, single whole nucleotide change may also be produced by chemical mutagens without in- volving breakage. It has been suggested ^ that chemical mutagens like the acridines insert themselves between the nucleotides of a chain which is subsequently to replicate. A molecule of a chemical mutagen, inter- calated this way between bases that are linear neighbors, could spread the chain 3.4A, and result in the addition of an entire nucleotide at this position to the complementary chain 2 As noted by I. H. Herskowitz (1955). 3 By L. S. Lerman (1961). made next. The possibility also exists that an unbound nucleotide or other normal sub- stance might intercalate with similar results. This mechanism would involve changes in the new genes formed. Before discussing the mechanisms possible for intra-nucleotide changes, consider what is known about the chemical behavior and mutagenicity of certain chemicals. Free T4 phage is known to be permeable to certain small molecules, such as nitrous acid (HNO2) and hydroxylamine (NH2OH), both of which are mutagenic. Nitrous acid removes NH2 from, or deaminates, purines and pyrimi- dines. Thus, when cytosine is deaminated it is converted to uracil and adenine is con- verted to hypoxanthine (the structural for- mulae for these compounds are shown in Figure 35-5), while deaminated guanine be- comes xanthine. Hydroxylamine acts in the reverse manner from nitrous acid, by adding an amino group, for example, to the 2 — C atom of cytosine, forming a molecule that may function like thymine. Such chemicals and others probably act as mutagens by producing intranucleotide changes. Such mutagens may ultimately cause one purine to be substituted by another purine (A <-> G) or one pyrimidine for another (T <-)•€). Replacement by another base of the same kind (purine or pyrimidine) can be called transition, while replacement of a purine by a pyrimidine or the reverse (for example, A <-^> C or T ^-^ G) can be called transversion^ Both transitions and transver- sions would act at the subnucleotide level. What sequence of events may be involved in transition and transversion? A particular base pair T : A exposed to a mutagen may become T : A' (see Figure 44-1). Suppose, at the time of chain separation A' specifies C (instead of T), and at the next division C acts normally to specify G. The net result is that the original A strand eventually produces a granddaughter strand carrying G, so that •* Following the terminology of E. Freese. 402 CHAPTER 44 FIGURE 44-1. One postulated sequence of events leading to transition or transversion. NORMAL MUTATED T:A A:T T:A / \, CHAIN SEPARATION T A' etc. REPLICATION 1 a' : C / \ CHAIN SEPARATION A' C etc. REPLICATION 2 C : G TRANSITION TRANSVERSION (A|G) (T^G) A:T / \, A T' etc. t':C T' C etc. C • G this is a transition. Or, given the original pair G ; C, a mutagen may produce C which specifies A (instead of G), which in turn speci- fies T. So the net change from C to T also is a transition. If A : T becomes A : T', after which T' specifies C, and C specifies G, the net result is that T has been replaced by G, which is a transversion. Such transitions and transversions would be expected to be fre- quent following treatment with certain chemi- cal mutagens. Penetrating radiations, es- pecially ionizing ones, would be expected to be less selective than chemical mutagens in the nucleotides attacked this way. Note, in the examples mentioned, that the transitions and transversions were initiated by a change in the old gene. A second mechanism can be hypothesized in which the initial change lead- ing to transition or transversion occurs in a base which is subsequently used in construct- ing a complementary DNA chain. A third possible mechanism for intra- nucleotide change has been suggested ^ in which the members of a base pair undergo rotational substitution by breaking their bonds to sugar, rotating 180°, and rejoining. Thus, following rotational substitution, which may 5 By H. J. MuUer, E. Carlson, and A. Schalet (1961). be a frequent consequence of ion action, C : G would become G \ C, the resultant double transversion being mutant. A fourth possible mechanism for intra- nucleotide change involves the fact that the bases in DNA may change their configura- tions without changing their chemical con- tent, that is, they can exist in several tauto- meric forms. In the double helix configura- tion of DNA, the most likely tautomers of each base were assumed to obtain, Tauto- mers of the bases can differ in the exact positions where the hydrogen atoms are attached, so that the pairing characteristics are changed. Thus, for example, while the usual tautomer of adenine pairs with thymine, one of its less common tautomers could pair with cytosine (Figure 44-2). In this way an incorrect complement may be specified, lead- ing to a transition or transversion.*' Such tautomeric shifts, causing changes in the new gene code, may play an important role in spontaneous mutation. You will agree that there would be no difficulty in identifying as mutant the com- pleted transition or transversion or whole 6 See reference on p. 318 (J. D. Watson and F. H. C. Crick, 1953c). Viruses: Bacterial, Animal, and Plant 403 ^ N- H Adenine CH, -N O H Thymine H H N Adenine ^ O H Cytosine FIGURE 44-2. Tautomeric shift of ade- nine which could change its complementary base from thymine to cytosine. Upper diagram shows adenine before, and lower diagram after, under- going a tautomeric shift of one of its hydrogen atoms. {After J. D. Watson and F. H. C. Crick.) nucleotide change. But at what point in a series of changes should one consider that a mutation has been first produced? The mu- tation can be said to be accomphshed when, as in one of the mechanisms discussed, A is permanently changed to A'. You might object to this answer on the basis that A' may never be reproduced in a future rephcation. But, the product of a novel change need not be replicated or transmitted in order to be considered mutant. Such a novel product need only be more or less permanent in order to qualify in this respect. A' may be demon- strated to be more or less permanently different from A in five ways (as identified by five different operational procedures). First, A' may have a different chemical composi- tion; second, it may have a different rate of change to a new chemical form ; third, it may not specify T at all, or to the same extent as A did; fourth, it may change the phenotypic effect of the cistron in which it is located; fifth, it may affect the recombination rate of itself or another recon. Operationally, then, it would seem desirable to classify as a mutation any one or a combination of novel identifiable changes in the chemical, muta- tional, replicative, phenotypical, or recombina- tional properties of one or more nucleotides. This new operational definition of mutation includes all aspects of the older one (a novel qualitative or quantitative change in the genetic material). Of course, at the present time, certain of the changes listed, which would identify a mutant, cannot be detected in specific individual nucleotides for technical reasons. Even so, it would seem fruitful to indicate even now all the possible operational ways we may be able to identify a mutant. It is also clear, from the preceding, that the smallest part of the genetic material whose change is mutant is presumably smaller than a nucleotide (and therefore smaller than a recon). Subnucleotide changes could in- volve the methyl, hydroxymethyl, or other groups or atoms in the base portion, as well as changes in the sugar or the phosphate portions of a nucleotide. Subnucleotide components should not be considered to be the smallest units of the genetic material capable of mutation, since the nucleotide is the smallest meaningful chemical unit of the genetic material. It is probably more fruitful to speak of subnucleotide parts as furnishing a number of mutational sites within the nucleo- tide. Until now, our discussion of viruses has been restricted almost completely to DNA- containing phages. (Genetic recombination also occurs in the more complex vaccinia virus, that causes smallpox, whose DNA is probably single-stranded in the infective stage.) There is another group of viruses which contains no DNA but is entirely, or mainly, ribonucleoprotein in content. These viruses include many of the smaller animal viruses (causing poliomyelitis, influenza, and encephalitis, for example), at least one bac- CHAPTER 44 teriophage,'' and many viruses attacking plants (notably including tobacco mosaic virus and turnip yellow mosaic virus). Before considering the genetics of RNA viruses, it will be desirable to describe briefly some features regarding their assay and life cycle. Although these comments will pertain specifically to the influenza virus, many of them apply also to other animal and, to some extent, some plant viruses. Because influenza virus does not usually lyse the cell it infects, it cannot be assayed, like phage, by its pro- duction of plaques. In the case of this virus, and others that produce no clear host-lethal effect, the technique of limit dilution, to be described, must be used for their detection. This detection is facilitated, in the case of influenza, by the use of a viral strain which is capable of growing in the cells that line the fluid-containing cavities of the chick embryo. The virus produces a pathogenic eff'ect in these cells. Accordingly, a sample to be assayed for influenza virus is sufficiently di- luted, and aliquots inoculated into a series of eggs. Two or so days later, the eggs are examined for pathogenic effect, to determine the fraction which contain virus. If near- limit dilutions are used, so that the proba- bility is low that an aliquot contains a single virus particle, one can estimate the virus content of the entire sample. Under these conditions, the progeny virus particles in a given egg comprise a clone. The mammalian cell, which usually serves as host for the influenza virus, typically possesses a flexible shape and has an ambigu- ous cell membrane surrounded by a mucoid coat. This coat acts as a virus receptor since it serves as a substrate for an enzyme located on the virus surface. The virus has an outer protein coat, containing the mucin-reacting enzyme, and an inner core of RNA. (The virus cannot attach to the cell if the mucoid coat is removed by special treatment.) After attachment, the particle enters the cell, per- ^ See reference on p. 305. haps by being engulfed via the cell's normal pseudopodial activity. Once inside the cell, the particle enters an eclipse phase and multi- plies vegetatively, during which period the cell's RNA content increases. Some hours after infection intact viral particles are hber- ated gradually, not in large spaced clusters. FIGURE 44-3. Electron micrographs of tobacco mosaic virus {TMV) slwwing its general configura- tion {top) and its liollow core {middle) . Tiie bottom photo shows a particle whose protein has been partially removed by treatment with detergent, leaving a thinner strand of RNA. {Courtesy of R. G^ Hart.) Viruses: Bacterial, Animal, and Plant 405 The evidence suggests that the viral coat, which contains some material made before infection (by the host alone) and some made after (by host and virus together), is added around the RNA as this emerges from the host cell. Two genetically different, haploid strains of influenza virus are available, SWE (having markers a c) and MEL (having markers A C). It is possible to multiply infect a chick's egg membranes with mixtures of the two strains. Such mixed infections give progeny particles which, when tested, yield pure clones, not only of the parental genotypes, but also of stable recombinant types (Ac or a C). Since other explanations can be excluded, the results prove that genetic recombina- tion occurs also between RNA-containing vi- ruses.^ No evidence has been obtained for the occurrence of genetic recombination among viruses attacking plants. In the case of the tobacco mosaic virus {TMV), infection is brought about experimentally by rubbing a sample of virus on the leaf surface. Even when a high concentration of virus is used, only a small fraction of the virus particles find and penetrate susceptible cells and give rise to a demonstrable lesion. For this reason, it is doubtful that one can multiply infect a tobacco cell, so that experiments that test for genetic recombination probably fail to find it because of the lack of mixed infec- tions. The TMV particle is a cylinder 3000 A long and about 160 A in diameter (Figure 44-3 A). It has a molecular weight of about 40 X 10^ of which 38 X lO" is protein and 2 X 10« is RNA.^ The outer dimensions of TMV are * Based upon the work of F. M. Burnet and others. ' The RNA content of different small viruses com- posed only of ribonucleoprotein is apparently con- stant, also contributing about 2 X lO""' to the molecu- lar weight of the virus particle. The protein contribu- tion varies from 2 X lO^ to 100 X IQb, depending upon the number of protein subunits present. due to the helical aggregation of about 2200 identical protein subunits; each subunit has a molecular weight of about 18,000 and con- tains 158 amino acids in a single polypeptide chain (Figure 44-4). In cross section, the TMV particle is seen to have a hollow core about 40 A in diameter (Figure 44-3B), so the protein subunit adds about 60 A to the radius. The RNA in a particle (Figure 44-3C) is typically a single, unbranched strand, consist- ing of some 6400 nucleotides behaving as a single molecule, which is threaded through the protein subunits at a radius of 40 A. Accordingly, the RNA is normally covered externally by about 40 A of protein subunit. Since the protein subunits are arranged in a gently pitched helix (49 subunits per three turns), the RNA forms a helix of the same pitch. When TMV is treated with phenol, the protein of the virus is destroyed, leaving the single RNA molecule intact. When tobacco is exposed to such RNA molecules, from which protein is removed, some infection occurs (the frequency is about 500 times less than that obtained using an equal number of whole virus particles), and typical TMV prog- eny (complete with TMV protein coats) are produced. Repeated phenol treatments do not decrease the infectivity of the RNA and no amount of protein can be detected chemi- cally in these preparations. On the other hand, RNAase destroys the infectivity of the RNA fraction completely. It must be con- cluded, therefore, that naked RNA is infective and carries all the genetic information to repli- cate itself}^ These experiments prove that TMV protein plays no part in the replication either of the RNA genetic material or of it- self. This is further illustrated by what may be called reconstitution experiments. It is pos- sible, under certain conditions, first to sepa- rate the protein and RNA of TMV, and then to have them recombine to produce the high 10 RNA isolated from a number of small animal viruses is also infective. 406 CHAPTER 44 ' 5 NH^ 10 15 Acetyl N-Ser*Tyr— ►•Sef-»'lleu— *'Thr-»-Thr— ►Pro— »-Ser-»>Glu-»>Phe-*'\tal-»-Phe-»Leu-*>Ser-».Sef -^^ 30 NH^ 25 NH^ 20 ) CAIa-*— Asp'*— Thr'*— CySH^-Asp-*— Leu'^-lleu-* — Leu-^— Glu-< — //ea/o-«— Try« — Alo ^ NH^ NH^ 35 NH^ NH^ NH^ 40 ^^ NH^ 45 Leu-^Gly— ♦Asp-*Glu— ►Phe— >Glu — ►Thr— ♦Glu— ♦Glu — ►Ala— •(Arg)— ►Thr— ►Vol— ►Glu— ♦Vbl ■^^ 60 NH^ 55 50 /V//, NH^ ) CVol"*— Thr<— Val-< — Glu-4-Pro-«— Ser^-Pro-* — Lys-<— Try<— Val-<— Glu-«— Ser-«— Phe-»-Glu*-(Arq) V (Ar^Phe-^Pro — ►Asp-»-Ser— ►Asp-^Phe— »{Lys)— ►Vol — ►Tyr— »^^)— ►Tyr— ►Asp— ►AlG-*Val ->. .90 85 80 ) C(Arqi)«— Thr-«— Asp-*— Phe*-Ala-*— Gly •♦- Leu-«— Leu^-Ala*— Thr<«— Vol-*— Leu*-Pro-*— Asp *— Leu V C ^— -. 95 NH^ IVMp Nflp lOU /V/^ I Asp-MArg)-^|leu — ►lleu-^GIu — ►Val->-Glu — ►Asp-^GIu— ►Alo— ►Asp-^Pro— ►Thr— ►Thr— ».Ala -^ Ala-«— Vol-*— Thr-< — Ala-4-Asp-*-Asp-#-Val-* — Arg-<-(Ar^>«— Thr-*— Ala<»-Asp-*-Leu-«— Thr-<— Glu 4r ^— V 125 /V//o 130 _ i^s lleu- -■ 115 ^-.^ 110 )<— Vol'* — Thr-* — Ala-*—Asp<— Asp*-Val'* — Arg-«-(Arq>«— Thr-*— Ale-*— Asp^-Leu-*— Thr-«— Glu /ir\ ^ '^^ '^"^ 130 ^_- 135 J— ►(Ar^)-^5er — ►Ala— ►Asp— ►lieu— ►Asc-^/«i^—^//ec/— ►vo/— ►G/t^ -►Leu —►lieu — »(Arg)-^GIy 150 CLeu*-biy«— ser-* — Ser-«-Ser-*— Glu-*-Phe*- 155 Vol— ►Try— ►Thr— ►Ser-*Gly— ► Pro-^AIa— ► 140 fi/M^ Leu'^-Gly'*— Ser-* — Ser-«-Ser-*— Glu-*-Phe*-Ser.«— Ser<-^ra)#— Asp. Leu— ►lieu — •^Arm— »oiy -v Tyr.«— Ser-*— Gly«— Thr V FIGURE 44-4. Amino acid sequence in the protein building block of the tobacco mosaic virus {TMV). There are 158 amino acids in the sub-unit, the encircled residues indicate the points of splitting by trypsin. {Courtesy of A. Tsugita, D. T. Gish, J. Young, H. Fraenkel-Conrat, C. A. Knight, and W. M. Stanley, Proc. Nat. Acad. Sci., U.S., 46:1465, 1960.) infectivity of the original virus. Using two genetically different strains of this virus, the standard (TMV) and Holmes rib grass (HR), it is possible to construct a highly infective virus containing the RNA of TMV and the protein coat of HR. The progeny obtained are typically TMV with respect to RNA and protein coat. The reciprocal construct, a virus with HR RNA and TMV protein pro- duces typical HR progeny in both their RNA and protein. Thus, // is only the RNA of a TMV particle which specifies the RNA and protein of the progeny virus. ^^ Mutations can be induced in TMV RNA by many of the agents which are mutagenic for DNA. For example, deamination of single bases by nitrous acid is mutagenic. " The genetic experiments described for TMV are based largely upon work of H. Fraenkel-Conrat and R. C. Williams (1955), A. Gierer (1960), G. Schramm, and others. Such results and others prove that the bio- logical activity of the RNA depends upon its primary (nucleotide content) and not its secondary (coiling pattern) structure. The results mentioned prove that RNA is the genetic material in RNA-containing vi- ruses. While the genetic information is carried by a single strand of ribotides within the RNA virus, we do not have any clear evidence with regard to the mode of replica- tion or of function of the RNA once it is inside its host. It may be noted, in the case of 0X174, whose DNA resembles RNA in being single-stranded, that there is some evi- dence suggesting that its replication involves the formation of a complementary DNA chain, which, however, does not become in- corporated into mature phage. Even if the formation of a complementary chain proves to be true for this phage, it still remains to be seen whether this also occurs during the replication of RNA genetic material. Viruses: Bacterial, Animal, and Plant 407 SUMMARY AND CONCLUSIONS There are mutational "hot spots" at the nucleotide level; these are different for mutants occurring spontaneously and for those induced by various chemical mutagens. Mutation is defined operationally as any detectable novel change affecting the chemical constitution, mutability, replication, phenotypic effect, or recombination of one or more nucleotides. Whole nucleotide mutations include additions and losses of single whole nucleotides; subnucleotide mutations may involve transitions and transversions. It is hypothesized that the components of a nucleotide serve as sites for mutation. RNA is the sole carrier of genetic properties in certain viruses. Some RNA viruses can undergo genetic recombination. REFERENCES Benzer, S., and Freese, E., "Induction of Specific Mutations with 5-Bromouracil," Proc. Nat. Acad. Sci., U.S., 44:112-119, 1958; reprinted in Papers on Bacterial Viruses, Stent, G. (Ed.), Boston, Little, Brown, 1960, pp. 220-227. Burnet, F. M., and Stanley, W. M. (Eds.), T/ie Viruses; Vol. 1, General Virology, 609 pp.; Vol. 2, Plant and Animal Viruses, 408 pp.; Vol. 3, Animal Viruses, 428 pp. New York, Academic Press, 1959. Fraenkel-Conrat, H., and Ramachandran, L. K., "Structural Aspects of Tobacco Mosaic Virus," Advances in Protein Chemistry, 14:175-229, 1959. Fraenkel-Conrat, H., and Williams, R. C, "Reconstitution of Tobacco Mosaic Virus from Its Inactive Protein and Nucleic Acid Components," Proc. Nat. Acad. Sci., U.S., 41:690-698, 1955; reprinted in Classic Papers in Genetics, Peters, J, A. (Ed.), Engle- wood Cliffs, N.J., Prentice-Hall, 1959, pp. 264-271. Freese, E., "The Difference Between Spontaneous and Base-Analogue Induced Mutations of Phage T4," Proc. Nat. Acad. Sci., U.S., 45:622-633, 1959. Freese, E., Bautz, E., and Freese, E. B., "The Chemical and Mutagenic Specificity of Hy- droxylamine," Proc. Nat. Acad. Sci., U.S., 47:845-855, 1961. Gierer, A., "Ribonucleic Acid as Genetic Material of Viruses," in Microbial Genetics, Hayes, W., and Clowes, R. C. (Eds.), Cambridge, Cambridge University Press, 1960, pp. 248-271. Herskowitz, I. H., "The Production of Mutations in Drosophila Melanogaster with Sub- stances Administered in Sperm Baths and Vaginal Douches," Genetics, 40:76-89 1955. Lerman, L. S., "Structural Considerations in the Interaction of DNA and Acridines," J. Mol. Biol., 3:18-30, 1961. Muller, H. J., Carlson, E., and Schalet, A., "Mutation by the Alteration of the Already Existing Gene," Genetics, 46:213-226, 1961. Tsugita, A., Gish, D. T., Young, J., Fraenkel-Conrat, H., Knight, C. A., and Stanley, W. M., "The Complete Amino Acid Sequence of the Protein of Tobacco Mosaic Virus," Proc. Nat. Acad. Sci., U.S., 46:1463-1469, 1960. QUESTIONS FOR DISCUSSION 44.1. Would you expect the mutational hot spots in the rll region to be different after exposing T4 to hydroxy lamine from what they are after T4 is exposed to nitrous acid? Why? 408 CHAPTER 44 44.2. How permanent need a change in a nucleotide be in order to qualify as being mutant? 44.3. Would you consider the substitution of P^'- for P in the phosphate of a nucleotide as a mutation? Why? 44.4. What do you now think of the statement on page 199 that the only way we have of detecting changes in individual recons is by the phenotypic changes these produce? 44.5. What is meant by limit dilution? Is this technique used in studying TMV? Why? 44.6. What conclusions can you draw from the observation that the number of nucleotides is approximately constant in small RNA protein viruses? 44.7. What conclusions can you reach from the fact that within about a day after infection with a single particle of TMV, the cell can produce about 50,000 viral nucleic acid molecules and about 100 X 10'' protein subunits? 44.8. How could you prove, using infections by naked RNA of TMV, that this RNA contains information for manufacturing TMV protein? 44.9. Compare transformation with infection by naked virus nucleic acid. 44.10. Discuss the view presented by F. Jacob that cancerous growths may originate because virus infection causes the activation of DNA replication. Chapter 45 EXTRANUCLEAR GENES AND THEIR INTERRELATIONS WITH NUCLEAR GENES WHEN a DNA phage enters the stage of vegetative replica- tion, the synthesis of host DNA and protein ceases and the cell pro- ceeds to make virus DNA and protein. While we do not know the detailed mechanism whereby this metabolic shift is produced, it could involve the utilization of the host's cistronic products as well as the direct sup- pression of host gene replication and/or cistronic functioning. It would seem of interest, in this connection, to discuss whether or not there is evidence of a direct inter- relationship between chromosomal and non- chromosomal genes. We have already pre- sented some evidence bearing on this during our study of episomes of F and temperate virus types. It should be recalled that when such episomes are associated with their chromosomal locus, replication, of the same episome located nonchromosomally, is re- pressed. Is there any evidence for the occurrence of genes which are always physically unassoci- ated with nuclear chromosomes, that is, genes which are not chromosomal or epi- somal but always extrachromosomal? It is possible that some virulent phages are com- posed of genes of this type. It is even possible that some of the genes in temperate phages are always extrachromosomal, in view of the fact that only the genes of the prophage are intimately associated with a chromosomal locus. We may now ask two questions: By 409 what means can we search for and prove the occurrence of extrachromosomal genes? If they exist, can we find any direct interactions between them and particular chromosomal genes? We can seek the answers to these two questions in investigations of organisms whose cells possess a definite nuclear mem- brane (and hence a definite nucleus). Ac- cordingly, we shall be concerned with the identification of extranuclear genes and their interrelations with nuclear genes. How are we going to recognize an extranu- clear component as being genie? We can do so by testing whether that component is oper- ationally genie — that is, by testing its chemi- cal, recombinational, mutational, phenotypi- cal. and replicative properties. If such studies reveal properties which satisfy our operational definitions of a gene, the component is genie. Let recombination be the first operational method used in the search for extranuclear genes. To detect recombination we shall require that the extranuclear gene produce a recognizable phenotypic effect. We shall also require that such a gene be capable either of mutation or recombination, or both. In other words, we need to have changes, in- volving either the kind or quantity of such a gene, or both, so that we are provided with phenotypic alternatives having an inborn basis. How would we actually proceed to look for an extranuclear gene in Drosophila'} We would initially observe the occurrence of (preferably, clearly) different phenotypic al- ternatives which occur generation after gener- ation under the same environmental condi- tions. By a series of crosses we would proceed to test whether the occurrence of the alternatives was associated with the presence of a particular chromosome (X, Y, II, III, IV) or a group of chromosomes. If it is, it is hkely that the phenotypic alternatives are due to some genie factor linked to (hence located on) a chromosome. (Then by additional appropriate crosses and/or cytological stud- 410 CHAPTER 45 ies, we could determine more about the precise nature of the nuclear gene change. As you may have surmised from the previous work discussed, the vast majority of gene- based traits that have been carefully analyzed are determined by genes contained in chromo- somes. Thus, we would usually fail to find the extranuclear gene we started out to de- tect.) But consider the genetic alternatives for resistance and susceptibility to CO 2 gas. One strain of Drosophila flies can be exposed to pure CO2 for as long as 15 minutes and recover without apparent eff"ect, while flies of another strain so exposed are almost invari- ably killed. Using marked chromosomes, it is found that C02-sensitivity is not linked to any chromosome of the normal genome. In fact, it is possible, by appropriate crosses, to replace each of the chromosomes present in the sensitive strain by a corresponding chro- mosome of the resistant strain. Yet, after this is done, the flies produced are still sensitive to COo! It is still possible, however, that the sensitive strain has somehow ob- tained an additional nuclear chromosome which, of course, would not be linked to any of the usual ones. Since the C02-sensitivity trait does not segregate in the progeny of hybrids between sensitive and resistant lines, this must mean that such a supernumerary chromosome cannot occur singly (in the individual that is hybrid for sensitivity) or as a pair (in flies of the pure sensitive strain). Moreover, cytological examination reveals no additional nuclear chromosome. Even so, the latter finding is not a conclusive argument against a nuclear locus for C02-sensitivity, since "chromosomes" so small that they es- cape cytological detection are known to exist from recombinational evidence. It is found, however, that while the sensi- tive female regularly transmits C02-sensi- tivity to some progeny, the sensitive male does so only under special circumstances. It is still possible to conceive that a nuclear gene for sensitivity might somehow be preferen- tially excluded from a nucleus destined for a sperm but not from one destined for an egg. It seems much more reasonable, how- ever, to attribute the nontransmission of CO2- sensitivity through the sperm as being the consequence of the relatively minute amount of cytoplasm included in a sperm, as com- pared with the amount present in an egg of Drosophila. It is, therefore, very probable that C02-sensitivity is due to the presence of a body, called sigma, which is mutable and proves to have many of the characteristics of a virus, including infectivity by experimental means. ^ Sigma is not visible within the cell, and so we do not know more about its location. Consider next another case, in Drosophila, already mentioned on p. 107. In this case, females mated to normal males give rise almost entirely to females. This trait has a genetic basis and also is not transmitted by males. Moreover, it is infective and un- hnked to the usual chromosomes. It turns out that this female-producing-female trait is intimately associated with the presence of a spirochaete which can be seen in the blood. We might mention also the occurrence in corn of a phenotypic change which is associ- ated with the presence of readily detectable, supernumerary, heterochromatic (so-called B) chromosomes. None of the three cases just described con- clusively demonstrates the existence of intra- cellular but extranuclear genes. However, they serve to illustrate the types of outcome which may be obtained, when investigations to prove the existence of such genes start with a study of genetic recombination. In view of such results, you can readily appreci- ate the advantage of correlating potentially extranuclear genes directly with objects observable in the cytoplasm. This advantage is held in the case of particles called kappa, ^ Sigma has been studied mostly by P. L'Heritier, G. Teissier, and coworkers. Extranuclear Genes and Nuclear Genes 411 FIGURE 45-1. Normal {above) and kappa-containing {right) Paramecium. {Courtesy ofT. M. Sonneborn.) which are located in the cytoplasm of certain strains of the protozoan, Paramecium. Hun- dreds of kappa particles can be seen un- stained in a single cell (Figure 45-1). They contain DNA and are self-reproducing. Individuals containing kappa are called killers, since animal-free fluid obtained from cultures of killer paramecia will kill sensitive (kappa-free) individuals. Mutant kappas are known which produce diff'erent poisons. Kappa is hberated into the medium once it develops a highly re- fractile granule, which sometimes appears as a "bright spot" under the microscope. One "bright spot" kappa particle is enough to kill a sensitive individual. Kappa has a specific relationship to its host, in that a particular dominant host gene must be present in order for kappa to maintain itself, i.e., reproduce. Killer individuals that are homozygotes for the recessive host allele partition the non- dividing kappa among the two daughter cells, and this continues in successive divisions until a daughter receives none and is a kappa- sensitive cell. Just what is kappa? In size and shape it resembles a bacterium and, like a bacterium, is known to be infective. But, kappa diff"ers from bacteria in certain staining reactions and in internal morphology, particularly when kappa develops the bright spot, the refractile granule. Even though kappa looks like no known bacterium, the fact that it is infective and is not typically found in para- mecia suggests that it is a foreign organism of some kind. Nevertheless, kappa furnishes our first example of genes which are extra- nuclear but intracellular in location. The special significance of kappa is that it furnishes a model of how a parasitic or 412 CHAPTER 45 FIGURE 45-2. Marcus M. Rhoades {in 1959) examines striped corn plants in the foreground. Unstriped corn plants are in the background. symbiotic microorganism can become so well- adapted to its host that it becomes a part of the host's genetic system and determines some of the traits of the host. Like kappa, the rickettsial organism that causes Rocky Mountain spotted fever is visible and in- herited through the cytoplasm. These organ- isms, as well as the virus and spirochaete we have already discussed in this Chapter, also determine certain traits of their hosts. Where- as each of the cases so far mentioned involves an organism which seems to be foreign to its host at present, we cannot be sure that the organism originated as a parasite or sym- biont. Could some of the now-foreign organisms located intracellularly originally Extranuclear Genes and Nuclear Genes 413 have been part of the normal gene content of a cell? This question is particularly pertinent when viruses are considered. From what has been learned in previous Chapters, we can no longer retain any preconceived notions either that viruses were always foreign infective agents, or that all presently known viruses are of this nature. Present-day virulent phages seem to be acting as foreign organisms when they lyse their bacterial hosts. But, the lytic capacity of phage depends upon its genotype and that of its host, so that under some genotypic conditions lysis is relatively rare. The decision as to normality or abnormality of present-day viruses is even more difficult when temperate phages are considered, for not only are they less lytic yet still capable of transduction, but the very genes characterizing their prophages seem to be associated with a part of a normal chromosome. As we learn more about viruses, particularly phage, our understanding of what is now genetically normal, and what is foreign, is undergoing radical revision.^ With the future increase in our knowledge of the genetics of viruses and their "hosts," we will also be in a better position to postulate how they originated. Let us continue our search for other extranuclear genes, restricting our attention now to cytoplasmic components which seem to be normal constituents of present-day cells, even though we shall make no decision as to their normality when they, or their precursors, first arose. Many plant cells contain cytoplasmic bodies called plastids, which when green because of the presence of chlorophyll are called chloroplasts, and when white are called leucoplasts. In the absence of sunlight, chloroplasts lose their pigment and become leucoplasts, the process being reversed when the plastids are again exposed to sunlight. 2 See A. Campbell (1961). Chromosomal genes are known in corn whose mutants affect the production of chlorophyll in plastids by interfering with the sequence of reactions leading to chlorophyll production. For example, such a nuclear gene may prevent plastids from producing any chlorophyll at all; thus there is a type of leucoplast which is incapable of becoming green for this reason. If a seedling possesses the appropriate nuclear genotype, it will be nongreen; it will grow until it exhausts the food supply in the seed, then die because in the absence of chlorophyll there is no photo- synthesis and no sugar manufactured. Such nuclear genes act as lethals when they produce albino seedlings. Corn plants are also known whose leaves are mosaic, having stripes of green and white (Figure 45-2), the white parts having only leucoplasts incapable of becoming green. ^ The white portions of the leaf survive be- cause they receive nourishment from the green parts. What is the basis for this mosaicism? Is it due to nuclear genes causing different paths of differentiation in different portions of the leaf? It is observed that the striping occurs not only within the leaves but also elsewhere, and seems to extend even into the reproductive organs, so that pollen and eggs can be ob- tained, derived both from green and from white parts. This may permit us to determine the answer to the question last posed. For, if the striping is due to a nuclear gene acting upon differentiation, such a gene should be transmissible through the male or female gamete, without relation to the whiteness or greenness of the tissue giving rise to the reproductive structures. What is done is to obtain an ear of corn derived from an ovary which was likely to be mosaic, having originated partly from green and partly from white tissue. The kernels in such an ear are the Fi, and are grown in rows ^ The following account is based primarily upon work of M. M. Rhoades. 414 CHAPTER 45 corresponding to their positions in the cob. What is observed is not all green seedlings, or ail white, or all striped, or a randomly distributed mixture of types; instead, groups of green and of albino seedlings are found (Figure 45-3). This suggests that striping actually was present in the ovary also and that it persisted in the cob. Other tests of this strain show that the greenness or whiteness of a seedling has nothing to do with the color of the parental part giving rise to the pollen used in the fertilization which produced the seed. The only deciding factor proves to be the color of the tissue giving rise to the ovary. The fact that this effect is independent of the pollen grain suggests that the effect is not due to a nuclear gene acting differently in different tissues, and by appropriate crosses it can be shown that none of the genes in the male chromosomes is involved. In other crosses this is also shown to be true for the nuclear genes contributed by the mother (a fact which you may have already suspected from the clustering of green and of albino seedlings already mentioned). We may con- clude, therefore, that, in the present case, it is only the nature of the plastids contained in different ova which is important. The fact that the pollen grain is not known to carry plastids, and, in the present case, has no influence on the type produced after fertilization, argues in favor of the view that plastids are derived only from pre-existing plastids, the color trait of the daughter plastids being determined only by the color potentiality of the parent plastid. This FIGURE 45-3. Groups of albino and non-albino seedlings from kernels planted in rows corresponding to their positions in the cob. Extranuclear Genes and Nuclear Genes 415 hypothesis may be subject to test in another way. What does one find in the cytoplasm of cells located at the border between white and green tissue? Here one finds cells which contain both fully green and completely white mature plastids, whereas cells within a green sector contain all green mature plastids, and cells in a white sector have only leuco- plasts. (It should be noted that, when im- mature, all plastids are smaller and colorless.) Thus, even when the two kinds of mature plastids are present in the same cell, they have no influence upon each other, but develop according to their innate capacities. If a zygote (or other cell) contains both kinds of plastids it will, by the accident of producing daughter cells having only "white" or only "green" plastids, give rise to stripes of white and of green, respectively. We can conclude from the results presented, amply supported by others not mentioned, that plastids are self-replicating, do not arise except from plastids, and are capable of innate transmis- sible changes. Accordingly, since plastids are self-replicating, mutable, and capable of repli- cating their mutant condition, they contain at least one extranuclear cytoplasmic gene. We have already mentioned that it was proven, from other work, that the chlorophyll trait is also influenced by nuclear cistrons. Thus, a trait of a self-replicating cytoplasmic body is subject to modification both by the extra- nuclear gene(s) it contains, as well as by nuclear genes. It has been found, after crossing two par- ticular all-green corn plants, that some of the progeny are green-and -white striped. The striped plants prove to be homozygous for a recessive nuclear gene, iojap (ij), for which their parents were heterozygous. That the striped phenotype is not due to some inter- ference by ij ij in the biosynthetic pathway leading to the production of chlorophyll pigment (or, in other words, that it is not due to some nuclear gene-caused error in metab- olism) is demonstrated by the fact that the leucoplasts in albino cells remain colorless in subsequent generations of corn plants, even after the iojap recon is replaced by its normal allele. The only simple explanation for this effect is that, in the presence of ij ij, an extranuclear gene, which is located in the plastid and which is responsible for chloro- phyll production, is caused to mutate to a form no longer capable of performing this function. This comprises proof that mutation of an extranuclear gene can be induced by a nuclear gene. A similar case, in which a nuclear gene controls chlorophyll production by mutating plastid genes, is known in the catnip, Nepeta. We have already mentioned * that the cytoplasmic particle kappa can be transmitted from one generation of Paramecium to the next. The distribution of kappa to the next generation depends upon the way the new generation initiates. Since a new generation can be formed in several ways, we shall understand kappa-transmission better after a brief description of two such mechanisms. One method of producing the next genera- tion of Paramecium is asexual. A typical Paramecium contains a diploid micronucleus and a highly polyploid (about lOOON) macro- nucleus (or meganucleus) . The individual can divide by fission to produce two daughter paramecia comprising the next generation. Both the micronucleus and macronucleus replicate and separate, so that when fission is completed, both daughter cells are chromo- somally identical to each other and to the mother cell from which they were derived. Even though the cytoplasmic contents are not equally apportioned to the daughters, a killer mother will normally produce two killer daughters, since both of these receive some of the hundreds of kappa particles distributed throughout the cytoplasm of the parent cell. Should the daughters undergo * The previous and following discussion of Para- mecium is based largely upon the work of T. M. Sonne- born and coworkers. 416 CHAPTER 45 FIGURE 45-4. Simplified representation of micronuclear events occurring during conjugation in Paramecium. Each conjugant has a single diploid micronucleus {A), which following meiosis produces four haploid nuclei {B) . Three of these disintegrate (C), and the remaining nucleus divides once mitotically (D). The conjugants ex- change one of the haploid mitotic products (E), after which fusion of haploid nuclei occurs (F) so that each of the conjugants, which later separate, contains a single diploid micronucleus. fission also, etc., a clone of chromosomally identical killer individuals will be produced. Successive fissions of a sensitive Paramecium produce, naturally, a clone of sensitive indi- viduals. A second process for forming new genera- tions is sexual. The members of a clone are all found to be of the same mating type. But when clones of different mating type are mixed together, there is a mating reaction. This involves the sticking together of indi- viduals of diff'erent mating types so that larger and larger clumps of paramecia are formed. This is followed by conjugation by pairs, each member of a pair being of a diff'erent mating type. During conjugation (Figure 45-4), the micronucleus of each mate undergoes meiosis, at the conclusion of which, three of the four haploid nuclei pro- duced disintegrate. The remaining nucleus divides mitotically once, producing two hap- loid nuclei. Then one of the two haploid nuclei in each mate migrates into the other mate and there joins the nonmotile haploid nucleus to form a single diploid nucleus in each conjugant. During conjugation the macronucleus disintegrates. After conjugation the two paramecia sepa- Extranuclear Genes and Nuclear Genes 417 rate, producing the exconjugants of what we shall consider to be the next generation. You will recognize that both exconjugants are chromosomally identical, since each con- jugant contributes an identical haploid nu- cleus to each fertilization micronucleus. The chromosomal identity of exconjugants can be proven by the use of various marker genes. (Of course, if the conjugants were homo- zygous for different alleles, the exconjugants would be identical heterozygotes.) The dip- loid micronucleus in each exconjugant divides once mitotically, one product forming a new macronucleus, the other remaining as the micronucleus. What would be the normal consequence of mating a killer with a sensitive individual? (Mating can occur before a killer can kill a sensitive individual; in fact, during conjuga- tion, all conjugants are resistant to killing action.) Normally, the cytoplasmic interiors of conjugants are kept apart by a boundary which is probably penetrated only by the migrant haploid nuclei. As a result, little or no cytoplasm is exchanged, so that, kappa- wise, the exconjugants are the same as the conjugants, namely, one is a killer and one is a sensitive individual. However, using certain experimental conditions, a wide bridge can be seen to form between the conjugants through which the cytoplasmic contents of both mates can flow and mix (Figure 45-5). Moreover, the extent of cytoplasmic mixing can be controlled experimentally. If one of the conjugants is killer and the other sensitive, and if cytoplasmic mixing is extensive enough, both exconjugants are found to be killers because of the flow of kappa particles into the sensitive conjugant. Consider now precisely how nuclear genes are distributed in conjugation. Suppose each conjugant is a micronuclear heterozygote, Aa. It would be a matter of chance in each mate which one of the four haploid nuclei produced by meiosis, A, A, a, a, fails to disintegrate, divides mitotically once, one of whose prod- ucts migrates to help form the fertilization nucleus in the other mate. Accordingly, both exconjugants will be AA 25% of the time, both exconjugants will be Aa 50% of the time, and both aa 25% of the time. This result would be obtained whether or not the conju- gants mix cytoplasms. Note again that, regardless of the genotype of the conjugants, the members of a pair of exconjugants are identical with respect to micronuclear genes and will give rise to clones phenotypically identical with respect to the micronuclear gene-determined trait under consideration. When dealing with a trait determined by a cytoplasmic particle hke kappa, on the other hand, we have seen that the results may be different. In this particular example, a cross of sensitive with killer produces exconjugants whose type depends upon the occurrence or nonoccurrence of cytoplasmic mixing. Suppose, to generalize, two paramecia, phenotypically different with respect to a given trait, are mated together. It may be found, when there has been no cytoplasmic exchange, that the two exconjugant clones remain different, each resembling its cyto- plasmic parent in this trait. Suppose, more- over, that only when there is cytoplasmic mixing are the two exconjugant clones found to be the same phenotypically. Such results would prove that the trait under test is determined at least in part by an extranuclear FIGURE 45-5. Silhouettes of conjugating Para- mecium. A. Normal, no cytoplasmic mixing. B. Wide bridge, permitting cytoplasmic mixing. 418 CHAPTER 45 gene. It becomes clear, therefore, that the occurrence and control of cytoplasmic mixing during conjugation provides a powerful tool for detecting and proving the occurrence of extranuclear genes in Paramecium. With this background regarding the differ- ential transmissive behavior of nuclear and extranuclear genes in Paramecium, consider the results obtained from the study of the genetic basis for two dijferent mating types, calling one alpha and the other beta. It can be proven that there is a gene basis for these mating types in the macronucleus. However, exconjugants from a mating of alpha by beta form clones of different mating type only if there is no cytoplasmic mixing, and clones of the same mating type only if the mates mix cytoplasms. There is clearly, then, also an extranuclear gene basis for the mating-type trait. The extranuclear genes involved in this case are invisible and seem to be a normal component of the cell. So, just as is the case for chlorophyll production in corn, the mating type phenotype in Paramecium is affected both by nuclear and extranuclear genes. Additional experiments clarify the role of both types of gene. Recall that the macro- nucleus degenerates during conjugation, and that one daughter nucleus produced by a mitosis of the fertilization nucleus forms the new macronucleus, which thereafter divides at every fission and goes to all daughter cells of a clone. At first this new macronucleus can be referred to as a young macronucleus, and later as an adult macronucleus. Experiments demonstrate that if a young macronucleus is located in cytoplasm con- taining the alpha extranuclear gene, then as an adult macronucleus it comes to determine the alpha mating type; a genetically identical young macronucleus placed in cytoplasm containing the beta extranuclear gene becomes an adult macronucleus determining beta type. Clearly the young macronucleus carries the potentiahty of producing either alpha or beta mating type in the form of one or more macronuclear genes. Which alternative comes to phenotypic expression is dependent, however, upon the type of extranuclear gene carried. Once the macronucleus is mature, that is, is determined as an alpha or beta type, thereafter it and its daughters will persist in this condition. So this fixation of the macronucleus is irreversible. On the other hand, it is found by suitable experimentation that a mature, fixed macro- nucleus produces, or determines the function- ing of, extranuclear genes of the same mating type. For example, an adult alpha macro- nucleus causes the alpha cytoplasmic effect to be produced, which, in turn, is ready to determine the mating type of the young macronucleus produced in the next sexual generation. This mutual, circular, depend- ency between extranuclear and nuclear genes is an example of what may be referred to as a feed-back system. The extranuclear gene feeds instructions to the nuclear gene, which in turn feeds back instructions to the extra- nuclear gene, which feeds back instructions to the nuclear gene, etc. Other traits in Paramecium are also known to be controlled by nuclear and extranuclear genes operating in feed-back systems. It should be noted, however, that when such systems operate, they may not always result in an irreversible fixation in the type of phenotypic alternative which either of the two kinds of genes may express. Moreover, it is not known whether the one kind of gene acts directly on the other kind of gene or on its products. Finally, it should be pointed out that in none of the cases mentioned, ignoring kappa, has it been proved that the extra- nuclear and the nuclear genes affecting the same trait differ except in location and in the consequences expected from this difference. Accordingly, we should not exclude the pos- sibility that extranuclear genes may some- times prove to be episomes or derivatives of episomes. Extranuclear Genes and Nuclear Genes 419 SUMMARY AND CONCLUSIONS The cytoplasm can contain extranuclear genes which are not known to be episomes. In some cases the extranuclear genes seem to be foreign organisms (sigma probably, kappa), in other cases they appear to be normal constituents of the cell (chloroplasts, mating type). Nuclear and extranuclear genes show the following interrelations: the former can mutate the latter; both may interact in the production of a particular phenotype, sometimes operat- ing as a feed-back system. REFERENCES Beale, G. H., The Genetics of Paramecium Aurelia, Cambridge, Cambridge University Press, 178 pp., 1954. Campbell, A., "Conditions for the Existence of Bacteriophage," Evolution, 15:153-165, 1961, Rhoades, M. M., "Plastid Mutations," Cold Spr. Harb. Sympos. Quant. Biol., 11:202-207, 1946. Rhoades, M. M., "Interaction of Genie and Non-Genie Hereditary Units and the Physiology of Non-Genie Inheritance," in Encyclopedia of Plant Physiology, Ruhland, W. (Ed.), Vol. 1, pp. 19-57, Berlin, Springer Verlag, 1955. Sonneborn, T. M., "The Role of the Genes in Cytoplasmic Inheritance," Chap. 14, pp. 291- 314, in Genetics in the 20th Century, Dunn, L. C. (Ed.), 1951. Sonneborn, T. M., "Kappa and Related Particles in Paramecium," Adv. Virus Res., 6:229- 356, 1959. Sonneborn, T. M., "The Gene and Cell Differentiation," Proc. Nat. Acad. Sci., U.S., 46:149-165, 1960. Hl^il Tracy M. Sonneborn, about 1960. 420 CHAPTER 45 QUESTIONS FOR DISCUSSION 45.1. What is revealed regarding nucleocytoplasmic interrelationships from the study of F? Of temperate phages? 45.2. What evidence can you present that COo-sensitivity is due to a virus rather than a normal chromosomal gene? 45.3. In proving the existence of extranuclear genes which operations (recombinational, mutational, functional, chemical) were we utilizing? Did we include their capacity for self-replication? Why? 45.4. Discuss the genetic control of chlorophyll production in corn. 45.5. Do you think the study of nucleocytoplasmic interrelations in Paramecium has any bearing upon differentiation processes in multicellular organisms? 45.6. What unique advantages does Paramecium have as an experimental organism for genetic investigations? 45.7. Certain paramecia are thin, due to a completely recessive nuclear gene, //;. What is the phenotypic expectation for the clones derived from exconjugants of a single mating of + + X + tIP. Is your expectation affected by the occurrence of cytoplasmic mixing? Why? 45.8. According to the definition of a chromosome given on page 19, would you consider kappa to be, or contain, a chromosome? Explain. 45.9. Keeping in mind the difficulties of proving the existence of extranuclear genes, which do you think represents the primary genetic material in cellular organisms, nuclear or extranuclear genetic material? Explain your decision. 45.10. Does adaptive enzyme formation provide an example of a feed-back system? Ex- plain. Chapter *46 GENE ACTION AND OPERONS T! |he extensive study of any or- ganism reveals a large number of alternative traits which have a genetic basis. Some of these traits de- scribe the presence or absence of genetic material (for example, the trait "cytoplasmic DNA" in Paramecium may be due to the presence of kappa). Other traits involve the relocation of genetic material (for example, changes in episomal state, or the inversion of a chromosomal segment). But such al- ternatives as the presence, absence, and movement of genetic material do not describe how the cell or organism is affected, or in what ways genetic material performs a func- tion. We are especially interested in studying those alternative traits which result from some action by, or involving, genetic material. One action, typical of what we have defined as genetic material, is self-replication. You will admit that self-replication must have some phenotypic consequences due to the removal of gene precursor material from the pool of metabolic substances and to the presence of new genetic material. Can all genie action be ascribed to the metabolic changes which take place because of genie self-replication? We know of several kinds of situations in which there is no evidence of gene replication, yet there is evidence of genie action. One example is found in the case of abortive transduction; another is provided by highly functional cells which never divide again, for example, neurons. We can conclude, therefore, that conserved 421 genetic material also functions by some mechanism other than self-replication. A study of inborn errors of metabolism, and of the pedigree of causes for pleiotropic ef- fects of mutants, led us to hypothesize (Chapter 32) that a gene has a single primary function. You realize now that this hypoth- esis refers to some action by genetic material other than self-replication. Depending upon the particular trait we consider to be primary, the scope of genetic material essential for this function, that is, the length of a cistron, will vary (Chapter 42). The general hypothesis of one cistron-one function (besides self- replication) was tested in the specific form of one cistron-one polypeptide chain (Chap- ter 32). The results demonstrated that the specific form of the general hypothesis is acceptable. It was mentioned, on p. 376, that Salmo- nella has at least eight closely hnked loci (Figure 46-1), all having an effect upon the sequence of chemical reactions leading to the biosynthesis of histidine. Already four of the eight loci have been correlated with specific enzymes,^ thereby providing addi- tional evidence for the hypothesis one poly- peptide-one cistron. Although it was also pointed out at that time that close linkage of genes controlling different parts of a biosyn- thetic sequence is not a universal phenom- enon, let us consider some finer genetic de- tails of the lactose, Lac, locus in E. coli which is, in this respect, similar to the histidine locus in Salmonella. You may remember, from p. 369, that the Lac segment contains three cistrons. The 7+ cistron specifies the structure of the enzyme galactoside permease, while z+ is the gene that specifies the structure of the enzyme /3-galactosidase. (Certain alleles of z result in the synthesis of a modified, enzymatically inactive, protein, called Cz, which can be identified by its specific antigenic charac- 1 See P. E. Hartman, J. C. Loper, and D. Serman (1960). 422 CHAPTER 46 I 1 O ilAXX 1 1 f^S to - AXX Str- O 1 00 AIXX as iiixxtl': Q IIXX ?^^ O IXX 80? - in - qXX z - OXX if- -o XIX 59- in O IIIAX 9S- CD J3 MAX £S - O lAX ti'. AX 11 ■ . CM U3 *^ AIX ^ qillX £92j_ DIIIX -~ Fl OIIX 2 < qilX 18 r — DUX 69_2i_ IX 90 i-l ox o£ ^ - O _^ qx 69 i Dx 8f ^ i^] " in iD qxi 9 H 0X1 16^ IIIA 60 i-| ^ qiA fri H D|A qA ^^~ I?] IT) - DA 85 ' ^ qAI 1 "Al ^o'lj LJ o III 5C - -Q II M - O 1 66- • • ^ 1 ■- ■= 2 _ o 2:-.- Gene Action and Operons 423 teristics.) The third cistron, /+, specifies the synthesis of a repressor substance which pre- vents j+ and z+ from producing the permease and galactosidase, respectively. When the substrate upon which these enzymes act is present, however, the repressor substance made by /+ is inactivated, so that the forma- tion of these enzymes becomes possible. Accordingly, E. coli of genotype 7+2+/+ can- not produce galactosidase or permease con- stitutively (in the absence of substrate for these enzymes) but can do so inductively (in the presence of enzyme substrate). This genotype provides us with an example of the genetic basis for the phenomenon of induced enzyme formation. Note, in this connec- tion, that it is often found that the formation of an enzyme is repressed by a high intra- cellular concentration of its product, so that a feed-back system is involved (cf. p. 418 and also Figure 46-1). The order of these genes relative to them- selves and others is: TL . . . Pro . . . (Lac) y z i . . . Ad . . . Gal. Note that all three Lac cistrons specify unique sub- stances. Because /+ produces a repressor substance, which in the absence of substrate is capable of pleiotropic effects — that is, of phenotypic suppression of both y^ and z+, /+ can be called a regulator gene. What is the precise mechanism by which /+ suppresses the action of two other genes which are them- selves nonallelic, even if they are part of the same biochemical sequence? With this question in mind, let us examine the consequences of certain mutations in the Lac region. Mutants capable of synthesiz- ing permease and galactosidase constitu- tively might be due to the genotype y+ z+ /, in which the specific repressor is not pro- duced, and the genes y^ and z+ are able to act under all circumstances. It is possible to construct E. coli which are hybrid for the Lac region, by introducing into F~ cells F particles containing the Lac region as chro- mosomal memory (see p. 369). Thus, one can obtain an E. coli whose chromosome has >'+ z / (which by itself would make per- mease and Cz protein constitutively) and whose F-Lac particle is y+ z+ /+ (which by itself would make permease and galactosidase only in the presence of substrate). In the hybrid no products are formed in noninduced bacteria (in the absence of inducing sub- strate), while all three (galactosidase, Cz pro- tein, and permease) are formed in induced bacteria (exposed to enzyme substrate). We must conclude from this that a single /+ gene can manufacture repressor substance which prevents the products of both normal and mutant y and z genes from being formed constitutively but not inductively, whether or not these genes are located on the same chromosome segment. In other Vv'ords, the repressor substance is diffusible and can act at a distance. There is some evidence that the repressor substance is RNA. Another mutant is found which also per- mits >'+ and z+ products to form constitu- tively, and is, therefore, presumably a muta- tion of /+, say to allele i"". When an ¥-Lac particle of the presumed genotype >'+ z+ /^ is placed in a cell whose chromosome is >'+ z / (which by itself is known to produce permease and Cz protein constitutively), no Cz pro- tein is formed constitutively in noninduced bacteria. Contrary to the assumption made, /^ must be /+ since it is producing repressor capable of repressing Cz protein formation constitutively. If so, in what respect is the ¥-Lac particle mutant? Let us make the supposition that the ¥-Lac particle is mutant at a locus, o+,.the new allele being o", which permits only the r and z loci in the same chro- mosome or particle to act constitutively re- gardless of which allele of / may be present in the cell. If this is so, then the F-Lac particle is genotypically y+ z+ o*" /+ while the genotype of the chromosome can be written y+ z 0+ /, ignoring gene order for the present. On the new hypothesis, the hybrid ought to produce permease and galactosidase consti- 424 CHAPTER 46 tutively, and to produce Cz protein also inductively. This is found. The results ob- tained with this genotype are summarized in Figure 46-2. It is possible to construct other hybrids containing both the o" and o+ alleles. These are also listed in Figure 46-2 with the results obtained. For example, y+ z 0+ i+/F-Lac y^ z+ C /+ produces y+ and z+ enzymes but no Cz sub- stance constitutively, and produces all three in induced bacteria. Partial phenotypic analysis was made for two other genotypes. Thus, ;; 2+ 0+ i^lF-Lac y+ z o' i+ produces Cz protein but no galactosidase in noninduced bacteria, but produces both of these in induced bacteria. Finally, j+ z 0+ i^l¥-Lac y z+ o" i^ produces galactosidase constitutively, and it and permease inductively. We conclude, therefore, that these results are consistent with the hypothesis that an operator gene, o+, exists and that it is this gene which is sensitive to the repressor substance produced by the regulator gene, /+. When the repressor substance is pro- duced and is not inactivated by the presence of substrate, it reacts with o+, and this pre- vents both z and y alleles from operating. When the mutant allele / is present, no re- pressor substance is produced, 0+ is not af- fected, and z and y alleles are capable of acting constitutively. However, a mutant allele of e»+, namely 0% is insensitive to the repressor substance, so that z and y alleles can act constitutively regardless of the geno- type with respect to /. Note that the be- havior of the y and z alleles is dependent upon the particular allele of o which is in the same chromosome or F particle — that is, which is linked in the cis position. Thus, the constitutive mutant of operator, o", has a pleiotropic effect only on other genes in the cis position. Other results demonstrate that the locus of o" is between z and /. Still other mutants have been obtained which prevent the syn- thesis of permease and galactosidase under all conditions. These mutants undergo re- GENOTYPE NON-INDUCED BACTERIA INDUCED BACTERIA Chromosome F-Lac + +. y z 0 1 + + C.+ y z 0 1 + + + y z 0 i + +c + y z 0 1 + +.+ y z 0 1 + C.+ y z 0 1 + + + y z 0 1 + C.+ y z 0 1 P G Cz P 33 36 nd 50 no nd — <1 30 nd 60 — P G Cz P 100 270 100 100 330 100 — 100 400 100 300 — P = Permease G = Galactosidase Cz P= Cz Protein FIGURE 46-2. Crosses, and their results, involving the Lac region of E. coli. nd = not detectable, — = not tested. Gene Action and Operons 425 REGULATOR GENE OPERON .A, Operator Structural gene genes O . A . B 1— t REPRESSOR SUBSTANCE "LTLT ' 1 I NAAA /\/\ 1 I Proteins FIGURE 46-3. Relationships be- tween regulator, operator, and struc- Metabolite tural genes. {After F. Jacob and J. removes Monod.) repressor verse mutation, show complementation neither with z nor with y mutants, are ex- tremely closely linked to the C mutant, and are located between z and /. These mutants are clearly alleles of o+, which we can call o". It should be noted that the operator gene does not seem to produce any unique product which can be detected cytoplasmically. In this respect it may be considered a gene whose primary job is not the specification of a chemical product, such as the amino acid sequence in a polypeptide, but one whose primary effect is to control the function of other genes. Accordingly, operator genes may be called genes for function in contrast to those which specify chemical structures and are, therefore, genes for structure (Fig- ure 46-3). It is possible to explain the action of an operator gene on the basis that it shares nucleotides with an adjacent cistron which is controlled by the operator gene. When the operator gene is functional the adjacent gene cannot be read correctly, since some of its nucleotides are unavailable for this usage. On another occasion, the operator gene might not be functional, permitting the adjacent gene to act. Moreover, the oper- ator gene could mutate in such a way that it is no longer ever functional, in which case the adjacent genes would always be functional, or in such a way that the opera- tor gene is always functional, in which case the adjacent genes could never be functional.) (See pp. 370-372 for other applications of the hypothesis of nucleotide-sharing.) We have seen that an operator gene coordi- nates the expression of adjacent genes. In the present case the genes controlled are related, in that they both affect the biochemi- cal pathway involving lactose utilization. This suggests that there is, at least in some cases, a unit of gene function which is inter- mediate in size between the cistron and the chromosome, and which we may call an operon. Operons, linear groups of genes whose structural activity is coordinated by a functional gene, or operator, may be common in microorganisms. They may also occur in other organisms and may be more frequent than one might at first suspect.^ 2 The preceding discussion of operons and operator genes is based upon the work of F. Jacob, D. Perrin, C. Sanchez, and J. Monod (1960), and F. Jacob and J. Monod (1961). 426 CHAPTER 46 SUMMARY AND CONCLUSIONS DNA has a function other than self-replication. This additional function is clearly de- pendent upon its sequence of nucleotides. In terms of this additional capacity, DNA cistrons can be classified as being genes for structure and/or genes for function. When acting as a gene for function, the cistron serves as an operator that controls the expression of structural genes which are its linear neighbors, the whole complex of genes comprising an operon. It is speculated that the operator gene may produce its different effects by sharing and not sharing nucleotides with structural genes in the operon. REFERENCES Gorini, L., "Antagonism Between Substrate and Repressor in Controlling the Formation of a Biosynthetic Enzyme," Proc. Nat. Acad. Sci., U.S., 46:682-690, 1960. Hartman, P. E., Loper, J. C, and Serman, D., "Fine Structure Mapping by Complete Trans- duction Between Histidine-Requiring Salmonella Mutations," J. Gen. Microbiol., 22:323-353, 1960. Jacob, F., and Monod, J., "Genetic Regulatory Mechanisms in the Synthesis of Proteins," J. Mol. Biol., 3:318-356, 1961. Jacob, F., Perrin, D., Sanchez, C, and Monod, J., "The Operon: A Group of Genes Whose Expression Is Coordinated by an Operator" (in French), C. R. Acad. Sci., Paris, 250:1727 1729, 1960; translated and reprinted in Papers on Bacterial Genetics, Adelberg, E. A. (Ed.), Boston, Little, Brown, 1960, pp. 395-397. McClintock, B., "Some Parallels Between Gene Control Systems in Maize and in Bacteria," Amer. Nat., 95:265-277, 1961. Yanofsky, C, and St. Lawrence, P., "Gene Action," Ann. Rev. Microbiol., 14:311-340, 1960. QUESTIONS FOR DISCUSSION 46.1. How does the phenotypic effect of o" differ from that of its alleles, o+ and o-? 46.2. How does an operator gene differ from a regulator gene? 46.3. Do you suppose the nucleotide sequence is longer in an operator gene than it is in the other genie members of an operon? Why? 46.4. Does prophage act as one or more regulator genes? Explain. 46.5. Some workers classify genes into three kinds: those for structure, for regulation, and for operation. Do you believe this distinction is basic? useful? Explain. 46.6. Hypothetically, what kinds of phenotypic effects might operons produce in man? 46.7. Discuss the hypothesis (of J. F. Danielli) that the nucleus controls the possibility of particular chemical substances appearing in cells, while the cytoplasm controls the way in which macromolecules are organized into functional units. 46.8. In what ways do genes function? 46.9. What is your present concept of the cistron? 3m-<\ Chapter *47 GENE ACTION AND AMINO ACID CODING li N THE last Chapter, we found that genes can act functionally, as .operator genes, or structurally, to cause the production of particular sub- stances. Restricting our attention to DNA, which seems to be the genetic material in most kinds of organisms, the question may be asked relative to structural gene action: What can DNA do, or have done to it, which would result in the formation of par- ticular polypeptide chains? Since we are dealing with conserved DNA, that is, DNA which remains part of a polynucleotide, whatever it does must be done in situ. Since DNA is not protein, it is probably not an enzyme, and probably does not act as a catalyst in producing its cistronic effect. Accordingly, being inactive in this respect, DNA is thought to serve as a kind of tem- plate, so that things are done to it or with it. (In this respect DNA serves as a better inert template than does RNA, whose ribose sugar is more reactive than the deoxy-D-ribose in DNA, so that, as a substance, RNA is less stable than DNA.) We know that following strand separation, each DNA strand apparently serves as a template for the formation of a complemen- tary chain. If DNA is also used as a tem- plate for cistron functioning, we may ask what kind of template information it may contain. This information must be con- tained in the fact that in a linear sequence of DNA, there are usually only four differ- ent base pairs, A : T, T : A, C ; G, G : C. 427 What information are these base pairs sup- posed to contain? Since their "primary" product is, at least in some cases, a specific polypeptide chain, consider what makes a polypeptide chain specific. Almost all poly- peptide chains contain one or more of each of the twenty amino acids commonly found in organisms. These amino acids are shown in Figure 32-2, p. 285. Polypeptides usually differ only in the number and sequence of these amino acid building blocks. Clearly, then, our problem is to understand how 20 amino acids and their sequence can be speci- fied by DNA. Both the polypeptide and DNA are linearly arranged, so this common trait helps us visualize the relation between the two. The sequence of nucleotides must be meaningful in specifying amino acid se- quence. But how can a linear template of nucleotides, of which there are usually only four kinds, determine the linear sequence of amino acids, of which there are 20 kinds? We are presented with a problem of DNA coding. Let us postpone further discussion of the nature of the genetic code, until we have completed a consideration of certain evi- dence regarding protein synthesis. Given the template of conserved DNA, the question can be asked: Where in the cell does the information contained in DNA become translated into a polypeptide se- quence? You might think that this specifi- cation takes place in the nucleus, using the DNA template directly. If so, it ought to be possible to demonstrate that the nucleus is the main or only site of protein synthesis. There is very good evidence, however, not only that some protein synthesis takes place in the nucleus isolated from its cytoplasm,^ but that protein synthesis also occurs in the cytoplasm in the absence of the nucleus. The evidence even suggests that the cytoplasm is the major site of protein synthesis. Let us 1 From work of A. E. Mirsky, V. G. Allfrey, and others. 428 CHAPTER 47 2 (30s) + 2 (50s); MOLECULAR WEIGHTS XIO* r>^ .8 1.8 2 (70s) 2.7 1 (100s) 5.4 FIGURE 47-1. Relation among ribosomes having different sedimentation rates. consider the nature of certain particular cytoplasmic components, because of the possibility that these may be concerned with protein synthesis. Electron micrographs of thin sections of cells reveal numerous ribosomes in the cyto- plasm of all cells which have been examined for them -^ plant, animal, or microorgan- ismal. They vary in size from 100-200 A in diameter and are particularly abundant in cells actively synthesizing protein. (Some are also found in the nucleus.) It is possible to rupture cells and characterize these ribo- somes by size according to their sedimenta- tion rate in the ultracentrifuge. Thus, in terms of sedimentation units, s (the smaller the number of units the smaller the particle, although the relationship is not linear), there are four discrete sizes in E. coli: 30s, 50s, 70s, and 100s. There are two basic sizes, 30s and 50s, the larger ones being composites of these units, as indicated in Figure 47-1. Both the 30s and 50s particles contain 63% RNA and 37% protein. Sev- eral enzymes appear to be attached to the ribosomes, including much, if not all, of the cell's RNAase and part of the cell's DN Aase.^ The smaller particles aggregate into the larger ones when Mg++ or other divalent cations are added. Most (about 80%) of the RNA in a cell is contained in ribosomes. (Small amounts of RNA are also reported in mitochondria and plastids.) Analysis shows that the RNA in ribosomes has the relatively high molecular weight of .56-1.1 X 10*^ (having about 1000-2000 bases). 2 See M. Tal and D. Elson (1961). A series of experiments can be performed ^ in which radioactive amino acids are injected into the body, and tissues rapidly synthe- sizing proteins examined at intervals. The first experiment involves injection of a large dose of labeled amino acid, and shows that the ribosomes become labeled almost im- mediately. A second experiment uses a very small dose of labeled amino acid, which is expected to be used up rapidly in protein synthesis. In this case, the label in the ribo- some increases quickly at first, and then de- creases. Finally, a third experiment can be performed which demonstrates that the labeled amino acid which moves out of the ribosomes is actually incorporated into pro- tein, for example, hemoglobin. Here, then, is clear evidence that ribosomes are asso- ciated with protein synthesis. Moreover, it would seem that the manufacture of hemo- globin takes place in the cytoplasm. But the amino acid sequence in hemoglobin is sup- posed to be determined as the primary func- tion of DNA cistrons (Chapter 34)! How can the DNA template, which apparently remains in the nucleus, function to order the amino acid sequence of hemoglobin, which is apparently manufactured in the cytoplasm? Clearly, in this respect, the DNA cannot be functioning as a template directly, but would have to be doing so indirectly. Suppose the DNA made another template which was neither DNA nor protein, which could leave the nucleus, enter the cytoplasm, and there serve as template for protein syn- thesis. A reasonable candidate for this func- 3 Following the work of P. C. Zamecnik and co- workers, and of M. Rabinovits and M. E. Olson. Gene Action and Amino Acid Coding 429 tion is RNA which is also a nucleic acid and also has a four symbol code, A, U, C, G, in which uracil (U) occurs in place of thymine (T). We would then require the four symbol code of DNA to be translated directly into the four symbol code of RNA. There are several questions we may ask, whose answers will serve to test the hypothesis that DNA nucleotide sequence specifies RNA nucleotide sequence which, in turn, specifies amino acid sequence. 1. Where is RNA synthesized? Several experiments support the view that much, if not all, RNA is synthesized in the nucleus, after which it can be detected, by radioactive tracer studies, to enter the cytoplasm. There is no evidence, on the other hand, of a flow of RNA from the cytoplasm to the nucleus. These results are consistent with the hypothe- sis under consideration. 2. What happens to nucleus-synthesized RNA? Since the typical chromosome con- tains RNA, chromosomal RNA, some of the newly synthesized RNA must be retained in the nucleus where it appears as part of daughter chromosomes. Most of the newly made RNA leaves the nucleus, and a con- siderable portion is believed to be used in the manufacture of new ribosomes. (In Neuro- spora, ribosomal RNA is known to be syn- thesized in the nucleus.) Already formed ribosomes do not accept large quantities of newly formed RNA, since it is found, by labeling experiments, that only a relatively small amount of a ribosome's RNA shows turnover. Accordingly, the greater part of the RNA in ribosomes, ribosomal RNA, is usually incorporated at the time of ribosome formation. Note that the mechanism by which such nuclear RNA becomes incor- porated as part of new ribosomes is still unknown. 3. What is the relationship between the RNA synthesized in the nucleus and the DNA in the nucleus? Bacteria manufacture RNA, and continue to do so even after being in- fected with phage. The base ratio in the DNA of a particular phage is known to differ from that of the DNA of its host cell. After phage infection it is found that the RNA manufactured is diiferent from that manu- factured prior to infection, having, in fact, the base ratio of phage (substituting U for T). Moreover, only the RNA synthesized after infection can base pair in vitro with phage DNA (made single-stranded by an- nealing) to form a double strand — one strand being RNA and one DNA. It is known that, under normal circum- stances, RNA is first synthesized in the chro- mosomes and is then transferred to the nucleolus. Yeast cells can be fed radioactive phosphorus so that the RNA synthesized shortly thereafter is labeled. When such labeled RNA is analyzed for its base ratio it is found to have the same one as has yeast DNA (making the substitution of U for T). Other work "* demonstrates that, in normal cells, freshly made nuclear RNA forms a complex with chromosomal DNA and pro- tein. (RNA in such a complex is resistant to RNAase.) Such results support the view that there is a direct base-for-base correspondence between DNA and nucleus- synthesized RNA. We can call this RNA, which has the base ratio equivalent of DNA, informational or template RNA. 4. How is informational RNA synthe- sized? In experiments dealing with the in vitro synthesis of DNA (Chapter 35), it was found that DNA can replicate in the absence of RNA. This is very likely to be true also in the nucleus, although there might be subtle secondary interactions with RNA. There is evidence, already mentioned in answer to the previous question, in favor of the view that RNA synthesis is intimately related to DNA. It is found ^ that the ■* By J. Bonner, R. C, Huang, and N. Maheshwari (1961). ^ From the work of J. Hurwitz, of A. Stevens, of S. B. Weiss, of their colleagues and of others. 430 CHAPTER 47 nucleus contains an enzyme, RNA poly- merase, which is necessary for RNA syn- thesis. This RNA synthesis can be per- formed in vitro, and requires also the pres- ence of DNA, as well as all four riboside triphosphates; the RNA synthesized proves to have the same base ratio as the DNA (except that T is U). This situation is remi- niscent of the synthesis of DNA, where the DNA polymerase takes directions from single-stranded DNA. Here RNA poly- merase takes directions from DNA in the making of RNA polymer. It has been found that single-stranded DNA can serve as tem- plate for the RNA polymerase, and that an RNA polymer probably cannot serve as primer. 5. How do the amino acids destined to form polypeptides arrive at the ribosomes? Whereas ribosomal RNA has a relatively high molecular weight (Jo to 1 million), as already mentioned, there is another kind of RNA in the cytoplasm which has the rela- tively low molecular weight of about 18,000 (having about 30 bases). Other evidence indicates that there are 80-90 bases per molecule. Since this RNA is soluble when ribosomal RNA is not, it is called soluble RNA or sRNA. There is also no experi- mental proof that sRNA is derived from nuclear RNA. Using radioactively labeled amino acids it can be demonstrated that the amino acids arrive at the ribosomes singly, each attached to a molecule of soluble RNA. All the soluble RNA molecules are similar in their terminal nucleotides, one end terminat- ing with the base G and the other end with the base sequence — C — C — A. Otherwise, they are dissimilar, being of about 20 dif- ferent types, each capable of carrying a differ- ent one of the amino acids typically found in protein. The transported amino acid is at- tached through its carboxyl ( — COOH) group to the 2' or 3' hydroxyl group of the terminal adenosine. Since sRNA serves to transfer the amino acids to the ribosomes, it can also be called transfer RNA. 6. What do we know about the formation of the amino acid-transfer RNA complex? It has been found that each of the 20 amino acids must be activated before it can be ac- cepted by its particular transfer RNA. Both the activation of an amino acid and its at- tachment to soluble RNA may involve the activity of a single enzyme. There is prob- ably a different activating enzyme for each kind of amino acid. Activation involves the combination of the amino acid at its carboxyl end to the adenosine triphosphate (ATP) located terminally, with the removal of two phosphates as pyrophosphate. This reaction can be summarized: amino acid + ATP ^ amino acid adenylate + pyro- phosphate. 7. What determines which transfer RNA molecules are to be attracted to a ribosome? It has been mentioned already that, after a phage infects its host, RNA is made having the base ratio of the phage DNA. It has been found that polynucleotides of this phage- specific RNA become attached to a small percentage of already formed ribosomes. This suggests that many ribosomes do not automatically carry a template of RNA, containing information for specifying amino acid sequence, which it obtained from the DNA template. Such ribosomes are capable of receiving segments of template RNA which carry the code for making phage- specific polypeptides. Thus, there is a third type of cytoplasmic RNA, messenger RNA, which carries information for cistronic action from phage DNA to the ribosome. It is the messenger RNA which attracts the various molecules of transfer RNA, each of which is carrying individual amino acids. It has also been shown ^ that messenger RNA is used in sending cistronic template information from the regular DNA content of a cell to its «" By M. Hayashi and S. Spiegelman (1961). Gene Action and Amino Acid Coding 431 Nucleus CGAACGUACUCA GCTTGCATGAGT CGAACGTACTCA GCUUGCAUGAGU /////// i I'^i Ig C G A A C G MESSENGER RNA TRANSFER RNA? RIBOSOMAL RNA? AMINO ACIDS + TRANSFER RNA AMINO ACID- TRANSFER RNA COMPLEX AMINO ///mmm acid CHAIN FIGURE 47-2. Hypothesized rela- tion between DNA ( — ) and nuclear and cytoplasmic RNA ( — ). 432 CHAPTER 47 ribosomes. (Guanosine triphosphate is re- quired in order that the labeled amino acids, carried by transfer RNA, appear attached to the ribosome.) It can be hypothesized (as illustrated in Figure 47-2) that each of the two DNA strands (which are complements) specifies an RNA complement (the two RNA strands would then also be complementary and com- prise informational or template RNA). Large segments of one of the informational RNA templates leave the nucleus as mes- senger RNA and become located in already formed ribosomes, while the complementary RNA template is broken into segments which are used to make transfer RNA and possibly other RNA's (such as ribosomal RNA). In this event, transfer RNA and messenger RNA would be complementary, they could pair at the ribosome in the cytoplasm, and, in doing so, would place the transported amino acids in proper sequence. These amino acids could then be enzymatically joined to form a polypeptide, which could be freed from transfer RNA, which, in turn, could be liberated from pairing with its messenger complement. The mechanism of protein synthesis can be studied using a suspension of ruptured cells. Such a cell-free system can be pre- pared from E. coli. The activity of the cell- free system can be preserved by the addition of mercaptoethanol. Also added to the mix- ture are the triphosphates of the ribo- nucleosides of A, G, C, and U, as well as all 20 of the amino acids in their L forms. The synthesis of protein can be readily de- tected if one of the added amino acids is radioactive. If the labeled amino acid is valine, for example, valine is found to be- come incorporated into protein. This in- corporation can be stopped by the addition of DNAase, which destroys the DNA, thereby halting the production of messenger RNA. In the absence of new messenger RNA, protein synthesis stops. That this effect of DNAase concerns the production of messenger RNA is demon- strated (1) by the absence of valine incorpora- tion when sRNA or ribosomal RNA is added to the system, and (2) by the resumption of valine incorporation when the RNA from washed ribosomes is added to the system. The informational RNA added may come from various sources. Some experiments have studied the in vitro conditions for the synthesis of /3-galactosidase ^ (see Chapter 46), using messenger RNA obtained from genetically different induced and noninduced E. coli. It is even possible to add TMV RNA to the bacterial cell-free system and detect the synthesis of TMV protein.*^ Using a bacterial cell-free system it is also possible to study whether the addition of synthetic polyribonucleotides has any ef- fect on protein synthesis. First, pure poly- ribotides containing only one base. A, or C, or U, are added. The first two fail to result in amino acid incorporation. However, the addition of polyuridylic acid causes L-phenylalanine to be incorporated into protein.^ It is found, moreover, that the protein formed is poly-L-phenylalanine, and that no other amino acid is incorporated. It is also found that phenylalanine linked to sRNA is an intermediate in this process. This surely means that wherever an appro- priate sequence of U's appears in normal messenger RNA, the protein being synthe- sized will incorporate L-phenylalanine. This is the first crack in the RNA code, that is, the first determination of a sequence of mes- senger RNA nucleotides which specifies the incorporation of a particular amino acid into protein. Note that the problem of DNA coding, mentioned on page 427, has become a problem of RNA coding. The results mentioned also tell us that certain ribonucleotide sequences do not have In work of G. D. Novelli and coworkers. M, W. Nirenberg and coworkers. Gene Action and Amino Acid Coding 433 amino acid meaning in the RNA code, since regardless of the length of the polyribotides of A or C added, no amino acid is incor- porated into protein. But even if each single ribonucleotide specified a different amino acid, only four amino acids would be en- coded. But 20 amino acids need be en- coded! Can we circumvent this difficulty by assuming that a sequence of two nucleotides specifies an amino acid? (This would be like having an alphabet of only four letters and a language composed only of two-letter words.) In this case, since the first letter can be any one of four, as can the second, there are 4x4, or 16, different doublets (or words) possible, if the presumption is made that the RNA code can be read only in one direction. Unidirectional reading seems reasonable, since a single strand of RNA is polarized, just as is a single strand of DNA. Still, this will not be enough to specify 20 amino acids, unless other assumptions are made. (For example, it could be hypothe- sized that a given doublet encodes more than one kind of amino acid, in which case we would be dealing with a degenerate code.) If, however, a sequence of three nucleotides, that is, a triplet, encodes an amino acid, there would be 4x4x4, or 64, different uni- directional sequences possible. This pro- vides more than enough triplets to encode 20 amino acids. (The code would also be degenerate if different triplets encoded the same amino acid.) There are, however, other characteristics of RNA which have a bearing on the nature of its code. Since the number of consecutive ribotides may be in the hundreds or thou- sands, there are no spaces (or non-nucleotide punctuation) to indicate where one triplet should stop and the next should begin. Ac- cordingly, we are dealing with what may be called a comma-free code. Suppose we had six ribotides arranged linearly, in positions called 123456. If triplet 123 specifies amino acid A and 456 amino acid B, errors would be possible if the overlapping triplets 234 or 345 served to encode different amino acids, and if the reading of different meaningful triplets were performed independently. In this event, the last two triplets would have to be eliminated as words in the code to avoid errors. Accordingly, the entire code has to be examined, and all overlapping triplets eliminated from being meaningful, that is, from specifying an amino acid. When, of the 64 possible triplets, all overlapping ones are eliminated, 20 nonoverlapping triplets (amino acid-specifying "words") remain. However, the question of overlapping triplets can be avoided to a great extent if the read- ing of the message can start only at a given place and triplets are read in succession. In this case, the punctuation is provided by the mechanism for reading the code. The least we can conclude from the present discussion is that it requires a sequence of more than one ribotide to encode an amino acid. When the synthetic polyribotide of U is mixed with the synthetic polyribotide of A, so that several strands are likely to base pair or wrap about one another, incorporation of phenylalanine is partially or completely re- duced. Thus, the synthetic polymer is most effective in synthesis when it is single- stranded, as is apparently also true for nor- mal messenger RNA. It is also possible to study the effect on amino acid incorporation into protein of the presence of different nucleotides in the same polyribonucleotide. Using polynucleotide phosphorylase, one can enzymatically syn- thesize in vitro polyribotides containing two or more different ribotides which are ap- parently in a random order. The analy- sis''^ is greatly expedited by the fact that polyphenylalanine is insoluble in the cell- free system. In practice, then, in forming any mixed polynucleotide, an excess of uridylic acid is used, in order to obtain the synthesized protein as a precipitate, which ' By S. Ochoa and coworkers. 434 CHAPTER 47 can then be analyzed for the amount and kind of amino acids, besides phenylalanine, which are present. Thus, to synthesize polyuridylic-adenylic acid, polyuridylic- cytidylic acid, and polyuridylic-guanylic acid, five times as much uridine diphosphate is used as the diphosphates of adenosine, cytidine, or guanosine, respectively. To make mixed polynucleotides containing UAC, UCG, or UGA, ten times as much uridine diphosphate is used as the nucleoside diphosphates of A, C, or G. For example, a mixed polyribotide com- posed of U and C is added to the cell-free system, which is then tested to determine whether any amino acid besides phenylala- nine is incorporated into protein. With this particular mixture, proline and serine are among the amino acids incorporated. The code letters for these amino acids include, therefore, at least one U and one C. One can also determine, in the same way, the ef- fects of other mixed polyribotides on amino acid incorporation. It is found that some amino acids, such as alanine and arginine, require three different nucleotides to be encoded, so that the coding ratio (the num- ber of nucleotides required to code one amino acid) is at least three. (No amino acid is found which requires the presence of all four types of nucleotides. Were the cod- ing ratio four, there would be 24 different sequences possible in quartets composed of AUGC, some of which might be expected to encode an amino acid.) In view of these results (and of others ^° suggesting a low coding ratio) it is hypothesized that triplets of nucleotides of messenger RNA have amino acid meaning, that is, that we are deal- ing with a triplet RNA code. By varying the proportions of uridylic acid and cytidylic acid in a mixed poly- nucleotide, it is discovered that less proline than serine is incorporated when there is an excess of polyuridylic acid. However, 10 Obtained by A. Garen (190). when there is a relative excess of cytidylic acid in the polymer, the reverse occurs, namely, more proline than serine is incor- porated. In terms of triplets, proline must be specified by UCC and serine as UUC (see Figure 47-3), although the sequence of nucleotides in the triplet and the order in which they are read are still undetermined. In other words, although the messenger RNA triplet code letters are UCC for proline, we cannot say whether the sequence is UCC, cue, or ecu. (The first and last triplets are different, since the single-stranded mes- senger RNA molecule is polarized.) Starting with ribotides of U and C in the relative frequencies 5 : 1, we can predict the relative frequencies of different triplets in the synthesized polymer. UUU should occur with a frequency of % X % X %, or i^s^ie. While there are three arrangements possible for the code letters UUC, any particular sequence should occur with a frequency of %X%X )i, or %6; any one of the three possible ar- rangements of UCC should occur with a fre- quency of %X%X %, or %i6, while CCC should occur with a frequency of Kie- These particular sequences are, respectively, in the relative frequencies 125, 25, 5, and 1. Accordingly, if a triplet code is the correct one, then when this particular polyribotide is studied for protein synthesis, one would expect five times more phenylalanine incor- porated than serine, and 25 times more phenylalanine incorporated than proline. Although the results actually obtained " using various synthetic polymers sometimes differ by a factor of two or so from those expected according to the present analysis, the over-all agreement is striking, and offers very strong support for a triplet RNA code. (If an amino acid is incorporated at a rate lower than expected, this may be due to the fact that a given nucleotide, U, is in a se- quence which can be read meaningfully in more than two ways. If the sequence is UUt/CCC, sometimes the triplet read may Gene Action and Amino Acid Coding 435 be UUf/ (phenylalanine), or UC/C (serine?), and still other times UCC (proline?). Thus, nucleotide-sharing may be partially respon- sible for actual incorporation rate deviating from that expected.) You should be able to work out that a polymer synthesized from ribotides of U, A, and C in the relative amounts of 6, 1, and 1, respectively, has the triplet code letters, UUU, UUA, AAU, UAC, AAA, CCC, in a given sequence in the relative frequencies 216 : 36 : 6:6:1 : 1, respectively. Using synthetic polyribotides, the triplet code letters have been determined for 19 amino acids ^^ and predicted for one amino acid, and it is these which are given in Figure 47-3. All meaningful triplets in the experi- mentally detected code contain U. This does not seem to be dependent solely upon the fact that, for technical reasons, each mixed polynucleotide contained U. For no amino acid was incorporated following treatment with pure polyribotides of A or of C. It seems reasonably certain that all meaningful triplets in the messenger RNA code for amino acids normally contain uracil. (This does not preclude the occurrence of mistakes, as the result of which a given amino acid is encoded by another triplet which may or may not contain U.) Of the 64 unidirectionally read triplets possible using A U G C, 37 have one or more U's and 27 have none. (The chance a triplet has no U is % X Ya X %, or %.) It is reasonable, therefore, to expect that triplets which do not contain U do not appear in messenger RNA, except possibly to inter- rupt the message in order to end a polypep- tide chain. If the presence of U in each trip- let is characteristic of messenger RNA, then this RNA must have been the complement of a DNA strand which characteristically has triplets containing A. Moreover, the complementary DNA chain would be charac- terized by the usual presence of T in its trip- le By S. Ochoa and coworkers. AMINO ACID TRIPLET CODE LETTERS (Sequence unknown) Alanine UCG Arginine UCG Asparagine UAA (UAC?) Aspartic acid UAG Cysteine UUG Glutamic acid UAG Glutamine UGC (predicted) Glycine UGG Histidine UAC Isoleucine UUA Leucine UUC (UUA?, UUG?) Lysine UAA Methionine UAG Phenylalanine UUU Proline UCC Serine UUC Threonine UAC (UCC?) Tryptophan UGG Tyrosine UUA Valine UUG FIGURE 47-3. Messenger RNA code for amino acids. (After Speyer, J. F. , et al. , Proc. Nat. Acad. Sci., U.S., 48:441-448, 1962.) lets and the template RNA made from it should contain A in almost every triplet. This RNA would not be used as messenger. We have already presumed (see Figure 47-2 and its discussion in the text) that part of this RNA is used to make sRNA. This is supported by the fact that although only one triplet of sRNA is known, it is ACC. We are led to suppose that sRNA has a different triplet which pairs with a triplet of messenger RNA, and that it contains at least one A. It is also supposed that this is the same triplet which directs a particular activating enzyme to join a particular kind of amino acid to a specific sRNA. Therefore, we expect that (1) if a portion 436 CHAPTER 47 of one DNA strand is rich in successive trip- used to make the same type of informational lets containing at least one A, this portion RNA (messenger RNA or sRNA). For it is is used to make U-containing messenger possible, via mutation, to invert a segment of RNA, and that (2) there is a corresponding double-stranded DNA; sometimes the length segment in the complementary DNA strand, of the inverted segment would correspond each of whose triplets contains T, which is exactly with a protein-specifying cistron or used not to make messenger RNA but A-con- operon. If this occurs, some parts of a given taining sRNA. It is not necessary to assume DNA strand may be used to make messenger that all portions of a given DNA strand are RNA, and other parts to make sRNA. SUMMARY AND CONCLUSIONS The translation of DNA nucleotide sequence into amino acid sequence involves the following events: Each of the single strands of double-helix DNA serves as template for RNA poly- merase, which synthesizes two complementary single strands of informational RNA. Seg- ments of informational RNA which are composed primarily of U-containing nucleotide triplets attach to ribosomes in the cytoplasm and function as messenger RNA. Segments of informational RNA which are complementary to messenger RNA, and hence have at least one A per triplet, are used to make transfer (soluble or s) RNA. Each kind of amino acid is individually activated and attached to a different kind of sRNA in the cytoplasm. The sRN A molecules, each carrying an amino acid, apparently base pair with complementary regions of messenger RNA, so that the transported amino acids are arranged in a specific linear sequence on the ribosome. The amino acids are then linked enzymatically to form a polypeptide which is freed from sRNA, after which the sRNA molecules are liberated from pairing with messenger RNA, and each is free to receive another amino acid for transfer. It is assumed that sRNA possesses a nucleotide triplet, containing at least one A, and that this triplet serves both to specifically attract a particular amino acid and to pair with its complementary triplet in messenger RNA. The code letters in this sRNA triplet and in messenger RNA would be complementary. The code letters in messenger RNA have been determined for almost all amino acids (each triplet contains at least one U), although the sequence of the letters within the triplet and the order in which they are read are still largely undetermined. The comma-free, triplet RNA code for specifying amino acids is essentially solved, al- though some of the details are unknown at this time. REFERENCES Allfrey, V. G., and Mirsky, A. E., "How Cells Make Molecules," Scient. Amer., 205:74-82, 1961. Bautz, E. K. F., and Hall, B. D., "The Isolation of T4-Specific RNA on a DNA-Cellulose Column," Proc. Nat. Acad. Sci., U.S., 48:400-408, 1962. Brenner, S., Jacob, F., and Meselson, M., "An Unstable Intermediate Carrying Information from Genes to Ribosomes for Protein Synthesis," Nature, London, 190:576-581, 1961. Crick, F. H. C, "Nucleic Acids," Scient. Amer., 197:188-200, 1957. Crick, F. H. C, "On Protein Synthesis," Symp. Soc. Exp. Biol., 12:138-163, 1958. Furth, J, J., Hurwitz, J., and Goldman, M., "The Directing Role of DNA in RNA Synthesis," Biochem. Biophys. Res. Commun., 4:362-367, 1961. Garen, A., "Genetic Control of the Specificity of the Bacterial Enzyme, Alkaline Phos- phatase," in Microbial Genetics, Hayes, W., and Clowes, R. C. (Eds.), Cambridge, Cambridge University Press, 1960, pp. 239-247. Gene Action and Amino Acid Coding 437 Speakers (/. to r.) M. W. Nirenberg, F. Lipmann, and S. OcHOA at a symposium on the RNA code held January, 1962 at Indiana University. Gay, H., "Nuclear Control of the Cell," Scient. Amer., 202 (No. I):126-136, 1960. Geiduschek, E. P., Nakamoto, T., and Weiss, S. B., "The Enzymatic Synthesis of RNA, Complementary Interaction with DNA," Proc. Nat. Acad. Sci., U.S., 47:1405-1415, 1961. Goldstein, A., "Chain Growing of Proteins: Some Consequences for the Coding Problem," J. Mol. Biol., 4:121-122, 1962. Gross, F., Hiatt, H., Gilbert, W., Kurland, C. G., Risebrough, R. W., and Watson, J. D., "Unstable Ribonucleic Acid Revealed by Pulse Labelling," Nature, London, 190:581- 585, 1961. Hall, B. D., and Spiegelman, S., "Sequence Complementarity of T2-DNA and T2-Specific RNA," Proc. Nat. Acad. Sci., U.S., 47:137-146, 1961. Hoagland, M. B., "Nucleic Acids and Proteins," Scient. Amer., 201 (No. 6):55-61, 1959. Hurwitz, J., and Furth, J. J., "Messenger RNA," Scient. Amer., 206 (No. 2):41-49, 1962. Kurland, C. G., "Molecular Characterization of Ribonucleic Acid from Esc/ierichia Coli Ribosomes," J. Mol. Biol., 2:83-91, 1960. Martin, R. G., Matthaei, J. H., Jones, O. W., and Nirenberg, M. W., "Ribonucleotide Compo- sition of the Genetic Code," Biochem. Biophys. Res. Commun., 6:410-414, 1962. Nirenberg, M. W., and Matthaei, J. H., "The Dependence of Cell-Free Protein Synthesis in E. Coli upon Naturally Occurring or Synthetic Polyribonucleotides," Proc. Nat. Acad. Sci., U.S., 47:1588-1602, 1961. 438 CHAPTER 47 Novelli, G. D., Kameyama, F., and Eisenstadt, J. M., "The Effect of Ultraviolet Light and X Rays on an Enzyme-Forming System," J. Cell. Comp. Physiol., 58 (Suppl.) :225-244, 1961. Schulman, H. M., and Bonner, D. M., "A Naturally Occurring DNA-RNA Complex from Neurospora crassa" Proc. Nat. Acad. Sci., U.S., 48:53-63, 1962. Speyer, J. F., Lengyel, P., Basilio, C, and Ochoa, S., "Synthetic Polynucleotides and the Amino Acid Code, II," Proc. Nat. Acad. Sci., U.S., 48:63-68, 1962. Stevens, A., "Net Formation of Polyribonucleotides with Base Compositions Analogous to Deoxyribonucleic Acid," J. Biol. Chem., 236 (No. 7), PC 44, 1961. Strauss, B. S., An Outline of Chemical Genetics, Philadelphia, Saunders, 1960. Tal, M., and Elson, D., "The Reversible Release of Protein, Ribonucleic Acid and Deoxy- ribonuclease from Ribosomes," Biochim. et Biophys. Acta, 53:227-229, 1961. Zamecnik, P. C, "The Microsome," Scient. Amer., 198 (No. 3) :1 18-124, 1958. Zamecnik, P. C, "Historical and Current Aspects of the Problem of Protein Synthesis," Harvey Lect., 54:256-281, 1960. QUESTIONS FOR DISCUSSION 47.1. Would you expect the RNA code for amino acids to be the same in all free-living organisms? Explain. 47.2. Compare the replication of RNA virus with that of polypeptide chains. 47.3. If a polypeptide chain is specified by a sequence 2000 or so nucleotides, what would you conclude regarding the presence of non-nucleotide material as punctuation between cistrons? 47.4. To what use can you put the fact that the spacing between nucleotides in a DNA chain is nearly the same as it is between amino acids in a polypeptide chain? 47.5. Discuss the hypothesis that ribosomes are viruses. 47.6. Make a report on advances in our understanding of the genetic code since the present account was written (February 1962). 47.7. What evidence can you present that the attachment of messenger RNA to the ribosome does not involve extensive complementary base pairing? 47.8. Give evidences that messenger RNA is single-stranded. Chapter 48 THE BIOCHEMICAL EVOLUTION OF GENETIC MATERIAL i; "n the course of studying the nature and effects of genes, you .may have wondered about the origin of genetic material on earth. This would involve the first occurrence of a chemi- cal substance capable of replicating itself and some of its modifications. At present we know of only two substances having these properties, DNA and RNA. Some under- standing of the problems involved in ex- plaining gene origin may be obtained from a consideration of our knowledge of present- day genes and of biological evolution. Let us assume that the first gene was either DNA or RNA. Given the presence of the first gene, how did it manage to replicate? We already know something regarding the mechanism of biological replication of DNA. The simplest DNA system capable of repli- cating in vitro requires the presence of (1) energy to separate the double-stranded helix, (2) four kinds of deoxyriboside tri- phosphates, and (3) the enzyme DNA polymerase, not to mention water, at the cor- rect pH, which contains the ions necessary to activate the enzyme. It does not seem very likely that the first gene was a DNA polymer which replicated in this manner. One reason for this opinion is based upon the essential role played by the enzyme. According to our present understanding, the amino acid sequence in such an enzyme is specified by a gene. It seems improbable that so complex an enzyme could be formed independently of gene action. However, one 439 might require that the first gene be capable of specifying DNA polymerase which would then be available for subsequent DNA syn- thesis. Even so, one would have to be con- vinced that the amino acids in the enzyme, the nucleoside triphosphate building blocks, and the energy for chain separation are com- ponents likely to be present in the environ- ment of that time. The difficulty of chain separation would be avoided if the first gene were RNA, since this nucleic acid is usually single-stranded. Unfortunately, we do not know nearly as much about the biological replication of RNA genes as we do about DNA genes. The complexity of present-day replication of DNA should not automatically exclude it or RNA as being the material basis of the first gene. Before making any decision on this matter, we would need to have more in- formation relative to DNA synthesis, includ- ing answers to such questions as: (1) Can any other simpler substance substitute, no matter how inefficiently, for the polymerase? (2) What is the shortest nucleotide sequence capable of self-replication? (3) What, if any, simpler substances can replace the triphos- phates? Although DNA and RNA are recognized as being genetic material today, it is possible that the first genes were of neither type, but of a related chemical composition. We may even wonder if, at present, there are genes which are neither DNA nor RNA. Note that we have thus far bypassed the problem of the origin of the first gene, and have con- sidered only the matter of its replication were it DNA or RNA. Although we initially inquired about the origin of genes, clearly the answer we suggest regarding the mechanism of its replication will depend upon the chemical identity we hypothesize for the first gene. In considering the origin of the first gene we should keep in mind the possibility that its nongenic predecessor might have been CHAPTER 48 capable of some degree of self-replication, but might not have been able to replicate any of its mutant forms. The search for infor- mation regarding nongenic systems having some but not all of the properties of genetic material is clearly highly desirable. Such information may be sought in studies of various polymers under test tube conditions. Subgenic chemicals may occur in present- day cells. Several constituents of the cyto- plasm other than plastids are able to self- replicate. These include the centriole and the kinetosome (which have already been discussed on pages 369-370 in connection with episomes). If these structures prove to be mutable and are still able to self-replicate, they can be classed as cytoplasmic or extra- nuclear genes. Experimental study of these organelles will be expected to reveal the details of their chemistry, and whether they possess only the self-replicating capacity of genes, never having had, or having lost, the ability to reproduce themselves after mutation. In any event, our knowledge of genes will be increased by such studies. We would also wish to know a great deal more about the synthesis of RNA genes, and whether ribosomes, which have the chemical compo- sition of RNA genes,' are self-replicating and mutable, in order to speculate fruitfully upon the nature of the pregenic and the first genie material. While we must conclude that we do not yet have sufficient information to decide which pathway led to the chemical evolution of the first gene, we do have some evidence on the subsequent history of genes. The only genetic material found exclusively in free- living organisms today is DNA, and this is found in all such organisms, whether they be unicellular or multicellular, plant, ani- mal, or microorganismal. Whether or not other types of genes exist or have existed, DNA genes must have a selective advantage for survival, having persisted as the main 1 See A. Van Kammen (1961). genetic material for about a billion years, which is approximately the period that plants and animals have been separate in their evolu- tion. Moreover, it is hkely that the forma- tion of chromosomes with telomeres and with centromeres, as well as the establishment of special methods of separating daughter and homologous chromosomes (by mitosis and meiosis), and of recombining them (fertiliza- tion), were innovations involving DNA which were established some time prior to the diver- gence of the plant and animal kingdoms. Not only must there have been a chemical evolution in DNA and the accessory ma- terial which packages and recombines it, but there probably was also an evolution in gene activity. It is likely that the primitive earth accumulated large amounts of different, more or less complex, organic materials which remained undegraded before the ad- vent of the first genetic material. As the first gene-containing organisms used up these resources in their metabolism, however, there would have been a selection in favor of mu- tants capable of synthesizing such organic materials from simpler organic, or from in- organic, components. This means that natural selection acted against those genes which functioned auxotrophically (that is, which were incapable of directing synthesis of nongenetic material or directed the forma- tion of no meaningful synthetic product) in favor of those genes which functioned proto- trophically (that is, which specified the syn- thesis of a component no longer available in the environment). Prototrophism would also be advanced by the physical association of genes involved in different portions of a given biochemical sequence. This would lead eventually to the selection of mutant genes whose function, other than self-replica- tion, was to regulate the functioning of other genes. Thus, in addition to genes for struc- ture, there would be expected to evolve genes for function — operator genes located in operons. The Biochemical Evolution of Genetic Material 441 A study of the comparative biochemistry of present higher plants and animals, bac- teria, and many viruses, shows that they all form or require the same 20 or so amino acids. Accordingly, nucleic acid and pro- tein are, perhaps, the most durable geo- chemical features of the earth, having per- sisted more than a billion years. Because of the intimate relationship between amino acids and genetic material, it would seem highly desirable to learn about the evolution of all kinds of organic (carbon-containing) compounds, including amino acids and poly- peptides, energy-rich compounds (like ATP), catalysts (like iron-containing compounds), and energy-capturing compounds (like chlorophyll). Our present understanding is that at an early stage in the history of the earth there was a reducing atmosphere, which was rich in water, hydrogen, methane, and ammonia, but poor in free oxygen and CO2. Using mixtures of gases predicated as being present in such a reducing atmosphere, and exposing such mixtures to heat, ultraviolet light, or electrical discharges (man-made lightning), it has been possible to produce large amounts and a large variety of amino acids. Other experiments have, by heat treatment, con- verted amino acids into polypeptides, and have produced purines like adenine from simpler components. Such experiments are expected to lead us to understand better the organic evolution which took place on earth prior to the formation of the first genes or organisms. We are not restricted to this planet, how- ever, in our search for information regarding either pregenic, preorganismal evolution, or postgenic, postorganismal evolution. The present universe is about ten billion years old, the earth being about half this age. Because the universe contains vast numbers of stars (suns) with planets, there must be numerous suns the size of our own that have planets which are about the same size as the earth and which are about the same dis- tance from their suns. Some of these planets are surely younger and others older than our own. What is the possibility that a chemical and biological evolution similar to that which occurred on earth would take place on these or other planets? The answer to this question will depend, of course, upon the chemical composition of these other planets. Most of the universe is composed of hydro- gen and helium (most of the earth's hydrogen having escaped from its atmosphere in the distant past). Of the remaining atoms, however, the universe has abundant oxygen and nitrogen and, in fact, is relatively richer than the earth in carbon, the atom essential for the organic compounds which have played so important a role in chemical and biological evolution on earth. It is, there- fore, likely that there are numerous places in the universe where the initiation of an or- ganic chemistry of biological interest would be possible. Since the earth is a relatively poor place for such an evolution (which nevertheless occurred), there are almost surely numerous planets in the universe like our own, which contain earher stages in chemical evolution, similar stages in biologi- cal evolution, as well as those which are older, and very probably have more ad- vanced types of organisms. We already have evidence for the occur- rence of organic radicals like CH, CN, CC, and CO in comets, and for organic molecules of an asymmetric type on Mars. Astrono- mers have also reported variations in ap- parent color and texture of Mars with changes in season. These evidences are very strong that Mars contains appreciable quantities of organic matter, although one cannot yet decide whether these have a preorganismal or an organismal origin. Further information about the chemistry of the sun and planets will doubtless be pro- vided by telescopes of various kinds placed 442 CHAPTER 48 into orbit above our atmosphere. In this position, such telescopes would not be im- peded, as they are now, by the absorption of energy by the contents of our own atmos- phere. Interplanetary research is now in progress to send instruments to or near various planets in the near future. Such missions will be capable of telling us the de- tailed chemistry of any of our neighboring planets, and will, of course, also be designed to detect the presence of organisms and of DNA. We are already sending radio sig- nals into space in an attempt to contact other organisms capable of replying. It will become apparent to you that in any space mission it is important to avoid the accidental transplantation of terrestrial geno- types to other planets. For, if a single bacterium, like E. coli, were to be placed on a planet containing a suitable medium, the progeny would occupy a volume the size of the earth in about 48 hours. Such an un- scheduled transplantation would be disas- trous to any plan we would have for a later study either of the preorganismal evolution of organic compounds or of any indigenous organisms. This is why objects sent beyond our atmosphere are carefully sterilized. Which heavenly objects, likely to be in- vestigated in the near future, are interesting from the point of view of preorganismal and organismal evolution? We have already mentioned the desirability of exploring Mars. Consider Venus, whose surface is unknown, being hidden completely by an opaque, highly reflecting, cloud layer, containing abundant CO2 and water. While estimates of Venus' temperature vary widely, and it is thought that its surface is dry and hot, we cannot assume that organic chemistry or even biological activity is impossible there. After studying its chemistry in sufficient de- tail, it is possible that we might wish to colonize Venus, perhaps first by placing a chlorophyll-containing microorganism in its outer atmosphere. In a very short period, such an organism, by using huge quantities of the atmospheric components for growth and reproduction, might change the entire climate on Venus. Missions to other planets in our solar system would be expected to reveal the kind of chemical evolution which occurs under other environmental conditions. Our own satellite, the moon, has no atmos- phere, and probably no water. Accordingly, the presence there today of earthlike life is out of the question. However, it is possible that the moon is just about as old as the earth and may have had an organic and even a biological evolution similar to our own before it lost its atmosphere. So, it will be interesting to obtain and analyze samples of its surface and, particularly, its subsurface material. It has been suggested that the moon might act as a gravitational trap for fossil spores which may have drifted between planets. Although improbable, the very possibility of an interplanetary gene flow is too important to ignore in our plans to ex- plore and exploit space. Planetary research has many motivations, but the search for evidence of chemical evolution, DNA, or- ganisms, and life would seem to be among the most significant. SUMMARY AND CONCLUSIONS DNA has been the primary genetic material on earth for about a billion years. During this time DNA together with accessory material has undergone a structural evolution leading to the establishment of chromosomes and of mechanisms for genetic recombination. There has probably been also a functional evolution of genes, which proceeded from those which serve as genes for structure (specifying the organization of nongenic compounds) to those which serve as genes for function (and act as operator genes in operons). The Biochemical Evolution of Genetic Material 443 Further information regarding pre- and postgenic evolution can be expected to be obtained from experiments with polymers, DNA, RNA, and various cell organelles, as well as from experiments involving other planets. It is expected that biochemical and biological evolution will also be found elsewhere in the universe. REFERENCES Abelson, P. H., "Extra-Terrestrial Life," Proc. Nat. Acad. Sci., U.S., 47:575-581, 1961. Blum, H. F., "On the Origin and Evolution of Living Machines," Amer. Sci., 49:474-501, 1961. Calvin, M., "The Origin of Life on Earth and Elsewhere," Ann. Int. Med., 54:954-976, 1961. Clark, F., and Synge, R. L. M., (Eds.), The Origin of Life on the Earth, New York, Pergamon Press, 1959. Huang, S.-S., "Life Outside the Solar System," Scient. Amer., 202 (No. 4):55-63, 1960. Lederberg, J., "Exobiology: Approaches to Life Beyond the Earth," Science, 132:393-400, 1960. Lederberg, J., and Cowie, D. B., "Moondust," Science, 127:1473-1475, 1958. Miller, S. L., and Urey, H. C, "Organic Compound Synthesis on the Primitive Earth," Science, 130:245-251, 1959. Oparin, A. L, Tlie Origin of Life on the Earth, 3rd Ed., New York, Academic Press, 1957. Penrose, L. S., "Self-Reproducing Machines," Scient. Amer., 200 (No. 6):105-114, 1959. Rose, H. H., A Synthesis of Evolutionary Theory, Englewood Cliffs, N.J., Prentice-Hall, 1962. Sagan, C, "The Planet Venus," Science, 133:849-858, 1961. Sagan, C, "On the Origin and Planetary Distribution of Life," Rad. Res., 15:174-192, 1961. Sinton, W. M., "Further Evidence of Vegetation on Mars," Science, 130:1234-1237, 1959. Tax, S. (Ed.), The Evolution of Life, Vol. 1 of Evolution after Darwin, Chicago, University of Chicago Press, 1960. Van Kammen, A., "Infectious Ribonucleic Acid in the Ribosomes of Tobacco Leaves Infected with Tobacco Mosaic Virus," (PN 941), Biochim. Biophys. Acta, 53:230-232, 1961. See Supplement VII. QUESTIONS FOR DISCUSSION 48.1. Which do you think came first in evolution, the gene or what we now consider "gene product"? Explain. 48.2. Do you consider it a fact that the genetic material on earth has undergone a biochemical evolution? A structural evolution? A functional evolution? Why? 48.3. Do you believe there are "superhumans" on other planets? Why? 48.4. Do you suppose, in the future, that we will need to be just as careful to avoid the accidental transplantation of genotypes from other planets to our own, as we now are to avoid the reverse? Why? 48.5. In what respects would you expect the environment of Venus to be changed by explan- tation of a photosynthesizing microorganism to its atmosphere? 48.6. What kind of information would you seek to discover from landings on the moon? Mars? Venus? 48.7. What characteristics would you expect genes from other planets to have? Chapter *49 GENES — NATURE AND CONSEQUENCE I "n the first Chapter, we postu- lated the occurrence of genetic .material responsible for pheno- typic similarities and differences between organisms. We later required that the genetic material be self-replicating and capa- ble of self-replicating some of its chemical changes. Most of this book has been con- cerned primarily with the further definition or delimitation of the nature of the genetic material. Each different way of studying segments of the genetic material, or genes, revealed additional properties of the genetic material which were expressed in terms of the operation used to detect them. Thus, in the study of the chemical basis of the genetic material, it was demonstrated that all known genes are inseparable from nucleotides of either RNA or DNA, the latter being the typical genetic substance of chromosomes and of free-hving organisms. RNA is usually single-stranded and DNA double-stranded. The biological replication of DNA involves strand separation and the action of a poly- merase that takes directions from the single DNA chain, in the utilization of deoxy- riboside triphosphates to manufacture the complementary DNA chain. Though DNA is self-replicating, this does not mean that the process is accomplished in one step. In fact, it requires two replications before a given strand can result in a copy of itself, the first replication producing the comple- mentary strand and the second replication 444 producing a copy of the first strand. It is probably justified to think of self-replication as occurring in this indirect way for several reasons. First, DNA replication is by single strands, each of which apparently acts inde- pendently of the other. Second, DNA oc- curs in organisms like 0X174 in the single- stranded condition, all strands being the same complement. Here self-replication must be considered a two-step process. Third, it is possible that one of the two strands in a double helix may be defective (mutant), so that it is, at least in some places, incapable of replicating a complement. Such a chain would be incapable of both replication and self-replication in its defective portion, while its normal, complementary chain would be capable of both. The preceding dealt with self-replication at the chemical level. When does self- replication occur at the informational level? The transforming ability of hybrid DNA double helices proves that a single DNA strand is capable, subsequent to replication, of providing all the information contained in a double-stranded helix possessing correct complementary pairs. It is likely that either single strand of a normal double helix can furnish this information after it has replicated once. However, all or part of the informa- tion used to make messenger RNA may be carried by only one DNA strand. There- fore, in order to carry the same functional (messenger, or sRNA, or other) information, DNA probably has to self-replicate. It would seem desirable, therefore, to recog- nize the difference between replication and self-replication, at both the chemical and in- formational levels. Does pure DNA have any of the proper- ties of the genetic material? It is still an assumption, however likely, that the DNA primer placed in vitro has come from the replication of DNA in vivo, rather than being the product of the activity of some other sub- stance, like protein or RNA. Accordingly, Genes — Nature and Consequence 445 we cannot use the origin of the DNA primer as part of a proof that it is genie. But, con- sider certain chemical characteristics pos- sessed by the DNA primer. It is self-replicat- ing under in vitro conditions, and, moreover, replicates precisely certain chemical modi- fications of itself. This is demonstrated directly by the extensive synthesis which oc- curs after uracil is substituted for the thymine in the substrate (cf. p. 325), and is demon- strated indirectly, using the normal substrate, by the synthesized product having the same A + T/C + G ratio as the primer, regard- less of what variation in this ratio the primer may show. We conclude, therefore, that DNA synthesis in vitro fulfills one of the re- quirements of our definition of genetic ma- terial. The detection of genetic material was originally dependent upon its presence in organisms and its production of a pheno- typic effect. In the course of this book, how- ever, we have apparently dispensed with these requirements. Pure virus DNA in a test tube is considered genetic material, even though it is no longer intraorganismal, re- combining, mutating, replicating, or per- forming any phenotypic function. This is valid on the basis that this DNA either is known or expected to possess such proper- ties when introduced into an organism, or is chemically indistinguishable from organis- mal DNA known or expected to have these properties. The DNA synthesized in vitro is chemically indistinguishable from organis- mal DNA, is capable of self-replicating itself and some of its modifications, and of under- going strand separation and recombination. Since it possesses these characteristics it would seem reasonable to consider that it, too, is genetic material, even though, until now, attempts to detect a cistronic effect via transformation have been unsuccessful. Ac- cording to this view, then, genetic material has already been synthesized in the test tube. We have just reasoned that DNA may be identified as genetic material, using solely the operation of chemical investigation. Using living cells there are other operations which can be employed to study and identify genes, including recombination, mutation, and phenotypic function. These other methods of studying genes have led to our present understanding of the recon and cis- tron. Chemically, we have found that the recon may be a single nucleotide, while the cistron is composed of several or many recons. What is the smallest chemical unit of genetic material capable of (chemical and informational) replication? We do not know. The phenotypical, mutational, and recom- binational operations employed to study gene properties are different from the study of genes by chemical methods in two respects. First, they require that the gene produce a phenotypic effect. Second, they require a genetic alternative which produces a detect- able change in phenotype. Let us make some additional observations regarding each of these three operations in turn. It should be made clear that just because all three of these operations depend upon phenotypic effects for their detection does not necessarily mean that these operations auto- matically characterize cistrons. For cistrons are genetic units as defined from the study of what are apparently the single primary phenotypic effects of genes. If we study the mutation from dull red to white eye in Dro- sophila, for example, or the recombination of such mutants, we are not necessarily con- cerned with whether we are dealing with the primary effects of genetic units; merely studying the effects of genes undergoing mutation and recombination does not auto- matically reveal information on how cistrons are related to the traits produced. The cis- tron has meaning only when it is hypothesized that the genetic material has a single effect other than replication, and only after the decision is made as to which effect is to be considered primary can the scope of the cis- 446 CHAPTER 49 tron be identified by experimental means. Let us examine the basic premise involved, which states that one genetic unit, or cistron, has one primary function besides replication. This was presented as a working hypothesis in the hope that its acceptance would lead to experiments which would result in a better understanding of the functional unit of genetic material. This hypothesis, however fruitful may have been the consequence of its ac- ceptance, has never been proved (refer to p. 278). What basis have we for such a conclusion? Suppose we start with a mor- phological or biochemical pleiotropism whose pedigree can be traced back to fewer and fewer causes. If, after tracing the pedigree back as far as our techniques permit, we find there are still two or more apparently un- related effects, the proponent in favor of the one cistron-one function hypothesis can always claim that had we searched longer with better techniques, we would find this pleiotropism has a single basis. Or, if we are able to trace a group of pleiotropic ef- fects back to a single origin, opponents of this hypothesis can still state that had we looked harder and better we would find still other, not yet known, effects which would be unrelated to the single factor which explains all the presently known pleiotropic effects. We must conclude, therefore, that we can- not decide what a primary effect of a gene is merely from the number of effects detected. However, we have used the idea, that if we find a one-to-one correlation between any effect and a gene change, the phenotypic effect is directly, hence primarily, the result of gene action. In this way, for instance, we tested the idea that polypeptide sequence is determined in a primary way by nucleotide sequence. We found, in the case of hemo- globin, that mutation was directly asso- ciated with a change in its amino acid com- position. Acceptance of the hypothesis one polypeptide-one cistron, however, does not mean that the reverse is true, namely, one cistron-one polypeptide, that is, that all cis- trons have as their primary function the specification of polypeptides. The reserva- tion was made that cistrons also might act in a primary way to specify other substances. In this connection, it should be mentioned that operator genes are recognized by having a primary effect upon the functioning of other genes, and that thus far such genes have not been demonstrated to specify any chemical product whatsoever. When we speak of a phenotypic result as being affected by a gene in a primary way, do we mean anything other than that there is a one-to-one relationship between gene and phenotype? Do we mean that the first action of a gene is, at least in some cases, the specification of amino acid sequence? No! For we have evidence from hemoglobin and other proteins that their synthesis takes place in the cytoplasm, physically isolated from conserved DNA. In these cases the amino acid sequence is specified by messenger RNA nucleotide sequence. In such cases, then, it is clearly more correct to consider RNA specification as being caused in a primary way by genie action than it is to claim the same for amino acid sequence. Let us discuss the matter of gene action by starting not from a phenotypic result and working back toward the gene, but from the other direction, starting with the gene. The genetic activity we know most about directly is replication. In DNA replication the single DNA strand is re- markably passive, in that it is neither energy- supplying, nor appreciably modified in its own structure, nor irreversibly changed in any directional manner, nor acting enzy- matically. What then are the properties of DNA which are responsible for its capacity to serve as template? These properties must include the physical configuration of linearly arranged nucleotides as well as the specific pattern of their electrical charges, both of which seem to act more in a physical than in Genes — Nature and Consequent 447 a chemical capacity. The utilization of the DNA template for DNA replication is, there- fore, a relatively passive process with regard to the DNA strand, and an active, chemical one relative to the highly specific action of DNA polymerase. If the DNA fiber which serves as a template for DNA poly- merase action is mostly a passive chemical, it should be not at all surprising that DNA serves as a template which is utilized by other enzymes, provided that the raw materials col- lected on the template are sufficiently similar to deoxyribotides in physical and electrical properties. This has, in fact, been found true for polyribotides synthesized by RNA polymerase which uses DNA as template. Whether nucleotides other than those in DNA and RNA, or still other substances, make use of the DNA template in a similar or dif- ferent manner has yet to be determined. Be that as it may, it is suggested that the simplest and broadest working hypothesis is that all the functional characteristics of genes de- pend upon the linear sequence of nucleotides and the ways this can be used as template by various substances and enzymes. In ac- cordance with this view, serving as template is the primary and only function of DNA. The DNA template would have at least two uses to which it is put, one involving the making of DNA and another the making of RNA polymers. We come then to a new definition of the gene, as being any template whose use even- tually results in self-replication of the tem- plate, and which either retains this property after mutation or is derived from a template which can do so. We can still consider as genetic any substance producing the same phenotypic effects as a known gene. In these terms, the RNA in plant viruses is genetic material, just as is conserved chromo- somal DNA. We do not know how viral RNA functions in replication and self-repU- cation, although there are several possibili- ties. The host cell may contain a special RNA polymerase which uses the virus RNA as template, the second replication being virus RNA. Or, the virus RNA may be used as template by a special host DNA poly- merase, and the DNA chain produced might then be used in a replication by regular RNA polymerase which would produce virus RNA. In either case, chemical and informational self-replication would be a two-step process, as is probably the case for DNA genes. Is chromosomal RNA genie? Only future experimentation will provide the answer. We need to know whether nuclear polymers of RNA (synthesized using DNA as tem- plate) can be used as template in a manner suggested for virus RNA. If it is, it can probably self-replicate some of its mutants. Because the DNA template contains par- ticular nucleotides, each individual one has meaning at least for the specification of other complementary nucleotides (of DNA and RNA type), and groups of these in some cases eventually have purpose in the align- ment of amino acids in polypeptides. It should be recalled that some protein syn- thesis apparently takes place in the nucleus. Does this synthesis involve the use of the RNA, or the DNA, or both, as template material? In this connection it may be re- marked that the hypothesis of nucleotide- sharing translates into a phenomenon in which a segment of DNA or RNA template is utilized for two different purposes. Such purposes may be to make more DNA (to be conserved or not), more RNA (to be con- served or not), or to order amino acids or other substances. We need not specify whether this template-sharing should occur in the nucleus or the cytoplasm. We have already been successful in the study of the replication and mutation of DNA in vitro, dissociated from protein. Recent studies ^ suggest that it may be pos- sible also to study the enzyme-specifying cistron in vitro. The one-to-one relation- ^ Ey D. Novelli and coworkers. 448 CHAPTER 49 ship between cistrons and polypeptides is also expected to be extremely useful for de- termining the details of the DNA and RNA codes, and the nature of mutation. We have discussed several genetic systems which seem of special interest in these respects. One involves the genetic determination of hemoglobin. In this case, however, while the amino acid sequence of some of the pro- tein is known, it is a difficult undertaking to determine the corresponding nucleotide se- quence because of the large amount of nu- cleotide material in the genome. Several other systems should be mentioned. All involve viruses, which have a relatively small number of nucleotides per genome. The amino acid sequence in the protein building block of tobacco mosaic virus is now known (see Figure 44-4). We need now to determine the sequence of ribotides in TMV. This is a formidable undertaking technically, whose success is hoped for, but is not assured. Progress along these lines is evidenced ^ by the finding that the termi- nal 5' linked nucleotide in isolated TMV- RNA is adenosine, unphosphorylated at the 2' and 3' positions. Another line of attack attempts to correlate the details of the pro- tein coat of phage with phage nucleotide sequence. Still another line of investigation ^ involves the rll cistron in phage, whose fine structure is analyzable down to the nucleotide level, but whose phenotypic effect is not under- stood in detail chemically. Nevertheless, it is possible to determine the nucleotide basis for certain point mutants in the rll region. Suppose, in r+, that the DNA strand "mak- ing" messenger RNA has a G in a certain triplet, and that this is replaced by A in a particular r point mutant. This mutant 2 From work of T. Sugiyama and H. Fraenkel- Conrat, in 1961. 3 Pursued by S. Benzer with S. P. Champe and other coworkers, and by others. (See reference in legend to Figure 43-5.) phage may not lyse the K12 strain of E. coli because its messenger RNA is abnormal and contains U instead of C, and wrongly made /•+ product results. It has been found that 5-fluoro uracil, FU, is not mutagenic, but can substitute for the U in RNA when added to the diet of K12. When FU substitutes for U in messenger RNA, the FU may some- times be mistaken for C by an sRNA mole- cule. So, in the case of the mutant under discussion, the sRNA paired with abnormal messenger RNA may be wrong, but it may be the one which carries the amino acid brought to this position in normal, r+, mes- senger RNA. As a result, the normal amino acid would be incorporated, r+ product would be formed, and the host cell would lyse. So, those r mutants, which can lyse only when FU is added, most probably have G on the /•+ DNA strand which "makes" mes- senger RNA, and C on the complementary DNA strand. Those mutants which do not lyse in the presence of FU may have T, A, or C at this locus in the DNA strand "making" messenger RNA. Using various chemical mutagens as well as FU, it is often possible to determine when T, or A, or C is pres- ent on the /•+ messenger-producing DNA strand. It is sometimes found that a single phage mutant may simultaneously suppress the ef- fects of point mutants at a number of other nucleotide sites. Suppose, in some of these cases, that all the latter point mutants have the same triplet modified by the same base substitution, and the result is that the same abnormal amino acid is incorporated into the different cistronic products. One way to suppress the effects of all of these mutants would be for a mutation to occur in the DNA which specifies the enzyme which determines which amino acid is transported by the specific sRNA which pairs with the abnormal triplet in the messenger RNA. In this case, an abnormal amino acid may be transported to the abnormal messenger RNA, and this Genes — Nature and Consequence 449 amino acid may be the one which is normally incorporated at that position in the cistronic product. So, mutants which make wrong messenger RNA may still form the correct protein product, if the additional, com- pensating error is made of having sRNA carry a specific wrong amino acid. Through these and other studies in phage we can ex- pect to learn a great deal about the sequence of nucleotides in DNA strands, the nature of the genetic code and of mutation. Further advances are expected from our increasing biochemical capabilities to determine nucleo- tide sequence in biologically made RNA and to synthesize specific sequences of ribonucleo- tides. In order to study mutation, it has always been necessary in the past to observe a change in the phenotype, that is, some mor- phological, physiological, or biochemical change in a trait. Since additions, subtrac- tions, or shifts of chromosomal material can be observed directly under the microscope, without any attempt being made to deter- mine whether an extrachromosomal trait is involved, you might at first think such changes are not changes in phenotype but only changes in genotype. But the chromo- some is surely just as much a part of the phenotype as is some nongenic portion of the organism. For example, the kappa particles (themselves genie) are properly described as being part of the Paramecium's phenotype (and also as part of its genotype). One can speak of the change in nuclear morphology (hence phenotype) which occurs when cells become polyploid or polytene (such changes having a genie basis). One can discuss changes in chromosomal phenotype during different stages of mitosis (even though there may be no gene change involved). It is clear, then, that the genotype refers to all the genes present, while the phenotype refers to all the genie and nongenic traits of the system. Of course, when one is working at the macroscopic level the phenotype cannot include chromosomal or other genie traits, and deals only with the consequences of the interactions between genes and their en- vironment. The phrase, a novel phenotype based upon genie change, partially defines what we have previously called a mutant (see p. 403). The word "novel" requires some additional consideration. It would have been entirely correct to consider the first case of segrega- tion as being a mutation, since it certainly was a novel change in the genetic material, one that had never before been recognized. When, however, other gene pairs were studied, it was found that segregation was not a novelty after all, but the rule for paired nuclear genes. We, therefore, include segre- gation as a means of genetic recombination, not of mutation. Similarly, genetic trans- formation was first considered to be a rare genetic change, and so was referred to as mutation. But once a variety of species ex- hibited this phenomenon, and once trans- formation was recognized as being a fre- quent event within certain species, it became clear that, usually, the process is better con- sidered as a mechanism for genetic recom- bination. Recall also the discussion of Modulator and Dissociation. Although the position effects these genes apparently cause were not considered mutations, the move- ments of Modulator and Dissociation were. More and more cases of the latter type of change are being discovered. Are we still justified in considering these as being muta- tional? We will probably soon consider cases like these to be examples of another mechanism for genetic recombination, at least in certain organisms. Finally, you may recall that the integration and deintegration of F were classified as genetic recombina- tions, and not as mutations. You may have been surprised at this at the time, but this interpretation was given in the light of knowl- edge (which had not yet been presented) that other types of episomes were known. In this 450 CHAPTER 49 case, the evolution in terminology, from mu- tation to recombination, was purposely short- circuited. Since what first appears to be a novel genetic change may prove, upon further in- vestigation, not to be novel, we are always subject to reclassifying mutation as genetic recombination. Under these circumstances, today's mutations are possibly tomorrow's new mechanisms for genetic recombination. The type of mutational change which seems to be most immune to reclassification as recombination is subnucleotide change. Clearly a substitution of 5-bromo uracil for thymine is a mutation. But even at this level, such immunity to reclassification is not absolute! Rotational substitution (A : T becomes T : A), now considered a possible type of mutation, may be found to be a normal mechanism of genetic recombination in some organisms. It seems desirable to restrict the term muta- tion to describe nucleotide changes which are unnatural rather than novel. You may re- call that we have already refrained from call- ing mutations certain genetic changes which occur naturally in the life cycle (polyploidy in liver cells, chromosome elimination in Sciara), although these same changes can be considered mutations at least when in- duced by extra-organismal factors. What have we learned about recombina- tion, the operation we used first to investi- gate the genetic material? We have found a variety of mechanisms which result in new genetic combinations, that is, changes in position or amount of naturally occurring nucleotide material. For chromosomal genes, these mechanisms include segregation, independent segregation, nondisjunction, crossing over, fertilization, polyploidy, poly- teny, aneusomy, structural changes in chro- mosomes caused by breakage (such as deletion, duplication, inversion, transloca- tion, transposition, and shift), transforma- tion, transduction, and strand recombina- tion in vitro. For extranuclear genes, we have found recombination to involve varia- tion in number or kind of these genes; for episomes, we have the same recombinations as for extranuclear genes plus the pairing of episomes with, or their integration in, the chromosome. In cases of transformation, strand recom- bination in vitro, and phage or plant virus infection, genetic recombination may re- quire the addition or presence only of DNA or RNA. In most cases, however, it is not possible to make such a direct association, since, at the time of genetic recombination, the nucleic acid is apparently bound to pro- tein in the form of nucleoprotein. Thus, for example, we cannot attribute chromosome breakage or crossing over to an action on or by DNA alone. It should also be reahzed that mutation (with the exception of certain virus-induced mutations) and template usage for gene action probably occur when the nucleic acid is in the form of nucleoprotein. For example, while the information may be carried solely by genetic RNA or DNA, this information is probably often used while the nucleic acid is bound to protein. Accord- ingly, our understanding of the usual recon and cistron will have to be expressed in terms of nucleoprotein activity, until such time as special materials and techniques are avail- able. Finally, you will realize that this book has been restricted largely to those consequences of genes which may reveal something of the nature of the genetic material. In compari- son with what is known, very little has been said about the applications of genetic princi- ples. We have had some brief discussions of how genetics plays a central role in our understanding of biological evolution, of diff'erentiation, and of development. We have mentioned briefly some of the uses of genetics in plant and animal breeding, and in the understanding of inborn and infectious diseases. Some discussion of the past, pres- Genes — Nature and Consequence 451 ent, and future uses and importance of game, which I trust you have found stimu- genetics is given in Supplement V. Some of lating. But I also hope the adventure is not the questions posed at the ends of various completed for you, as it surely is not for me. Chapters also indicate various uses to which Let us look to a future in which we continue genetics has been or may be put. rapidly to increase our knowledge about the We have come to the epd of our thinking nature and consequence of genes, REFERENCES Allen, J. M. (Ed.), The Molecular Control of Cellular Activity, New York, McGraw-Hill, 1961. Cellular Regulatory Mechanisms, Cold Spr. Harb. Symp. Quant. Biol., 26, 1962. Champe, S. P., and Benzer, S., "Reversal of Mutant Phenotypes by 5-Fluorouracil: An Approach to Nucleotide Sequences in Messenger-RNA," Proc. Nat. Acad. Sci., U.S., 48:532-546, 1962. Fresco, J. R., and Straus, D. B., "Biosynthetic Polynucleotides: Models of Biological Templates," Amer. Sci., 50:158-179, 1962. Mitchell, J. S. (Ed.), The Cell Nucleus, New York, Academic Press, 1960. Muller, H. J., "Genetic Nucleic Acid: Key Material in the Origin of Life," Perspectives in Biol, and Med., 5 (No. 1, Autumn) :l-23, 1961. Niu, M. C, Cordova, C. C, and Niu, L. C, "Ribonucleic Acid-Induced Changes in Mam- malian Cells," Proc. Nat. Acad. Sci., U.S., 47:1689-1700, 1961. Sager, R., and Ryan, F. J., Cell Heredity, New York, Wiley, 1961. Strauss, B. S., An Outline of Chemical Genetics, Philadelphia, Saunders, 1960. QUESTIONS FOR DISCUSSION 49.1. Of what value are operational definitions of a gene? 49.2. What is your present definition of a gene? 49.3. In what respects has the concept of the gene been static and in what respects has it been dynamic, in the course of this book? 49.4. Define the recon and the cistron in chemical terms. 49.5. Ignoring its chemical composition, what are the other characteristics of a cistron? A recon? 49.6. How would you now define a mutant? 49.7. Is the recon a single nucleotide or a single nucleotide pair? Explain. 49.8. Discuss two areas of future investigation which you believe might provide basic information relative to the nature of the gene. 49.9. If a "replicon" is defined as the smallest unit of the genetic material capable of replica- tion, discuss what is already known about it. What questions can you ask about it; whose answers are yet unknown? 49.10. The work of R. W. Briggs and T. J. King on nuclear transplantation proves that development involves irreversible changes in the nucleus. Should such changes be described as mutations? Explain. 49.11. Compare the terminal nucleosides of TMV and transfer RNA. What can you imply from this comparison? 49.12. What do you consider to be the essential characteristics of genetic material? AUTHOR INDEX Italicized page numbers refer to photographs. Abelson, P. H., 443 Adams, J. N., 378 Adelberg, E. A., 292, 318, 338, 348, 354, 359, 360, 361, 362, 363, 364, 367, 372, 380, 426, s-49, s-76 Adler, J., s-64 Alexander, P., 184, 202 Allen, J. M., 451 Allfrey, V. G., 427, 436 Allison, A. C, 236 Amano, T., s-38 Ames, B. N., s-49 Anderson, T. F., 357, s-74 Arber, W., 379, 380, s-74 Auerbach, C, 250 Avery, O. T., 340, 348, s-63 Bacq, Z. M., 184 Baglioni, C, 288 Bailey, W. T., 386 Bangham, A. D., 109 Barclay, R. K., s-63 Barnett, L., 389 Baron, L. S., s-74 Basilio, C, 438 Bateson, W., 55 Baumiller, R. C, 242, 370. Bautz, E. K. F., 407, 436 Beadle, G. W., 726, 272, 281, 292, s-J, s-38, s-39, s-49, s-63 Beale, G. H., 419, s-38 Beam, A. G., 161, 280 Becker, E., s-38 Belling, J., 139, 144, 162 Bennett, D., 266 Bensch, K. G., 317 Benzer, S., 389, 392, 396, 397, 400, 407, 448, 451, s-49, s-75 Berg, P., s-77 Bergmann, F. H., s-77 Bessman, M. J., s-64 Beutler, E., 269 Binnington, J. P., 184 Blakeslee, A. F., 139, 144, 162 Blum, H. F., 443 Boedtker, H., s-75 Bollum, F. J., s-64 453 Bonner, D. M., 438, s-49 Bonner, J., 429 Brachet, J., 21, 31 Brehme, K. S., 148 Brenner, S., 382, 389, 436 Bridges, C. B., 87, 92, 104, 109, 142, 148, 186, 195 Briggs,R., 271,451,5-75 Brink, R. A., 216, 217, 222, 223 Brown, B., s-49 Bryson, V., s-75 Burnet, F. M., 405, 407, s-75 Burnham, C. R., 126 Burns, S. N., 362, 363, 367, 372 Burrous, J. W., s-76 Butenandt, A., s-38 Calef, E., s-49 Calvin, M., 443 Campbell, A., 379, 391, 413, 419, s-75 Canellakis, E. S., 382, 389 Carey, W. F., s-74 Carlson, E., 402, 407 Carlson, J. G., 179 Case, M. E., s-49 Cavalli, L. L., s-76 Cavalli-Sforza, L. L., s-75 Champe, S. P., 389, 396, 448, 451 Chargaff, E., 305, 372, s-63 Chase, M., 383, 387, 389, s-75 Chesley, P., 266 Chevais, S., s-49 Chu, E. H. Y., 184, 250 Clark, F., 443 Claus, W. D., 184 Cleland, R. E., 24, 25, 26, 162, 166, 167, 172 Clowes, R. C, 363, 436 Cohen, S. S., s-64, s-75 Cohn, M., s-49 Cordova, C. C, 451 Correns, C, 14 Cowie, D. B., 443 Crawford, I. P., 292, s-77 Creighton, H. S., 125, 126 Crick, F. H. C, 308, 309, 312, 318, 402, 403, 436, s-63, s-75, s-77 Crow, J. F., 235, 236, 241, 242, 250 Darlington, C. D., 370 Davidson, J. N., 305 Davidson, P. F., 392, 398 Davis, B. D., s-49 Dawson, M. H., 340 DeGeorge, F. V., 79 Delbruck, M., 386, s-75 Dellweg, Z., s-64 Demerec, M., 158, 376, s-38, s-75 DeRobertis, E. D. P., 31 DeVries, H., 14, 162, 172 Dewey, V. C, s-64 Dieckmann, M., s-77 Dobzhansky, Th., 7, 14, 60, 68, 73, 227, 236, 239, 240, 250, 253, 254, 261 Dodge, B. O., s-49 Dodson, E. O., 14, 261 Doty, P., 345, 348, s-75 Doudney, C. O., s-75 Dunn, D. B., s-64 Dunn, L. C, 7, 14, 60, 261, 263, 266, 268, 419 du Vigneaud, V., s-49 Eagle, H., s-49 Edwards, P. R., s-76 Ehrensvard, G., s-49 Eigner, J., 345, 348 Eisenstadt, J. M., 438 Elson, D., 428, 438 Emerson, R. A., 126 Emerson, S., 162 Ephrussi, B., 272, 280, s-38, s-49 Ephrussi-Taylor, H., 280, 341, 348 Epling, C, 254 Fairbanks, V. F., 269 Falconer, D. S., 60 Ferguson-Smith, M. A., 147 Feughelman, M., s-63 Fincham, J. R. S., s-49 Flaks, J. G., s-64 Flemming, W., 21 Fling, M., s-38, s-39, s-49 Fogel,S.,21,31,98, 115, 126,202, 227 Fox, S. W., s-38 Fraenkel-Conrat, H., 406, 407, 448 Francis, T., 266 Eraser, D. K., 384, s-76 Freese, E., 400, 401, 407, s-75 Freese, E. B., 407 Freifelder, D., 392, 398 Freiman, A. E., s-49 Freire-Maia, N., 250 Fresco, J. R., 451, s-75 Fries, N., s-49 Furth, J. J., 436, 437 Gabriel, M. L., 21, 31,98, 115, 126, 202, 227 Garen, A., 434, 437 Garnjobst, L., s-49 Garrod, A. E., 275, s-38 Gay, H., 437 Geiduschek, E. P., 437 German III, J. L., 161 Gierer, A., 406, 407 Gilbert, W., 437 Giles, N. H., 184, 250, s-38, s-49 Gish, D. T., 406, 407 Glass, B., 381, 398, 399, s-76 Glover, S. W., s-38, s-75 454 INDEX Gluecksohn-Waelsch, S., 269 Goldman, M., 436 Goldschmidt, R. B., 73, 109, 269 Goldstein, A., 437 Golomb, S. W., s-75 Gorini, L., 426, s-49 Gots, J. S., s-38, s-75 Gowen, J. W., 236 Griffith, F., 340, s-75 Gross, F., 437 Gross, S. R., s-49 Guthrie, G. G., 384 Haagen-Smit, A. J., s-49 Haas, F. L., s-75 Hadorn, E., 67, 69, 73, 269 Haldane, J. B. S., s-38 Hall, B. D., 436, 437 Hallmann, G., s-38 Hamburger, V., 263 Hamilton, L. D., s-63 Hannah-Alava, A., 98 Hardy, G. H., 227 Harford, C. G., s-64 Harris, H., 280, 292 Hart, R. G., 404 Hartman, P. E., 376, 377, 421, 422, 426, s-38, s-49, s-75 Hartman, Z., s-38, s-75 Haselkorn, R., s-75 Hayashi, M., 430 Hayes, W., 356, 358, 363, 364, 436, s-77 Hecht, L. I., s-75 Hede, R., 392, 398 Heinrich, M. R., s-64 Helinski, D. R., 395, 399 Herriot, R. M., 348 Hershey, A. D., 383, 386, 387, i5S, 389, 392, 399, s-63, s-75 Herskowitz, I., 76 Herskowitz, I. H.. 55, 209, 242, 250, 370, 372, 401, 407 Herskowitz, J., 76 Hiatt, H., 437 Hiraizumi, Y., 223 Hirota, Y., s-75 Hoagland, M. B., 437 Hogness, D. S., 380, 381 Hollaender, A., 161,212 Holtz, A. M., 73 Hooper, C. W., s-63 Home, R. W., 389 Horowitz, N. H., s-38, s-49 Hotchkiss, R. D., 348, s-75 Houlahan, M. B., s-49 Hsia, D. Y.-Y., 280, 292 Hsu, T. C, 146 Huang, R. C, 429 Huang, S.-S., 443 Hurwitz, J., 429, 436, 437 Hutner, S. H., s-77 lijima, T., s-75 lino, T., s-76 Ingram, V. M., 286, 288, 292, s-49 Iritani, H., s-38 Itano, H. A., 286, 292 Jacob, F., 357, 359, 362, 363, 364, 367, 369, 372, 379, 380, 381, 391, 399,425,426,436, s-75, s-77 Jesaitis, M. A., s-64 Johannsen, W. L., 1,6 Johnston, A. W., 147 Jones, O. W., 437 Josse, J., s-64 Kaiser, A. D., 380, 381, 391, 399 Kallman, F. J., 79 Kameyama, F., 438 Kammen, H. O., 382, 389 Karpechenko, G. D., 260 Kaufmann, B. P., 142, 159, 160 Kellenberger, E., 330, 379, 380, 384, 388, 389 Kempthorne, O., 60 Khouvine, Y., s-49 Kidder, G. W., s-64 King, D. W., 317 King, T. J., 271,451,5-75 Kjeldgaard, N., s-76 Klein, E., s-49 Klein, G., s-49 Kluyver, A. J., s-75 Knight, B. C. J. G., s-75 Knight, C. A., 406, 407 Knox, W. E., s-39 Kogl, F., s-49 Kornberg, A., 321, 328, s-^, s-63, s-64, s-75 Kornberg, S. R., s-64 Koshland, D. E., Jr., s-64 Kossikov, K. V., 196 Krakow, J. S., 382, 389 Krieger, H., 250 Kurek, L. I., s-49 Kurland, C. G., 437 Landauer, W., 263, 269 Landsteiner, K., 35 Lane, D., 345, 348 Langridge, R., s-63 Laughlin, J. S., 209 Law, L. W., s-49 Lederberg, E. M., 334, 335, 338, 339, 379, 381, s-75, s-76 Lederberg, J., 334, 335, 338, 339, 349, 353, 354, 374, 377, 379, 381, 443, %-4, s-49, s-75, s-76 Lehman, I. R., s-64 Lehrmann, H., 286 Lengyel, P., 438 Lennox, E., 376 Leopold, U., s-39 Lerman, L. S., 341, 345, 348, 401,407 Levene, H., 7, 60 Levine, P., 35 Levinthal, C., 387, 392, 398 Levy, M., s-49 Lewis, E. B., 191, 193, 194, 196 L'Heritier, P., 410 Li, C. C, 227 Lichtenstein, J., s-64 Lima de Faria, A., 370 Lindegren, C. C, s-49, s-77 Lindegren, G., s-77 Lipmann, F., 431 Litt, M., s-75 Lively, E. R., s-76 Loeb, T., 304, 305, 357 Loper, J. C, 421,426 Luria, S. E., 378, s-76 Lwoff, A., s-39 Maal0e, O., 392, 399 Maas, W. K., s-49 McCarty, M., 340, 348, s-63 McClintock, B., 125, 126, 214, 215, 222,426 McCoy, A., s-76 MacDowell, E. C., 266 McElroy, W. D., 381, 398, 399, s-76 MacLeod, C. M., 340, 348, s-63 Maheshwari, N., 429 Mahler, H., 384 Marmur, J., 345, 348 Martin, R. G., 437 Matthaei, J. H., 437, 438 Mazia, D., 21 Melechen, N. E., 332, 335 Mendel, G., 8, 14, 39, 47, s-/ Merrill, D. J., 261 Meselson, M., 312, 314, 318, 388, 389, 436, s-76 Metz, C. W., 173 Metzenberg, R. L., s-49 Miller, S. L., 443 Mirsky, A. E., 31,427,436 Mitchell, H. K., 280, s-39, s-49 Mitchell, J. S., 451 Mohr, O. L., 37 Monod, J., 391, 399, 425, 426, s-49, s-76 Montagu, A., 79 Moore, J. A., 7 Morgan, D. T., Jr., 158 Morgan, T. H., 83,92, 115, s-2 Morse, M. L., 379, 381, s-76 Morton, N. E., 241, 242, 250 Muller, H. J., vi, 55, 161, 182, 186, 196, 202, 203, 204, 209, 210, 212, 241, 242, 250, 402, 407, 451, s-2, s-39, s-76 Nagel, E., s-76 Authors 455 Nageli, C, 14 Nakamoto, T., 437 Nanney, D. L., s-76 Neel, J. v., 37, 286 Nelson, N. J., s-49 Newman, H. H., 79 Nirenberg, M. W., 432, 437, 438 Niu, L. C, 451 Niu, M. C, 451 Novelli, G. D., 432, 438. 447 Novick, A., 336. 338, s-76 Nowinski, W. W., 31 Nye, J. F., s-49 Ochoa, S., 433, 435, 437, 438 O'Conner, C. M., s-77 Oehlkers, F., 162 Ofengand, E. J., s-77 Olson, M. E., 428 Oparin, A. I., 443 Prskov, F., s-77 0rskov, I., s-77 Osborn, F., 79 Oyama, V. I., s-49 Ozeki, H., 377, 381, s-38, s-75 Pardee, A. B., s-49 Parks, R. E., Jr., s-64 Partridge, C. W. H., s-49 Passano, K., 184, 250 Pateman, J. A., s-49 Pauling, L., 286, s-76 Penrose, L. S., 443 Perrin, D., 425, 426 Perutz, M. F., 286 Peters, J. A., 6, 14, 31, 126, 136, 196. 202, 222, 227, 292, 318, 348. 407 Peterson, P. A., 222 Peterson, W. H., s-49 Pintner, 1. J., s-77 Pirie. N. W., s-77 Pollister, A. W.. 7, 370, 372 Poliister, P. F., 370, 372 Pond, v., 184 Potter, V. R., 305, s-64 Preiss, J., s-77 Prokofyeva-Belgovskaya. A. A., 196 Provasoli, L., s-77 Puck, T. T., 177, 184, s-49 Rabinovits, M., 428 Race. R. R.. 67 Radding, C. M., s-64 Ramachandran, L. K., 407 Rasmuson, M., 228 Rees, M. W., 389 Renner, O., 162 Revesz, L., s-49 Rhoades, M. M., 29. 31, 126, 160,4/2,413,419 Richter, A. A., s-77 Risebrough, R. W., 437 Robinson, E. A., 292 Rose, H. H., 443 Rotman, R., 386 Rous, P., s-49 Rubenstein, I., 392, 399 Rudkin, G., 316 Rudnick, D., 263 Ruhland, W., 419 Ryan, F. J., 7, 451, s-77 Saez. F. A., 31 Sagan, C, 443 Sager, R., 451 St. Lawrence, P., 426 Sanchez, C. 425, 426 Sandler, I., 223 Sandler, L., 223 Sanger, F., s-77 Schachman, H. K., s-64 Schalet. A., 212,402,407 Schildkraut, G., 345, 348 Schlossberger, H., s-38 Schrader, F., 7, 21, 370 Schramm, G., 406 Schreil, W. H. G., 330 Schrodinger, E.. s-77 Schull, W. J., 37 Schulman, H. M., 438 Schultz, J., 294 Seeds, W. E., s-63 Serman, D., 421, 426 Shettles, L. B., 107, 108, 109 Shug, A., 384 Sia, R. H., 340 Siminovitch, L., s-76 Simms, E. S., s-64 Sinnott, E. W., 14, 60 Sinsheimer, R. L.. 384, s-64 Sinton, W. M., 443 Slatis, H., 242 Smith, J. D., s-64 Smith, P. E., 266 Snell. G. D., 266 Sonneborn, T. M., 41 1, 415, 4/9 Sparrow, A. H., 184 Spencer, J. H., 372 Speyer, J. F., 435, 438 Spiegelman, S., 430, 437, s-77 Spilman, W. M., s-74 Spizizen, J., 384 Stadler, L. J., vi, 203 Stahl. F. W., 312, 314, 318, s-76 Stanley, W. M., 406, 407 Stebbins, G. L., 261 Stent. G. W., 318, 380, 381, 398, 399, 407, s-75, s-77 Stephenson, M. L., s-75 Stern. C., 37, 64, 78, 125, 126, 210, 228, 242 Stevens, A., 429. 438 Stocker, B. A. D.. 377, s-77 Stokes, A. R., s-63 Straus, D. B., 451 Strauss, B. S., 438, 451 Streisinger, G., 389. s-64 Sturtevant, A. H., 100, 109, 136, 162, 186, 196, s-39 Sueoka, N., 318 Sugiyama, T., 448 Suskind, D. R., s-49 Sutton, W. S., 31 Swanson, C. P., 21, 31 Swartz, M. N., 328 Synge, R. L. M., 443 Szilard, L., 280, 336, 338 Szybalski, W., s-75 Tal, M., 428, 438 Tatum, E. L., 281, 292, 349, 354, s-3, s-38, s-39, s-49, s-76, s-77 Tax, S., 443 Taylor, A. L., 359, 360, 361, 364 Teissier, G., 410 Tessman, 1., s-77 Thomas, C. A., Jr., 384, 392, 399 Todd, A., s-77 Tolmach, L. J., 341, 345, 348 Torii, M., s-38 Trautner, T. A., 328 Tschermak, E.. 14 Tsugita, A., 406, 407 Umbarger, H. E., s-49 Urey, H. C. 443 Van Beneden. E.. 31 Van Kammen. A.. 440, 443 van Niel. C. B., s-75 van Schaik, N. W., 223 Vogel, H. J., s-49 Votkin, E., s-64 von Euler, H., s-75 Wacker, A., s-64 Waddington, C. H., 269 Wagner, R. P., 280. s-39 Wallace, E. M., 52. 81, 102, 103, 143 Watson, J. D., 308, 309, 312, 318, 392, 399, 402, 403, 437, s-63, s-77 Weidel, W., s-38 Weigle, J. J., 379, 380, 388, 389, s-64 Weinberg, W., 228 Weiss, S. B., 429, 437 Welch, L. R., s-75 Wexler, I. B., 67 Weygand, F., s-64 Wiener, A. S., 67 Wilkins, M. H. F., s-63 Williams, R. C, 406, 407 Wilson, E. B., 31, 98 Wilson, H. R., s-63 WoUman. E. L., 357, 359, 362, 363, 364, 369, 372. 379, 380, 381, 391, 399, s-75, s-77 Wolstenholme, G. E. W., s-77 456 Wood, H. G., s-49 Yates, R. A., s-49 Zelle, M., 354 Wright, S., 227, 169, s-39 Yeh, M., 269 Zichichi, M. L., 388, 389 Wyatt G R s-64 Young, J., 406, 407 Zimmerman, S. B., s-64 Yanofsky c! 286, 292, 395, Yura, T., s-38, s-49, s-75 Zinder, N. D., 304, 305, 357, 399 426 s-39, s-49, s-77 Zamecnik, P. C, 428, 438, s-75 374, 377, 381, s-76, s-77 SUBJECT INDEX Italicized page numbers refer to illustrations. abnormal abdomen in Drosophila, 72 abortion, 106, 243 absorbency, 302, 312, 314, 315, 345 achondroplastic (chondrodystrophic) dwarfism, 230, 242 acridine, 356, 362, 363, 368, 401 activation, 176, 430 Activator (Ac), 2\5, 360 adaptive value (see fitness, biological) adenine (A), 284, 291, 296, 297, 304, 306, 337, 371, 401, 402, 403, 441 adenosine, 430, 448 adenosine triphosphate (ATP), 320, 321, 441 adenylosuccinase, 284, 290-29/ adolescence, 234 age, 72, 106, 130, 147, 201, 245, 249, 336, 341 agglutination, 70, 340 air, 178,275 albinism, 32, 34, 49, 63, 68, 277, 414 alcapton (homogentisic acid), 275, 276 allele, 10, 185, 191-195, 287-291, 395 iso-, 65, 214, 239 multiple, 35-36, 62-65, 191, 198 allopatric, 255 America, 252 amino acid, 69, 231, 262, 276, 281, 285, 332, 395, 406, 431, 435, 441 amorph, 210, 371 amphiploidy (allopolyploidy), 143, 255, 258, 259 anaphase, 17, 18, 25, 26, 28, 29, 370 anemia, 36, 69, 70, 234 sickle cell, 70, 242, 269, 287, 289, 290 aneusomy, 214 Angstrom unit (A), 286 anlage (imaginal disc), 265, 272 annelids, 94, 95 anthranilic acid, 376 anthropology, 253 antibiotics, 244, 284 antibodies and antigens, 34, 62, 68, 340, 421 Antirrhinum (snapdragon), 65 antiserum, 35, 340, 375 apricot eye color (vv, apr), 62, 122, 191, 204, 21 1 arginine, 285, 287 Arizona, 253, 254 Arrowhead inversion, 253, 254 Artemia (water shrimp), 140, 317 arthritis, 275 457 Ascaris, 140, 197, 198, 213 ascospore and ascus, 121, 123, 283, 291 Asia, 252 Aspergillus, 193 assay, 384-385, 404 aster, 257 asynaptic gene in com, 198, 213 ataxia, 34 atmosphere, 441 atom, 177,247, 303 attached-X's, 121, 122, 191, 192 autopolyploidy, 140 autosome, 81, 101 auxotrophy, 333, 349-354, 350, 356, 440 azaguanine, 337 Bacillus, 341 backcross, 43, 44, 82, 11 1 bacteria, 214, 222, 312, 329-iJ5, 332, 369 conjugation in, 349-354 bacteriophage (phage, 0), 296, 304, 374-380, 382- 405, 429^30, 448 lambda or 434, 378-380, 388, 390, 391 PI or P22, 375, 376 pro-, 375, 378, 390, 391, 392 T series, 307, 311, 336-338, 353, 382-388, 393-i97, 448 XI 74 or SI 3, 311, 325, 347, 384, 406, 444 balanced lethals, 163, 164-165, 207 balancers (halteres), 193, 194 Bar (B) or Bar-like eye, 186, 187, 188-190, 206, 207 Basic technique, 204, 205, 206 base, organic, 295 bean plant, 1 benzene, 294, 295 biochemistry, 69, 173, 266, 269 biophysics, 269 biotin, 281, 349-354 birds, 12, 84 (see also chicken) bithorax (bx), 193, 194, 195 bivalent, 24, 25 black body color (b), 116 Blendor, 358, 362, 374, 383 blood, 69, 275 blood eye color (wm), 62 blood type or group, 34, 62, 234 ABO, 35, 36, 49, 75, 252, 253 MN, 35, 49, 68, 75 Rh (Rhesus), 36, 75 bobbed bristles (bb), 114, 208, 210 bone, 95, 247, 275 Bonellia, 95 Brachyury (Brachy) (T), 266-268 brain, 69 branched track method, 40, 42 bridge-breakage-fusion-bridge cycle, 151, 152 brown eye color (bw), 272-274 Brownian movement, 150 buff eye color (w''0, 62 cabbage, 259, 260 caff"eine, 337 calcium chloride, 384 458 California, 253, 254, 260 cancer, 213, 306 carbamates, 200 carbon, 247-248, 293, 294, 338, 441 carbon dioxide, 147, 410, 441, 442 carmine eye color (cm), 206, 207 carnation eye color (car), 189, 206, 207 cartilage, 265, 275 catalase, 337 catnip (Nepeta), 415 cattle, 257 cell and its division, 5, 16, 201, 245, 383 centrifugal forces, 181 centriole and centrosome, 369, 440 centromere, 16, 27, 120, 130, 152, 169, 181, 182, 197, 282, 283, 369, 370 cesium, 247-248,312 chain of chemical reactions, 273 chemical messenger, 95, 266, 267 chemorecovery, 200 chiasma, 24-25, 26, 30, 44, 116-126, 120, 128-135, 282, 283 chiasmata, 100, 131, 133, 134 chicken, 12, 53, 54, 84, 263, 264, 265, 306, 307, 404 Chlamydomonas, 96, 315 chloramphenicol, 336 chlorophyll, 65, 96, 412-415, 441 chromatid, 16, 18, 360 chromatophore, 96 chromocenter, 181 chromosome, 16, 19, 174, 181, 293, 410, 440 {see also gene) acentric, 150, 151 aneucentric, 154, 755 bacterial, 329, 330, 391 balance, 104, 158 blocks in, 181 breakage, 150-153, 151, 157, 173, 176-/79, 181, 197,214, 343, i44, 360,450 bridge, 151 buckles and loops, 160 changes, 137, 150-160, 180, 186, 257, 343, 344, 450 natural and induced, 20, 173-183, 249 coils or gyres, 141, 179, 180, 181 complexes, 171 cross-bands in, 141, 142 cytology, 86, 249 dicentric, 150, 757, 177, 214 disjunction, 157, 167 DNA content, 300, 341, 360 doublet in, 195 doubling of, 141 duplicate parts of, 756 ends, 150, 179, 180, 181, 197 eucentric, 154, 755 eutelomeric, 156 fragment, 150, 198 as genetic material, 20, 90-91 heteromorphic, 87, 140 homologous, 19, 102, 103, 125, 157 human, 705, 146, 147, 177, 249 hydration, 181 iso-, 757 loss, 97, 106, 159,209 maternal and paternal, 27 metaphase plates, 174 movement, 767, 181 and nondisjunction, 89, 90 number, 21, 98, 106, 146, 181, 182 polycentric, 197, 214 puffs in, 316 ring and rod, 152, 153, 174, 775, 359- J67 salivary gland, 20, 141, 142, 158, 159, 160, 173, 195, 198, 213, 294, 302,316 segregation, 90, 168 shape or size, 173-775, 179, 181, 182 shift and transposition, 158, 159, 173, 220 sister strand, 120 sphnt effect in, 181 structural linearity, 19, 197 volume, 179 chymotrypsin, 287 cilia, 95, 370 cinnabar eye color {en), 272-274 cinquefoil {Potentilla), 254-255 circle of chromosomes, 766, 167, 168 cis and trans positions, 189, 190, 193 cistron, 279, 281-291, 293, 304, 317, 347, 368, 370-371, 377, 395-i96-i97-398, 415, 411^16, 430^36, 445^48, 450 civilization and races, 256 climate and mutability, 214 clone, 96, 329-338, 332, 334, 349-354, 416 cloud, 442 clubfoot, 77 "cn+ substance, 273 coding of amino acids, 427^36 coincidence, coefficient of, 132 colchicine, 141, 259 cold and autopolyploidy, 141 colicine, 369 collochore, 182,213 color-blindness, 87, 114 comb types, 53, 54 comet, 441 competence, 215, 267-268, 341 complementation, i9i-395 concordance vs. discordance, 76, 77 congenital abnormalities, 146, 233 conjugation, 349-354, 356, 357, 416 constitutive enzyme, 423 continuous traits, 56-60, 104, 108, 262 Cooley's anemia (thalassemia major), 36, 289, 290 copy-choice, 344, 387, 388 coral eye color {w"^), 62 core, 382, 383 corn, Indian (maize, Zea mays), 19, 29, 126, 755, 760, 193, 214-222, 235-237, 302, 360, 369, 370, ^72^15 cotton, 193, 259 coupling, 114 crab, 371 Subjects 459 Creeper (Cp), 263, 264, 265 crops, 236 crossing over, 112, 116-126, 120, 130, 131, 167, 182, 186, 259, 343,^4^,450 and cis-trans positions, 189, 190 within cistron, 290-291 frequency, 114, 116, 128 crossovers, 112, 124, 128, 130, 133, 189 genetical and cytological, 125 crossveinless wiii^^s (cv), 117, 119, 128 cubitus interrupt us wing (ci), 64, 81 curled wing fly, 52 cut wings ict), 117, 119, 128, 129, 206, 207 cyclosis, 181 cystine, 69, 285, 349-354 cytochemistry, 300 cytogenetics, 125, 141, 159, 162-171, 199, 357 of sex, 90, 100 cytoplasm and cytosome, 16, 141, 427 cytoplasmic mixing, 417 cytosine (C), 296, 304, 306, 400-403 derivatives of, 296, 325, 326, 400 Datura (Jimson weed), 139, 140, 144, 145 deamination, 401 death, 233, 240, 241, 242-246 DDT, 244 deficiency, \51-153, 159, 173, 179, 185, 395 deletion, 175,214,401 Delphinium (larkspur), 259, 260 density gradient ultracentrifugation, 312, 314, 388 deoxyad'enosine, 298, 299, 301, 304 deoxycytidine, 298, 299, 301, 304 deoxyguanosine, 298, 299, 304 2'-deoxy-D-ribose (deoxyribose), 298, 304, 427 deoxyribonuclease (DNAase), 302, 320, 323, 327, 340, 342-346, 352, 361, 375, 428 deoxyribonucleic acid (DNA), 324, 340-348, 356, 368, 369, 375, 379, 382, 383, 384, 387, 388, 411, 427, 439^50 (see also gene, recon, cistron, mutant) bases in, 306, 309, 310, 315, 325, 327, 345 biological synthesis, s-50-s-64 chain growth, 323 chemical composition, 294-303 coiling, 309 conserved vs. nonconserved, 316-317, 345, 392 denatured or renatured, 315, 324, 345 density, 345 enzymatic degradation, 323, 324 evolution of, 439-440 H-bonding in, 309 homologous vs. heterologous, 346, 349, 374 hybrid, 313, 346-347, 429 molecular weight, 324, 342, 345, 346, 360, 392 nearest nucleotide neighbor in, 327 nucleoside and nucleotide ends, 322, 323 polymerase, 325, 328, 430, 447 primer, 322, 325, 445 replication, 312-313-314-315, 320-328 and RNA interrelationship, 427-436, 431 single-stranded, 311 strand recombination, 345-347, 450 structure, 294-302, 308, 309, 311 synthesis, 316, 322, 327,352 unnatural bases in, 325, 326 in vitro, 320-328 /// vivo, 306-317 Watson-Crick model, 306-312, 308, 311, 318 deoxyribonucleoprotein, 294 deoxyribonucleotide (deoxyribotide), 299, 300, 301. 302, 304, 320 deoxyriboside, 298, 299, 300, 304, 321, 322 deoxyuridine, 5-bromo, 371 detergent, 404 detrimental equivalents, 241 development, 74, 141, 201, 245, 257, 266, 272, 316 diabetes, 243 diakinesis, 25, 26, 28 differentiation, 95, 100, 105, 265, 268, 316 diffuse (growth) stage, 26 Diplococcus (see Pneumococcus) diploid. 10, 353, 379 diplonema, 25, 26, 28, 123 Diptera, 159,213 Dissociation (Ds), 214, 215, 216, 360, 371, 449 dogs, 255 dominance, 12, 13, 47, 49, 53, 67, 111, 138 complete or nearly complete, 210 and mutation, 185 phenotypic effect of, 58, 59, 72 in populations, 225, 252 double cross, 236, 237 doubling dose, 247 drift, random genetic, 226, 242, 245, 253 Drosophila, 52, 53, 69, 70, 72, 116, 121, 122, 140, 173, 174, 193, 214, 222, 294, 360, 369, 371, 409^10 bibliographies, 55 eye, 62, 68, 83, 114, 186, 271-272-27i-274 larva and pupa, 69, 141, 144, 272 melano^aster, 81, 128, J42, J 43, 151, 176, 182, 204-209 persimilis, 257 pseudoobscura, 234-235, 239, 240, 253, 254 and sex, 83, 96, 100, 101, 106 wing, 6< 116, 117, 193 drug, 141, 259, 332-336,400 duplicated parts and penetrance, 72 duplication, 156, 159, 173, 179, 185, 186, 195, 206, 207, 370, 387, 388 dusky wings (dy), 206, 207 dwarfism, 230, 242, 265, 266, 267 dyad, 26, 88, 120, 124 ear, 275 Earth, 224, 439, 441, 442 earthquakes, 256 earthworm, 94 ebony body color (e), 81 Echinus (sea urchin), 140, 306, 307 echinus, rough eyes (ec), 206, 207 eclipse phase, 284, 404 ecology, 173, 254, 256 egg, 5, 91, 95, 97, 124, 249, 370, 404 460 electricity, 108, 441 electron, 142, 176, Ml, 209 electron microscope, 330, 345, 357 electrophoresis, 340 embryo, 263, 264, 265-268, 289 encephalitis, 304, 403 endolysin, 384 energy, 179, 320 England, 87 environment, 1, 56, 72, 79, 95, 252 and mutants, 200, 241, 244 enzyme, 68, 70, 274, 277, 281-286, 293, 302, 320, 328, 404, 421^25 episome, 356-372, 374-380, 382, 409, 418, 421, 449 epistasis, 53, 72, 74, 101, 252 epoxide, 200 equivalents, lethal or detrimental, 241 Escherichia coli, 286, 307, 321, 329, 330, 341 , 346, 349- 369, 361, 315-385, 393, 421-425, 428, 432, 442, 448 ethology, 256 euchromatin, 181, 186, 194, 198, 316, 370-371 Europe, 252 evening primrose {see Oenothera) evolution, 143, 147, 158, 195, 208, 213-214, 224-226, 244, 245, 255 biochemical, 439-442 biological, 94, 226, 450 in various species, 170, 173, 176 exogenote, 377 expressivity, 70-73, 241 eye, 186,206,272-274 color in Drosophila, 53, 62, 68, 69, 83, 117, 186, 189, 224, 271,272, 27i, 274 F„ F.,, 9 F episome, 356-369, 380, 381, 382 fallout, 247-248 family method, 32, 275 feather, 84 feed-back system, 418, 422-425 ferns, 222 fertility and fecundity, 9, 90, 208 fertilization, 1, 5, 8, 97, 232, 256, 302 random, 42, 74, 90 cross-, in populations, 224-226 Feulgen-Rossenbeck technique, 19, 20, 108, 300, 316 fibroblast, 777, 317 filtration, 284, 352, 375 "fingerprints," 287, 288, 382 fingers, 71, 265 fish, 293, 296, 306, 307 fission, 415 fitness, biological (adaptive value, reproductive poten- tial), 208, 211, 224-226, 229, 245, 253-254, 329 flagellum, 96, 330, 370 flavin mononucleotide, 320, 321 flight, insect, 244 flowers, 8,9, 39, 114, 139 forked bristles if) 97, 118, 119, 722, 128, 189,206,207 fowl (see chicken) France, 240 frogs, 140, 271 fruiting bodies, 121, 72i fungi, 222, 254, 302 fungus gnat (Sciara), 316, 450 galactose locus (Gal), 358, 378, 390, 391 /3-galactosidase, 369, 421-424, 425 gametes, 5, 88, 94, 152, 166, 246, 302 gametogenesis, 106, 157, 166, 214, 248, 317 gametophyte, 95, 163 garnet eye color (_^), 206, 207 gene (see also mutant, DNA, RNA), 10, 14, 63, 91, 100, 101, 120, 137, 164, 185, 262 action, 70, 152, 185, 186, 193, 215, 262, 271-292, 395, 398, 409-440 arrangement, 128-135, 185, 189 balance, 104, 144 chemical nature of, 293-304, 341 and chemical reactions, s-27-s-49 and chromosomes, 44—46, 90, 198 code, 402 defined, 199, 278-279, 447 dominant and recessive, 12, 138 essential vs. nonessential, 392 extrachromosomal or extranuclear, 363, 408-418, 450 for function vs. structure, 425, 440 functional, 278-279 interaction, 49-54, 56-60, 104, 236 linear order of, 182, 197, 354 linked, 112, 113, 116, 128, 197 material basis of, 19, 30, 44, 90 modifier, 210 mutability controlled, 213-222 mutable, 200 and mutation, 197-202, 216 mutator, 214 nature of, 278-279, 317, 444-451, s-ll-s-14 normal. 111, 210 number, 116, 158, 159, 195 operational, 137, 199 origin of, 439-442 pairs, 10, 40 phenotypic effect, 65-70, 210-211, 224, 262 polarity, 197 pool, 213, 224-226, 229, 242-243, 253-255 precursor and mutation, 201-202 recombinational, 199 regulator, 423-^25 segregation, 8-14 self-replication of, 20, 137, 213, 320-328, 363, 368, 377, 405, 415, 421, 439^140, 444-445, 447 self-sterility, 63 size, 195, 198,279 and speciation, 257 stable, 199 suppressor, 214-216 as template, 446-447 transformer, 101, 105 transmission, 73 untainted, 10, 20 genetic death, 242-246 Subjects 461 genetic drift, random, 226, 242, 245, 253 genetic equilibrium, 224-226, 246 genetic factor, 1 genetic fine structure, 394-i97, 448 genetic imbalance, 144 genetic information, 347, 383-384, 405, 427-436, 450 genetic material, 1-6,9, 10, 14, 116, 137, 199,278,293, 363, 367, 377, 384, 403, 406, 421, 439-442, 444-451 genetic polymorphism in races, 252 genetic stability and variability, 137, 213, 239, 240 genetic units, 10 genetics, applied, 236, 450 bacterial, 329-380 biochemical, 69-70, 208, 271-292 comparative, 173 developmental, 262-268 pheno-, 262 physiological, 269 a view of, s-65-s-77 genitalia, 105, 257 genome, 30, 103, 137-143, 345, 448 genotype, 1, 72, 74, 130, 147, 208, 258, 294, 449 geography, 253-256, 254 germ line, 106, 118, 146, 152, 197, 205, 293 Globe seed capsule, 144, 145 glutamic acid, 285, 287 glutamine, 69 glycine, 285, 1%1 goatsbeard, 259 gonad, 94, 105, 246 grasshopper, 96, 179 gravity, 181 growth, 26, 265, 267, 284, 337-338, 349-354 of virus, 384, 385 guanine (G), 297, 298, 304, 306, 401, 432 guinea pigs, 72 gynandromorph (gynander), 96, 97 half-life, 248 halteres (balancers), 193, 194 haploid (monoploid), 10, 105, 139, 141, 144, 353, 379 Hardy-Weinberg equilibrium principle, 225n head, 34, 252, 382, 383 heart, 69, 265 helium, 441 //^//.v (snail), 94, 95, 317 helix, 286, 308 hemizygous, 84 hemoglobin, 70, 238, 271, 286-255-259-290, 428, 446, 448 hemophilia, 87, 114 Hemophilis, 341 heredity, 1 5 hermaphrodite, 94 heterochromatin, 181, 186, 194, 198, 204, 214, 316, 370-371,410 heterogametic females, 92 heterogenote, 377, 379 heterosis (hybrid vigor), 233-237 heterozygote (hybrid), 11, 42, 43, 130, 134, 135, 163, 207, 232-236, 256, 354, 387 Hexaptera, 137, 138 histidine, 285, 376, 421, 422 histochemistry, 300, 306, 307 histone, 293-294 homogenote, 381 homozygote, 11, 232-233 hormones, 98, 105, 266, 267, 268, 271 horse, 257, 286 host range, 386, 393 "hot spots" in mutation, 400 human genetics, 19, 32-37, 71-72, 74-79, 98, 105-108, 146-147, 177, 224-226, 229-234, 240-249, 252- 253, 274-278, 286-290, 307, 315 hybrid vigor (heterosis), 233-237 hybrids, 234-237, 258-260 hydrogen, 309, 441 hydroxylamine, 401 hyperploid vs. hypoploid, 152 hypomorph, 210 hypostasis, 53, 72, 101 hypoxanthine and its derivatives, 326, 401 Iceland, 252 imaginal disc (anlage), 265, 272 imidazole ring, 295, 297 immunity, 375, 377, 379, 380, 390, 391 inborn error of metabolism, 275, 450 inbreeding, 32, 232-237, 233, 241 India, 252 Indians, American, 252 indol, 284, 376 induction, 267-268, 378, 390, 423-425 influenza, 304, 403-404 insects, 296 {see specific types) insulin, 243 integration, 343-i44, 349, 353, 357, 360, 363, 366, 382,391,449 interference, 131, 132, 134 International Commission on Radiological Protec- tion, 248 interphase, 17, 18, 25, 26, 29, 302 intersex, 102, 103, 105, 140 interspecific hybridization, 258-260 intestine, small, 245 introgression, 260 inversion, 153-158, 155, 173, 179, 204, 206, 207, 214, 235, 253, 254 iodine-131, 251 iojap (ij), 415 ions, 176-178, 345-346, 401-402 Ireland, 252 iron, 441 isogenic strains, 69 isotope, 303, 312, 388 Italy, 37 Japan, 32, 233 Jimson weed (Datura), 139, 140, 144, 145 juvenile amaurotic idiocy, 230, 242 kappa, 410, 411, 415^17, 449 keto form, 296 kidney, 69 kinetosome, 370, 440 462 Klinefelter's type male, 98, 146 knuckle, 275 kynurenine and its derivatives, 273, 274 lactate, 336 lactose locus {Lac), 358, 363, 366-369, 421^25, 424 larkspur {Delphinium), 259, 260 law of parsimony (Occam's rule), 10, 62, 199 lawn, bacterial, 384, 386 leptonema, 24, 28 lethal equivalents, 241 leucine, 285, 349-354 life, expectancy or span, 201, 249 light, ultraviolet, 176, 178, 200, 201, 302, 312, 314, 336, 337, 349, 357, 441 visible, 176, 200, 301 lily meiosis, 24, 25, 26 limit dilution, 404 linear gene order, 128, 354 linkage, 111-//7, 169, i67, 376 sex-, 81-92 lipid, 340, 369 lithium, 308 liver, 141, 213, 275, 277,450 load of mutants, 239-249 locus (loci), 120, 121, 193, 222, 343, 366 locust, 306, 307 lysine, 285, 287 lysis, 352, 353, 363, 375, 378, 384 lysogeny, 375, 377, 378, 390 lysozyme, 382 macromolecule, 307, 315 magnesium, 320, 321, 322, 336, 428 maize {Zea; see corn) malaria, 234 male, Lfr, Hfr, or Vhf, 357, 359, 360, 361 Malpighian tubules, 211 mammal, 296, 345, 404 manifold effects {see pleiotropism) mannitol. 343 maps, 124, 135, 182, 183, 189, 361, 390, 391, 395-397, s-12 markers, selective vs. unselected, 352-353 marriages, cousin, 32, 233, 241 nonrandom vs. random, 231-236, 233, 256 Mars, 224,441,442 mathematics, 227 mating, assertive, 231-232 interspecific, 342 in microorganisms, 387, 416, 418 reciprocal, 9, 82, //7 maximal permissible concentration, 248 Maxy technique, 206, 207 mean, statistical, 58 measles, 78 medicine, 241, 243, 244, 247, 404, 450 medium, basic minimal, 281, 332 meiosis, 23-30, 24, 25, 95, 121, 122, 124, 213, 416^11, 440 in various organisms, 29, 88, 96, 154, 167, 282, 283 melanin, 50, 277 "memory," 367, 423 mentality, 78, 79, 231,277 mercaptoethanol, 432 meromixis and merozygote, 359, 368, 379 metabolism, 266, 271, 215-278, 303, 332, 440 and mutability, 181, 214, 337-338 of phenylalanine, 231 metafemale and metamale, 103 metaphase, 17, 18, 25, 26, 28, 29, 86, 87 methane, 441 methionine, 285, 332, 353, 375-376 methods for detecting point mutants, 204-208 methyl green, 301 metronome, 78 Mexico, 239 Micrococcus, 346 microcytemia, 36 micromanipulation, 271, 331, 357 micron, 178 microorganisms, 193, 244 microspectrophotometry, 301 migration, 226, 242, 245, 253 miniature wings {m), 1 14, 1 17, 1 19, 128, 129 mitochondria, 428 mitosis, 16, / 7, 18-1\, 24, 147, 213, 302, 440, 449 Modulator {Mp), 218-222, 449 moisture and penetrance, 72 mold (see template) mold, bread {see Neurospora) mollusc, 370 monad, 26, 120 Mongolism, 146, 147 monoecious, 94 monoploid {see haploid) monosomic (haplosomic), 143, 145, 147 Moon, 442 morphology, 68, 173, 256-257, 262 mosaics, 96, 105, 186, 268, 329, 354, 371, 412^\5 mosquito, 257 mosses, 95 moths, 87, 98, 140 mountains, 256 mouse, 65-68, 162, 193, 201, 248, 257, 263, 264, 266, 267, 268 mucoid coat, 404 muscle, 95, 269 mustard gas, 200 mutability, 181, 200, 245, 248, 249, 336, 415 genetic control of, 213-222 mutagens, 200, 214, 245-249, 281, 302, 332, 333, 336, 340, 349 ami-, 337-338 types of, 338, 400-402 mutant {see also gene, DNA, RNA), 3, 5, 111, 137, 206, 241, 245,248-249 clearing, 390, 391, 392 detection of, 138, 199, 204-206, 205, 281-282 detrimental, 206, 208, 210, 229-231, 241 dosage, 208 intra-nucleotide, 400-402 lethal, 62, 65-66, 69, 152, 156, 163, 208, 229, 234, 239, 240, 241 Subjects 463 balanced, 163, 164-165,207 recessive, 65, 68, 152, 163, 204, 205, 206, 207, 209, 211, 229, 230, 231, 263, 264, 265-268 phenotypic effect of, 66, 185, 206, 208, 210, 211, 239, 240, 244, 246 point, 204-208, 448 pre- vs. postadaptive, 333-336 rare, in population, 229-231, 242-243 semilethal, 239, 240 twin, 218 "visible," 204, 206, 207, 208 mutation (see also mutant), 5, 13, 71, 75, 87, 94, 137, 138, 150, 158, 188, 304, 340-341, 349, 400- 402-403, 406, 415, 440, 447, 449^50, s-15- s-26 in bacteria, 329-338 in bacteriophage, 392-394 and gene precursor, 201-202 germinal, 246-249 nature of, 144, 199-200, 201 point and gene, 197-202, 204-208, 216 and populations, 225 prevention or recovery from, 200, 213 rate, 200, 209, 214, 229-230, 247, 336, 386 and selection, 229-231 somatic, 245-246 and speciation, 256 at specific locus, 204, 340 spontaneous, 200, 333, 350-354, 394 and time, 201, 248 units of, 213 mutational loads, 239-249 mutational site, 199, 400-403, 450 mutational spectrum, 200-201, 400-401 National Research Council, 248 nature vs. nurture, 74 Neisseria, 341 neomorph, 210 nerve cell or tissue, 245, 266, 269, 421 Neurospora, 121, 123, 124, 132, 154, 281-286, 231, 332, 429 neutrons, 176, 178-180 New Mexico, 253, 254 Nicotiana (tobacco), 63 nicotinamide mononucleotide, 320, 321 nitrogen and its compounds, 179, 281, 294, 296, 312- 2>\5,314, 336, 346, 388,441 nitrous acid, 401, 406 nondisjunction, 59-91, 106, 143, 146, 147, 188, 207, 214, 247 nose, 275 notochord, 266-267 novel phenotype, 137, 138, 403, 449 no-wing fly, 52 nuclear war, 249 nucleoprotein, 303, 450 nucleoside, 303, 304 nucleotide, 303, 304, 307, 368, 371, 380, 394-395, 400- 403, 426-436, 450 di-, 320, 321, 327 -sharing, 370-372, 376, 425, 435, 447 nucleus and its parts, 16, 20, 121, 152, 181, 271, 300, 307, 329, 359, 370, 409, 429 macro- (mega-) vs. micro-, 415, 416, 418 nurse cell, 317 nutrition, 130, 265, 266, 332, 337-338, 349-354 ocelliless {oc), 206, 207 C>e/7o//;£'ra (evening primrose), 139, 762-171, 176, 194- 195 ommatidia (eye facets), 186, 188 one polypeptide-one cistron view, 286 onion, 17, 18 operations (techniques), vi, 7, 13, 137, 278, 293, 403, 444 operon and operator gene, 421-425, 440, 446 Ophryotrocha, 95 organelle, 274, 369, 370, 371, 440 outstretched win^, small eye (odsy), 206, 207 ovary and ovules, 63, 105, 163, 414 oxygen, 178, 179, 200, 201, 214, 287, 337, 441 Pi, P>, 9 pachynema, 24, 25, 28, 158, 160 panmixis, 238 paracentric vs. pericentric, 152 paralysis, 69 Paramecium, 410, 411, 415-418, 449 parasite, 95, 107,411 parsimony, law of, 10, 62, 199 parthenogenesis, 140, 141 pea, garden, 8, 12, 39, 81, 1 1 1, 162, 163 sweet, 114 pedigree, 32, 34, 71, 87, 231, 233, 275 of causes, 70, 244, 271, 278, 287, 421 penetrance, 70-73, 241 penicillin, 341, 343 pentose sugar, 298, 304 peptides, 69, 287, 288 pericarp variegation, 216-222 persistence, 242 personality, 78 phagocytosis, 317, 342 phenogenetics, 262 phenol, 384, 405 phenotype, 2, 57, 70, 111, 116, 138, 185, 210-211, 2ii, 278, 394, 449 phenotypic expression, 49-54, 63, 137 phenylalanine, 231, 276, 211, 285, 349-354, 433-436 phenylketonuria, 231, 233, 277 phenylpyruvic acid, 277 phenyl thiocarbamide (PTC), 228 phokomelia, 265 phosphate, 299, 304, 320, 345, 403, 430 phosphorus, 281, 341, 383, 392, 429 photorecovery, 200 photosynthesis, 65, 413 physics, 189 physiology, 173, 198, 254, 256, 257, 266 Pikes Peak inversion, 253, 254 pigment, 214-222, 271-274, 277 pistil, 63 pituitary, 266, 267, 271 placenta, 266 464 INDEX planet, 441^42 plants, flowering, 222, 259 plaque, 386, 387 plastid, 413^15, 428, 440 plate, hexagonal, 382, 383 pleiotropism (manifold eff"ects), 68-70, 163, 208, 210, 234, 244, 265, 271, 275, 277, 279, 287, 421, 446 ploidy, 139, 181,245, 246 eu- vs. aneu-, 143 Pneumococcus (Diplococcus), 340-342, 345, 346 point mutation, 197-202 polar bodies, 88 polarity, 197, 300, 303 poliomyelitis, 77, 304, 403 pollen, 63, 114, 121, 152 polyamine, 382 Polydactyly, 71, 74 polydeoxyribonucleotide, 300, 302, 303, 308, 311 polygenes, 56, 108 polymer, 300, 303, 371, 433^140 polymorphism, balanced, 242 polynucleotide phosphorylase, 433^36 polypeptide, 286-290, 289, 293, 309, 382, 395 polyploidy, 139-141, 147, 213, 302, 306, 449, 450 polyribonucleotide (polyribotide), 303, 433^36 polysaccharide, 340, 342 polysomy, 143, 145, 249 polyspermy, 97, 317 polyteny, 141, 213, 303, 316, 317, 449 population, 32, 210, 254, 275, 330 genetics, 224-226, 229-236, 239-249 natural, 170, 173, 176,253,254 position eff"ect, 185-195, 215, 235, 371, 449 and mutation, 185, 222, 245 postbitlwrax {pbx), 193, 194 potassium, 345 Potentilla (cinquefoil), 254-255 precursors, 273, 278, 282 probability, 11, 130, 132 proboscis, 95 proline, 69, 285, 353, 369 prophase, 16, 77, 18, 24, 25, 26, 28, 29 protamine, 293-294 protein, 262, 277, 284, 293, 340, 382, 406, 421-425, 424 synthesis, 427-436, 447 Proteus, 380 proton, 176 protoplasm, 150, 181, 274, 281, 293 protoplast, 384 prototrophy, 332, 349-354, 351, 356, 440 prune eye color (pn), 206, 207 Pseudomoims, 366, 380 pseudopod, 404 pure line. 2, 3, 8, 84, 138, 162, 171, 252 purine and its derivatives, 281, 295-298, 297, 304, 337, 401,402,441 putrescine, 382 pyrimidine, 281, 282, 294, 295, 296, 304, 337, 401, 402 quadrivalent, 259 quadruplets, identical, 74 quantitative traits, 56-60, 104, 108, 262 quantum, 201 r region in T4, 393-i97, 448 r (roentgen) vs. rad unit, 178 rabbit, 4, 35, 62, 63, 68, 141 race, 170,226,252-258 radiation and mutation, 141, 147, 176-180, 200, 201, 245-249, 336-337, S-15-S-26 radicals, 441 radio, 442 radioactivity, 147, 247-248, 303, 321-323, 383, 392, 428-432 radish, 259, 260 raspberry eye color (,ras), 206, 207 ratios, phenotypic and genotypic, 49, 51 ravelase, 347 reaction, norm or range of, 4 receptor, 357, 404 recessive, 1 2, 43 {see also dominance) recombination, genetic, 13, 79, 94, 112, 134, 137, 245, 290-291, 329, 409-410, 449^50 in bacteria, 340-347, 349-354, 356-380 in viruses, 382-405 recon, 279, 290-291, 303, 304, 345-346, 395, 445, 450 reconstitution, 13, 405 red blood cells (corpuscles), 34, 70, 287 regression, 58 replica, partial, 387, 388 -plating, 349-354, 350, 351 replication, 9, 181, 344, 444 {see also gene) replicon, 451 repressor, 369, 391, 423-425 reproduction, asexual or sexual, 5, 74, 94, 329, 330 reproductive barriers, 256, 257 reproductive disadvantage of mutants, 154-157 reproductive generation, 247 reproductive isolation, 255-260 reproductive potential {see fitness, biological) repulsion, 114 research, 442, 45 1 respiration, 247 retinoblastoma, 229, 242 Rhyncosciara, 316 ribonuclease (RNAase), 303, 340, 375, 405, 428 ribonucleic acid (RNA), 303-304, 309, 382, 403-406, 427^36, 431, 439, 441, 447, 448 coding, 431^36 polymerase, 430, 447 various types of, 429-436 ribonucleoprotein, 303, 304, 403, 405 ribonucleoside (riboside), 303, 304, 430-432 ribonucleotide (ribotide), 303, 304, 320, 406 D-ribose (ribose), 298, 303, 304, All ribosome, 428-436 rickettsia, 412 rotational substitution, 402, 450 roundworm, 140, 197 ruby eye color {rb), 206, 207 rye, blue wild, 257 Saint Louis, Mo., 247 salamander, 140, 141 Salmonella, 341, 346, 366, 375-379, 377, 421-422 Subjects 465 schizophrenia, 79 Sciara (fungus gnat), 316, 450 scute bristles (sc), 204, 206, 207 sea urchin (Echinus), 140, 306, 307 sedimentation, 324, 375, 428 seed, 39, 111, 139, 144, 145, 216, 260 seedling, 414 segregant, 353 segregation, 10, 168, 282-283, 360, 449, s-5-s-lO gene, 8-14 independent, 39^7, 74, 82, 111, s-5-s-lO in man, 32-37 selection, 2, 59, 106, 195, 210, 226, 231, 245, 248-249, 256, 440 coefficient, 229-234 and mutants, 208, 213, 229-231, 242-243 serine, 284, 285 serology, 173, 340 sex, 5, 94, 95, 103, 104, 114, 201, 213, 256 in bacteria, 349-354, 356-368 chromosomes, 81, 90, 104, 179, 207 in man, abnormal, 98, 106, 146 determination, 90, 94-98, 100-109 -duction, 381 genetic basis for, 81, 100, 101 -linked, 83, 112 ratio, 81, 100, 101, 105-108 super-, 103 sheath, 382, 383 Siamese cat, 4 siblings, 74, 232, 289 sigma, 410 sit{^ed bristles (sn), 206, 207 sister strands, 120, 131, 150, 151 site, 199, 342 size, 1, 95, 252 skin, 69, 252 smallpox, 403 snail {Helix), 94, 95, 317 snake venom diesterase, 324 snapdragon {Antirrhinum), 65 Solenobia, 140, 141 somatic line, 23, 118, 141, 152, 197, 293 sonication, 315, 322, 343 Spain, 252 species, 5, 173, 200, 226, 255-260, 342 DNA in different, 306, 307 spectroscopy, 340 sperm, 5, 69, 95, 96, 106, 707, 108, 121, 124, 178, 180, 201,205,293,336,370,401 spermadine, 382 spermatheca, 69 spermatids, 106, 201, 336 spindle, 17, 141, 369 spirochaete, 107, 410 spleen, 69 splenic phosphodiesterase, 323, 327 split bristles {spD, 128, 189 spore, 121, 123, 124, 28\-283, 442 spotted fever. Rocky Mountain, 412 Standard gene arrangement, 253, 254 Staphylococcus, 380 star (sun), 441 starchy, 221 starvation, 141, 265 statistical methods, 58, 60, 249 sterility, 102, 239, 240, 256 stigma, 63 stillbirths, 233 stratosphere, 248 streaking method, 332 Streptococcus, 346 streptomycin, 333-336, 334, 335, 342, 343, 346, 347, 356, 357 strontium-90, 247-248 style, plant, 63 succinic acid, 284 sugar, 281, 298, 304,403 sulfanilamide, 400 sulfur, 281, 336, 383 superfemale vs. supermale, 102, 103 superposition, 50 suppression, 371, 448 surface-volume relationships, 141 survival of human species, 249 Sweden, 32, 108 symbiont, 412 symbols, 32, ii, 62, 111, 206 sympatric, 255 synapsis, 24, 100, 157, 181, 757, 198, 213, 258, 259, 342, 343 eu- vs. aneu-, 757 somatic, 141, 7-^2, 194 tail and tail fibers, 382, 383 tarweeds, 257 tasters, 228 tautomeric shift, 402, 403 taxonomy, 226 telescope, 441 telomere, 150, 181, 197,440 telophase, 77, 18, 25, 26, 29 temperate (nonlysogenic), 375 temperature, 63, 68, 130, 176, 200, 442 template, 312, 313, 320, 344, 427^35, ^A6-4A1 tempo preference, 78 tendon, 275 tendrils, 1 1 1 territory, 253-255 test cross, 43, 44, 84 tetrad, 24, 25, 88, 720, 160 tetraploid, 104, 139, 140, 145 Texas, 253, 254 Thailand, 238 thalassemia, 36, 289, 290 theophylline, 337 thiamin, 281, 349-354 thiazole, 281,282 threonine, 285, 332, 349-354, 375-376 thymidine, 298, 299, 304 thymine (T), 296, 304-306, 338, 371, 400-40i, 445 thymus, calf, 324, 346 Tibet, 257 466 INDEX tissue, 176, 178, 265, 267-268 culture, 141,265,267, 317 tobacco (Nicotiana), 63 tobacco mosaic virus (TMV), 304, 382, 404-406, 448 toes, 71, 265 traits, continuous (quantitative), 56-60, 104, 108, 262 transduction, genetic, 374-380, 377, 421 transformation, genetic, 340-347, 352, 379-380, 444- 445, 449^50 transformer (tra), 101, 105 transition vs. transversion, 401, 402 translocation, 156-757, 760, 769, 173, 775-777, 180, 209 transmission genetics, 8, 56, 66, 71, 73, 84, 112, 162, 340-341 transplantation, 265, 111-21A, 442 transposition, 158, 173, 218, 220 triplet code, 434-435, 448 triplets, identical, 74 triploidy, 702, 139, 140, 144, 147 trivalent homologs, 102, 103, 259 trypsin, 287, 288, 328, 382, 406 tryptophan, 284, 285, 336, 376 synthetase, 284, 286, 291 tuberculosis, 77, 78, 306, 307 Turner's type female, 98, 146 turnip yellow mosaic virus, 383, 404 turnover, atomic, 303 twin mutant spots, 275 twins, human, 32, 33, 74-79, 76 typhoid, 375 tyrosine, 276, 277, 285 ultracentrifugation, 312, 314, 340, 346 union, cross- (nonrestitutional, exchange), 150, 180, 181, 185,371 unit. Angstrom (A), 286 chemical, 403 map, 124, 353-354 mutational, 197,213,403 sedimentation, 428 United Nations' report, 247 U.S.S.R., 108, 248 United States, 239, 247, 248, 253, 254 univalent, 26, 120 uracil, 296, 303, 304, 325, 326, 401, 433^36, 445 5-bromo or 5-fluoro, 325, 326, 400-401, 448, 450 urine, 274, 275, 277 v+ substance, 273 vaccinia, 307, 403 valine, 285, 287 variance, 58 variation, 2, 7, 73, 78, 235 variegation, 186, 214, 275, 276, 277, 275, 371 vegetative phage, 384 Venus, 442 vermilion eye color (v), 193, 195, 206, 207, 111-114 vertebrae, 267 vestigial win^s i^vg), 116, 244 viabihty, 9, 66, 128, 129, 239, 240 Vibrio, 380 virulence, 384, 390 virus, 304, 332, 341, 352, 353, 356, 357, 381, 403-404, 410,412^13,441,447 water, 214, 256, 281, 307, 308, 441, 442 water shrimp (Artemia), 140, 317 Watson-Crick model of DNA, 308 (see also DNA) waxy, 221 wheat, 143, 296 white eye (w), 62, 65, 69, 83, 114, 117, 119, 122, 128, 186,206,207 wild-type, 65 X chromosome, 81, 90, 91, 97, 100, 146, 204, 205, 206, 207 Xanthomonas, 341 X ray, 176-180, 779, 200, 206, 349 {see also radiation, mutation) X ray diffraction pattern, 307, 308, 340 Y chromosome, 81, 90, 91, 100, 106, 1 14, 146, 175, 182, 186,206, 371 yak, 257 yeast, 307, 429 yellow body color {y), 1 17, 1 19, 128, 188, 189, 206, 207 Zea {see corn) zygonema, 24 zygote, 5, 354 Irwin H. Herskowitz GENETICS SUPPLEMENTS SUPPLEMENT Part of a Letter (1867) from Gregor Mendel to C. Nageli Translated from German by Leonie Kellen Piter nick and George Piternick. Reprinted from The Birth of Genetics, Supplement to Genetics, Vol. jj. No. 5, Part 2 {1950), by permission of Genetics, Inc. Gregor Mendel (i 822-1 884) SUPPLEMENT II Nobel Prize Lecture (1934) of Thomas Hunt Morgan Reprinted by permission of The Nobel Foundation for Les Prix Nobel. The complete lecture is published in The Scientific Monthly /or July igj^. Only the first portion is reprinted here. Thomas Hunt Morgan (1866- 1945) By permission of The American Genetic Association, The Journal of Heredity, frontispiece, Vol. 24, No. 416, 1933- SUPPLEMENT III Nobel Prize Lecture (1946) of Hermann Joseph Muller Reprinted by permission of The Nobel Foundation for Les Prix Nobel. Published in The Journal of Heredity, 38: 259-270, 1947. Hermann Joseph Muller (li SUPPLEMENT IV Nobel Prize Lecture (1958) of George Wells Beadle Reprinted by permission of The Nobel Foundation for Les Prix Nobel. Published in Science, izg-.i^is-iJiQ^ 1959- George Wells Beadle (1903- SUPPLEMENT V Nobel Prize Lecture (1958) of Edward Lawrie Tatum Reprinted by permission of The Nobel Foundation for Les Prix Nobel. Published in Science, 129:1715-1719, 1959- Edward Lawrie Tatum (1909- SUPPLEMENT VI Nobel Prize Lecture (1959) of Arthur Kornberg Reprinted by permission of The Nobel Foundation for Les Prix Nobel. Published in Science, 131 :i503-i5o8, i960. Arthur Kornberg (191 8- ) SUPPLEMENT VII Nobel Prize Lecture (1959) of Joshua Lederberg Reprinted by permission of The Nobel Foundation for Les Prix Nobel. Published in Stanford Med. Bull., ly. -120-132, 1959; and in Science, 131:269-276, i960. Joshua Lederberg (1925- SUPPLEMENT I PART OF A LETTER (1867) from GREGOR MENDEL to C. NAGELI Highly esteemed Sir: My most cordial thanks for the printed matter you have so kindly sent me! The papers "die Bastardbildung im Pflanzenreiche," "liber die abgeleiteten Pflanzenbastarde," "die Theorie der Bastardbildung," "die Zwischenformen zwischen den Pflanzenarten," "die systematische Behandlung der Hieracien riicksichtlich der Mittelformen und des Umfangs der Species," especially cap- ture my attention. This thorough revision of the theory of hybrids according to contemporary science was most welcome. Thank you again! With respect to the essay which your honor had the kindness to accept, I think I should add the following information: the experiments which are dis- cussed were conducted from 1856 to 1863. I knew that the results I obtained were not easily compatible with our contemporary scientific knowledge, and that under the circumstances publication of one such isolated experiment was doubly dangerous; dangerous for the experimenter and for the cause he repre- sented. Thus I made every effort to verify, with other plants, the results ob- tained with Pisum. A number of hybridizations undertaken in 1863 and 1864 convinced me of the difficulty of finding plants suitable for an extended series of experiments, and that under unfavorable circumstances years might elapse without my obtaining the desired information. I attempted to inspire some control experiments, and for that reason discussed the Pisum experiments at the meeting of the local society of naturalists. I encountered, as was to be expected, divided opinion; however, as far as I know, no one undertook to s-5 GREGOR MENDEL repeat the experiments. When, last year, I was asked to publish my lecture in the proceedings of the society, I agreed to do so, after having re-examined my records for the various years of experimentation, and not having been able to find a source of error. The paper which was submitted to you is the un- changed reprint of the draft of the lecture mentioned; thus the brevity of the exposition, as is essential for a public lecture. I am not surprised to hear your honor speak of my experiments with mis- trustful caution; I would not do otherwise in a similar case. Two points in your esteemed letter appear to be too important to be left unanswered. The first deals with the question whether one may conclude that constancy of type has been obtained if the hybrid Aa produces a plant A , and this plant in turn produces only A . Permit me to state that, as an empirical worker, I must define constancy of type as the retention of a character during the period of observation. My statements that some of the progeny of hybrids breed true to type thus in- cludes only those generations during which observations were made; it does not extend beyond them.- For two generations all experiments were conducted with a fairly large number of plants. Starting with the third generation it became necessary to limit the numbers because of lack of space, so that, in each of the seven experiments, only a sample of those plants of the second generation (which either bred true or varied) could be observed further. The observations were extended over four to six generations (p. 13). Of the varieties which bred true (pp. 15-18) some plants were observed for four generations. I must further mention the case of a variety which bred true for six generations, although the parental types differed in four characters. In 1859 I obtained a very fertile descendent with large, tasty, seeds from a first generation hybrid. Since, in the following year, its progeny retained the desirable characteristics and were uniform, the variety was cultivated in our vegetable garden, and many plants were raised every year up to 1865. The parental plants were bcDg and BCdG: B. albumen yellow b. albumen green C. seed-coat grayish-brown c. seed-coat white D. pod inflated d. pod constricted G. axis long g. axis short The hybrid just mentioned was BcDG. The color of the albumen could be determined only in the plants saved for seed production, for the other pods were harvested in an immature condition. Never was green albumen observed in these plants, reddish-purple flower color (an indication of brown seed-coat), constriction of the pod, nor short axis. This is the extent of my experience. I cannot judge whether these findings would permit a decision as to constancy of type; however, I am inclined to regard the separation of parental characteristics in the progeny of hybrids in Pisum as complete, and thus permanent. The progeny of hybrids carries one or the other of the parental characteristics, or the hybrid form of the two; I have s-6 LETTERS TO CARL NAGELI, 1866-1873 never observed gradual transitions between the parental characters or a pro- gressive approach toward one of them. The course of development consists simply in this; that in each generation the two parental characteristics ap- pear, separated and unchanged, and there is nothing to indicate that one of them has either inherited or taken over anything from the other. For an example, permit me to point to the packets, numbers 1035-1088, which I sent you. All the seeds originated in the first generation of a hybrid in which brown and white seed-coats were combined. Out of the brown seed of this hybrid, some plants were obtained with seed-coats of a pure white color, with- out any admixture of brown. I expect those to retain the same constancy of character as found in the parental plant. The second point, on which I wish to elaborate briefly, contains the following statement: "You should regard the numerical expressions as being only empirical, because thev can not be proved rational." My experiments with single characters all lead to the same result: that from the seeds of the hybrids, plants are obtained half of which in turn carry the hybrid character {Aa), the other half, however, receive the parental charac- ters A and a in equal amounts. Thus, on the average, among four plants two have the hybrid character A a, one the parental character A, and the other the parental character a. Therefore 2ylc+^+fl or ^-|-2.4a+ais the empirical simple, developmental series for two differentiating characters. Likewise it was shown in an empirical manner that, if two or three differentiating characters are combined in the hybrid, the developmental series is a combination of two or three simple series. Up to this point I don't believe I can be accused of having left the realm of experimentation. If then I extend this combination of simple series to any number of differences between the two parental plants, I have indeed entered the rational domain. This seems permissible, however, because I have proved by previous experiments that the development of any two differentiating characteristics proceeds independently of any other differences. Finally, regarding my statements on the differences among the ovules and pollen cells of the hybrids; they also are based on experiments. These and similar experiments on the germ cells appear to be important, for I believe that their results furnish the explanation for the development of hy- brids as observed in Pisum. These experiments should be repeated and verified. I regret very much not being able to send your honor the varieties you desire. As I mentioned above, the experiments were conducted up" to and including 1863; at that time they were terminated in order to obtain space and time for the growing of other experimental plants. Therefore seeds from those experiments are no longer available. Only one experiment on differences in the time of flowering was continued; and seeds are available from the 1864 harvest of this experiment. These are the last I collected, since I had to aban- don the experiment in the following year because of devastation by the pea beetle, Bruchus pisi. In the early years of experimentation this insect was only rarely found on the plants, in 1864 it caused considerable damage, and ap- peared in such numbers in the following summer that hardly a 4th or 5th s-7 GREGOR MENDEL of the seeds was spared. In the last few years it has been necessary to dis- continue cultivation of peas in the vicinity of Briinn. The seeds remaining can still be useful, among them are some varieties which I expect to remain constant; they are derived from hybrids in which two, three, and four differ- entiating characters are combined. All the seeds were obtained from members of the first generation, i.e., of such plants as were grown directly from the seeds of the original hybrids. I should have scruples against complying with your honor's request to send these seeds for experimentation, were it not in such complete agreement with my own wishes. I fear that there has been partial loss of viability. Further- more the seeds were obtained at a time when Bruchus pisi was already ram- pant, and I cannot acquit this beetle of possibly transferring pollen; also, I must mention again that the plants were destined for a study of differences in flowering time. The other differences were also taken into account at the harvest, but with less care than in the major experiment. The legend which I have added to the packet numbers on a separate sheet is a copy of the notes I made for each individual plant, with pencil, on its envelope at the time of harvest. The dominant characters are designated as A, B, C, D, E, F, G and as concerns their dual meaning please refer to p. 11. The recessive characters are designated a, b, c, d, e, f, g; these should remain constant in the next genera- tion. Therefore, from those seeds which stem from plants with recessive char- acters only, identical plants are expected (as regards the characters studied). Please compare the numbers of the seed packets with those in my record, to detect any possible error in the designations — each packet contains the seeds of a single plant only. Some of the varieties represented are suitable for experiments on the germ cells; their results can be obtained during the current summer. The round yellow seeds of packets 715, 730, 736, 741, 742, 745, 756, 757, and on the other hand, the green angular seeds of packets 712, 719, 734, 737, 749, and 750 can be recommended for this purpose. By repeated experiments it was proved that, if plants with green seeds are fertilized by those with yellow seeds, the albumen of the resulting seeds has lost the green color and has taken up the yellow color. The same is true for the shape of the seed. Plants with angular seeds, if fertilized by those with round or rounded seeds, produce round or rounded seeds. Thus, due to the changes induced in the color and shape of the seeds by fertilization with foreign pollen, it is possible to recognize the constitution of the fertilizing pollen. Let B designate yellow color; b, green color of the albumen. Let A designate round shape; o, angular shape of the seeds. If flowers of such plants as produce green and angular seeds by self-fertiliza- tion are fertilized with foreign pollen, and if the seeds remain green and angular, then the pollen of the donor plant was, as regards the two characters ab If the shape of the seeds is changed, the pollen was taken from Ab If the color of the seeds is changed, the pollen was taken from aB If both shape and color is changed, the pollen was taken from AB LETTERS TO CARL NAGELI, 1866-1873 The packets enumerated above contain round and yellow, round and green, angular and yellow, and angular and green seeds from the hybrids ab-\-AB. The round and yellow seeds would be best suited for the experiment. Among them (see experiment p. 15) the varieties AB, ABb, Aab, and AaBb may occur; thus four cases are possible when plants, grown from green and angular seeds, are fertilized by the pollen of those grown from the above mentioned round and yellow seeds, i.e. I. ab+AB II. ab-\-ABb III. ab-{-AaB IV. ab+AaBb If the hypothesis that hybrids form as many types of pollen cells as there are possible constant combination types is correct, plants of the makeup AB produce pollen of the type AB ABb " " " " " AB and Ab AaB " " " " " AB and aB AaBb " " " " " AB, Ab, aB, and ab Fertilization of ovules occurs: I. Ovules ab with pollen AB II. " ab " " AB and Ab III. " ab " " AB and aB IV. " ab " " AB, Ab, aB, and ab The following varieties may be obtained from this fertilization: I. AaBb 11. AaBb and Aab III. AaBb and aBb IV. AaBb, Aab, aBb, and ab If the different types of pollen are j^roduced in equal numbers, there should be in I. All seeds round and yellow II. one half round and yellow one half round and green III. one half round and yellow one half angular and yellow IV. one quarter round and yellow one quarter round and green one quarter angular and yellow one quarter angular and green Furthermore, since the numerical relations between AB, ABb, AaB, AaBb are 1:2:2:4, among any nine plants grown from round yellow seed there should be found on the average AaBb four times, ABb and AaB twice each, and AB s-9 GREGOR MENDEL once; thus the IVth case should occur four times as frequently as the 1st and twice as frequently as the Ilnd or Illrd. If on the other hand, plants grown from the round yellow seeds mentioned are fertilized by pollen from green angular plants, the results should be exactly the same, provided that the ovules are of the same types, and formed in the same proportions, as was reported for the pollen. I have not performed this experiment myself, but I believe, on the basis of similar experiments, that one can depend on the result indicated. In the same fashion individual experiments may be performed for each of the two seed characters separately, all those round seeds which occurred to- gether with angular ones, and all the yellow ones which occurred with green seeds on the same plant are suitable. If, for instance, a plant with green seeds was fertilized by one with yellow seeds, the seeds obtained should be either 1) all yellow, or 2) half yellow and half green, since the plants originating from yellow seeds are of the varieties B and Bb. Since, furthermore, B and Bb occur in the ratio of 1:2, the 2nd fertilization will occur twice as frequently as the 1st. Regarding the other characters, the experiments may be conducted in the same way; results, however, will not be obtained until next year. . . , As must be expected, the experiments proceed slowly. At first beginning, some patience is required, but later, when several experiments are progressing concurrently, matters are improved. Every day, from spring to fall, one's in- terest is refreshed daily, and the care which must be given to one's wards is thus amply repaid. In addition, if I should, by my experiments, succeed in hastening the solution of these problems, I should be doubly happy. Accept, highly esteemed Sir, the expression of most sincere respect from Your devoted, G. Mendel (Altbriinn, Monastery of St. Thomas) Brtinn, 18 April, 1867 SUPPLEMENT II THE RELATION OF GENETICS TO PHYSIOLOGY AND MEDICINE NOBEL LECTURE, PRESENTED IN STOCKHOLM ON JUNE 4, 1934 By Dr. THOMAS HUNT MORGAN DIRECTOR OF THE WM. G. KERCKHOFF LABORj^TORIES, CALIFORNIA INSTITUTE OF TECHNOLOGY The study of heredity, now called genetics, has undergone such an ex- traordinary development in the present century, both in theory and in practice, that it is not possible in a short address to review even briefly all its outstanding achievements. At most I can do no more than take up a few topics for discussion. Since the group of men with whom I have worked for tAventy years has been interested for the most part in the chro- mosome-mechanism of heredity, I shall first briefly describe the relation between the facts of heredity and the theory of the gene. Then I should like to discuss one of the physiological problems im- plied in the theory of the gene ; and finally, I hope to say a few words about the applications of genetics to medicine. The modern theorj^ of genetics dates from the opening years of the present century, vrith the discovery of Mendel's long-lost paper that had been overlooked for thirty-five years. The data obtained by de Vries in Holland, Correns in Ger- many and Tschermak in Austria showed that Mendel's laws are not confined to garden peas, but apply to other plants. A year or two later the work of Bateson and Punnett in England and Cuenot in France made it evident that the same laws apply to animals. In 1902 a young student, William Sutton, working in the laboratory of E. B. Wilson, pointed out clearly and completely that the known behavior of the chromosomes at the time of matura- tion of the germ-cells furnishes us with a mechanism that accounts for the kind of separation of the hereditary units postulated in Mendel's theory. The discovery of a mechanism, that suffices to explain both the first and the second law of IMendel, has had far-reach- ing consequences for genetic theory, espe- cially in relation to the discovery of addi- tional laws ; because the recognition of a mechanism that can be seen and followed demands that any extension of Mendel's theories must conform to such a recog- nized mechanism ; and also because the apparent exceptions to Mendel's laws, that came to light before long, might, in the absence of a known mechanism, have called forth purely fictitious modifica- tions of Mendel's laws or even seemed to invalidate their generality. We now know that some of these "exceptions" are due to newly discovered and demon- strable properties of the chromosome mechanism, and others to recognizable irregularities in the machine. Mendel knew of no processes taking place in the formation of pollen and egg- S-I] THE SCIENTIFIC MONTHLY GENETICS, PHYSIOLOGY AND MEDICINE cell that could furnish a basis for his primary assumption that the hereditary elements separate in the germ-cells in such a way that each ripe germ-cell comes to contain only one of each kind of ele- ment : but he justified the validity of this assumption by putting it to a crucial test. His analysis was a w^onderful feat of reasoning. He verified his reasoning by the recognized experimental procedure of science. As a matter of fact it would not have been possible in Mendel's time to give an objective demonstration of the basic mechanism involved in the separation of the hereditary elements in the germ-cells. The preparation for this demonstration took all the thirty-five years between Mendel's paper in 1865 and 1900. It is here that the names of the most promi- nent European cytologists stand out as the discoverers of the role of the chromo- somes in the maturation of the germ-cells. It is largely a result of their work that it was possible in 1902 to relate the well- known eytological evidence to Mendel's laws. So much in retrospect. The most significant additions that have been made to Mendel's two laws may be called linkage and crossing over. In 1906 Bateson and Punnett reported a two-factor case in sweet peas that did not give the expected ratio for two pairs of characters entering the cross at the same time. By 1911 two genes had been found in Drosophila that gave sex-linked inheri- tance. It had earlier been shown that such genes lie in the X-chromosomes. Ratios were found in the second genera- tion that did not conform to Mendel's second law when these two pairs of char- acters are present, and the suggestion was made that the ratios in such cases could be explained on the basis of inter- change between the two X-chromosomes in the female. It was also pointed out that the further apart the genes for such characters happen to lie in the chromo- some, the greater the chance for inter- change to take place. This would give the approximate location of the genes with respect to other genes. By further extension and clarification of this idea it became possible, as more evidence ac- cumulated, to demonstrate that the genes lie in a single line in each chromosome. Two years previously (1909) a Bel- gian investigator, Janssens, had de- scribed a phenomenon in the conjugating chromosomes of a salamander, Batraco- seps, which he interpreted to mean that interchanges take place between homol- ogous chromosomes. This he called chiasmatypie — a phenomenon that has occupied the attention of cytologists down to the present day. Janssens' ob- servations were destined shortly to sup- ply an objective support to the demon- stration of genetic interchange between linked genes carried in the sex chromo- somes of the female Drosophila. To-day we arrange the genes in a chart or map, Fig. 1. The numbers attached express the distance of each gene from some arbitrary point taken as zero. These numbers make it possible to fore- tell how any new character that may appear will be inherited with respect to all other characters, as soon as its cross- ing over value with respect to any other two characters is determined. This abil- ity to predict would in itself justify the construction of such maps, even if there were no other facts concerning the loca- tion of the genes ; but there is to-day direct evidence in support of the view that genes lie in a serial order in the chromosomes. What are the Genes? What is the nature of the elements of heredity that Mendel postulated as purely theoretical units? What are genes? Now that we locate them in the chromosomes are we justified in regard- ing them as material units ; as chemical bodies of a higher order than molecules? S-I3 THE SCIENTIFIC MONTHLY Frankly, these are questions with which the working geneticist has not much con- cern himself, except now and then to speculate as to the nature of the postu- lated elements. There is no consensus of opinion amongst geneticists as to what the genes are — whether they are real or purely fictitious — because at the level at which the genetic experiments lie it does not make the slightest difference whether the gene is a hj'pothetical unit or whether the gene is a material particle. In either case the unit is associated with a specific chromosome, and can be localized there by purely genetic analysis. Hence, if the gene is a material unit, it is a piece of a chromosome; if it is a fictitious unit, it must be referred to a definite location in a chromosome — the same place as on the other hypothesis. Therefore, it makes no difference in the actual work in genetics which point of view is taken. Between the characters that are used by the geneticist and the genes that the theory postulates lies the whole field of embryonic development, where the prop- erties implicit in the genes become ex- plicit in the protoplasm of the cells. Here we appear to approach a physio- logical problem, but one that is new and strange to the classical physiology of the schools. We ascribe certain general properties to the genes, in part from genetic evi- dence and in part from microscopical ob- servations. These properties we may next consider. Since chromosomes divide in such a way that the line of genes is split (each daughter chromosome receiving exactly half of the original line) we can scarcely avoid the inference that the genes divide into exactly equal parts; but just how this takes place is not knoAvn. The anal- ogy of cell-division creates a presumption that the gene divides in the same way, but we should not forget that the rela- tively gross process involved in cell-divi- sion may seem quite inadequate to cover the refined separation of the gene into equal halves. As we do not know of any comparable division phenomena in or- ganic molecules, we must also be careful in ascribing a simple molecular constitu- tion to the gene. On the other hand, the elaborate chains of molecules built up in organic material may give us, some day, a better opportunity to picture the mo- lecular or aggregate structure of the gene and furnish a clue concerning its mode of division S-14 SUPPLEMENT III THE PRODUCTION OF MUTATIONS IF, as Darwin maintained, the aclap- tiveness of living things results from natural selection, rather than from a teleological tendency in the process of variation itself, then heritable variations must, under most conditions, occur in numerous directions, so as to give a wide range of choice for the selective process. Such a state of affairs seems, however, in more or less contradiction to the com- monly held idea, to which Darwin also gave some credence, that heritable varia- tions of given kinds tend to be produced, in a fairly regular way, by given kinds of external conditions. For then we are again confronted with the difficulty, how- ls it that the "right kinds" of variations (i.e. the adaptive ones) manage to arise in response to the "right kinds" of con- ditions (i.e. those they are adapted to) ? Moreover, the de Vriesian notion of mu- tations does not help us in this connec- tion. On that view, there are sudden jumps, going all the way from one "ele- mentary species" to another, and involv- ing radical changes in numerous charac- ters at once, and there are relatively few different jumps to choose between. This obviously would fail to explain how, through such coarse steps, the body could have come to be so remarkably streamlined in its internal and external organization, or, in other words, so thoroughly adaptive. The older selectionists, thinking in terms of chemical reactions on a molar scale when they thought in terms of chemistry at all, did not realize suffi- ciently the ultramicroscopic random- ness of the processes causing inherited variations. The earliest mutationists failed, in addition, to appreciate the qualitative and quantitative multiplicity of mutations. It was not long, however, before the results of Baur on Antirrhi- mon and of Morgan on Drosophila, sup- plemented by scattered observations on other forms, gave evidence of the occur- rence of numerous Mendelizing muta- tions, many of them small ones, in varied directions, and they showed no discover- erable relation between the type of mu- tation and the type of environment or condition of living imder which it arose. These observations, then, came closer to the statistical requirements for a process of evolution which has its basis in acci- dents. In what sense, however, could the events be regarded as accidental? Were they perhaps expressions of veiled forces working in a more determinate manner? It was more than ever evident that further investigation of the manner of occurrence of mutations was called for. If the mutations were really non-teleo- logical, with no relation between type of environment and type of change, and above all no adaptive relation, and if they were of as numerous types as the theory of natural selection would de- mand, then the great majority of the changes should be harmful in their ef- fects, just as any alterations made blind- ly in a complicated apparatus are usually detrimental to its proper functioning, and many of the larger changes should even be totally incompatible with the functioning of the whole, or, as we say, lethal. That is, strange as it may seem at first sight, we should expect most mu- tations to be disadvantageous if the the- ory of natural selection is correct. We should also expect these mainly disad- vantageous changes to be highly diversi- fied in their genetic basis. Frequency of Mutations To get exact evidence on these points required the elaboration of special genet- S-15 The Journal of Heredity ic methods, adapted to the recognition of mutations that ordinarily escape de- tection— (1) lethals, (2) changes with but small visible eiifects, and (3) changes without any externally visible effects but influencing the viability more or less unfavorably. It would take us too far afield to explain these techniques here. Suffice it to say that they made use of the principle according to which a chro- mosome is, as we say, "marked," by hav- ing had inserted into it to begin with one or more known mutant genes with conspicuous visible effects, to differenti- ate it from the homologous chromosome. An individual with two such differenti- ated chromosomes, when appropriately bred, will then be expected to give two groups of visibly different offspring, holding certain expected ratios to one another. If, however, a lethal mutation has occurred in one of the two chromo- somes, its existence will be made evident by the absence of the corresponding ex- pected group of offspring. Similarly, a mutated gene with invisible but some- v;hat detrimental action, though not fully lethal, will be recognized by the fact that the corresponding group of offspring are found in smaller numbers than ex- pected. And a gene with a very small visible effect, that might be overlooked in a single individual, will have a great- ly increased chance of being seen because the given group of offspring as a whole will tend to be distinguished in this re- gard from the corresponding group de- rived from a non-mutant. In this way, it was possible in the first tests of this kind, which Alten- burg and the writer conducted, part- ly in collaboration, in 1918-19, to get definite evidence in Drosophila that the' lethal mutations greatly outnumbered those with visible effects, and that among the latter the types having an obscure manifestation were more numerous than the definite conspicuous ones used in ordinary genetic work. Visible or not, the great majority had lowered viability. Tests of their genetic basis, using the newly found facts of linkage, showed them to be most varied in their locus in the chromosomes, and it could be calcu- lated by a simple extrapolative process that there must be at least hundreds, and probably thousands, of different kinds arising in the course of spontaneous mu- tation. In work done much later, employ- ing induced mutations, it was also shown (in independent experiments both of the present writer and Kerkis, and of Timofeeff and his co-workers, done in 1934) that "invisible" mutations, which by reason of one or another physiologi- cal change lower viability without being fully lethal, form the most abundant group of any detected thus far, being at least two to three times as numerous as the complete lethals. No doubt there are in addition very many, perhaps even rnore, with effects too small to have been detected at all by our rather crude meth- ods. It is among these that we should be most apt to find those rare accidents which, under given conditions or in given combinations with others, may happen to have some adaptive value. Tests of Timofeeff, however, have shown that even a few of the more conspicuous visible mutations do in certain combina- tions give an advantage in laboratory breeding. Because of the nature of the test whereby it is detected — the absence of an entire group of offspring bearing cer- tain conspicuous expected characters — a lethal is surer of being detected, and detected by any observer, than is the inconspicuous or invisible, merely detri- mental, mutation. Fortunately, there are relatively few borderline cases, of nearly but not quite completely lethal genes. It was this objectivity of recognition, com- bined with the fact that they were so much more numerous than conspicuous visible mutations, that made it feasible for lethals to be used as an index of mutation frenuency, even though they suffer from the disadvantage of requir- ing the breeding of an individual, rather than its mere inspection, for the recogni- tion that it carries a lethal. In the earli- est published work, we (Altenburg and the author) attempted not only to find a quantitative value for the "normal" mu- tation frequency, but also to determine whether a certain condition, which we s-i6 Muller: Production of Mutations considered of especial interest, affected the mutation frequency. The plan was ultimately to use the method as a gen- eral one for studying the effects of vari- ous conditions. The condition chosen for the first experiment was tempera- ture, and the results, verified by later work of the writer's, indicated that a rise of temperature, within limits normal to the organism, produced an increase of mutation frequency of about the amount to be expected if mutations were, in essentials, orthodox chemical reactions. Mutations as Chemical Reactions On this view, however, single muta- tions correspond with individual molecu- lar changes, and an extended series of mutations, in a great number of identi- cal genes in a population, spread out over thousands of years, is what cor- responds with the course of an ordinary chemical reaction that takes place in a whole collection of molecules in a test tube in the course of a fraction of a second or a few seconds. For the indi- vidual gene, in its biological setting, is far more stable than the ordinary chemi- cal molecule is, when the latter is ex- posed to a reagent in the laboratory. Thus, mutations, when taken collective- ly, should be subject to the statistical laws applying to mass reactions, but the individual mutation, corresponding to a change in one molecule, should be sub- ject to the vicissitudes of ultramicro- scopic or atomic events, and the appari- tion of a mutant individual represents an enormous amplification of such a phe- nomenon. This is a principle which gives the clue to the fact, which otherwise seems opposed to a rational, scientific and molarly deterministic point of view, that differences in external conditions or conditions of living do not appear to af- fect the occurrence of mutations, while on the other hand, even in a normal and sensibly constant environment, muta- tions of varied kinds do occur. It is also in harmony with our finding, of about the same time, that when a mutation takes place in a given gene, the other gene of identical type present nearby in the same cell usually remains unaft'ected though it must of course have been sub- jected to the same macroscopic physico- chemical conditions. On this conception, then, the mutations ordinarily result from submicroscopic accidents, that is, from caprices of thermal agitation, that occur on a molecular and submolecular scale. More recently Delbriick and Timofeeff, in more extended work on temperature, have shown that the amount of increase in mutation frequen- cy with rising temperature is not merely that of an ordinary test-tube chemical reaction, but in fact corresponds closely with that larger rise to be expected of a reaction as slow in absolute time rate (i.e. with as small a proportion of mo- lecular changes per unit of time) as the observed mutation frequency shows this reaction to be, and this quantitative cor- respondence helps to confirm the entire conception. Now this inference concerning the non-molar nature of the individual mu- tation process, which sets it in so differ- ent a class from most other grossly ob- servable chemical changes in nature, led naturally to the expectation that some of the "point effects" brought about by high-energy radiation like X-rays would also work to produce alternations in the hereditary material. For if even the rela- tively mild events of thermal agitation can, some of them, have such conse- quences, surely the energetically far more potent changes caused by power- ful radiation should succeed. And, as a matter of fact, our trials of X-rays, car- ried out with the same kind of genetic methods as previously used for tempera- ture, proved that such radiation is ex- tremely effective, and inordinately more so than a mere temperature rise, since by this method it was possible to obtain, by a half hour's treatment, over a hun- dred times as many mutations in a group of treated cells as would have occurred in them spontaneously in the course of a whole generation. These mutations, too, were found ordinarily to occur pointwise and randomly, in one gene at a time, without affecting an identical S-17 The Journal of Heredity gene that might be present nearby in a homologous chromosome. Radiation-Effects In addition to the individual gene changes, radiation also produced rear- rangements of parts of chromosomes. As our later work (including that with co-workers, especially Raychaudhuri and Pontecorvo) has shown, these latter were caused in the first place by break- ages of the chromosomes, followed after- wards by attachments occurring between the adhesive broken ends, that joined them in a different order than before. The two or more breaks involved in such a rearrangement may be far apart, caused by independent hits, and thus re- sult in what we call a gross structural change. Such changes are of various kinds, depending upon just where the breaks are and just which broken ends become attached to which. But, though the effects of the individual "hits" are rather narrowly localized, it is not un- common for two breaks to be produced at nearby points by what amounts to one local change (or at any rate one lo- calized group of changes) whose influ- ence becomes somewhat spread out. By the rejoining, ''n a new order, of broken ends resulting from two such nearby breaks, a minute change of sequence of the genes is brought about. More usual- ly, the small piece between the two breaks becomes lost (a "deficiency"), but sometimes it becomes inverted, or even becomes transferred into a totally different position, made available by a separate hit. Both earlier and later work by collabo- rators (Oliver, Hanson, etc.) showed definitely that the frequency of the gene mutations is directly and simply propor- tional to the dose of irradiation applied, and this despite the wave length used, whether X- or gamma or even beta rays, and despite the timing of the irradiation. These facts have since been established with great exactitude and detail, more especially by Timofeeff and his co- workers. In our more recent work with Raychaudhuri these principles have been extended to total doses as low as 400r, and rates as low as .Olr per min- ute, with gamma rays. They leave, we believe, no escape from the conclusion that there is no threshold dose, and that the individual mutations result from in- dividual "hits", producing genetic ef- fects in their immediate neighborhood. Whether these so-called "hits" are the individual ionizations, or may even be the activations that occur at lower ener- gy levels, or whether, at the other end of the scale, they require the clustering of ionizations that occurs at the termini of electron tracks and of their side branches (as Lea and Fano point out might be the case), is as yet undecided. But in any case they are, even when microscopically considered, what we have termed "point mutations," as they involve only disturbances on an ultra- microscopically localized scale. And whether or not they are to occur at any particular point is entirely a matter of accident, using this term in the sense in which it is employed in the mathematics of statistics. Naturally, other agents than photons which produce effects of this kind must also produce mutations, as has been shown by students and collaborators working under Altenburg in Houston for neutrons (Nagai and Locher) and for alpha rays (Ward) and confirmed by Timofeeff and his co-workers (Zim- mer, et al). Moreover, as Alten- burg showed, even the smaller quan- tum changes induced by ultraviolet exert this effect on the genes. They cause, however, only a relatively small amount of rearrangement of chromosome parts (MuUer and Mackenzie), and, in fact, they also tend to inhibit such rearrange- nient, as Swanson, followed by Kauff- mann and Hollaender. has found. Since the effective ultraviolet hits are in the form of randomly scattered single-atom changes in the purines and pyrimidines of the chromosome, rather than in groups of atom changes, it seems likely that clusters of ionizations are not neces- sary for the gene mutation effects, at any rate, although we cannot l>e sure of this until the relation of mutation frequency to dosage is better known for this agent. S-i8 Muller: Production of Mutations Induced and Natural Mutations Inasmuch as the changes brought about in the genes by radiation must certainly be of an accidental nature, un- premeditated, ateleological, without ref- erence to the value of the end result for the organism or its descendants, it is of interest to compare them with the so- called spontaneous or natural mutations. For in the radiation mutations we have a yardstick of what really random changes should be. Now it is found in Drosophila that the radiation-induced mutations of the genes (we exclude here the demonstrable chromosome rear- rangements) are in every respect which has been investigated of the same es- sential nature as those arising naturally in the laboratory or field. They usually occur in one gene without affecting an identical one nearby. They are distrib- uted similarly in the chromosomes. The effects, similarly, may be large or small and there is a similar ratio of fully lethal to so-called visible gene mutations. That is, the radiation mutations of the genes do not give evidence of being more dele- terious. And when one concentrates at- tention upon given genes one finds that a whole series of different forms, or al- leles, may be produced, of a similar and in many cases sensibly identical nature in the two cases. In fact, every natural mutation, when searched for long enough, is found to be producible also by radiation. Moreover, under any given condition of living tried, without radia- tion, the effects appear as scattered as when radiation is applied, even though of much lower frequency. All this sure- ly means then, does it not, that the natu- ral mutations have in truth no innate tendency to be adaptive, nor even to be different, as a whole group, under some natural conditions than under others? In other words, they cannot be determi- nate in a molar sense, but must them- selves be caused by the ultramicroscopic accidents of the molecular and submolec- ular motions, i.e. by the individual quan- tum exchanges of thermal agitation, tak- ing this word in a broad sense. The only escape from this would be to suppose that they are caused by the radiation present in nature, resulting from natural radioactive substances and cosmic rays, but a little calculation (by Mott-Smith and the writer, corroborated by others) has shown that this radiation is quite inadequate in amount to account for the majority of mutations occurring in most organisms. But to say that most natural muta- tions are the results of the quantum ex- changes of thermal agitation, and, fur- ther, that a given energy level must be reached to produce them, does not, as some authors have seemed to imply, mean that the physicochemical condi- tions in and around the organism, other than temperature, have no influence upon their chance of occurrence. That such circumstances may play a decided role was early evident from the studies of spontaneous mutation frequency, when it was found (1921, reported 1928) that the frequency in one ex- periment, with one genetic stock, might be ten times as high as in another, with another stock. And more recently we have found that, in different portions of the natural life cycle of the same in- dividual, the mutation frequency may be very dift'erent. Finally, in the work of Auerbach and Robson, with mustard gas and related substances, it has been proved that these chemicals may induce mutations at as high a frequency as a heavy dose of X-rays. In all these cases, however, the effects are similarly scat- tered at random, individually uncon- trolled, and similarly non-adaptive. It should also be noted in this con- nection that the genes are not under all conditions equally vulnerable to the mutating effects of X-rays themselves. Genes in the condensed chromosomes of spermatozoa, for example, appear to be changed more easily than those in the more usual "resting" stages. W'e have mentioned that, as Swanson has shown, ultraviolet exerts besides its own mutat- ing effect an inhibition on the pro-cess of chromosome breakage, or at any rate on that of reunion of the broken parts in a new viable order, while infrared, in Hol- laender's and Kaufmann's recent experi- S-19 The Journal of Heredity ments, has a contrary action. And Stadler, in his great work on the pro- duction of mutations in cereals, started independently of our own, has obtained evidence that in this material X-radiation in the doses used is unable to produce a sensible rise in the gene mutation fre- quency, though numerous chromosome breakages do arise, leading to both gross and minute rearrangements of chromo- some parts. Either the genes are more resistant in this material to permanent changes by X-rays as compared with their responsiveness to thermal agita- tion, or a break or loss must usually be produced by X-rays along with the gene change. The milder ultraviolet quanta, on the other hand, do produce gene mu- tations like the natural ones in these plants. Such variations in effectiveness are, I believe, to have been expected. They do not shake our conclusion as to the acci- dental, quantum character of the event which usually initiates a gene mutation. But they ,f;ive rise to the hope that, through further study of them, more may be learned concerning the nature of the mutation process, as well as of the genetic material that undergoes the changes. Controlled Mutation? No one can answer the question whether some special means may not be found whereby, through the application of molar influences, such as specific anti- bodies, individual genes could be changed to order. Certainly the search for such influences, and for increasing control of things on a microscopic and submicro- scopic scale as well, must be carried further. But there is as yet no good evi- dence that anything of the sort has been done artificially, or that it occurs natu- rally. Even if possible, there could be no generalized method of control of gene composition without far greater knowledge than we now have of the in- timate chemical structure and the mode of working of the most complicated and diverse substances that exist, namely, nucleoproteins, proteins in general, and enzymes. The works of Sumner. North- rup and Stanley, together with those of other protein chemists, point the way in this direction, but everyone will agree that it is a long and devious system of roads which is beginning here. It is true that some cases are known of mutable genes which change selec- tively in response to special conditions. Such cases may be very informative in shedding light on gene structure, but we have as yet no indication that the al- terations of these genes, which in the great majority of instances known are abnormal genes, have anything in com- mon with ordinary natural mutations. It is also true that cases are known among bacteria and viruses of the induction of particular kinds of hereditary changes by application of particular substances, but here the substances applied are in each case the same as those whose pres- ence is later found to have been induced, and so there is every reason to infer that they have in fact become implanted in some way, that is, that we do not really have a specifically induced mutation. So far, then, we have no means, or prospect of means, of inducing given mutations at will in normal material ; though the production of mutations in abundance at random may be regarded as a first step along such a path, if there is to be such a path. So long as we cannot direct mutations, then, selection is indispensable, and progressive change in the hereditary constitution of a living thing can be made only with the aid of a most thoroughgoing selection of the mu- tations that occur since, being non-adap- tive except by accident, an overwhelming majority is always harmful. For a sen- sible advance, usually a considerable number of rare steps must be accu- mulated in this painful selective pro- cess. By far the most of these are indi- vidually small steps, but, as species and race crossings have shown, there may be a few large distinctive steps that have been, as Huxley terms it. "buiTered", by small changes that readjust the organ- ism to them. Not only is this accumula- tion of many rare, mainly tiny changes the chief means of artificial animal and plant improvement, but it is, even more. Muller: Production of Mutations the way in which natural evolution has occurred, under the guidance of natural selection. Thus the Darwinian theory becomes implemented, and freed from the accretions of directed variation and of Lamarckism that once encumbered it. It is probable that, in a state of nature, most species have a not very much (though somewhat) lower frequency of gene mutation than would be most ad- vantageous for them, in consideration of the degree of rigor of the natural selec- tion that occurs in the given species. A much higher frequency would probably lead to faster genetic degenerative pro- cesses than the existing selection could well cope with. But, under conditions of artificial breeding, where selection can be made more effective, a higher mutation frequency can for a time at least be tolerated in some cases, and larger mutations also can be nursed through to the point where they become suitably buffered. Here it may become of practical use to apply X-rays, ultra- violet, or other means of inducing mu- tations, as Gustafsson especially has demonstrated for X-rays. This will be especially true in species which natural- ly undergo much inbreeding, or in which there is a well expressed haploid phase, or a considerable haploid portion of the genotype, for under these circumstances many of the spontaneous mutations that might otherwise have accumulated in the population and that could be brought to light by inbreeding, will have become eliminated before they could be found, and the natural mutation rate itself will be lower. We have above largely confined our- selves to considering the relation of the production of gene mutations to the problems of the general method of evo- lution, including that of the nature of hereditary variation, because this has been, historically, the main line of ap- proach to the subject of artificial muta- tions. It was from the first evident, how- ever, that the production of mutations would, as we once stated, provide us with tools of the greatest nicety, where- with to dissect piece by piece the physio- logical, embryological, and biochemical structure of the organism and to analyze its workings. Already with natural mu- tations, such works as those of Bon- nevie, Grueneberg, Scott-Moncrief, Eph- russi and Beadle, etc., have shown how the intensive tracing of the effects, and interrelations of effects, of just one or a few mutations, can lead to a deeper un- derstanding of the complex processes whereby the genes operate to produce the organism. But there are thousands of genes, and it is desirable to be able to choose them for study in an orderly fash- ion as we proceed with our dissection process. For this purpose we have thought that it would often be advan- tageous to produce mutations artificially in abundance, so as then to take our pick of those more suited for successive steps in our analysis. The work of Bea- dle and his co-workers on Ncurospora in recent years, followed by similar work of Malin and Fries and of others, has brilliantly shown the applicability of this method for studies of the paths of bio- chemical synthesis of amino-acids, vita- mins, purines and pyrimidines. And yet, in a sense, the surface of the subject as a whole has barely been scratched, and we may look forward with confidence to the combination of this technique with that of tracer substances and with all the other techniques of biochemistry, physi- ology and experimental embryology, for the increasing tmravelling of that sur- passingly intricate tangle of processes of which the living organism is constituted. There is no time, however, to go further into this subject here. Chromosome Analysis For we cannot neglect here a brief outline of another phase of the artificial mutation work, more specifically of in- terest to geneticists : that is, the further analysis of the properties of the chromo- somes and their parts, gained chiefly from studies in which parts have been removed, added, or rearranged. We have already spoken, in passing, of the studies of the mechanism of such structural change, in which a relatively simple gen- eral scheme lying at the basis of all such alterations has emerged: namely, break- S-2I The Journal of Heredity age first, followed by adhesion of broken ends. It was early evident that by the use of such rearranged chromosomes ad- ditional proof of the physical validity of the linkage maps could be obtained, and this was done (Muller and Painter). Furthermore, it has been possible to throw light on problems of crossing over, as in the demonstration (Muller, Stone, and Offermann) that to whatever posi- tion the centromere is moved, it causes a strong inhibition of crossing over, the strenth of which gradually diminishes with distance. Moreover, the same proves to be true of any point of dis- continuity in pairing, caused by hetero- zygosity in regard to a structural change. Such studies on crossing over, and on the pairing forces that afifect segregation. are still capable of considerable exten- sion. We must remember, in speaking of the centromere and other apparently distinc- tive chromosome parts, that we have no right to infer that they are autonomous, locally determined structures, dependent on the genes of the regions in which they are seen to lie, before observations have been made that show the effects of removing or displacing those regions. Therefore, it has in the main been neces- sary to wait for the study of induced inversions, deletions and translocations of chromosomes, before the inference could be secure that the centromere is, in most instances, such an autonomous organelle, dependent upon a gene or genes in the immediate neighborhood (but not in all instances in the neighbor- hood, as Rhoades has recently shown in a special strain of maize). Similarly, it has been possible to show (despite some contrary claims, the validity or invalidi- ty of which cannot be discussed here) that the free end of the chromosome, or telomere, constitutes in much material a locally determined distinctive structure. By a combined genetic and cytolos^ical analvsis of various cases of breakage and rearrangement of parts, it was found that there are distinctive, largely locally determined, regions of the chromosomes, usually most markedly develooed near the centromeres, which we at first called "inactive" but which are now usually referred to as "heterochromatic." These were also found independently in pure- ly cytological studies by Heitz. It would be fascinating to enter here into a dis- cussion of the remarkable peculiarities which the cytogenetic studies have shown these regions to have — the evidence of repetition of more or less similar parts, of a tendency to conjugation between the differently placed parts, of distinc- tive cytological appearance correlated with whether or not such conjugation occurs, of inordinately high tendency to structural change, of strong influence of certain of their genes upon segregation, etc., — and then to go on to discuss hy- potheses of their evolutionary origin and their functions. This would unfortunate- ly take us too far afield. We must, how- ever, insist upon one point — as it is not yet eenerally enough recognized, — name- ly, that the evidence is very strong that what, in the Drosophila chromosome as seen at mitosis, is called "the heterochro- matic region," is simply a large tempo- rary body of accessory, non-genic nuc- leoprotein, produced under the influence of one or two particular genes from among the dozen or more that constitute the whole heterochromatic region, as de- tected by genetic analysis and by the chromosome as seen at the resting stage (as in the salivary gland). And it is not these conspicuous non-genic blocks which are responsible for the other known peculiarities of the heterochro- matin, above mentioned — the function of the blocks is still undetermined. In other words, the so-called "heterochro- matin" with which the cytologist deals in studying mito^^ic chromosomes is a nuite different thing from, although in the neighborhood of. the heterochro- matin proper having the above described complex of properties. Moreover, it has been possible to show (Sutton-Gersh in collaboration with the author, unnub- lished) that the conspicuous nucleoli often associated with the heterochromat- in are produced under the influence of still other autonomous eenes in it. that are senara+e from those for the mitotical- ly visible blocks. Mullen Production of Mutations One of the most interesting findings which has come out of the study of Dro- sopliila chromosomes that underwent re- arrangement of parts as a result of ir- radiation has been the generaHzation of the existence of the phenomenon known as "position effect." This effect was first found by Sturtevant in the case of the spontaneous mutant known as Bar eye, but it was not known to what extent the effect might be a special one until numerous rearrangements could be studied. The term position effect im- plies that the functioning of a gene is to a certain extent at least dependent upon what other genes lie in its neighborhood. There is now adequate evidence that this is a general principle, applying to very many if not all the genes in Droso- phila, and that their functioning can be qualitatively as well as quantitatively conditioned by the character of the genes in their vicinity, some of the genes hav- ing much more effect than others and different genes working in different ways and to different extents. It is possible that, as Sturtevant sug- gested, the position effect is caused by the interaction between gene products in the vicinity of the genes producing them, assuming that such products are more concentrated there and under such cir- cumstances tend to react more with one another than when dispersed. However, the interpretation which we favor is that the functioning of the gene is affected by its shape and that this, in turn, varies with the strength and nature of synaptic forces acting on the region of the chro- mosome in which it lies. These might consist of forces directly exerted on the gene by other genes, whether allelic or not (MuUer), or they might be resul- tants of the state of spiralization, etc., of the chromosome region, circumstances which in their turn are in part dependent on synaptic forces (Ephrussi and Sut- ton). This interpretation, in either of its variants, would explain why position effects are so much more general in Drosophila, an organism in which the synaptic forces are known to operate strongly even in somatic cells, than in other organisms tested, in which such forces are much weaker or absent in somatic 'cells. It would also fit in with the author's findings that the hetero- chromatic regions tend to have especial- ly strong, extensive, and distinctive kinds of position effects, effects varying in degree with tlie total amount of heter- ochromatin present in a cell, as well as with vacillating embryological factors. For these genetic findings are in con- formity with the cytological effects of heterochromatin, observed first by Pro- kofyeva, on the degree of extension, synaptic properties, etc., of euchromatin in its neighborhood, effects which she showed to be subject to similar vacilla- tions, that are correlated with the varia- tions in the phenotypically observed po- sition effects. Recent observations, both by Ephrussi and by Sutton, following suggestions of the author, and by Stern, also seem to point in this direction, for they show an influence, on the position effects exhibited by given parts, of the arrangement of homologous chromosome parts. If this interpretation based on gene shape should hold, it would open up a new angle of attack on the struc- ture and method of functioning of the gene, perhaps ultimately relating it to nucleoprotein composition and proper- ties. Another use to which the process of breakage and rearrangement of chromo- some parts by irradiation has been put is for the study of the effects of adding and of subtracting small pieces of chro- mosomes, in order to determine the re- lation of gene dosage to gene expression. In this way, it has been found out (1) that most normal genes are, even in sin- gle dose, near the upper limit of their effectiveness, and (2) that most mutant genes have a final effect qualitatively similar to but auantitatively less than that of their allelic normal gene. The dominance of normal genes over their mutant alleles, then, turns out in most instances to be a special ca=e of the prin- ciple that one dose of a normal gene usu- ally produces nearly though not quite as much effect as two doses. This in turn is best understood as resulting from a long course of selection of the normal s-23 The Journal of Heredity gene and its modifiers for stability of ex- pression, when under the influence of environmental and genetic conditions which would afifect the gene's operation quantitatively, i.e. in a manner similar to that of dosage changes. This does not mean that selection has specifically worked to produce dominance of the normal gene over its alleles, however, because (3) not all mutant genes be- have merely like weaker normal genes, and (4) those which the dosage tests show to produce qualitatively difTerent effects from the normal genes seem oft- ener to escape from the principle of be- ing dominated over by the normals, just as would be expected on our hypothesis. Among the further results of gene dosage studies carried out by the use of chromosome fragments produced by irradiation, attention should be especial- ly called to the findings coming under the head of "dosage compensation." These have shown (1) that, when the dosage of virtually all genes in the X chromosome except a given one is held constant, the expression of that one is usually so very nearly the same when present in one dose as in two that no difference in the character can ordinarily be seen, and (2) that nevertheless this invisible difference has been so impor- tant for the organism that, in the course of the past natural selection, a system of modifying genes, called compensators, has been established, having the function of making the effects of the one and two doses nor- mally present in the two respective sexes much more nearly equal still, when these dosage differences in the given genes are present simultaneously with those in all the other X-chromosomal genes. Each gene seems to have acquired a different system of compensators, the interrela- tions of all together being extremely complicated. This then gives evidence from a new angle of the meticulousness of natural selection, of the very precise adaptiveness of the characters existing in a species, and of the final grade of a character having ordinarily become es- tablished through the accumulation of numerous small mutations having very complex functional relations with one another. It is in line with our previous thesis of evolution through the selection of multitudinous tiny accidental changes. When attention is concentrated on a given very circumscribed region of a chromosome, by a comparison of vari- ous induced rearrangements all of which have a point of breakage within that re- gion, other facts come to light, bearing on the problems of chromosome and gene divisibility. By means of special genetic methods, which cannot be de- tailed here, evidence has been obtained that the breaks in any such limited re- gion tend to occur at specific points, giving indication that discrete units or segments lie between these points, and thus arguing against the idea of the chro- mosome being a continuum and in favor of its genes corresponding to physical entities rather than merely to concepts arbitrarily set up for the convenience of geneticists. We are also enabled in this way to make estimates of the probable number of genes in the chromosome, as well as to get maximally limiting figures for their size. These estimates agree as closely as could have been expected with those based on previous genetic work, using entirely different methods, al- though not with the c^tima+es based on the "sensitive volume" hypothesis. Duplications and Evolution Another finding made in studies of cases having a small fragment of chro- mosome moved, as a result of irradia- tion, to another position, was that indi- viduals are frequently able to survive and reproduce even when they have the given chromosome part present in its orieinal position as well as in the new position. In fact, it was in work of this kind that the effect of extra doses of genes was determined. Now, in some of these cases stocks could even be obtained which were homozygous for the dupli- cated piece as well as for the original piece. This led to the idea that duplica- tions of chromosome material might in this manner have become established in the previous course of evolution. When, in the analysis of a limited region of the S-24 MuUer: Production of Mutations A' chromosome, including the locus of the so-called "scute" etTect, it was found that there are in fact, within the normal X chromosome, two genes of closely re- lated effect ("achaete" and "scute") very close or adjacent to one another, it became evident that this was in all probability an example of the above postulated occurrence. This then showed the way, and apparently the main if not the only way (aside from the far rarer phenomena of polyploidy and "tetra- somy"), by which the number of genes has become increased during the course of evolution. By a curious coincidence. Bridges was at the same time making his studies of salivary chromosomes and finding direct cytological evidence for the existence of such "repeats," as he called them, in the normal chromosome, and he interpreted these in the same manner. In the twelve years since that time, various other clear cases of the same kind have been demonstrated. Thus, increase in gene number, brought about by the duplication of small parts of chromosomes, more usually in posi- tions near their original ones, must be set down as one of the major pro- cesses in evolution, in addition to the mutations in the individual genes. By itself, this process would not be of great importance, but it becomes important because, by allowing gene mutations to come afterwards that differentiate the genes in one position from the originally identical ones in the other position, the number of different kinds of genes is in- creased and so the germ plasm, and with it the processes of development and the organism as a whole, are eventuallv en- abled to grow more complex. Rearrangements of chromosome parts which do not lead to an increase in gene number can of course also occur in evo- lution, although it is unlikely that their role is so fundamental. By producing such changes in the laboratory it has been possible to find out a good deal more about what types can arise, and what their properties are. Various in- ferences can then be drawn concerning the viability and fertility that the differ- ent types would have, under varied ge- netic circumstances, and whether they would tend to become eliminated or to accumulate in a population of a given type. Some of them can be shown to have, under given conditions, an evolu- tionary survival value, both by aiding in the process of genetic isolation and in other ways, as by affecting heterosis. In this manner, evolutionary inferences have been drawn which have later been confirmed by comparison of the chromo- some differences actually existing be- tween related races, sub-species, and species. Probably of greater ultimate interest will be the results of studies of gene mu- tations occurring at individual loci. Radi- ation mutations are frequent enough to lend themselves to comparisons of the potentialities of different loci, although not nearly enough has yet been done along these lines. Similarly, a compari- son of the different mutations which can occur at the same locus can lead to very important results, especially since it has been shown that the different alleles may have every complex relationships to one another, so as even, in some cases, to reconstitute the normal type when they are crossed together. The way in which genes may change as a result of succes- sive mutations remains to be gone into at much greater length. So. too, does the question of changes in gene muta- bility, brought about by gene mutation itself. Somatic Radiation Effects The further the analysis of the genetic effects of irradiation has gone, particu- larly of the breakage *and rearrangement of chromosome parts, the more does our conviction grow that a large proportion if not the great majority of the somatic effects of irradiation that have been ob- served by medical men and by students of embryology, regeneration, and gen- eral biology, arise secondarily as conse- quences of genetic effects produced in the somatic cells The usefulness of this interpretation has been shown in recent studies of Koller, dealing with improved methods of irradiation of mammalian carcinoma. This is too large a subject S-25 The Journal of Heredity to digress upon here, but it is to be noted that it has been the analyses based in the first place on genetic and cytogenetic studies of the reproductive cells, as shown by subsequent generations, which are thus helping to clear the way for an understanding of the mechanism by which radiation acts in inhibiting growth, in causing sterilization, in pro- ducing necrosis and burns, in causing recession of malignant tissue, and per- haps also, on occasion at least, in induc- ing the initiation of such tissue. During the war years, a curious con- firmation of the correctness of the above inference regarding the nature of the somatic effects of irradiation has come to light. While working with mustard gas in Edinburgh, J. H. Robson was struck with the remarkable similarity be- tween the somatic effects of this agent and those produced by X-ray and radi- um irradiation. This led him to wonder whether perhaps mustard gas might pro- duce genetic changes of essentially the same kind as those known to be brought about by irradiation. Comprehensive ex- periments were thereupon undertaken by C. Auerbach, working in collabora- tion with Robson, and (as mentioned on p. 263) she succeeded in showinqj that in fact this substance does produce mutations, both in the individual genes and by breakage and rearrangement of chromosome parts, such as X-rays and radium do, and in similar abundance. Other substances of the same general group were then found to have a similar effect. This constitutes the first decided break in the chemical attack on muta- tion. The fact that these findings were made as a direct result of the above in- ference, when so many previous at- tempts to produce mutations by chemi- cal means had failed, appears to provide strong evidence that these peculiar so- matic effects are in truth consequences of the more underlving ones which, when occurring in the germ cells, are analvzed bv the geneticist in his breeding tests. There are, however, some very interest- ing differences between the nature of the genetic effects of irradiation and of these chemicals, which we cannot go into here, but which give promise of allowing an extension of the genetic and somatic analyses. We see then that production of muta- tions by radiation is a method, capable of being turned in various directions, both for the analysis of the germ plasm itself, and of the organism which is in a sense an outgrowth of that germ plasm. It is to be hoped that it may also, in certain fields, prove of increasing prac- tical use in plant and animal improve- ment, in the service of man. So far as direct practical application in man him- self is concerned, however, we are as yet a long way from practicing any in- tentional selection over our own germ plasm, although like most species we are already encumbered by countless un- desirable mutations, from which no in- dividual is immune. In this situation we can, however, draw the practical lesson, from the fact of the great majority of mutations being undesirable, that their further random production in ourselves should so far as possible be rigorously avoided. As we can infer with certainty from experiments on lower organisms that all high-energy radiation must pro- duce such mutations in man, it becomes an obligation for radiologists — though one far too little observed as yet in most countries — to insist that the simple pre- cautions are taken which are necessary for shielding the gonads, whenever peo- nle are exposed to such radiation, either in industry or in medical practice. And, with the coming increasing use of atomic energy, even for peacetime purposes, the problem will become very important of insuring that the human germ plasm — the all-important material of which we are the temporarv custodians — is effec- tively protected from this additional and Dotent source of permanent contamina- tion. S-26 SUPPLEMENT IV GENES AND CHEMICAL REACTIONS IN NEUROSPORA by George W. Beadle. Pasadena, California, California Institute of Technology. Nobel Lecture, December ii, 1958. On this occasion of sharing the high honor of a Nobel Award with Edward L. Tatum for our ". . . discovery that genes act by regulating chemical events", and with Joshua Lederberg for his related ". . . discoveries concerning the organization of the genetic material of bacteria", it seems appropriate that I sketch briefly the background events that led to the work on Neurospora thaf Tatum and I initiated in 1940. I shall leave to my co-recipients of the Award the task of describing in detail the developments in Neurospora that followed our first success, and the relation of this to the rise of bacterial genetics, which has depended largely on studies of genetic recombination following con- jugation and transduction. I shall make no attempt to review the entire history of biochemical genetics, for this has been done elsewhere (2, 13, 22, 23). Anthocyanins and Alcaptonuria. Soon after de Vries, Correns and Tschermak "rediscovered" Mendel's 1865 paper and appreciated its full significance, investigators in the exciting new field, which was to be called genetics, naturally speculated about the physical nature of the "elements" of Mendel and the manner of their action. Renamed genes, these units of inheritance were soon found to be carried in the chromosomes. One line of investigation that was destined to reveal much about what genes do was started by Wheldale (later Onslow) in 1903. It began with a genetic study of flower pigmentation in snapdragons. But soon the genetic observations began to be correlated with the chemistry of the anthocyanin and related pigments that were responsible. The material was favorable for s-27 both genetic and chemical studies and the work has continued to yield new information ever since and almost without interruption. Many workers and many species of plants have been involved (2, 4, 13, 22, 23). It became clear very soon that a number of genes were involved and that they acted by somehow controlling the onset of various identifiable and specific chemical reactions. Since an understanding of the genetics helped in interpreting the chemistry and vice versa, the anthrocyanin work was well known to both geneticists and biochemists. It significantly influenced the thinking in both fields and thus had great importance in further develop- ments. A second important line of investigation was begun even earlier by the Oxford physician-biochemist Sir Archibald E. Garrod. At the turn of the century he was interested in a group of congenital metabolic diseases in man, which he later named, "inborn errors of metabolism". There are now many diseases described as such; in fact, they have come to be recognized as a cate- gory of diseases of major medical importance. One of the first inborn errors to be studied by Garrod was alcaptonuria. Its most striking symptom is blackening of urine on exposure to air. It had been recorded medically long before Garrod became interested in it and important aspects of its biochemistry were understood. The substance respon- sible for blackening of the urine is alcapton or homogenetisie acid (2,5-dihydr- oxyphenylacetic acid). Garrod suggested early that alcaptonuria behaved in inheritance as though it were differentiated by a single recessive gene. By 1908 a considerable body of knowledge about alcaptonuria had accumu- lated. This was brought together and interpreted by Garrod in his Croonian lectures and in the two editions of his book, "Inborn Errors of Metabolism", which were based on them (11). It was his belief that alcaptonuria was the result of inability of affected individuals to cleave the ring of homogentisic acid as do normal individuals. He believed this to be due to absence or inactivity of the enzyme that normally catalyzes this reaction. This in turn was depen- dent on the absence of the normal form of a specific gene. Thus Garrod had clearly in mind the concept of a gene-enzyme-chemical- reaction system in which all three entities were interrelated in a very specific way. In the 1923 edition of "Inborn Errors" (11) he wrote: "We may further conceive that the splitting of the benzene ring of homo- gentisic acid in normal metabolism is the- work of a special enzyme, that in congenital alcaptonuria this enzyme is wanting ..." Failure to metabolize an intermediate compound when its normal pathway s-28 is thus blocked by a gene-enzyme defect was a part of the interpretation and accounted for the accumulation and excretion of homogentisic acid. Garrod recognized this as a means of idantifying an intermediate compound that might otherwise not appear in sufficient amounts to be detected. He also clearly appreciated that alcaptonurics would be used experimentally to explore the metabolic pathways by which homogentisic acid was formed. He summarized a large body of evidence indicating that when normal precursors of homogentisic acid are fed to alcaptonurics there is an almost quantitative increase in homogentisic acid excretion. In this way evidence was accumulated that phenylalanine, tyrosine and the keto acid analog of the latter were almost certainly the direct precursors of homogentisic acid. Despite the simplicity and elegance of Garrod's interpretation of alcap- tonuria and other inborn errors of metabolism as gene defects which resulted in inactivity of specific enzymes and thus in blocked reactions, his work had relatively little influence on the thinking of the geneticists of his time. Bate- son's ''Mendel's Principles of Heredity" and a few other books of its time discuss the concept briefly. But up to the 1940's, no widely used later text book of genetics that I have examined even so much as refers to alcaptonuria. It is true that a number of other workers had seriously considered that genes might act in regulating chemical reactions by way of enzymes (2, 13, 17, 21, 23). But there was no other known instance as simple as alcaptonuria. It is interesting — and significant, I think — that it was approximately 50 years after Garrod proposed his hypothesis before it was anything like fully verified through the resolution into six enzymatically catalyzed steps of phenyl- alanine-tyrosine metabolism via the homogentisic acid pathway, and by the clear demonstration that homogentisate oxidase is indeed lacking in the liver of an alcaptonuric (17). Perhaps it is also well to recall that it was not until 1926 that the first enzyme was isolated in crystalline form and shown in a convincing wa}' to consist solely of protein. Eye Pigments of Drosophila. I shall now shift to a consideration of an independent line of investigation that ended up with conclusions very much like those of Garrod and which led directly to the work with Neurospora that Tatum and I subsequently began. In 1933, Boris Ephrussi came to the California Institute of Technology to work on developmental aspects of genetics. During his stay he and I had many long discussions in which we deplored the lack of information about s-29 the manner in which genes act on development. This we ascribed to the fact that the classical organisms of experimental embryology did not lend them- selves readily to genetic investigation. Contrariwise, those plants and animals about which most was known genetically had been little used in studies of development. It would be worth-while, we Ix^lieved, to attempt to remedy this situation by finding new ways experimentally to study Drosophila melanogaster — which, genetically, was the best understood organism of the time. Tissue culture technics seemed to offer hope. In the spring of 1935 we joined forces in EuPHRUSSi's section of ITnstitut de Biologic physio-chimique in Paris, resolved to find ways of culturing tissues of the larvae of Drosophila. After some discouraging preliminary attempts, we followed Ephrussi's suggestion and shifted to a transplantation technic. It was our hope that in this way we could make use of non-autonomous genetic characters as a means 01 investigating gene action in development. Drosophila larvae are small. And we were told by a noted Sorbonne authority on the development of diptera that the prospects were not good. In fact, he said, they were terrible. But we were determined to try, so returned to the laboratory, made micro- pipettes, dissected larvae and attempted to transfer embryonic buds from one larva to the body cavity of another. The results were discouraging. But we persisted, and finally one day discovered we had produced a fly with three eyes. Although our joy was great with this small success, we immediately began to worry about three points: First, could we do it again? Second, if we could, would we be able to characterize the diffusible substances responsible for interactions between tissues of different genetic types? And, third, how many non-autonomous characters could we find? We first investigated the sex-linked eye-color mutant vermilion because of the earlier finding of Sturtevant that in gynandromorphs genetically vermihon eye tissue often fails to follow the general rule of autonomy (20). Gynandromorphs may result if in an embryo that begins development as a female from an egg with two X chromosomes, one X chromosome is lost during an early cleavage, giving rise to a sector that has one X chromosome and is male. If the original egg is heterozygous for a sex-linked gene, say ver- milion, and the lost chromosome carries the normal aueie, the male sector will be genetically vermilion, whereas the female parts are normal or wild type. (Other sex-linked characters like yellow body or forked bristles can be used as markers to independently reveal genetic constitution in most parts of the body.) s-30 Yet in Sturtevant's gynandromorphs in which onty a small part of the body including eye tissue was vermilion, the appearance of that tissue was usually not vermilion but wild type — as though some substance had diffused from wild-type tissue to the eye and caused it to become normally pigmented. It was on the bsisis of this observation that Ephrussi and I transplanted vermilion eyes into wild type larvae. The result was as expected — the trans- planted eyes were indeed wild type. At that time there were some 26 separate eye-color genes known in Uroso- phila. We obtained stocks of all of them and made a series of transplants of mutant eyes into wild-type hosts. We found only one other clear-cut non- autonomous eye character. This was cinnabar, a bright red eye color, like ver- milion but differentiated by a second chromosome recessive gene. We had a third less clear case, claret, but this was never entirely satisfactory from an experimental point of view because it was difficult to distinguish claret from wild-type eyes in transplants. The vermilion and cinnabar characters are alike in appearance; both lack the brown pigment of the wild-type fly but retain the bright red component. Were the diffusible substances that caused them to develop brown pigment when grown in wild-type hosts the same or different? If the same, reciprocal transplants between the two mutants should give mutant transplanted eyes in both cases. If two separate and independent substances were involved, such reciprocal transplants should give wild-type transplanted eyes in both in- stances. We made the experiment and were much puzzled that neither of these results was obtained. A cinnabar eye in a vermilion host remained cinnabar, but a vermilion eye in a cinnabar host became wild type. To explain this result we formulated the hypothesis that there must be two diffusible substances involved, one formed from the other according to the scheme: ^ Precursor -> t;+ substance -^ cn+ substance -+ Pigment . . . where ^' + substance is a diffusible material capable of making a vermilion eye become wild type and cw+ substance is capable of doing the same to a cinnabar eye (9). The vermilion {v) mutant gene blocks the first reaction and the cinnabar {en) mutant gene interrupts the second. A vermilion eye in a cinnabar host makes pigment because it can, in its own tissues, convert the r + substance into CM + substance and pigment. In it, the second reaction is not blocked. This scheme involves the following concepts: a. A sequence of two gene-regulated chemical reactions, one gene identified with each. s-3] b. The accumulation of intermediates prior to blocked reactions. c. The ability of the mutant blocked in the first reaction to make use of an intermediate accumulated as a result of a genetic interruption of the second reaction. The principle involved is the same as that employed in the cross- feeding technic later so much used in detecting biosynthetic intermediates in microorganisms. What was later called the one gene-one enzyme concept was clearly in our minds at this time although as I remember, we did not so designate it. Ours was a scheme closely similar to that proposed by Garrod for alcap- tonuria, except that he did not have genes that blocked an adjacent reaction in the sequence. But at the time we were oblivious of Garrod's work, partly because geneticists were not in the habit of referring to it, and partly through failure of ourselves to explore the literature. Garrod's book was available in many libraries. We continued the eye-color investigations at the California Institute of Technology, Ephrussi having returned there to spend part of 1936. Late in the year, Ephrussi returned to Paris and I went for a year to Harvard, both continuing to work along similar lines. We identified the source of dif- fusible substances — fat bodies and malpighian tubercules — and began to devise ways of determining their chemical nature. In this I collaborated to some extent with Professor Kenneth Thimann. In the fall of 1937 I moved to Stanford, where Tatum shortly joined me to take charge of the chemical aspects identifying the eye-color substances. Dr. Yvonne Khouvine worked in a similar rcle with Ephrussi. We made progress slowly. Ephrussi and Khouvine discovered that under certain conditions feeding tryptophane had an effect on vermilion eye color. Following this lead, Tatum found — through accidental contamination of an asceptic culture containing tryptophane and test flies — an aerobic Bacillus that converted tryptophane into a substance highly active in inducing formation of brown pigment in vermilion flies. He soon isolated and crystallized this, but its final identification was slowed down by what later proved to be a sucrose molecule esterified with the active compound. Professor Butenandt and co-workers (6) in Germany who had been col- laborating with Professor Kuhn on an analogous eye-color mutant in the meal moth Ephestia, and Amano et al. (i), working at Osaka University, showed that t;+ substance was kynurinine. Later, Butenandt and Hallmann (5), and Butenandt et al. (7) showed that our original cw+ substance was 3-hydroxy kynurenine . s-32 Thus was established a reaction series of the kind we had originally con- ceived. Substituting the known chemical, it is as follows: H H I I -C— C— COOH I I H NHj Tryptophan / O H H il I I -C— C— C— COOH yCVfCt N-Formylkynurenine NH 0 H H I ! i -C— C— C— COOH I i H NH2 Kynurenine NH, en O H H II I I — C— C— C — COON I 1 H NH 04 H.. 3-hydroxykynurenine OH 1 Brown Pigment A Neiv Approach. Isolating the eye-pigment precursors of Drosophila was a slow and dis- couraging job. Tatum and I realized this was likely to be so in most cases of attempting to identify the chemical disturbances underlying inherited ab- normalities; it would be no more than good fortune if any particular example chosen for investigation should prove to be simple chemically. Alcaptonuria was such a happy choice for Garrod, for the chemistry had been largely s-33 worked out and the homogentisic acid isolated and identified many years before. Our idea — to reverse the procedure and look for gene mutations that in- fluence known chemical reactions — was an obvious one. It followed logically from the concept that, in general, enzymatically catalyzed reactions are gene- dependent, presumably through genie control of enzyme specificity. Although we were without doubt influenced in arriving at this approach by the antho- cyanin investigations, by Lwoff's demonstrations that parasites tend to become specialized nutritionally through loss of ability to synthesize sub- stances that they can obtain readily from their hosts (i8), and by the specula- tions of others as to how genes might act, the concepts on which it was based developed in our minds fairly directly from the eye-color work Ephrussi and I had started five years earlier. The idea was simple: Select an organism like a fungus that has simple nutri- tional requirements. This will mean it can carry out many reactions by which amino acids and vitamins are made. Induce mutations by radiation or other mutagenic agents. Allow meiosis to take place so as to produce spores that are genetically homogeneous. Grow these on a medium supplemented with an array of vitamins and amino acids. Test them by vegetative transfer to a medium with no supplement. Those that have lost the ability to grow on the minimal medium will have lost the ability to synthesize one or more of the substances present in the supplemented medium. The growth requirements of the deficient strain would then be readily ascertained by a systematic series of tests on partially supplemented media. In addition to the above specifications, we wanted an organism well suited to genetic studies, preferably one on which the basic genetic work had already been done. Neurospora. As a graduate student at Cornell, I had heard Dr. B. 0. Dodge of the New York Botanical Garden give a seminar on inheritance in the bread mold Neurospora. So-called second division segregation of mating types and of albinism were a puzzle to him. Several of us who had just been reviewing the evidence for 4-strand crossing over in Drosophila suggested that crossing over between the centromere and the segregating gene could well explain the result. Dodge was an enthusiastic supporter of Neurospora as an organism for genetic work. "It's even better than Drosophila", he insisted to Thomas Hunt Morgan, whose laboratory he often visited. He finally {)ersuaded Morgan s-34 to take a collection of Neurospora cultures with him from Columbia to the new Biology Division of the Cahfornia Institute of Technology, which he established in 1928. Shortly thereafter when Carl C. Lindegren came to Morgan's laboratory to become a graduate student, it was suggested that he should work on the genetics of Neurospora as a basis for his thesis. This was a fortunate choice, for Linde- gren had an abundance of imagination, enthusiasm and energy and at the same time had the advice of E. G. Anderson, C. B. Bridges, S. Emerson, A. H. Sturtevant and others at the Institute who at that time were actively interested in problems of crossing over as a part of the mechanism of meiosis. In this favorable setting, Lindegren soon worked out much of the basic genetics of Neurospora. New characters were found and a good start was made toward mapping the chromosomes. Thus, Tatum and I realized that Neurospora was genetically an almost ideal organism for use in our new approach. There was one important unanswered question. We did not know the mold's nutritional requirements. But we had the monograph of Dr. Nils Fries, which told us that the nutritional requirements of a number of related filamentous fungi were simple. Thus encouraged, we obtained strains of Neurospora crassa from Lindegren and from Dodge. Tatum soon discovered that the only growth factor required, other than the usual inorganic salts and sugar, was the recently discovered vitamin, biotin. We could not have used Neurospora for our purposes as much as a year earlier, for biotin would not then have been available in the quantities we required. It remained only to irradiate asexual spores, cross them with a strain of the opposite mating type, allow sexual spores to be produced, isolate them, grow them on a suitably supplemented medium and test them on the un- supplemented medium. We believed so thoroughly that the gene-enzyme- reaction relation was a general one that there was no doubt in our minds that we would find the mutants we wanted. The only worry we had was that their frequency might be so low that we would get discouraged and give up before finding one. We were so concerned about the possible discouragement of a long series of negative results that we prepared more than thousand single spore cultures on supplemented medium before we tested them. The 299th spore isolated gave a mutant strain requiring vitamin B6 and the i 085th one required Bi. We made a vow to keep going until we had 10 mutants. We soon had dozens. Because of the ease of recovery of all the products of a single meiotic process s-35 n Neurospora, it was a simple matter to determine whether our newly induced nutritional deficiencies were the result of mutations in single genes. If they were, crosses with the original should yield four mutant and four non-mutant spores in each spore sac. They did (3, 21). In this long, roundabout way, first in Drosophila and then in Neurospora, we had rediscovered what Garrod had seen so clearly so many years before. By now we knew of his work and were aware that we had added little if any- thing new in principle. We were working with a more favorable organism and were able to produce, almost at will, inborn errors of metabolism for almost any chemical reaction whose product we could supply through the medium. Thus we were able to demonstrate that what Garrod had shown for a few genes and a few chemical reactions in man was true for many genes and many reactions in Neurospora. In the fall of 1941 Francis J. Ryan came to Stanford as a National Research Council Fellow and was soon deeply involved in the Neurospora work. A year later David M. Bonner and Norman H. Horowitz joined the group. Shortly thereafter Herschel K. Mitchell did hkewise. With the collaboration of a number of capable graduate students and a group of enthusiastic and able research assistants the work moved along at a gratifying pace. A substantial part of the financial support that enabled us thus to expand our efforts was generously made available by the Rockefeller Foundation and the Nutrition Foundation. The directions of our subsequent investigations and their accomplishments I shall leave to Professor Tatum to summarize. One Gene — One Enzyme. It is sometimes thought that the Neurospora work was responsible for the one gene — one enzyme hypothesis — the concept that genes in general have single primary functions, aside from serving an essential role in their own replication, and that in many cases this function is to direct specificities of enzymatically active proteins. The fact is that it was the other way around — the hypothesis was clearly responsible for the new approach. Although it may not have been stated explicitly, Ephrussi and I had some such concept in mind. A more specific form of the hypothesis was suggested by the fact that of all the 26 known eye-color mutants in Drosophila, there was only one that blocked the first of our postulated reactions and one that similarly interrupted the second. Thus it seemed reasonable to assume that s-36 the total specificity of a particular enzyme might somehow be derived from a single gene. The finding in Neurospora that many nutritionally deficient mutant strains can be repaired by supplying single chemical compounds was a verification of our prediction and as such reinforced our belief in the hypoth- esis, at least in its more general form. As I hope Professor Tatum will point out in detail, there are now known a number of instances in which mutations of independent origin, all abolishing or reducing the activity of a specific enzyme, have been shown to involve one small segment of genetic material (8, 12, 24). To me these lend strong support to the more restricted form of the hypothesis. Regardless of when it was first written down on paper, or in what form, I myself am convinced that the one gene-one enzyme concept was the product of gradual evolution beginning with Garrod and contributed to by many, including Moore, Goldschmidt, Troland, Haldane, Wright, Gruneberg and many others (2, 13, 19, 22, 23). Horowitz and his co-workers (15, 16) have given it, in both forms referred to above, its clearest and most explicit formulation. They have summarized and critically evaluated the evidence for and against it, with the result that they remain convinced of its continued value. In additition Horowitz has himself made an important appHcation of the concept in arriving at a plausible hypothesis as to how sequences of biosyn- thetic reactions might originally have evolved (14). He points out that many biologically important compounds are known to be synthesized in a stepwise manner in which the intermediate compounds as such seem not to serve useful purposes. How could such a synthetic pathway have evolved if it serves no purpose unless complete? Simultaneous appearance of several independent enzymes would of course be exceedingly improbable. Horowitz proposes that the end product of such a series of reactions was at first obtained directly from the environment, it having been produced there in the first place by non-biological reactions such as have been postu- lated by a number of persons, including Darwin, Haldane, Oparin and Urey and demonstrated by Miller, Fox and others (10). It is then possible reasonably to assume that the ability to synthesize such a compound biologically could arise by a series of separate single mutations, each adding successive enzy- matically catalyzed steps in the synthetic sequence, starting with the one im- mediately responsible for the end product. In this was each mutational step could confer a selective advantage b}^ making the organism dependent on one less exogenous precursor of a needed end product. Without some such mechanism, by which no more than a single gene mutation is required for the s-37 origin of a new enzyme, it is difficult to see how complex synthetic pathways could have evolved. I know of no alternative hypothesis that is equally simple and plausible. The Place of Genetics in Modern Biology, In a sense genetics grew up as an orphan. In the beginning botanists and zoologists were often indifferent and sometimes hostile toward it. "Genetics deals only with superficial characters", it was often said. Biochemists like- wise paid it little heed in its early days. They, especially medical biochemists, knew of Garrod's inborn errors of metabolism and no doubt appreciated them in the biochemical sense and as diseases; but the biological world was inadequately prepared to appreciate fully the significance of his investigations and his thinking. Geneticists, it should be said, tended to be preoccupied mainly with the mechanisms by which genetic material is transmitted from one generation to the next. Today, happily, the situation is much changed. Genetics has an estabhshed place in modern biology. Biochemists recognize the genetic material as an integral part of the systems with which they work. Our rapidly growing knowl- edge of the architecture of proteins and nucleic acids is making it possible — for the first time in the history of science — for geneticists, biochemists and biophysicists to discuss basic problems of biology in the common language of molecular structure. To me, this is most encouraging and significant. REFERENCES. 1. Amano, T., M. Torii, and H. Iritani, Med. J. Osaka Univ., 2, 45 (1950). 2. Beadle, G. W., Chem. Rev. 37, 15 (1945)- 3. — and E. L. Tatum, Proc. Nat. Acad. Sci. (U. S. A.), 27. 499 (1941)- 4. Beale, G. H. J. Genetics, 42, 196 (1941)- 5. BuTENANDT, A. and G. Kallmann, Z. Naturforsch., 5b, 444 (1950)- 6. — W. Weidel, and E. Becker, Naturwiss., 28, 63 (1940). 7. and H. Schlossberger, Z. Naturforsch., 4b, 242 (1949). 8. Demerec, M., Z. Hartman, P. E. Hartman, T. Yura, J. S. Gots, H. Ozeki, and S. W. Glover, Publication 612, Carnegie Inst. Wash. (1956). 9. Ephrussi, B., Quart. Rev. Biol. 17, 327 (1942). 10. Fox, S. W., Amer. Sci. 44, 347 (1956). 11. Garrod, a. E., Inborn Errors of Metabolism, O.xford Univ. Press (i9-23)- 12. Giles, N. H., Proc. X Int. Cong. Genetics (in Press). 13. Haldane, J. B. S., The Biochemistry of Genetics, London, Allen & Unwin (1954)- 14. Horowitz, N. H., Proc. Nat. Acad. Sci. (U. S. A.), 31, 153 (i945)- 15. Horowitz, N. H., and M. Fling, In '''Enzymes", p. 139 (Gaebler, O. H., Ed.), New York, Academic Press (1956). S-38 i6. Horowitz, N. H., and U. Leopold, Cold Spring Harbor Symp. Quant. Biol., i6- 65 (1951)- 17. Knox, W. E., Am. J. Human Genetics, 10, 95 (1958). 18. LwoFF, A., L'evolution physiologique, Paris, Hermann et Cie {1944). 19. MuLLER, H. J., Proc. Royal See. (London) B 134, i (1947). 20. Sturtevant, a. H., Proc. VI Int. Cong. Genetics, i, 304 (1932). 21. Tatum, E. L., and Beadle, G. W., Proc. Nat. Acad. Sci. (U. S. A.), 28, 234 (1942). 22. Wagner, R. P. and H. K. Mitchell, Genetics and Metabolism, New York, Wilev (1955)- 23. Wright, S., Physiol. Rev., 21, 487 (1941). 24. Yanofsky, C, In '"Enzymes", p. 147 (Gaebler, O. H., Ed.), New York, Academic Press I 11956). s-39 SUPPLEMENT V A CASE HISTORY IN BIOLOGICAL RESEARCH. By E. L. Tatum. Nobel Lecture, December ii, 1958. In casting around in search of a new approach, an important consideration was that much of biochemical genetics has been and will be covered by Pro- fessor Beadle and Professor Lederberg, and in many symposia and reviews, in which many aspects have been and will be considered in greater detail and with greater competence than I can hope to do here. It occurred to me that perhaps it might be instructive, valuable, and interesting to use the approach which I have attempted to define by the title "A Case History in Biological Research". In the development of this case history I hope to point out some of the factors involved in all research, specifically the dependence of scientific progress: on knowledge and concepts provided by investigators of the past and present all over the world; on the free interchange of ideas within the international scientific community; on the hybrid vigor resulting from cross-fertilization between disciphnes; and last but not least, also depend- ent on chance, geographical proximity, and opportunity. I would like finally to complete this case history with a brief discussion of the present status of the field, and a prognosis of its possible development. Under the circumstances, I hope I will be forgiven if this presentation is given from a personal viewpoint. After graduating from the University of Wisconsin in chemistry, I was fortunate in having the opportunity of doing graduate work in biochemistry and microbiology at this University under the direction and leadership of W. H. Peterson and E. B. Fred. At that time, in the early 30's, one of the exciting areas being opened concerned the so- called "growth-factors" for microorgaisms, for the most part as yet mysterious and unidentified. I became deeply involved in this field, and was fortunate to have been able, in collaboration with H. G. Wood, then visiting at Wiscon- sin, to identify one of the required growth-factors for propionic acid bacteria, as the recently synthesized vitamin Bi or thiamine (i). This was before the s-40 universality of need for the B vitamins, and the enzymatic basis of this require- ment, had been clearly defined. The vision of Lwoff and Knight had already indicated a correlation of the need of microorganisms for "growth-factors" with failure of synthesis, and correlated this failure with evolution, particularly in relation to the complex environment of "fastidious" pathogenic micro- organisms. However, the tendency at this time was to consider "growth- factors" as highly individual requirements, peculiar to particular strains or species of microorganisms as isolated from nature, and their variation in these respects was not generally considered as related to gene mutation and varia- tion in higher organisms. Actually my ignorance of and naivete in genetics was probably typical of that of most biochemists and microbiologists of the time, with my only contact with genetic concepts being a course primarily on vertebrate evolution. After completing .graduate work at Wisconsin I was fortunate in being able to spend a year studying at the University of Utrecht with F. Kogl, the dis- coverer of the growth factor biotin, and to work in the same laboratory with Nils Fries, who already had contributed significantly in the field of nutrition and growth of fungi. At this time. Professor Beadle was just moving to Stanford University, and invited me as a biochemist to join him in the further study of the eye- color hormones of Drosophila, which he and Ephrussi in their work at the California Institute of Technology and at Paris had so briUiantly established as diffusible products of gene-controlled reactions. During this, my first con- tacts with modem genetic concepts, as a consequence of a number of factors — the observation of Khouvine, Ephrussi and Chevais (2) in Paris that dietary tryptophane was concerned with Drosophila eye-color hormone pro- duction; our studies on the nutrition of Drosophila in aseptic culture (3); and the chance contamination of one of our cultures of Drosophila with a particular bacterium — we were able to isolate the v+ hormone in crystalline state from a bacterial culture supplied with tryptophane (4), and with A. J. Haagen- Smit to identify it as kynurenine (5); originally isolated by Kotake, and later structurally identified correctly by Butenandt. It might be pointed out here that kynurenine has since been recognized to occupy a central position in tryptophane metabolism in many organisms aside from insects, including mammals and fungi. At about this time, as the result of many discussions and considerations of the general biological applicabihty of chemical genetic concepts, stimulated by the wealth of potentialities among the microorganisms and their variation S-41 in nature with respect to their nutritional requirements, we began our work with the mold Neurospora crassa. I shall not renumerate the factors involved in our selection of this organism for the production of chemical or nutritionally deficient mutants, but must take this opportunity of reiterating our indebtedness to the previous basic findings of a number of investigators. Foremost among these, to B. O. Dodge for his establishment of this Ascomycete as a most suitable organism for genetic studies (6); and to C. C. Lindegren (7), who became interested in Neurospora through T. H. Morgan, a close friend of Dodge. Our use of Neurospora for chemical genetic studies would also have been much more difficult, if not impossible, without the availability of synthetic biotin as the result of the work of Kogl (8) and of du Vigneaud (9). In addi- tion, the investigations of Nils Fries on the nutrition of Ascomycetes (10) were most helpful, as shown by the fact that the synthetic minimal medium used with Neurospora for many years was that described by him and supple- mented only with biotin, and has ordinarily since been referred to as "Fries medium". It should also be pointed out that the experimental feasibihty of producing the desired nutritionally deficient mutant strains depended on the early pioneering work of Roentgen, with X-Rays, and on that of H. J. Muller, on the mutagenic activity of X-Rays and ultraviolet light on Drosophila. All that was needed was to put these various facts and findings together to produce in the laboratory with irradiation, nutritionally deficient (auxotrophic) mutant strains of Neurospora, and to show that each single deficiency produced was associated with the mutation of a single gene (11). Having thus successfully tested with Neurospora the basic premise that the biochemical processes concerned with the synthesis of essential cell constituents are gene controlled, and alterable as a consequence of gene mutation, it then seemed a desirable and natural step to carry this approach to the bacteria, in which so many and various naturally occurring growth-factor requirements were known, to see if analogous nutritional deficiencies followed their exposure to radiation. As is known to all of you, the first mutants of this type were successfully produced in Acetohacter and in E. coli (12), and the first step had been taken in bringing the bacteria into the fold of organisms suitable for genetic study. Now to point out some of the curious coincidences or twists of fate as in- volved in science: One of the first series of mutants in Neurospora which was studied intensily from the biochemical viewpoint was that concerned with the biosynthesis of tryptophan. In connection with the role of indole as a precursor s-42 of tryptophan, we wanted also to study the reverse process, the breakdown of tryptophan to indole, a reaction typical of the bacterium E. coli. For this purpose we obtained, from the Bacteriology Department at Stanford, a typical E. coli culture, designated K-12. Naturally, this strain was later used for the mutation experiments just described so that a variety of biochemically marked mutant strains of E. coli K-12 were soon available. It is also of interest that Miss Esther Zimmer, who later became Esther Lederberg, assisted in the production and isolation of these mutant strains. Another interesting coincidence is that F. J. Ryan spent some time on leave from Columbia University at Stanford, working with Neurospora. Shortly after I moved to Yale University in 1945, Ryan encouraged Lederberg, then a medical student at Columbia who had worked some with Ryan on Neurospora, to spend some time with me at Yale University. As all of you know, Lederberg^ was successful in showing genetic recombination between mutant strains of E. coli K-12 (13) and never returned to medical school, but continued his brilliant work on bacterial recombination at Wisconsin. In any case, the first demonstration of a process analogous to a sexual process in bacteria was successful only because of the clear-cut nature of the genetic markers available which permitted detection of this very rare event, and because of the combination of circumstances which had provided those selec- tive markers in one of the rare strains of E. coli capable of recombination. In summing up this portion of this case history, then, I wish only to emphasize again the role of coincidence and chance played in the sequence of develop- ments, but yet more strongly to acknowledge the even greater contributions of my close friends and associates. Professor Beadle and Professor Lederberg, with whom it is a rare privilege and honor to share this award. Now for a brief and necessarily somewhat superficial mention of some of the problems and areas of biology to which these relatively simple experiments with Nerospora have led and contributed. First, however, let us review the basic concepts involved in this work. Essentially these are (i) that all bio- chemical processes in all organisms are under genie control, {2) that these overall biochemical processes are resolvable into a series of individual stepwise reactions, (3) that each single reaction is controlled in a primary fashion by a single gene, or in other terms, in every case a i : i correspondence of gene and biochemical reaction exists, such that (4) mutation of a single gene results only in an alteration in the ability of the cell to carry out a single primary chemical reaction. As has repeatedly been stated, the underlying hypothesis, which in a number of cases has been supported by direct experimental evidence, s-43 is that each gene controls the production, function and specificity of a particular enzyme. Important experimental implications of these relations are that each and every biochemical reaction in a cell of any organism, from a bacterium to man, is theoretically alterable by gene mutation, and that each such mutant cell strain differs in only one primary way from the non-mutant parental strain. It is probably unnecessary to point out that these experimental ex- pectations have been amply supported by the production and isolation, by many investigators during the last 15 or more years, of biochemical mutant strains of microorganisms in almost every species tried, bacteria, yeasts, algae, and fungi. It is certainly unnecessary for me to do more than point out that mutant strains such as those produced and isolated first in Neurospora and E. colt have been of primary utility as genetic markers in detecting and elucidating the details of the often exotic mechanisms of genetic recombination of micro- organisms. Similarly, it seems superfluous even to mention the proven usefulness of mutant strains of microorganisms in unraveling the detailed steps involved in the biosynthesis of vital cellular constituents. I would like to list, however, a few of the biosynthetic sequences and biochemical interrelationships which owe their discovery and elucidation largely to the use of biochemical mutants. These include: the synthesis of the aromatic amino acids via dehydroshikimic and shikimic acids (14, 15), by way of prephenic acid to phenylalanine (16), and by way of anthranilic acid, indole glycerol phosphate (17), and conden- sation of indole with serine to give tryptophan (18); the conversion of trypto- phan via kynurenine and 3-OH anthranilic acid to niacin (19, 20); the bio- synthesis of histidine (21); of isoleucine and valine via the analogous di-OH and keto acids (22); the biosynthesis of proline and ornithine from glutamic acid (23); and the synthesis of pyrimidines via orotic acid (24). If the postulated relationship of gene to enzyme is correct, several conse- quences can be predicted. First, mutation should result in the production of a changed protein, which might either be enzymatically inactive, of inter- mediate activity, or have otherwise detectably altered physical properties. The production of such proteins changed in respect to heat stability, enzymatic activity, or other properties such as activation energy, by mutant strains has indeed been demonstrated in a number of instances (25 — 31). Recognition of the molecular bases of these changes must await detailed comparison of their structures with those of the normal enzyme, using techniques similar to the elegant methods of Professor Sanger. That the primary effect of gene mutation s-44 may be as simple as the substitution of a single amino acid by another and may lead to profound secondary changes in protein structure and properties has recently been strongly indicated by the work of Ingram on hemoglobin (32). It seems inevitable that induced mutant strains of microorganisms will play a most important part in providing material for the further examination of these problems. A second consequence of the postulated relationship stems from the con- cept that the genetic constitution defines the potentialities of the cell, the time and degree of expression of which are to a certain extent modifiable by the cellular environment. The analysis of this type of secondary control at the biochemical level is one of the important and exciting new areas of biochemistry. This deals with the regulation and integration of biochemical reactions by means of feed-back mechanisms restricting the synthesis or activities of en- zymes {^^ — 36) and through substrate induced biosynthesis of enzymes (37). It seems probable that some gene mutations may affect biochemical activities at this level, (modifiers, and suppressors) and that chemical mutants will prove of great value in the analysis of the details of such control mechanisms. An equally fascinating newer area of genetics, opened by Benzer (38) with bacteriophage, is that of the detailed correlation of fine structure of the gene in terms of mutation and recombination, with its fine structure in terms of activity. Biochemical mutants of microorganisms have recently opened this area to investigation at two levels of organization of genetic material. The higher level relates to the genetic linkage of non-allelic genes concerned with sequential biosynthetic reactions. This has been shown by Demerec and by Hartmann in the biosynthesis of tryptophan and histidine by Salmonella (39)- At a finer level of organization of genetic material, the biological versatility of Neurospora in forming heterocaryotic cells has permitted the demonstration (40 — 42) that genes damaged by mutation in different areas, within the same locus and controlling the same enzyme, complement each other in a hetero- caryon in such a way that synthesis of enzymatically active protein is restored, perhaps, in a manner analogous to the reconstitution of ribonuclease from its a and b constituents, by the production in the cytoplasm of an active protein from two gene products defective in different areas. This phenomenon of complementation, which appears also to take place in Aspergillus (43), permits the mapping of genetic fine structure in terms of function, and should lead to further information on the mechanism of enzyme production and clarifica- tion of the role of the gene in enzyme synthesis. s-45 The concepts of biochemical genetics have already been, and will un- doubtedly continue to be, significant in broader areas of biology. Let me cite a few examples in microbiology and medicine. In microbiology the roles of mutation and selection in evolution are coming to be better understood through the use of bacterial cultures of mutant strains. In more immediately practical ways, mutation has proven of primary impor- tance in the improvement of yields of important antibiotics — such as in the classic example of penicillin, the yield of which has gone up from around 40 units per ml. of culture shortly after its discovery by Fleming to approximately 4 000, as the result of a long series of successive experimentally produced mutational steps. On the other side of the coin, the mutational origin of an- tibiotic resistant microorganisms is of definite medical significance. The therapeutic use of massive doses of antibiotics to reduce the numbers of bac- teria which by mutation could develop resistance, is a direct consequence of the application of genetic concepts. Similarly, so is the increasing use of com- bined antibiotic therapy, resistance to both of which would require the si- multaneous mutation of two independent characters. As an important example of the application of these same concepts of microbial genetics to mammalian cells, we may cite the probable mutational origin of resistance to chemotherapeutic agents in leukemic cells (44), and the increasing and effective simultaneous use of two or more chemotherapeutic agents in the treatment of this disease. In this connection it should be pointed out that the most effective cancer chemotherapeutic agents so far found are those which interfere with DNA synthesis, and that more detailed information on the biochemical steps involved in this synthesis is making possible a more rational design of such agents. Parenthetically, I want to emphasize the analogy between the situation in a bacterial culture consisting of two or more cell types, and that involved in the competition and survival of a malignant cell, regardless of its origin, in a population of normal cells. Changes in the cellular environment, such as involved in chemotherapy, would be expected to affect the metabolic efficiency of an altered cell, and hence its growth characteristics. However, as in the operation of selection pressures in bacterial populations, based on the interaction between cell types, it would seem that the effects of chemotherapeutic agents on the efficiency of selective pressures among mammalian cell populations can be examined nxusL effectively only in controlled mixed populations of the ceU types concerned. In other areas in cancer, the concepts of genetics are becoming increasingly important, both theoretically and practically. It seems probable that neoplastic s-46 changes axe directly correlated with changes in the biochemistry of the cell. The relationships between DNA, RNA, and enzymes which have evolved during the last few decades, lead one to look for the basic neoplastic change in one of these intimately interrelated hierarchies of cellular materials. In relation to DNA hereditary changes are now known to take place as a consequence of mutation, or of the introduction of new genetic material through virus infection (as in transduction) or directly (as in transformation). Although each of these related hereditary changes may theoretically be involved in can- cer, definite evidence is available only for the role of viruses, stemming from the classic investigations of ROUS on fowl sarcoma (45). At the RNA level of genetic determination, any one of these classes of change might take place, as in the RNA containing viruses, and result in an heritable change, perhaps of the cytoplasmic type, semi-autonomous with respect to the gene. At the protein level, regulatory mechanisms determining gene activity and enzyme synthesis as mentioned earlier, likewise provide promising areas for exploration. Among the many exciting applications of microbial-genetic concepts and techniques to the problems of cancer, may I mention in addition the explora- tion by Klein (46) of the genetic basis of the immunological changes which distinguish the cancer cell from the normal, and the studies on the culture, nutrition, morphology and mutation of isolated normal and malignant mam- malian cells of Puck (47) and of Eagle (48). Such studies are basic to our exploration and to our eventual understanding of the origin and nature of the change to malignanc}'. Regardless of the origin of a cancer cell, however, and of the precise genetic level at which the primary change takes place, it is not too much to hope and expect eventually to be able to correct or alleviate the consequences of the metabolic defect, just as a closer understanding of a heritable metabolic defect in man permits its correction or alleviation. In terms of biochemical genetics, the consequences of a metabolic block may be rectified by dietary limitation of the precursor of an injurious accumulation product, aromatic amino acids in phenylketonuria; or by supplying the essential end-product from without the cell, the specific blood protein in hemophilia, or a specific essential nutrient molecule such as a vitamin. Time does not permit the continuation of these examples. Perhaps, however, I will be pardoned if I venture briefly on a few more predictions and hopes for the future. It does not seem unrealistic to expect that as more is learned about control S-47 of cell machinery and heredity, we will see the complete conquering of many of man's ills, including hereditary defects in metabolism, and the momentarily more obscure conditions such as cancer and the degenerative diseases, just as disease of bacterial and viral etiology are now being conquered. With a more complete understandig of the functioning and regulation of gene activity in development and differentiation these processes may be more efficiently controlled and regulated, not only to avoid structural or metabolic errors in the developing organism, but also to produce better organisms. Perhaps within the lifetime of some of us here, the code of life processes tied up in the molecular structure of proteins and nucleic acids will be broken. This may permit the improvement of all living organisms by processes which we might call biological engineering. This might proceed in stages from the in vitro biosynthesis of better and more efficient enzymes, to the biosynthesis of the corresponding nucleic acid molecules, and to the introduction of these molecules into the genome of organisms, whether via injection, viral introduction into germ cells, or via a process analogous to transformation. Alternatively, it may be possible to reach the same goal by a process involving directed mutation. As a biologist, and more particularly as a geneticist, I have great faith in the versatiHty of the gene and of living organisms in providing the material with which to meet the challenges of life at any level. Selection, survival and evolution take place in response to environmental pressures of all kinds, in- cluding sociological and intellectual. In the larger view, the dangerous and often poorly understood and controlled forces of .modern civilization, including atomic energy and its attendant hazards, are but more complex and sophis- ticated environmental challenges of life. If man cannot meet those challenges, in a biological sense he is not fit to survive. However, it may confidently be hoped that with real understanding of the roles of heredity and environment, together with the consequent improvement in man's physical capacities and greater freedom from physical disease, will come an improvement in his approach to, and understanding of, sociological and economic problems. As in any scientific research, a problem clearly seen is already half solved. Hence, a renaissance may be foreseen, in which the major sociological problems will be solved, and mankind will take a big stride towards the state of world brotherhood and mutual trust and well- being envisaged by that great humanitarian and philanthropist Alfred Nobel. s-48 BIBLIOGRAPHY. 1. E. L. Tatum, H. G. Wood and W. H. Peterson, Biochem. J., jo, 1898, 1936. 2. Y. Khouvine, B. Ephrussi and S. Chevais, Biol. Bull., 75, 425, 1938. 3. E. L. Tatum, Proc. Nat. Acad. Sci., U. S., 27, 193, 1941. 4. — and G. W. Beadle, Science, pj, 458, 1940. 5. — and A. J. Haagen-Smit, J. Biol. 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The knowledge drawn in recent years from studies of bacterial transfor- mation (i) and viral infection of bacterial cells (2, 3) combined with other evi- dence (3), has just about convinced most of us that deoxyribonucleic acid (DNA) is the genetic substance. We shall assume then that it is DNA which not only directs the synthesis of the proteins and the development of the cell but that it must also be the substance which is copied so as to provide for a similar development of the progeny of that cell for many generations. DNA, like a tape recording, carries a message in which there are specific instructions for a job to be done. Also like a tape recording, exact copies can be made from it so that this information can be used again and elsewhere in time and space. Are these two functions, the expression of the code (protein synthesis) and the copying of the code (preservation of the race) closely integrated or are they separable? What we have learned from our studies over the past five years and what I shall present is that the replication of DNA can be examined and at least partially understood at the enzymatic level even though the secret of how DNA directs protein synthesis is still locked in the cell. DNA structure. First I should like to review very briefly some aspects of DNA structure which are essential for this discussion. Analysis of the composition of samples of DNA from a great variety of sources and by many investigators (4) revealed the remarkable fact that the purine content always equals the pyrimidine content. Among the purines, the adenine content may differ considerably from the guanine, and among the pyrimidines, the thymine from the cytosine. s-50 %^^c-c / " THYMINE GUANINE ^^,h ^ CTTOI— Fig. I. Hydrogen Bonding of Bases. However, there is an equivalence of the bases with an amino group in the 6-position of the ring, to the bases with a keto group in the 6-position. These facts were interpreted by Watson and Crick (5) in their masterful hypothesis on the structure of DNA. As shown in Fig. i, they proposed in connection with their double-stranded model for DNA, to be discussed presently, that the 6-amino group of adenine is linked by hydrogen bonds to the 6-keto group of thymine and in a like manner guanine is hydrogen-bonded to cytosine, thus accounting for the equivalence of the purines to the pyrimidines. On the basis of these considerations and the results of X-ray crystallographic measurements by WiLKiNS and associates (6), Watson and Crick proposed a structure for DNA in which two long strands are wound about each other in a helical manner. Fig. 2 is diagrammatic representation of a fragment of a DNA chain about ten nucleotide units long. According to physical measurements, DNA chains are on the average 10 000 units long. We see here the deoxypentose rings linked by phosphate residues to form the backbone of the chain; the purine and pyrimidine rings are the planar structures emerging at right angles from the main axis of the chain. Fig. 3 is a more detailed molecular model (7) and gives a better idea of the packing of the atoms in the structure. The purine and pyrimidine bases of one chain are bonded to the pyrimidine and purine bases of the complementary chain by the hydrogen bonds described in Fig. I. The X-ray measurements have indicated that the space between the opposing chains in the model agrees with the calculated value for the hydrogen-bond linkage of a purine to a pyrimidine; it is too small for two purines and too large for two pyrimidines. Most rewarding from the biological point of view, the structure provides a useful model to explain how cellular replication of DNA may come about. For, if you imagine that these two chains separate and that a new chain is formed complementary to each of them, the result will be two pairs of strands, each pair identical to the original parent duplex and identical to each other. S-51 Fig. 2. Double Helical Structure of DNA (Watson and Crick Mo- del). Enzymatic approach to the problem of DNA replication. Although we have in the Watson and Crick proposal a mechanical model of replication, we may at this point pose the question: "What is the chemical mechanism by which this super molecule is built up in the cell?" Some sixty years ago the alcoholic fermentation of sugar by a yeast cell was a "vital" process inseparable from the living cell, but through the Buchner discovery of fermentation in extracts and the march of enzymology during the first half of this century we understand fermentation by yeast as a, now familiar, sequence of integrated chemical reactions. Five years ago the synthesis of DNA was also regarded as a "vital" process. Some people considered it useful for biochemists to examine the combustion chambers of the cell, but tampering with the very genetic apparatus itself would surely produce nothing but dis- order. These gloomy predictions were not justified then nor are similar pessi- mistic attitudes justified now with regard to the problems of cellular structure s-52 O Hydrogen ^B Oxygen ^^ Carbon in ^B phosphate-ester ^^ chain ( ; Guanine ^m Cytosine ( . ) Adenine { : Thymine Fig. 3. Molecular Model of DNA ^^B Phosphorus (After M. Feughelman, et al. (7)). and specialized function which face us. High adventures in enzymology lie ahead and many of the explorers will come from the training fields of carbo- hydrate, fat, amino acid and nucleic acid enzymology. I feel now, as we did then, that for an effective approach to the problem of nucleic acid biosynthesis it was essential to understand the biosynthesis of the simple nucleotides and the coenzymes and to have these concepts and methodology well in hand. It was from these studies that we developed the conviction that an activated nucleoside 5 '-phosphate is the basic biosynthetic building block of the nucleic acids (8). You will recall that the main pathways of purine and pyrimidine biosynthesis all lead to the nucleoside 5 '-phosphate (8); they do not, except as salvage mechanisms, usually include the free bases or nucleosides. While the 2' and 3' isomers of the nucleotides are known, they probably arise mainly from certain types of enzymatic degradation of the nucleic acids. You will also recall from the biosynthesis of coenzymes (9), s-53 o o o II II 11 Adenosine -O-P tOP-O-P-O /> o" I o o o I Nucleoside - O - P - O' II O Fig. 4. Nucleophilic Attack of a Nucleoside Monophosphate on ATP. the simplest of the nucleotide condensation products, that it is ATP which condenses with nicotinamide mononucleotide to form diphosphopyridine nu- cleotide, with riboflavin phosphate to form FAD, with pantetheine phosphate to form the precursor of coenzyme A and so forth. This pattern has been amplified by the discovery of identical mechanisms for the activation of fatty acids and amino acids and it has been demonstrated further that uridine, cytidine and guanosine coenzymes are likewise formed from the respective triphosphates of these nucleosides. This mechanism (Fig. 4), in which a nucleophilic attack (10) on the pyro- phosphate-activated adenyl group by a nucleoside monophosphate leads to the formation of a coenzyme, was adopted as a working hypothesis for studying the synthesis of a DNA chain. As illustrated in Fig. 5, it was postulated that the basic building block is a deoxynucleoside 5 '-triphosphate which is attacked by the 3'-hydroxyl group at the growing end of a polydeoxynucleotide chain; inorganic pyrophosphate is eliminated and the chain is lengthened by one unit. The results of our studies on DNA synthesis, as will be mentioned presently, are in keeping with this type of reaction. Properties of the DNA-synthesizing enzyme. First let us consider the enzyme and comment on its discovery (8, 11, 12). Mixing the triphosphates of the four deoxynucleosides which commonly occur in DNA with an extract of thymus or bone-marrow or of Escherichia coli S-54 0 0-^-0'" I I HoC (T K^' O-P-O ^o i,.. HO H C^t: 0 0 o-p4o-^-o-f-o" Poly- nucleotide HO H XTP Fig.' 5- Postulated Mechanism for Extending a DNA Chain. would not be expected to lead to the net synthesis of DNA, Instead, as might be expected, the destruction of DNA by the extracts of such cells and tissues was by far the predominant process and one had to resort to the use of more subtle devices for detection of such a biosynthetic reaction. We used a C^*- labeled substrate of high specific radioactivity and incubated it with ATP and extracts of Escherichia coli, an organism which reproduces itself every 20 minutes. The first positive results represented the conversion of only a very small fraction of the acid-soluble substrate into an acid-insoluble fraction (50 or so counts out of a million added). While this represented only a few jufimoles of reaction, it was something. Through this tiny crack we tried to drive a wedge and the hammer was enzyme purification (13). This has been and still is a major preoccupation. Our best preparations are several thousand- fold enriched with respect to protein over the crude extracts, but there are still contaminating quantities of one or more of the many varieties of nuclease s-55 + DNA; DNA TP dGP dAP dCP •— — ' n + 4(n)PP Fig. 6. Equation for Enzymatic Synthesis of DNA. n TPPP n dGPPP n dAPPP n dCPPP and diesterase present in the coli cell. The occurrence of what appears to be a similar DNA synthesizing system in animal cells as well as in other bacterial species has been observed (14). We must wait for purification of the enzymes from these sources in order to make valid comparisons with the coli system. The requirements for net synthesis of DNA with the purified coli enzyme (15) are shown in the equation in Fig. 6. All four of the deoxynucleotides which form the adenine-thymine and guanine-cytosine couples must be present. The substrates must be the tri- and not the diphosphates and only the deoxy sugar compounds are active. DNA which must be present may be obtained from animal, plant, bacterial or viral sources and the best indications are that all these DNA samples serve equally well in DNA synthesis provided their molecular weight is high. The product, which we will discuss in further detail, accumulates until one of the substrates is exhausted and may be 20 or more times greater in amount than the DNA added and thus is composed to the extent of 95 % or more of the substrates added to the reaction mixture. Inorganic pyrophosphate is released in quantities equimolar to the deoxy- nucleotides converted to DNA. Should one of these substrates be omitted, the extent of reaction is di- minished by a factor of greater than 10* and special methods are now required for its detection. It turns out that when one of the deoxynucleotide substrates is lacking, an extremely small but yet significant quantity of nucleotide is linked to the DNA primer. We have described this so-called "limited reac- tion" (16), and have shown that under these circumstances a few deoxy- nucleotides are added to the nucleoside ends of some of the DNA chains but S-56 P-P: (P ^A A T (p) "-A c G (p) ^A T A (p) ^A G c X Y Fig. 7. Mechanism for Enzymatic DNA Replication. that further synthesis is blocked for lack of the missing nucleotide. Current studies suggest to us that this limited reaction represents the repair of the shorter strand of a double helix in which the strands are of unequal length, and that the reaction is governed by the hydrogen bonding of adenine to thymine and of guanine to cytosine. When all four triphosphates are present, but when DNA is omitted, no reaction at all takes place. What is the basis for this requirement? Does the DNA function as a primer in the manner of glycogen or does it function as a template in directing the synthesis of exact copies of itself? We have good reason to believe that it is the latter and as the central and restricted theme of this lecture I would like to emphasize that it is the capacity for base pairing by hydrogen bonding between the preexisting DNA and the nucleotides added as substrates that accounts for the requirement for DNA. The enzyme we are studying is thus unique in present experience in taking directions from a template — it adds the particular purine or pyrimidine sub- strate which will form a hydrogen-bonded pair with a base on the template (Fig. 7). There are five major lines of evidence that I would like to present to support this thesis. s-57 Physical properties of enzymatically synthesized DNA. The first line of evidence is derived from studies of the physical nature of the DNA produced by the enzyme. It may be mentioned again that in these descriptions as in those of the chemical nature of DNA, to be discussed shortly, go — 95 % of the DNA sample comes from the substrates used in the reaction. From collaborative studies with Dr. Howard K. Schachman, to whom we are greatly indebted, it can be said that the enzymatic product is indistinguish- able from high-molecular weight, double-stranded DNA isolated from nature (17). It has sedimentation coefficients in the neighbourhood of 25, reduced viscosities of 40 deciliters per gram and, on the basis of these measurements, it is beheved to be a long, stiff rod with a molecular weight of about 6 million. Upon heating the DNA, the rod collapses an'd the molecule becomes a compact, randomly coiled structure; it may be inferred that the hydrogen bonds holding the strands together have melted and this is borne out by characteristic changes in the viscometric and optical properties of the molecule. Similar results are found upon cleavage of the molecule by pancreatic deoxyribo- nuclease. In all these respects the enzymatically synthesized DNA is indis- tinguishable from the material isolated from nature, and may thus be presumed to have a hydrogen-bonded structure similar to that possessed by natural DNA. Would one imagine that the collapsed jumbled strands of heated DNA would serve as a primer for DNA synthesis? Very hkely one would think not. Guided by intuition derived from everyday experience with a jumbled strand of twine one might regard this as a hopeless template for replication. It turns out that the collapsed DNA is an excellent primer and the nonviscous, ran- domly coiled, single-stranded DNA leads to the synthesis of highly viscous, double-stranded DNA (18). Sinsheimer has isolated from the tiny 0X 174 virus a DNA which appears to be single-stranded (19). Like heated DNA it has proved to be an excellent primer (18) and a favorable material in current studies (20) for demonstrating in density gradient sedimentations that it is progressively converted to a double-stranded condition during the course of enzymatic synthesis. While a detailed discussion of the physical aspects of replication is not feasible in this lecture, it should be mentioned that the DNA in the single- stranded condition is not only a suitable primer but is the only active form when the most purified enzyme preparations are used. With such coli prepa- rations, the native, double-stranded DNA is inert unless it is heated or pre- treated very slightly with deoxyribonuclease. Bollum has made similar ob- servations with the enzyme that he has purified from calf thymus (21). s-58 Substitution of analogues in DNA synthesis. The second line of evidence is derived from studies of the activity of the substrates when substitutions are made in the purine and pyrimidine bases. From the many interesting reports on the incorporation of bromouracil (22), azaguanine (23) and other analogues into bacterial and viral DNA, it might be surmised that some latitude in the structure of the bases can be tolerated provided there is no interference with their hydrogen bondings. When experi- ments were carried out with deoxyuridine triphosphate or 5-bromodeoxy- uridine triphosphate, it was found that they supported DNA synthesis when used in place of thymidine triphosphate but not when substituted for the triphosphates of deoxyadenosine, deoxyguanosine or deoxycytidine. As already described (24), 5-methyl- and 5-bromocytosine specifically replaced cytosine; hypoxanthine substituted only for guanine; and, as just mentioned, uracil and 5-bromouracil specifically replaced thymine. These findings are best interpreted on the basis of hydrogen bonding of the adenine-thymine and guanine-cytosine type. Along these lines it is relevant to mention the existence of a naturally oc- curring "analogue" of cytosine, hydroxymethyl cytosine (HMC), which is found in place of cytosine in the DNA of the coli bacteriophages of the T-even series (25). In this case the DNA contains equivalent amounts of HMC and guanine and, as usual, equivalent amounts of adenine and thymine. Of addi- tional interest is the fact that the DNA's of T2, T4 and T6 contain glucose linked to the hydroxymethyl groups of the HMC in characteristic ratios (26, 27, 28) although it is clear that in T2 and T6 some of the HMC groups contain no glucose (27). These characteristics have posed two problems regarding the synthesis of these DNA's which might appear to be incompatible with the simple base-pairing hypothesis. First, what mechanism is there for preventing the inclusion of cytosine in a cell which under normal conditions has deoxy- cytidine triphosphate and incorporates it into its DNA? Secondly, how does one conceive of the origin of the constant ratios of glucose to HMC in DNA if the incorporation were to occur via glucosylated and non-glucosylated HMC nucleotides? Our recent experiments have shown that the polymerase reaction in the virus-infected cell is governed by the usual hydrogen-bonding restric- tions but with the auxiliary action of several new enzymes developed specifi- cally in response to infection with a given virus (29, 30). Among the new enzymes is one which splits deoxycytidine triphosphate and thus removes it from the sites of polymerase action (30). Another is a type of glucosylating s-59 DNA A T G C A + G T + C A + T G+C M, phle primer product 0.65 0.66 0.66 0.65 1-35 1-34 1-34 1-37 1. 01 0.99 0.49 0.48 E, coli primer product I. GO 1.04 0.97 I. DO 0.98 0.97 1.05 0.98 0.98 1. 01 0.97 1.02 Calf thymus primer product 1. 14 1. 12 1.05 1.08 0.90 0.85 0.85 0.85 1.05 1.02 1.25 1.29 Becteriophage Tz primer product 1-33 1.32 1.29 0.67 0.69 0.70 0.70 0.98 1.02 1.92 1.90 A-T Copolymer 1.99 1-93 4o Fig. 8. Chemical Composition of Enzymatically Synthesized DNA with Different Primers, enzyme which transfers glucose from uridine diphosphate glucose directly and specifically to certain HMC residues in the DNA (30). Chemical composition of enzymatically synthesized DNA. The third line of evidence is supplied by an analysis of the purine and pyrimidine base composition of the enzymatically synthesized DNA. We may ask two questions. First, will the product have the equivalence of adenine to thymine and of guanine to cytosine that characterize natural DNA? Sec- ondly, will the composition of the natural DNA used as primer influence and determine the composition of the product? In Fig. 8 are the results which answer these two questions (31). The experiments are identical except that in each case a different DNA primer was used: Mycobacterium phlei, Escherichia coli, calf thymus and phage T2 DNA. In answer to the first question it is clear that in the enzymatically synthesized DNA, adenine equals thymine and guanine equals cytosine so that the purine content is in every case identical to the pyrimidine. In answer to the second question it is again apparent that the characteristic ratio of adenine-thymine pairs to guanine-cytosine pairs of a given DNA primer is imposed rather faithfully on the product that is synthe- sized. Whether these measurements are made with isotopic tracers when the net DNA increase is only i % or if it is i 000 % the results are the same. It can be said further that it has not been possible to distort these base ratios by using widely differing molar concentrations of substrates or by any other means. In the last line of Fig. 8 is a rather novel "DNA" which is synthesized under conditions that I will not describe here (18, 32). Suffice it to say that after very long lag periods a copolymer of deoxyadenylate and thymidylate s-60 (A-T) develops which has the physical size and properties of natural DNA and in which the adenine and thymine are in a perfectly alternating sequence. When this rare form of DNA-Hke polymer is used as a primer, new A-T polymer synthesis starts immediately and even though all four triphosphates be present, no trace of guanine or cytosine can be detected in the product. The conclusion from these several experiments thus seems inescapable that the base composition is replicated in the enzymatic synthesis and that hydrogen-bonding of adenine to thymine and guanine to cytosine is the guiding mechanism. Enzymatic replication of nucleotide sequences. The fourth line of evidence which I would like to cite is drawn from current studies of base sequences in DNA and their replication. As I have suggested already, we believe that DNA is the genetic code; the four kinds of nucleotides make up a four-letter alphabet and their sequence spells out the message. At present we do not know the sequence; what Sanger has done for peptide sequence in protein remains to be done for nucleic acids. The problem is more difficult, but not insoluble. Our present attempts at determining the nucleotide sequences (33) will be described in detail elsewhere and I will only summarize them here. DNA is enzymatically synthesized using P^^ ^s label in one of the deoxynucleoside triphosphates; the other three substrates are unlabeled. This radioactive phosphate, attached to the 5-carbon of the deoxyribose, now becomes the bridge between that substrate molecule and the nucleotide at the growing end of the chain with which it has reacted (Fig. 9). At the end of the synthetic reaction (after some 10^® diester bonds have been formed), the DNA is isolated and digested enzymatically to yield the 3' deoxynucleotides quantitatively. It is apparent (Fig. 9) that the P atom formerly attached to the 5-carbon of the deoxynucleoside triphosphate substrate is now attached to the 3-carbon of the nucleotide with which it reacted during the course of synthesis of the DNA chains. The P^^ content of each of the 3' deoxynucleotides, isolated by paper electrophoresis, is a measure of the relative frequency with which a particular substrate reacted with each of the four available nucleotides in the course of synthesis of the DNA chains. This procedure carried out four times, using in turn a different labeled substrate, yields the relative frequencies of all the sixteen possible kinds of dinucleotide (nearest neighbor) sequences. Such studies have to date been carried out using DNA primer samples from six different natural sources. The conclusions are: i) All 16 possible dinucleotide sequences are found in each case. S-61 X>Q Fig. 9. Method lor Determining Sequences in DNA. SYNTHESIS (by polymerose) DEGRADATION (by micrococcol DNose and splenic diesterose) 2) The pattern of relative frequencies of the sequences is unique and re- producible in each case and is not readily predicted from the base compo- sition of the DNA. 3) Enzymatic replication involves base pairing of adenine to thymine and guanine to cytosine and, most significantly: 4) The frequencies also indicate clearly that the enzymatic replication produces two strands of opposite direction, as predicted by the Watson and Crick model. These studies and anticipated extensions of them should yield the dinucleo- tide frequencies of any DNA sample which can serve as an effective primer for enzymatic replication and thus provide some clues for deciphering the DNA code. Unfortunately this method does not provide information about trinucleotide frequencies but we are hopeful that with the improvement of enzymatic tools for analysis and chromatographic techniques for isolation some start can be made in this direction. Requirement for four triphosphates and DNA for DNA synthesis. Returning to the earlier-stated requirement for all four deoxynucleoside triphosphates and DNA in order to obtain DNA synthesis, we can now regard and understand these requirements as another and final line of evidence for hydrogen bonding. Without added DNA there is no template for hydrogen bonding and without all four triphosphates synthesis stops early and abruptly for lack of a hydrogen bonding mate for one of the bases in the template. s-62 Summary. The enzymatic approaches to the problem of DNA replication and the properties of the DNA-synthesizing enzyme purified from Escherichia coli have been sketched. The unifying and basic generalization about the action of this enzyme is that it catalyzes the synthesis of a new DNA chain in re- sponse to directions from a DNA template; these directions are dictated by the hydrogen bonding relationship of adenine to thymine and guanine to cytosine. The experimental basis for this conclusion is derived from the ob- servations of: (i) The double-stranded character of the enzymatically synthe- sized DNA and its origin from a single stranded molecule, (2) the pattern of substitution of analogues for the naturally-occurring bases, (3) the replication of the chemical composition, (4) the rephcation of the nucleotide (nearest neighbor) sequences and the antiparallel direction of the strands, and (5) the requirement for all four deoxynucleoside triphosphates (adenine, thymine, guanine and cytosine) and DNA for DNA synthesis. 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Griboff, Nature ^74' 306 (1954)- 23. M. R. Heinrich, V. C. Dewey, R. E. Parks, Jr., and G. W. Kidder, J. Biol. Chem. 197. 199 (1952). 24. M. J. Bessman. I. R. Lehman, J. Adler, S. B. Zimmerman, E. S. Simms and A. Kornberg, Proc. Nat. Acad. Sci. (U. S. A.) 44, 633 (1958). 25. G. R. WvATT and S. S. Cohen, Biochem. J. 55, 774 (1953) • 26. R. L. Sinsheimer, Science 120, 551 (1954); E. Volkin, J. Am. Chem. Soc. 76, 5892 (1954)- 27. R. L. Sinsheimer, Proc. Nat. Acad. Sci. (U. S. A.) 42, 502 (1956); M. A. Jesaitis, J. Exp. Med. 106, 233 (1957); Federation Proc. 17, 250 (1958). 28. G. Streisinger and J. Weigle, Proc. Nat. Acad. Sci. (U. S. A.) 42, 504 (1956). 29. J. G. Flaks and S. S. Cohen, J. Biol. Chem. 234, 1501 (1959); J. G. Flaks, J. Lich- TENSTEiN and S. S. Cohen, J. Biol. Chem. 234, 1507 (1959). 30. A. Kornberg, S. B. Zimmerman, S. R. Kornberg and J. Josse, Proc. Nat. Acad. Sci. (U. S. A.) 45. 772 (1959)- 31. I. R. Lehman, S. B. Zimmerman, J. Adler, M. J. Bessman, E. S. Simms and A. Kornberg, Proc. Nat. Acad. Sci. (U.S.A.) 44, 1191 (1958). 32. C. M. Radding, J. Adler and H. K. Schachman, Federation Proc , 79,307 (i960). 33. J. Josse and A. Kornberg, Federation Proc, 79,305 (i960). S-64 SUPPLEMENT VII A VIEW OF GENETICS* Joshua Lederberg Department of Genetics, Stanford Vnirersity School of Medicine The Nobel Statutes of 1900 charge each prize-winner to give a pubUc lecture in Stock- holm within six months of Commemoration Day. That I have fully used this margin is not altogether ingenuous, since it furnishes a pleas- ant occasion to revisit my many friends and colleagues in your beautiful city during its best season. The charge might call for a historical ac- count of past "studies on genetic recombina- tion and organization of the genetic material in bacteria," studies in which I have enjoyed the companionship of many colleagues, above all my wife. However, this subject has been reviewed regularly (36, 37, 3S, 41, 42, 45, 49, 54' 55' 5^) -^^^ ^ hope you will share my own inclination to assume a more speculative task, to look at the context of contempf)rary science in which bacterial genetics can be better under- stood, and to scrutinize the future prospects of experimental genetics. The dispersion of a Nobel award in the field of genetics symbolizes the convergent efforts of a world-wide community of investigators. That genetics should now be recognized is also timely — for its axial role in the conceptual structure of biology, and for its ripening yield for the theory and practice of medicine. How- ever, experimental genetics is reaching its full powers in coalescence with biochemistry: in principle, each phenotype should eventually be denoted as an exact sequence of amino acids in protein (79) and the genotype as a corre- sponding sequence of nucleotides in DNA (a, 63). The precise demarcation of genetics from biochemistry is already futile: but when genetics has been fully reduced to its molecular foundations, it may continue to serve in the same relation as thermodynamics to mechan- ics (69). The coordination of so many adja- • Received for publication May 14, 1959. Nohci Prize lecture given at the Rova! Caroline Meclicf)-SurRical Insti- tute, Stockholm, May 29, 1959. The Nobel Prize in Physi- ology or Medicine was awarded December H), 1958, jointly to G. W. Beadle, E. L. Tatum, and J. Lederberg. cent sciences will be a cogent challenge to the intellectual powers of our successors. That bacteria and their genetics should now be so relevant to general biology is already a fresh cycle in our scientific outlook. When thought of at all, they have often been relegat- ed to some obscure byway of evolution, their complexity and their homology with other or- ganisms grossly underrated. "Since Pasteur's startling discoveries of the important role played by microbes in human affairs, micro- biology as a science has always suffered from its eminent practical applications. By far the majority of the microbiological studies were undertaken to answer questions connected with the well-being of mankind" (30). The pedagogic cleavage of academic biology from medical education has helped sustain this dis- tortion. Happily, the repatriation of bacteria and viruses is only the first measure of the re- payment of medicine's debt to biology (6, 7, 8) . Comparative biochemistry has consum- mated the unification of biology revitalized by Darwin one hundred years ago. Throughout the living world we see a common set of struc- tural units — amino acids, coenzymes, nucleins, carbohydrates and so forth — from which every organism builds itself. The same holds for the fundamental process of biosynthesis and of energy metabolism. The exceptions to this rule thus command special interest as mean- ingful tokens of biological individuality, e.g., the replacement of cytosine by hydroxymethyl cytosine in the DNA of T2 phage (12). Nutrition has been a special triumph. Bac- teria which required no vitamins had seemed simpler than man. But deeper insights (32, 61) interpret nutritional simplicity as a greater power of synthesis. The requirements of more exacting organisms comprise just those me- tabolites they cannot synthesize with their own enzymatic machinery. Species differ in their nutrition: if species are delimited by tlieir genes, tfien genes must con- trol tlie hiosyntfietic steps tvfiicli are reflected S-65 Led er berg: Genetics in nutritional patterns. This syllogism, so evi- dent once told, has been amplified by Beadle and Tatum from this podium. Its implications for experimental biology and medicine are well known: among these, the methodology of bacterial genetics. Tatum has related how his early experience with bacterial nutrition reinforced the foundations of the biochemical genetics of Neurospora. Then, disregarding the common knowledge that bacteria were too simple to have genes, Tatum took courage to look for the genes that would indeed control bacterial nutrition. This conjunction marked the start of my own happy association with him, and with the fascinating challenges of bacterial genetics. Contemporary genetic research is predicated on the role of DNA as the genetic material, of enzyme proteins as the cell's working tools, and of RNA as the communication channel between them (63). Three lines of evidence substantiate the genetic function of DNA. Two are related to bacterial genetics; the third and most general is the cytochemical observa- tion of DNA in the chromosomes, which are undeniably strings of genes. But chromo- somes also contain other constituents besides DNA: we want a technique to isolate a chro- mosome or a fragment of one, to analyze it, and to retransplant it to verify its functional capacity. The impressive achievements of nu- clear transplantation (29) should encourage the audacity needed to try such experiments. The constructive equivalent to chromosome transplantation was discovered by a bacteriolo- gist thirty years ago (20). The genetic impli- cations of the "pneumococcus transformation" in the minds of some of Griffith's successors were clouded by its involvement with the gummy outer capsule of the bacteria. How- ever, by 1943, Avery and his colleagues had shown that this inherited trait was transmitted from one pneumococcal strain to another by DNA. The general transmission of other traits by the same mechanism (25) can only mean that DNA comprises the genes (b). To reinforce this conclusion, Hershey and Chase (23) proved that the genetic element of a bacterial virus is also DNA. Infection of a host cell requires the injection of just the DNA content of the adsorbed particle. This DNA controls not only its own replication in the production of new phage but also the speci- ficity of the protein coat, which governs the serological and host range specificity of the intact phage. At least in some small viruses, RNA also displays genetic functions. However, the he- reditary autonomy of gene-initiated RNA of the cytoplasm is now very doubtful — at least some of the plasmagenes that have been pro- posed as fulfilling this function are now better understood as feedback-regulated systems of substrate-transport (81, 65, 72). The work of the past decade thus strongly supports the simple doctrine that genetic infor- mation is nucleic, i.e., is coded in a linear se- quence of nucleotides. This simplification of life may appear too facile, and has/urnished a tempting target for agnostic criticism (37, 41, 44, 74). But, while no scientific theory would decry continual refinement and amplification, such criticism has little value if it detracts from the evident fruitfulness of the doctrine in ex- perimental design. The cell may, of course, carry information other than nucleic either in the cytoplasm or, accessory to the polynucleotide sequence, in the chromosomes. Epinucleic information has been invoked, without being more precisely defined, in many recent speculations on cyto- differentiation and on such models of this as^ antigenic phase variation in Salmonella (ji, 52, 56, 47). Alternative schemes have so much less information capacity than the nucleic cycle that they are more likely to concern the regula- tion of genie functions than to mimic their specificities. DNA AS A SUBSTANCE The chemistry of DNA deserves to be ex- posed by apter craftsmen (86, 31, 13) and I shall merely recapitulate before addressing its biological implications. A segment of DNA is illustrated in Fig. i. This shows a linear poly- mer whose backbone contains the repeating unit: — O — PO,-— O — CH, diester phosphate C.-/ CH C/ CH- The carbon atoms are conventionally num- bered according to their position in the furan- s-66 Stanford Medical Bulletin \,/^ X./ X./l-' ,-( \ f ;-< \-Z.. I 1 I I y-^y o^\ / \ xl Fig. I. — Primary structure of DNA — a segment of a polynucleotide sequence CGGT. From (13). ose ring of deoxyribose, which is coupled as an N-glycoside to one of the nuclein bases: adenine, guanine, cytosine, or thymine, sym- boUzed A, G, C, or T, the now well-known alphabet in which genetic instructions are com- posed. With a chain length of about 10,000 residues, one molecule of DNA contains 20,000 "bits of information," comparable to the text of this article, or in a page of newsprint. Pyrophosphate - activated monomer units (e.g., thymidine triphosphate) have been iden- tified as the metabolic precursors of DNA (31). For genetic replication, the monomer units must be assembled in a sequence that re- flects that of the parent molecule. A plausible mechanism has been forwarded by Watson and Crick (87) as a corollary to their struc- tural model whereby DNA occurs as a two- stranded helix, the bases being centrally ori- ented. When their relative positions are fixed by the deoxyribose-phosphate backbones, just two pairs of bases are able to form hydrogen bonds between their respective NH and CO groups; these are A : T and G : C. This pairing of bases would tie the two strands together for the length of the helix. In conformity with this model, extensive analytical evidence shows a remarkable equality of A with T and of G with C in DNA from various sources. The two strands of any DNA are then mutually complementary, the A, T, G, and C of one strand being represented by T, A, C, and G, respectively, of the other. The information of one strand is therefore equivalent to, because fully determined by, the other. The determi- nation occurs at the replication of one parent strand by the controlled stepwise accretion of monomers to form a complementary strand. At each step, only the monomer which is com- plementary to the template would fit for a chain-lengthening esterification with the adja- cent nucleotide. The model requires the un- raveling of the intertwined helices to allow each of them to serve as a template. This might, however, occur gradually, with the growth of the daughter chain — a concept em- bedded in Fig. 2 which symbolizes the new Cabala. The discovery of a single-stranded configuration of DNA (85) makes complete unraveling more tenable as an alternative model. For the vehicle of life's continuity, DNA may seem a remarkably undistinguished mole- cule. Its over-all shape is controlled by the uni- form deoxyribose-phosphate backbone whose monotony then gives X-ray diffraction pat- terns of high crystallinity. The nucleins them- selves are relatively unreactive, hardly differ- ent from one to the other, and in DNA intro- verted and mutually saturated. Nor are any of the hydroxyls of deoxyribose left unsubstituted in the polymer. The structure of DNA befits the solipsism of its function. The most plausible function of DNA is ultimately to specify the amino acid sequence in proteins. However, as there are twenty amino acids to choose among, there cannot be a one: one correspondence of nucleotide to amino acid. Taking account of the code dupli- cation in complementary structures and the need to indicate spacing of the words in the code sequence, from three to four nucleins S-67 Lederberg: Genetics may be needed to spell one amino acid (19). While a protein is also defined by the se- quence of its monomeric units, the amino acids, the protein molecule lacks the "aperiodic cryitallinity" (80) oi\Ji^\.l^\\t differentiae oi the amino acids vary widely in size, shape, and ionic charge (e.g., H.N-CHvCH.-CH/CH^-; COOHCH.-CHo-; HOCoH4-CH/; CHa'; H") and in the case of proline, bond angles. Fig. 2. — The scheme of Watson and Crick for DNA replication. "Unwinding and replication proceed pari passu. All three arms of the Y ro- tate as indicated" (14). The biological action of a protein is, therefore, attributable to the shape of the critical surface into which the polypeptide chain folds (73). The one-dimensional specificity of the DNA must therefore be translated into the three-di- mensional specificity of an enzyme or anti- body surface. The simplest assumption would be that the amino acid secjuence of the ex- tended polypeptide, as it is released from the protein-building template in the cytoplasm, fully determines the folding pattern of the complete protein, which may, of course, be stabilized by nonpeptide linkages. If not, we should have to interpose some accessory mech- anism to govern the folding of the protein. This issue has reached a climax in speculations about the mechanism of antibody formation. If antibody globulins have a common se- quence on which specificity is superimposed by directed folding, an antigen could directly mold the corresponding antibody. However, if sequence determines folding, it should in turn obey nucleic information. As this should be independent of antigenic instruction, we may look instead to a purely selective role of antigens to choose among nucleic alternatives which arise by spontaneous mutation (8, 50). The correspondence between amino acids and clusters of nucleotides has no evident basis in their inherent chemical make-up and it now appears more probable that this code has evolved secondarily and arbitrarily to be trans- lated by some biological intermediary. The coding relationship would then be analogous to, say, Morse-English (binary linear) to Chi- nese (pictographic). Encouragingly, several workers have reported the enzymatic reaction of amino acids with RNA fragments (22, 75). Apparently each amino acid has a diflferent RNA receptor and an enzyme whose twofold specificity thus obviates any direct recognition of amino acid by polynucleotides. The align- ment of amino-acyl residues for protein syn- thesis could then follow controlled assembly of their nucleotidates on an RNA template, by analogy with the model for DNA replication. We then visualize the following modes of in- formation transfer: (i) DNA replication — assembly of comple- mentary deoxyribonucleotides on a DNA template. (2) Transfer to RNA by some comparable mechanism of assembling ribonucleotides. Our understanding of this is limited by uncertainties of the structure of RNA (16). (3) Protein synthesis: {a) Aminoacylation of polynucleotide fragments; (h) Assembly of the nucleotidates on an RNA template bv analogy with step (c) Peptide condensation of the amino acid residues. Some workers have suggested that RNA is s-68 Stanford Medical Bulletin replicated in step (3) concurrently with pro- tein synthesis, in addition to its initiation from DNA. The chief difference in primary structure be- tween DNA and RNA is the hydroxylation of C2' in the ribose, so that a reactive sugar hy- droxyl is available in RNA. This may prove to be important in the less ordered secondary structure of RNA, and in its function as an intermediary to protein. It remains to be de- termined whether the aminoacyl nucleotidates are escerified at d' or at C3' which is also avail- able in the terminal residue. From this resume we may observe that the DNA backbone con- stitutes an inert but rigid framework on which the differential nuclcins are strung. Their spa- tial constraint lends specificity to the pattern of hydrogen bonding exposed at each level. This extended pattern is a plausible basis for replication; it is difficult to visualize any re- agents besides other nucleotides to which this pattern would be relevant. These conditions are quite apt for a memory device — rubber and guncotton are poor choices for a computing tape. DNA AND BACTERIAL MUTATION The ignis fatuus of genetics has been the specific mutagen, the reagent that would pene- trate to a given gene, recognize and modify it in a specific way. Directed mutation has long been discredited for higher organisms and the "molar indeterminacy" of mutation estab- lished both for its spontaneous occurrence and for its enhancement by X-rays (68) . However, the development of resistance apparently in- duced by drugs revived illusions that bacterial genes might be alterable, an inference that would inevitably undermine the conception of "gene" for these organisms. No wonder that the mechanism of drug resistance has excited so much controversy (89) ! What sort of molecule could function as a specific mutagen, a reagent for a particular one of the bacterium's complement of genes, which can hardly number less than a thousand targets? On the nucleic hypothesis, the small- est segment capable of this variety would be a /2^A-tmucleotide, all possible configurations of which must be discriminated by the specific mutagen. How could this be generally accom- plished except by another molecule of con- forming length and periodicity, that is, an analogous polynucleotide? Certainly there is nothing in the chemistry of penicillin or strep- tomycin to support their direct intervention in nucleic instructions. In addition, we recognize no chemical re- agent capable of substituting one nuclein for another in the structure of existent DNA. However, as the modification of a nuclein, even to give an unnatural base, could have mutagenic effect, the chief limitation for spe- cific mutagenesis is the recognition of the ap- propriate target. Of course the origin of drug resistance, for all its theoretical implications, poses an experi- mental challenge of its own. Concededly, ex- periments cannot decide untried situations. Nevertheless, the mechanism whereby resist- ant mutants arise spontaneously and are then selected by the drug can account for every well-studied case of inherited resistance (10, 5). Furthermore, in favorable instances the spontaneous origin of drug-resistant mutants can be verified unambiguously by contriving to isolate them without their ever being ex- posed to the drug. One method entails indi- rect selection. To illustrate its application, con- sider a culture of Escherichia coli containing 10^ bacteria per ml. By plating samples on agar containing streptomycin, we infer that one bacterium per million or 10^ per ml pro- duce resistant clones. But to count these clones they were selected in the presence of strepto- mycin which hypothetically might have in- duced the resistance. We may however dilute the original bacteria in plain broth to give samples containing 10^ per ml. Since 10"® of the bacteria are resistant, each sample has a mathe- matical expectation of o.i of including a resist- ant bacterium. The individual bacterium be- ing indivisible by dilution, nine samples in ten will include no resistants; the tenth will have one, but now augmented to io~^. Which one this is can be readily determined by retrospec- tive assay on the incubated samples. The pro- cedure can be reiterated to enrich for the resist- ant organisms until they are obtained in pure culture (11). The same result is reached more conveniently if we spread the original culture out on a nutrient agar plate rather than dis- s-69 Lederberg: Genetics tribute samples into separate test tubes. Rep- lica plating, transposing a pattern of surface growth from plate to plate with a sheet of velvet, takes the place of assaying inocula dis- tributed in tubes (53). Dilution sampling and replica plating are, then, alternative methods of indirect selection whereby the test line is spared direct contact with the drug. Selection is accomplished by saving sublines whose sib- ling clones show the resistant reaction. This proof merely reinforces the incisive arguments that had already been forwarded by many other authors. If mutations are not specific responses to the cellular environment, how do they arise? We still have very little information on the proxi- mate causes of spontaneous, even of radiation and chemically induced, mutation. Most mu- tagenic chemicals are potent alkylating agents, e.g., formaldehyde or nitrogen mustard, which attack a variety of reactive groups in the cell. Similar compounds may occur in normal me- tabolism and account for part of the spontane- ous mutation rate; they may also play a role as chemical intermediates in radiation effects. For the most part, then, studies on mutagene- sis, especially by the more vigorous reagents, have told us little about the chemistry of the gene. Probably any agent that can penetrate to the chromosomes and have a localized chemical effect is capable of introducing ran- dom errors into the genetic information. If the cell were not first killed by other mechanisms most toxic agents would then probably be mu- tagenic. Another class of mutagenic chemicals prom- ises more information: analogues of the natural nucleins which are incorporated into DNA, For example, bromouracil specifically replaces thymine in phage DNA when fur- nished as bromodeoxyuridine to infected bac- teria. Freese has shown, by genetic analyses of the utmost refinement, that the loci of result- ing mutations in T4 phage are distributed dif- ferently from the mutants of spontaneous ori- gin or those induced by other chemicals (18). This method presumably maps the locations of thymine in the original DNA. In order to ac- count for wide variations in mutation rate for different loci, further interactions among the nucleotides must be supposed. So far, these studies represent the closest approach to a ra- tional basis for chemical mutagenesis. How- ever, every gene must present many targets to any nuclein analogue and the specificity of their mutagenesis can be detected only in sys- tems where the resolution of genetic loci ap- proximates the spacing of single nucleotides (4) . At present this is feasible only in micro- organisms; similar studies with bacteria and fungi would be of the greatest interest. More specific effects might result from the insertion of oligo- and polynucleotides, a pro- gram which, however, faces a number of tech- nical difficulties: even if the requisite polymers were to be synthesized, there are obstacles to their penetration into cells. The use of DNA extracted from mutant bacteria to transfer the corresponding genetic qualities is discussed as "genetic transduction." RNA is the one other reagent that may be expected to recognize particular genes. As yet we have no direct evidence that the transfer of information from DNA to RNA is reversible. However, the anti-mutagenic effect of nuclein ribosides (21, 71) may implicate RNA in mu- tation. The reversibility of DNA «=^ RNA in- formation is also implicit in Stent's closely reasoned scheme for DNA replication (82). The needed experiment is the transfer of DNA information by some isolated RNA. Al- though not reported, this has probably not been fairly tried. One motivation for this approach is the diffi- cult problem of finding sources of homogene- ous nucleic acids. DNA occurs biologically as sets of different molecules presumably in equi- molar proportions. (A useful exception may be a remarkably small phage which seems to be unimolecular [85]). The species of RNA, however, may vary with the predominant met- abolic activity of the cells. If so, some molecu- lar species may be sufficiently exaggerated in specialized cells to facilitate their isolation. A purified RNA would have many potential ap- plications, among others as a vehicle for the recognition of the corresponding DNA im- plied by our theory of information transfer. Pending such advances, specific mutagenesis is an implausible expectation. Adaptive mutations, of which drug resist- ance is a familiar example, are crucial to the S-70 Stanford Medical Bulletin methodology of microbial genetics. Once hav- ing connected adaptive variation with gene mutation (78), we could proceed to exploit these systems for the detection of specific geno- types in very large test populations. The geno- types of interest may arise, as in the previous examples, by mutation: the most extensive studies of the physiology of mutation now use these methods for precise assay. For, in order to count the number of mutants of a given kind, it suffices to plate large numbers of bac- teria into selective media and count the surviv- ing colonies which appear after incubation. In this way, mutation rates as low as one per 10^ divisions can be treated in routine fashion. GENETIC RECOMBINATION IN BACTERIA The selective isolation of designed genotypes is also the most efficient way to detect genetic recombination. For example, the sexual mecha- nism of Escherichia coli was first exposed when prototrophic (nutritionally self-sufficient) re- combinants arose in mixed cultures of two auxotrophic (nutritionally dependent) mu- tants (35, 57, 84). At first only one recombin- ant appeared per million parental bacteria and the selective procedure was quite obliga- tory. Later, more fertile strains were discov- ered which have been most helpful to further analysis (45, 51). This has shown that typical multinucleate vegetative bacteria unite by a conjugation bridge through which part or all of a male genome migrates into the female cell (43). The gametic cells then separate. The ex- conjugant male forms an unaltered clone, sur- viving by virtue of its remaining nuclei. The exconjugant female generates a mixed clone including recombinants (46, i). Wollman, Jacob, and Hayes (88) have since demon- strated that the paternal chromosome migrates during fertilization in an orderly, progressive way. When fertilization is prematurely inter- rupted, the chromosome may be broken so that only anterior markers appear among the re- combinants. All of the genetic markers are arranged in a single linkage group and their order can be established either by timing their passage during fertilization or by their statisti- cal association with one another among the re- combinants. Finally, the transfer of genetic markers can be correlated with the transfer of DNA as inferred from the lethal effect of the radioactive decay of incorporated P^' (27). Sexual recombination is one of the methods for analyzing the gene-enzyme relationship. The studies so far are fragmentary but they support the conception that the gene is a string of nucleotides which must function as a co- herent unit in order to produce an active en- zyme (4, 33, 67, 15, 90). However, metabolic blocks may originate through interference with accessory regulatory mechanisms instead of the fundamental capacity to produce the enzyme. For example, many "lactase-nega- tive" mutants have an altered pattern of en- zyme induction or a defective permease system for substrate transport (55, 65) . Several labora- tories are now working to correlate the relative sequence of genetic defects with the sequence of corresponding alterations in enzyme pro- teins; this may be the next best approach to the coding problem short of a system where a pure DNA can be matched with its protein pheno- type. At first these recombination experiments were confined to a single strain of E. coli, K-12. For many purposes this is a favorable choice of material — perhaps the main advantage is the accumulation of a library of many thou- sands of substrains carrying the various mark- ers called for by the design of genetic tests. However, strain K-12 is rather unsuitable for serological studies, having lost the character- istic surface antigens which are the basis of serological typing. In any event it would be important to know the breeding structure of the group of enteric bacteria. Systematic studies have therefore been made of the inter- fertility of different strains of bacteria, princi- pally with a convenient tester of the K-12 strain (39, 93). About one-fourth of the serotype strains of E. coli are fertile with strain K-12, and in at least some instances with one another. Whether the remaining three-fourths of strains are completely sterile, or whether they include different, closed, breeding groups (i.e., differ- ent genetic species) has not been systematically tested, partly because of the preliminary work needed to establish suitable strains. E. coli K-12 is also interfertile with a num- ber of strains of Shigella spp. (59). Finally al- though attempted crosses of E. coli with many s-7] Lederberg: Genetics Salmonella types and of Salmonellas with one another have usually failed, Baron has demon- strated crosses of E. colt with a unique strain of Salmonella typhimurium (3). This may be especially useful as a means of developing hy- brids which can be used to bridge the studies of sexuality in E. colt and transduction in Sal- monella. GENES AND VIRUSES Bacteria furnish a unique opportunity to Study the genetic relationships with their host cells. Another treasure of strain K-12 was for a time hidden: it carries the temperate bacte- riophage, 'k, which is technically quite favor- able for genetic work. In accord with Burnet's early predictions, we had anticipated that the provirus for A. would behave as a genetic unit, but Dr. Esther Lederberg's first crosses were quite starding in their implication that the pro- phage segregated as a typical chromosomal marker (34). This was shown quite unambig- uously by the segregation of lysogenicity versus sensitivity from persistent heterozygous cells, a test that bypassed the then controversial de- tails of fertilization. The viabihty of such het- erozygous cells supports the hypothesis that lysogenicity depends in part on the develop- ment of a cytoplasmic immunity to the cyto- pathic effects of infecting phage as a secondary result of the establishment of the prophage in a bacterial chromosome. This picture is also brought out by zygotic induction (26) where- by the fertilization of a sensitive cell by a pro- phage-bearing chromosome may provoke the maturation and progressive growth of the phage and the lysis of the complex. On the other hand, the introduction of a sensitive chromosome into a lysogenic bacterium does not result in this induction. The mode of at- tachment of prophage to its chromosomal site is as unsettled as the general picture of the higher organization of DNA, but most stu- dents favor a lateral rather than an axial rela- tionship for the prophage. The isolation of in- tact chromosomes of bacteria would give a new approach to this question but has so far been inconclusive. Another infectious particle that has jumped out of our Pandora's box determines the very capacity of E. coli to function as a male partner in fertilization (51). For lack of a better in- spiration, we call this particle "F." Two kinds of male strains are now recognized according to whether the F particle has a chromosomal or a cytoplasmic location. F+ strains, like the original K-12, are highly contagious for F and will rapidly convert populations of female, F — strains in which they are introduced. Hfr males, on the other hand, have a chromosomal localization of the F factor resulting from oc- casional transpositions in F+ strains. The dif- ferent localization of the F particle in the two cases is diagnosed primarily by the behavior of the particle in crosses. In addition, Hirota and lijima (24) found that the F particle could be eliminated from F+ strains by treatment with acridine dyes. Hfr clones are unaffected by acridine orange, but when they revert to the F+ state, as occasionally happens, the F par- ticle again becomes vulnerable to the dye. The accessibility of extrachromosomal F is paral- leled by several other examples of plasmid dis- infection (reviewed in 40) ; perhaps the most notable is the bleaching of green plant cells by streptomycin (17, 76). No reagent is known to inactivate F or prophage while bound to the chromosome. The virus A. and the plasmagene F are analo- gous in many features (28, 48). Their main differences are: (i) Cytopathogenicity. A bacterium cannot long tolerate A in its cytoplasmic slate and remain viable. The vegetative A. must promptly reduce itself to a chromosomal state or multiply aggressively and lyse the host bacterium. F has no known cyto- pathic effect. (2) Maturation. Vegetative A, organizes a pro- tein coat and matures into an infective phage particle. F is known only as an intracellular vegetative element; however, the coat of the F+ cell may be analogous to that of the phage. (3) Transmission. A. is infective, i.e., forms a free particle which can penetrate suscepti- ble cells. F is transmitted only by cell-to- cell conjugation. (4) Fixation, A. has a foreordained site of fixa- tion on the bacterial chromosome; F has been identified at a variety of sites. How- s-72 Stanford Medical Bulletin ever, this difference may be illusory. In special situations, F does have preferential sites of fixation (77), and generally, trans- locations of F to different sites are more readily discovered than those of A, would be. (5) Induction. Exposure of lysogenic bacteria to small doses of ultraviolet light causes the prophage to initiate a lytic cycle with the appearance first of vegetative, then of ma- ture phage (62). Hfr bacteria make no analogous response. However, the kinetics of the reversion, Hfr ~^ F+, has not been carefully studied. The genetic function of bacteriophages is further exemplified by transduction whereby genes are transferred from cell to cell by the intervention of phage particles (42, 91 ) . In our first studies we concluded that the bacterial genes were adventitiously carried in normal phage particles (92, 66, 83). Further studies favor the view that the transducing particle has a normal phage coat but a defective phage nucleus. This correlation has suggested that a gene becomes transducible when a prophage segment is translocated to its vicinity (2, 9, 60). Transduction focuses special attention on the phenomenon of specific pairing of homol- ogous chromosome segments. Howsoever a transduced gene is finally integrated into the bacterial genome, at some stage it must locate the homologous gene in the recipient chromo- some. For in transduction, as in sexual recom- bination, new information is not merely added to the complement; it must also replace the old. This must involve the confrontation of the two homologues prior to the decision which one is to be retained. Synapsis is even more puzzling as between chromosomes whose DNA is in the stabilized double helix and then further contracted by supercoiling. Conceivably gene products rather than DNA are the agency of synaptic pairing. The integration of a transduced fragment raises further issues (41). The competing hy- potheses are the physical incorporation of the fragment in the recipient chromosome, or the use of its information when new DNA is repli- cated. The same issues still confound models of crossing over at meiosis in higher forms; once again the fundamentals of chromosome structure are needed for a resolution. VIRUS VERSUS GENE The homology of gene and virus in their fundamental aspects makes their overt differ- ences even more puzzling. According to the simplest nucleic doctrine, DNA plays no active role in its own replication other than furnish- ing a useful pattern. Various nucleotide se- quences should then be equally replicable. What then distinguishes virus DNA, which replicates itself at the expense of the other path- ways of cellular anabolism? For the T-even phages, the presence of the unique glucos- ylated hydroxymethylcytosine furnishes a partial answer (12). However, other viruses such as A, display no unique constituents; fur- thermore, as prophage they replicate coordi- nately with bacterial DNA. Does the virus have a unique element of structure, either chemical or physical, so far undetected? Or does it instruct its own preferential synthesis by a code for supporting enzymes .-^ THE CREATION OF LIFE The mutuahsm of DNA, RNA, and pro- teins as just reviewed is fundamental to all contemporary life. Viruses are simpler as in- fective particles but must, of course, parasitize the metabolic machinery of the host cell. What would be the least requirements of a primeval organism, the simplest starting point for pro- gressive replication of DNA in terms of pres- ently known or conjectured mechanisms? They include at least: (i) DNA. (2) The four deoxyribotide pyrophosphates in abundance. (3) One molecule of the protein, DNA po- lymerase. (4) Ribotide phosphates as precursors for RNA. (5) One molecule of the protein RNA po- lymerase. (6) A supply of the twenty amino acyl nucle- otidates. (a) Failing these, each of the twenty en- zymes which catalyze the condensa- tion of an amino acid and correspond- s-73 Lederberg: Genetics ing RNA fragments together with sources of these components. (7) One molecule of the protein aminoacyl- RNA polymerase. In principle, this formidable list might be reduced to a single polynucleotide polymer- ized by a single enzyme. However, any scheme for the enzymatic synthesis of nucleic acid calls for the coincidence of a particular nucleic acid and of a particular protein. This is a far more stringent improbability than the sudden emergence of an isolated DNA such as many authors have suggested, so much more so that we must look for alternative solutions to the problem of the origin of Hfe. These are of two kinds. The primeval organism could still be a nucleic cycle if nucleic replication oc- curs, however imperfectly, without the inter- vention of protein. The polymerase enzyme, and the transfer of information from nucleic acid to protein, would then be evolved refine- ments. Alternatively, DNA has evolved from a simpler, spontaneously condensing polymer. The exquisite perfection of DNA makes the second suggestion all the more plausible. The nucleoprotein cycle is the climax of bio- chemical evolution. Its antiquity is shown by its adoption by all phyla. Having persisted for '-^ 10® years, nucleoprotein may be the most durable feature of the geochemistry of this planet. At the present time, no other self-replicat- ing polymers are known or understood. Never- theless, the nucleic system illustrates the basic requirements for such a polymer. It must have a rigid periodic structure in which two or more alternative units can be readily substituted. It must allow for the reversible sorption of spe- cific monomers to the units in its own se- quence. Adjacent, sorbed monomers must then condense to form the replica polymer, which must be able to desorb from the tem- plate. Primitively, the condensation must be spontaneous but reliable. In DNA, the sorp- tion depends on the hydrogen bonding of nu- clein molecules constrained on a rigid helical backbone. This highly specific but subtle de- sign would be difficult to imitate. For the more primitive stages, both of biological evolution and of our own experimental insight, we may prefer to invoke somewhat cruder techniques of complementary attachment. The simplest of these is perhaps the attraction between ionic groups of opposite charge, for example, NHs"" and COO" which are so prevalent in simple organic compounds. If the ingenuity and craftsmanship so successfully directed at the fabrication of organic polymers for the practi- cal needs of mankind were to be concentrated on the problem of constructing a self-replicat- ing assembly along these lines I predict that the construction of an artificial molecule hav- ing the essential function of primitive life would fall within the grasp of our current knowledge of organic chemistry. CONXLUSIONS The experimental control of cellular geno- type is one of the measures of the scope of genetic science. However, nucleic genes will not be readily approached for experimental manipulation except by reagents that mimic them in periodic structure. 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There is increasing evidence that such a molecule is a natural unit rather than an artefact of fragmentation. c The experimental work from my laboratory summarized in this paper has been generously supported by research grants from the National Institutes of Health, U.S. Public Health Service, the National Science Foundation, the Rockefeller Foundation, the Wisconsin Alumni Research Foundation, the University of Wisconsin, and, most recendy, Stanford University. It is also a pleasure to record my thanks to the Jane Coffin Childs Fund for Medical Research for a research fellowship which supported my first association with Professor E. L. Tatum. ''.\'>L S-77