Advances in Genetics Volume 16

ADVANCES IN GENETICS VOLUME 16 Edited by E. W. CASPARI Department of Biology University of Rochester Rochester, New York

1971 ACADEMIC PRESS

NEW YORK AND LONDON

COPYRIGHT 0 1971, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED NO PART OF THIS BOOK MAY BE REPRODUCED IN A N Y FORM, BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, OR ANY OTHER MEANS, WITHOUT WR-N PERMISSION FROM THE PUBLISHERS.

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DEDICATION M. Demerec Editor of Advances in Genetics

About two years ago, I was approached by some of his former collaborators with the suggestion to dedicate a volume of Advances in Genetics to the memory of M. Demerec. I gladly accepted this proposal since it seems to me fitting that Advances in Genetics honor in this way the memory of its founder and the Editor of its first nine volumes. Demerec became concerned about the “information explosion” in the sciences long before it became a popular topic of complaint. H e was possibly more aware than others of the resulting lack of communication between scientists in different though related fields, because he himself refused to be confined by artificial boundaries, and changed the object of his research several times in his life. H e had thus to get acquainted with new techniques and with a completely new literature. Part of his prodigious energy was therefore devoted to various means by which communication between scientists active in different fields could be established and extended. The founding of Advances in Genetics was one of his main activities in this direction. It was particularly designed to make it possible for geneticists working in a particular area and with a particular organism to get acquainted with work done in other fields of genetics. H e writes in the Preface to Volume I (1947): This series of review articles, Advances in Genetics, has been started in order that critical summaries of outstanding genetic problems, written by competent geneticists, may appear in a single publication.The articles are expected to deal with both theoretical and practical problems, and to cover plant breeding, animal breeding, and human heredity, as well as the related fields of biophysics, biochemistry, physiology, and immunology. The aim is to have the articles written in such form that they will be useful as reference material for geneticists and also as a source of information to nongeneticists.

The way these stated goals were implemented can be seen from the contents of the volumes which appeared under Demerec’s editorship. They have continued to serve as guidelines for the editorial policies of Advances in Genetics. Y

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The review papers contained in this volume have been prepared by friends and former collaborators of Demerec. They reflect, therefore, to a certain degree, his interests during the last half of his scientific life. But since his interests were always wide ranging, and encompassed the whole range of genetics, the articles contained in this volume are not much more uniform than those in other volumes of this series. We hope that this volume constitutes a fitting memorial to a great geneticist who, by his own work, and by stimulating and facilitating the work of other scientists, has deeply influenced the direction genetics has taken in the last twenty years, and has contributed greatly to the conspicuous progress of genetics during his lifetime. I want to thank sincerely those geneticists who, by their contributions to this volume or by service on the Editorial Committee, have helped to make the appearance of this volume possible. Particular recognition is due to Dr. A. Eisenstark who, as chairman of the Editorial Committee, contributed a great deal to the organization of this volume. It gives me great personal satisfaction to have collaborated on this tribute to a man I have always admired and esteemed as a great investigator, as an original thinker, as a resourceful and indefatigable organizer of scientific activities, and as a staunch, reliable friend. Ernst Caspari Rochester, New Yo& June 1971

CONTRIBUTORS TO VOLUME 16 Numbers in parentheses indicate the pages on which the authors’ contributions begin.

BBUCEN. AM= ( l ) , Department of Biochemistry, University of California, Berkeley, California

L. ELIZABETH BERTANI(199) , Microbial Genetics Laboratory, Karolinska Institutet, Stockholm, Sweden

GIUSEPPM BERTANI(199) , Microbial Genetics Laboratory, Karolinska Institutet, Stockholm, Sweden

DONJ. BRENNER(81), Division of Biochemistry, Walter Reed Army and the Department of Institute of Research, Washington, D.C., Microbiology, Schools of Medicine and Dentistry, Georgetown University, Washington, D.C.

H. J. CURTIS(305) , Biology Department, Brookhaven National Laboratory, Upton, New York

A. EISENSTARK (167) , Division of Biology, Kansas State University, Manhattan., Kansas STANLEY FALKOW (81), Division of Biochemistry, Walter Reed Army Institute of Research, Washington, D.C., and the Department of Microbiology, Schools of Medicine and Dentistry, Georgetown University, Washington, D.C. PHILIPE. HARTMAN ( l ) , Department of Biology, The Johns Hopkins University, Baltimore, Maryland

ZLATAHARTMAN (1), Department of Biology, The Johns Hopkins University, Baltimore, Maryland

ROLLIND. HOTCHKISS (325) , The Rockefeller University, New Yorlc, New York RICHARD B. MIDDLETON* (53), Department of Biology, McGill University, Montreal, Quebec, Canada

* Present address: Faculty of Medicine, Memorial University, St. John’s, Newfoundland, Canada. xi

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16

TOBIASMOJICA-A*(53), Department of Biology, McGill University, Montreal, Quebec, Canada HOWARD B. NEWCOMBE (239), Biology and Health Physics Division, Atomic Energy of Canada Limited, Chalk River, Ontario KENNETHE. SANDERSON (35),Department of Biology, T h e University of Calgary, Calgary, Alberta, Canada D. A. SMITH(141), Genetics Department, University of Birmingham, England RUTHC. STAHL( l ) ,Department of Biology, T h e Johns Hopkins University, Baltimore, Maryland H. E. UMBARGER (119) , Department of Biological Sciences, Purdue University, Lafayette, Indiana

* Present address: Polish Academy of Sciences, Institute of Biochemistry and Biophysics, 36 Rakowiecka Street, Warszawa 12, Poland.

M. Demerec

MlLlSLAV DEMEREC* (1895-1966) To Milislav Demerec, research in genetics was far more than a profession; it was his way of life. He lived it to his last hour. On April 12, 1966, he submitted a paper by two colleagues for publication in the Proceedings of the National Academy of Sciences. This day he spent mostly in discussing with a younger colleague the plans of work in his new laboratory a t the C. W. Post College of Long Island University. They parted around 8 P . M . ; a t about a quarter past nine Demerec was found dead in his study and bedroom. Milislav Demerec was born on January 11, 1895, at Kostajnica in Croatia (Yugoslavia), the third of eight children in a family of a school teacher, later a n inspector of schools. He attended a school in the town of Petrinja, where he lived with his grandfather and his aunts. Later he attended a high school (Realschule) in Zagreb, and the College of Agriculture a t Krifevci, near Zagreb. H e was an excellent student i n both, and upon his graduation was given the position of adjunct at the Krifevci Experiment Station, where he worked with the pioneer Croatian plantbreeder Professor B. Bohutinsky. The First World War was still going on, but Demerec was released from military service to work for a commission charged with purchasing foodstuffs for the civilian population. After the Armistice, he obtained a small scholarship t o study at the College of Agriculture in Grignon, France. He attended the lectures of Professors Ducomet, Scribeaux, and Crepin, and made short visits to the Institute for Agricultural Research in Versailles and the famous plant-breeding establishment of Vilmorin in Verier, France. I n 1919 Demerec became a graduate student a t the Department of Plant Breeding, Cornell University. He joined the group of investigators working on the genetics of maize under the leadership of Professor R. A. Emerson. This was one of the most active centers of research in genetics in the United States. It produced several outstanding geneticists in addition to Demerec, among them G. W. Beadle, M. M. Rhoades, and B. McClintock. Demerec was awarded his Ph.D. degree in 1923, having by then published four articles and notes on the genetics of maize, the number rising to fifteen by 1927. These early publications

* Reprinted through the courtesy of the Year Book of the American Philosophical Society, 1966,pp. 115-121. xv

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already show the personal style which was to be the earmark of Demerec’s writing to the end of his career-abundance of exact and carefully recorded observations and experiments, thoughtful analysis, avoidance of hasty speculation and theorizing, and austerely economical use of words, which made his writings err occasionally on the side of abridgment but never on the side of prolixity. I n summers of 1920 and 1921 Demerec visited the Marine Biological Laboratory in Woods Hole, where he met C. W. Metz and members of the school of T. H. Morgan, working on the genetics of Drosophila. He was fascinated with this, new to him, material for genetic experimentation. In 1923 he joined Metz, as resident investigator a t the Department of Genetics, Carnegie Institution of Washington, at Cold Spring Harbor, on Long Island. He became assistant director in 1942, and was its director from 1943 to 1960. In 1941 he added also the directorship of the neighboring, in fact the adjacent, research institution, the Biological Laboratory of the Long Island Biological Association, remaining in this post likewise until 1960. Demerec’s dedication to, and his identification with Cold Spring Harbor and with the scientific research institutions of which he became a member and eventually a leader, were quite extraordinary. The genius loci took firm possession of him, or it is perhaps more accurate to say that he became that genius personified. Although, especially in later years, Demerec traveled rather widely in the United States and abroad, no spot anywhere in the world was to him a worthy rival of Cold Spring Harbor in attractiveness as a place to live and to work (and, of course, to live and to work were synonyms). No work hours were too onerous, no kind of work and no detail were to be avoided or overlooked, if they helped his beloved laboratories. His directorships were, quite literally, full-time occupations. Demerec worked successfully on many research problems and on diverse materials. After his early studies on maize, he took up the study of mutable genes in Drosophila virilis and in the larkspur, Delphinium ajacis. Some of these genes were observed to mutate only in somatic cells, others only in germ cells, and still others in both. This led logically to a series of investigations of radiation-induced mutations in the genes and the chromosomes in Drosophila melanogaster. Together with H. Fricke he studied the mutability under the influence of X-rays of different wave lengths, with M. E. Hoover the effects of deficiencies of small groups of genes or of single genes (discovering the so-called “cell lethals”), with B. P. Kaufmann and H . Bauer the nature and frequency of radiation-induced chromosomal changes. The Second World War presented the challenge of research to help the war effort. With Demerec’s help, E. R. Sansome undertook induction of mutations in the mold Peni-

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cillium, selecting those which were increasing the yield of penicillin. They were quite successful in this endeavor. I n the late 1930’s and early 1940’s Demerec began to look for materials for mutation studies in which very large numbers of individuals could be observed. Microorganisms naturally suggested themselves. Max Delbruck and Salvador Luria, then “enemy aliens,” came to Cold Spring Harbor in 1941 to work jointly on bacteria and bacteriophages. I n 1943 they published their classical studies on mutations which confer a resistance to the attack of bacteriophages on colon bacteria, Escherichia coli. Demerec and Fano were quick to appreciate the advantages of the Luria-Delbruck experimental procedures, and published in 1944 preliminary accounts and in 1945 detailed reports of their experiments on bacteriophage resistance in bacteria. From then on, Demerec worked almost exclusively in the rapidly growing field of the genetics of microorganisms. Mutations which make bacteria resistant to such antibiotics as penicillin and streptomycin, then coming into general use in medical practice, engaged his attention during the late forties. Somewhat later came a series of works on the influence of certain chemical substances on the mutability of bacteria. The main result was that different mutagens (mutation-inducing agents) increase the mutability of different genes in different proportions. This phenomenon still awaits its explanation. During the last decade of his life, Demerec showed again his undiminished ability to shift to new objects and new methods. His main and enduring interest, which runs through his entire scientific life, remained the same: The phenomena of mutation and the gene theory. However, he now worked with biochemical mutants of Salmonella typhimurium, using the method of transduction to analyze the hereditary materials in this organism. Together with numerous collaborators, among them one of his two daughters, Zlata Demerec (Mrs. P. E. Hartman), he made elegant studies of complex loci in Salmonella. Most interesting has been the discovery that, at least in this organism, the functionally related genes are often clustered together in the same part of the chromosome. The list of Demerec’s publications up to 1965 contains some 205 titles, and some papers are still in press. Demerec was Secretary-Treasurer of the Genetics Society of America, 1935-1937, its Vice-President in 1938, and President in 1939; Treasurer of the American Society of Naturalists in 1933-1935, Vice-president in 1947-1949, and President in 1954; member of eight different committees of the National Research Council at various times between 1940 and 1953 ; member of the International Committee of the International Genetics Congress from 1939 to 1953; Chairman of the section of Zoology

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and Anatomy of the National Academy of Sciences from 1958 to 1961; member of various other committees, editorial boards of scientific journals, and active participant in meetings, symposia, and congresses too numerous to mention. Together with C. B. Bridges he founded “Drosophila Information Service,” the prototype of several such bulletins sent periodically, usually free of charge, to workers studying a given material or problem. He continued to be the editor of this “D.I.S.” long after he himself abandoned active research on Drosophila, in fact until his retirement from Cold Spring Harbor in 1960. Already in the 1930’s Demerec planned a book, Biology of Drosophila, chapters of which were to be written by a series of authors, some of whom came to Cold Spring Harbor to do the necessary studies. As so often happens with such collective works, some chapters were delayed so long that the book was published only in 1950 (by John Wiley, New York). Yet the book had a success, being a manual which any student of Drosophila needs. The book soon went out of print, and a new printing was issued in 1965. From 1947 to 1958, Demerec edited nine volumes of Advances in Genetics, a series of publications of considerable importance in the development of modern genetics. Another undertaking which was destined to play an important role in the development of genetics started modestly in 1945, when M. Delbruck gave his first “bacteriophage course” to summer visitors, some of whom were coming regularly or occasionally to Cold Spring Harbor to work at the Biological Laboratory, using the laboratory facilities and the helpful hospitality extended by Demerec. This and related courses soon became a regular feature of the summer activities a t this Laboratory, together with the more formal Symposia on Quantitative Biology. These Symposia were initiated earlier, in 1933, but they developed under Demerec’s sponsorship and have been held annually since 1941, each symposium resulting in the publication of a rather awkwardly large red-bound volume, now found in libraries of most institutions where genetics research is carried on, as well as in the personal libraries of many geneticists. Demerec’s organizing ability and good judgment attracted to these symposia at one time or another probably all, or a t least most, of the outstanding geneticists and evolutionists living in the United States, and many foreign ones. Topics ranging from biophysics, biochemistry, embryology, molecular, microbial, population, and evolutionary genetics, to ecology, demography, and anthropology have all had their places in these symposia. Many an important discovery, including the now celebrated Watson-Crick model of the structure of DNA, was first discussed in these Cold Spring Harbor Symposia and the summer courses following the Symposia.

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For nineteen years, from 1941 to 1960, Demerec carried a heavy load of administrative duties, directing two scientific institutions, as well as the manifold responsibilities of editorships, committee memberships, etc. What is most remarkable and admirable is that, carrying this load, he not only did not abandon his personal research work but much of this research continued to be done with his own hands and eyes, rather than by technicians and assistants. Demerec was a master of the gentle art of “grantsmanship”; in a sense he was, fashionably, one of the scientific “empire builders”; but he was one of the few of these (personally, I know of no other) who managed at the same time to remain an active, practicing, research worker in his own right, as well as a busy administrator. Some of his friends wondered how he accomplished this feat, which many beginning administrators hope to accomplish but few actually do. A part of the answer is that the field of his interests became narrower and more specialized as time went on. I n his thirties, Demerec had a living interest in all branches of genetics and a broad understanding of the whole field. As genetics grew by leaps and bounds, he was able to learn new ideas and new methods; a t the same time he developed some blind spots. This was, however, not the whole story. By far the most important component of this story was Demerec’s total dedication to science, which dominated his personality to the end of his life. So complete it was, that it virtually eliminated small talk; Demerec was able to spend an evening in a friendly gathering without uttering a word, and yet enjoying the company! He was not a t all taciturn in scientific or organizational discussions. I n 1921 Demerec married Mary Alexander Ziegler, who survives him, as do their two daughters, Mrs. Philip E. Hartman and Mrs. Neville Dyson-Hudson. One of Demerec’s most engaging personal qualities was his kindness, which led him to trust people sometimes rather indiscriminately. H e made some unfortunate appointments and overoptimistic plans. Upon his retirement from administrative posts in 1960, he was unable t o continue his work in his beloved Cold Spring Harbor laboratories. To terminate his research was to him unthinkable. The Brookhaven National Laboratory came to the rescue, made him Senior Geneticist, and enabled him to work happily with a small group of enthusiastic collaborators for five years (1960-1965). He continued, however, to live in his house near Cold Spring Harbor, and the daily commuting to Brookhaven was increasingly becoming a strain, especially as his health began to weaken. His spirit was not weakening, however, and neither was his enthusiasm for genetics research. He accepted a research professorship a t the C. W. Post College, and proceeded to organize another new laboratory; the

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laboratory was beginning to function when death came with merciful suddenness. Demerec had his share of academic honors. He was, in addition to being a member in the American Philosophical Society (elected in 1952), a member of the National Academy of Sciences, the Yugoslav Academy of Sciences, Royal Danish Academy of Sciences, Genetics Society of Japan, Society of Biology of Chile, and served his turn as president of the Genetics Society of America and the American Society of Naturalists. He received the Kimber Genetics Medal of the National Academy of Sciences, and honorary doctorates from Hofstra College, the University of Zagreb, and Long Island University.

Theodosius Dobzhanslcy

With his daughter, Mrs. Rada Dyson-Hudson

MlLlSLAV DEMEREC* (1895-1966)

I n 1919, when a certain young Yugoslav agricultural student came

to Cornell University to pursue graduate study in genetics under R. A.

Emerson, the first great decade of American genetical studies was just drawing to a close. The ensuing four and a half decades, to the very time of his recent death, have been identified with Milislav Demerec’s own career and contributions as with few others. H e was among the dozen or so giants whose work has made the United States preeminent in this biological science. His own contributions reflected not only good judgment in recognizing the most important problems but astounding initiative and flexibility in shifting from one area of significance to another, a t the cost of abandoning tried techniques and acquiring skill in entirely new ones. Yet equally important to the advancement of science, have been his services as scientific leader and administrator. Even the briefest account must not ignore their value. Demerec began his genetic investigations with a study of the basis of variable phenotypic traits. The striping of leaves and the variegations of maize seeds and the virescence of seedlings that at first are albino but later turn green evoked in him a deep interest in the basis of somatic mosaicism. Suspecting somatic mutation to be a prevalent cause of such changes, he very early directed attention to this phenomenon, and shifted from maize to Drosophila virilis and to delphiniums as appropriate organisms for probing the nature of the factors that control mutation rates. His classic papers on the unstable genes of Drosophila virilis remain basic to an understanding of the mutation process. A second period in Demerec’s genetic investigations began with the study of X-ray-induced mutations and deficiencies in Drosophila in the early 1930’s. Here, too, an interest in the role of gene mutation in ontogeny was evident, since one of his most important early contributions to this new area was the relation of induced deficiencies to cell lethality. With the introduction of salivary gland chromosome analysis, the work on deficiencies and other types of chromosome aberrations advanced *Reprinted through the courtesy of the Cold Spring Harbor Symposium on Qwntitative Biology ~~:XXI-XXII. xxiii

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rapidly, in collaboration first with Margaret Hoover, and later with B. P. Kaufmann, Eileen Sutton, and others. Demerec’s interest in the factors controlling spontaneous mutability was not lost, however. From this period date also those extremely significant studies of the differences in mutability in various wild-type strains of Drosophila melanogaster, and the identification of a mutability-stimulating gene in the Florida stock. By the 1940’s, Demerec was studying the effects of ultraviolet radiation and of neutrons in inducing gene mutations and chromosome aberrations, but the advent of the phage group, which summered in Cold Spring Harbor for the first time in 1941, and the practical demands of World War 11, brought about a shift of activity in his laboratory a t Cold Spring Harbor. Of enormous practical importance was the work performed to induce, in Penicillium, mutations that would increase the yield of penicillin. Of greater long range value were the parallel studies of mutations evoking resistance on the part of Staphylococcus to penicillin and other antibiotic agents or to drugs. The 1945 paper of Demerec and Ugo Fano on bacteriophage-resistant mutants in Escherichia coli marked a definite change in direction in Demerec’s work. B y the end of the 1940’s Drosophila studies had been fully replaced by studies of chemical mutagens and their effects on the genes of bacteria, especially in relation to mutations promoting bacterial resistance. This work in turn led, in the 1950’s, to the studies that were to occupy Demerec until the end, namely, the investigation of recombination and the fine structure of the gene in Salmonella. Independently, and in collaboration with Philip Hartman and others, Demerec analyzed the functional organization of the bacterial chromosome and discovered a remarkable parallelism between the sequence of genes and the sequence of steps in a biosynthetic chain. Aside from his earliest scientific studies while a graduate student, and his years following retirement, which were spent a t the Brookhaven National Laboratory and a t C. W. Post College, all of Demerec’s long career was identified with Cold Spring Harbor, from the first day in 1923 when he arrived as a fresh Ph.D. to join the staff of the Department of Genetics of the Carnegie Institution of Washington, to the day in 1960 when he retired as Director of both the Carnegie Institution’s Department of Genetics and of the Long Island Biological Laboratory. I n the span of nearly forty years of activity, Demerec had not only made scientific contributions that earned him membership in the National Academy of Sciences, the American Academy of Arts and Sciences, the American Philosophical Society; and honorary memberships in the British Genetical Society, Yugoslav Academy of Sciences, and the

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Royal Danish Academy, as well as the highest award among geneticists, the Kimber Medal (1962), he had also made Cold Spring Harbor a worldwide focus of inspiration and leadership in genetics. This role began when he became Director of the Biological Laboratory in 1941, and in 1943 Director of the Carnegie Institution’s Department of Genetics. The close and fruitful collaboration of the two neighboring biological institutions thus started toward the fusion that eventually took place. The outstanding series of Cold Spring Harbor Symposia on Quantitative Biology brought scientists from all over the world to engage in relaxed, informal conversations on the sandspit and the veranda of Blackford Hall, as well as in the formal sessions where interesting and sometimes epochal papers were presented and discussed. From 1941, when the symposium on “Genes and Chromosomes” was held, until the year of his retirement, it was chiefly the foresight and wisdom of Milislav Demerec in choosing subjects of most timely interest, selecting participants of worldwide eminence, organizing the program, and editing the proceedings that made Cold Spring Harbor in June the mecca of genetic biology, where genetics, evolution, and biochemistry came together in fruitful interaction. Stimulating contacts spread through the summer as visiting research workers exchanged ideas with each other and with members of the permanent staffs of the Biological Laboratory and the Carnegie Institution. At least one visitor can testify that he never worked harder than during those summers and never had a better vacation for himself and his family than in those years when Cold Spring Harbor was a summer home and laboratory. By judicious appointments to the research staffs of the two institutions, Demerec kept the spearheads of investigation probing into significant new fields. Especially noteworthy was the arrival of A. D. Hershey in 1950. Cold Spring Harbor had already become a center of the phage group. Summer meetings had led in 1945 to the establishment of a special summer course for instruction in the theory and techniques of genetic investigation with bacterial viruses. A parallel summer course in the genetics of bacteria was started in 1955. These and later additions to the summer courses have made Cold Spring Harbor the biaological center where germinated much of the work that resulted in present-day microbial and viral genetics. Somehow, year after year, Demerec made this possible on a financial shoestring. The Carnegie Institution provided new laboratories for its own staff, and grants were obtained to support the permanent staff of the Biological Laboratory and the summer courses, and to maintain the charming but ancient buildings. Year after year local friends of the Cold Spring Harbor community were encouraged to help maintain the Laboratory through their gifts. The scientific community throughout

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the United States was canvassed. I n mysterious ways, and with real courage, for two decades Demerec found the means not only to keep the summer programs and the year-round research going, but to expand them in significant directions of the new molecular biology that was springing into being. Demerec served the scientific community in many ways. For twenty years he helped to organize the International Congresses of Genetics, both as a member of the Permanent International Committee and as a member of Organizing and Program Committees. H e filled every office, including that of president, in the Genetics Society of America. H e was treasurer and later president of the American Society of Naturalists. He served on the U.S. Committee of the International Union of Biological Sciences and on the Committee on the Genetic Effects of Atomic Radiation of the National Academy of Sciences. H e served as chairman of the Section of Zoology and Anatomy of the National Academy of Sciences. He started Drosophila Information Service, the first of the professional newsletters and information services in genetics; and he established the Drosophila stock center a t Cold Spring Harbor in the Carnegie Department of Genetics, the first of such stock centers to maintain the important experimental stocks of Drosophila and to supply them to Drosophila workers around the world. H e founded the series Advances in Genetics (Academic Press) and served as editor for nine volumes, from 1947 through 1958; and he served on the editorial boards of several genetical journals. Yet in the end those of us who knew him well will remember him as the characteristic spirit and impulse of Cold Spring Harbor, the man who made it a nerve center of modern genetics.

H . Bentley Glass

With Dr. Richard 0. Goldschmidt

MlLlSLAV DEMEREC* (1895-1966)

We mourn Dr. M. Demerec’s sudden death. It was reported that Dr. Demerec had passed away suddenly, though it was a cheerful day in the middle of April. We have lost another very great geneticist who was one of the pioneers in genetics. This follows Dr. F. Ryan’s sudden death of just three years ago. Dr. Demerec has made a great contribution to the Genetics Society of Japan as an honorary member. Also, many Japanese researchers working a t the present time were recipients of his kind and valuable comments. With these persons, we would like to look back upon Dr. Demerec’s biographical sketch, scientific achievements, and so on . . . with prayers for the repose of his soul from the bottom of our hearts. The New York Times reported Dr. Demerec’s death with this headline : Dr. Milislav Demerec, 71, Dies; Discoverer of Mutable Genes. H e was over 70 years old, although it was quite difficult t o realize this since he still looked very active. Dr. Demerec was born in Kostajnica in Yugoslavia in 1895. He was educated a t an agricultural college in KriBevci in that country and then studied in France. When he was 24 years old, in 1919, he went to the United States to study the genetics of corn as a Research Assistant a t Cornell University. When he was awarded his Ph.D. from Cornell University, in 1923, he was appointed Resident Investigator in the Genetics Department at the Carnegie Institution a t Cold Spring Harbor i n New York State. He had worked a t the Institution for 37 years, until he retired a t the age of 65, in 1960. During this time, he received United States citizenship, in 1931, and he served as Director of the Institution for 17 years, 1943-1960. Furthermore, he served as a Director of the Long Island Biological Laboratories adjacent t o the Carnegie Institution from 1941 to 1960, and held an additional post as an Associate in Genetics at Columbia University. Retiring from the Carnegie Institution in 1960, he was appointed Senior Geneticist a t Brookhaven National Laboratory in New York State, and worked solely as a researcher for the rest of his life, leaving most of his other responsible positions. And he just recently organized a new laboratory at C. W. Post *Reprinted through the courtesy of the Japanese J o u m l of Genetics, 41:25& 251. Translation by Ikuo Ino. xxix

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College on Long Island, planning to continue his research activity in the future. However, unfortunately, he passed away on the night of April 12th a t his peaceful home, surrounded by trees, near Cold Spring Harbor. It was said, also, he was so active that he even attended a party taking place just before he passed away. The time must have come suddenly and peacefully. Dr. Demerec was member of the National Academy of Sciences, the American Philosophical Society, the American Academy of Arts and Sciences, the Genetics Society of America, and other societies and academies. He was also awarded many honors for his remarkable studies in genetics. It might be appropriate to mention the following honors: Honorary Degree of Doctor of Humanities, given by Hofstra College in 1957 for his role in developing techniques to obtain higher yields of penicillin through mutation ; the Kimber Genetics Medal, awarded by the National Academy of Sciences in 1962. As we can see in Dr. Demerec’s research achievements, he has changed his experimental materials from corn to Drosophila to microbes, e.g., E . coli and Salmonella, each of which had advantages for study a t the time. However, his main interest was concentrated on one main problem, that is, the clarification of the nature of the gene. As we can see from the headline of the New York Times mentioned above, he discovered and studied mutable genes. He also made studies of mutation and of fine structure of genes in bacteria by using transduction techniques, and he greatly contributed toward establishing a concept of the gene as a functional unit (i.e., cistron) through pseudoallelism. Furthermore, he found that genes with related biochemical functions tend to be adjacent on the chromosome, and these findings were linked to the Operon Theory proposed later. In this manner, with some resolution of gene structure, one of his interests moved to the genetic structure of the whole bacterial chromosome. Comparing the genetic maps of the chromosomes of E. coli and Salmonella, he found that the arrangements of the genes are similar; nevertheless there are various grades in homology among species for genes with corresponding functions. In his last paper, he discussed the problems of evolution in bacteria by summarizing these results. This relatively short paper, evidence of a distinguished career in research, must have stimulated new ideas (Demerec, M. 1965. Gene Differentiation, Nat. Cancer Inst. Monogr. 18, 15-20). Furthermore, we cannot remember Dr. Demerec without thinking of the many young Japanese researchers, including the authors, who, not long after graduation from college, were introduced to microbial genetics by his kindness a t either the Carnegie Institution or a t Brookhaven National Laboratory. We will list these Japanese researchers in our

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memory. I n 1953, Takashi Yura (1953-1955) was first introduced to Dr. Demerec by Prof. Hitoshi Kihara who was then a t Kyoto University, and was followed by Haruo Ozeki (1955-1957), Tadashi Miyake (1957-1959), Kazuo Hashimoto (1957-1958), Jun-ichi Ishidsu (19591961) , Kiyoshi Mizobuchi (1960-1961), Yasuo Nishioka (1961-1963), Norika Ohta (1962-1964), Ikuo Ino (1965 to the present), Hiraku Itikawa (1965 to the present). I n this way, we have been obliged to him always. We should like to thank Dr. Demerec sincerely for giving an opportunity to study abroad to these young, unknown researchers, for introducing them to the study of microbial genetics, and also for giving them an opportunity to see personally many internationally outstanding scientists. When he was the Director a t the Carnegie Institution, visiting Japanese researchers a t Cold Spring Harbor were too plentiful to enumerate. I n any event, we will make a list of the names of visitors who stayed for several weeks or months and who we can recall when the authors were staying there: Drs. Itaru Watanabe (1955), Tetsuo Iino (1956), Hisao Uetake (1957), Tsunehisa Amano (1957), Tsutomu Watanabe (1957) , Chozo Oshima (1957) , Hideo Kikkawa (1958), Tadao Horiuchi (1959) , and also Jun-ichi Tomizawa (1956-1958) and Teiichi Minagawa (1958-1959), both of whom were working on phages in the laboratory of Dr. A. D. Hershey, and Japanese researchers in Dr. B. P. Kaufmann’s laboratory. These persons have a pleasing memory of Cold Spring Harbor. All those listed above, and many others, have been obliged to Dr. Demerec directly or indirectly. We would like to pray for the repose of Dr. Demerec’s soul with these persons again. We were looking forward to seeing Dr. Demerec and to talking with him cheerfully again a t the International Congress of Genetics which will take place two years from now in Japan. Unfortunately, it is now impossible. Nevertheless, we should like to wish sincerely that Dr. M. Demerec could watch the development of genetical studies in the future, if possible even from the other world.

Jun-ichi Ishidsu Haruo Ozeki Tadashi Miyalce Takashi Yura

With Sir Alexander Fleming

MlLlSLAV DEMEREC* (1895-1966) Milislav Demerec died suddenly on April 12, 1966, a t his home in Laurel Hollow, New York. He was seventy-one years old. Dr. Demerec was born in Yugoslavia, and began his scientific training there. His graduate work was done at Cornell University, in Ithaca, New York, where he obtained the Ph.D. degree in 1923. I n the same year, he joined the staff of the Department of Genetics of the Carnegie Institution of Washington, in Cold Spring Harbor, New York, remaining there until his retirement in 1960. In 1941, he was appointed Director of the neighboring Biological Laboratory, and Acting Director of the Carnegie Institution’s Department of Genetics. Two years later, he became Director of the Department of Genetics, and served as head of both units until 1960. After his retirement, Dr. Demerec continued to live in the Cold Spring Harbor area, while carrying on his research a t the Brookhaven National Laboratory, in Upton, New York. In January, 1966, he became Research Professor in Biology a t the C. W. Post College of Long Island University, and was engaged in organizing his new laboratories there a t the time of his death. Dr. Demerec contributed richly to the growth of genetics, not only through his own extensive research, but also through his key role in the development of the Cold Spring Harbor laboratories as a world center for progress in genetics. It was an extraordinary accomplishment to combine an active career as an investigator with the burden of administering as complex an institution as the Cold Spring Harbor laboratories. Even for a man of Dr. Demerec’s unusual stamina, the normal working day could not encompass all that he had to do. His solution was to arise, most days, a t about four A. M., and to make use of the early morning hours for tasks requiring concentration-planning research, writing papers, studying data. By the time most of the other staff members were starting work, Dr. Demerec had been in action for four or five hours. Dr. Demerec’s characteristic style of approaching scientific or administrative problems was a unique blend of optimism and energy that often

* Reprinted through the courtesy of Mutation Research 4, 237-239. xxxvii

xxxviii

OBITUARY

worked wonders. Not many other geneticists of his generation, trained and experienced entirely in the methods and materials of classical genetics, were intrepid enough to plunge into work with microorganisms in the forties. Dr. Demerec had sensitive antennae for new directions in genetics, and his appreciation of the possibilities of work with bacteria and viruses helped to catalyze the explosive development of microbial genetics that began about twenty-five years ago. He was not content, however, with merely encouraging and providing facilities for such pioneers as Max Delbriick and Salvador Luria. Converting a part of his Drosophila quarters into a microbiological laboratory, he was soon himself entrenched in the new avant garde, carrying on an active program of experiments with Escherichia coli, and going on to make some of his most important scientific contributions as a bacterial geneticist. Dr. Demerec’s earliest work was on the inheritance of chlorophyll characters in maize, initially as part of the intensive investigation of linkage relationships being carried out by Professor R. A. Emerson a t Cornell University. While still engaged in this work, he began to look for an experimental approach to the nature of the gene, always to him the most fascinating question of all. He was attracted by the possibilities of variegated characters in plants, which Emerson had proposed might be due to “unstable genes.” Since no way was yet known to alter genes experimentally, and since changes in ordinary “stable” genes were rare, the study of variegation seemed to Dr. Demerec a promising approach. At Cold Spring Harbor, while continuing his work with maize, he began an intensive study of “unstable genes” in Delphinium, and later in Drosophilia virilis. At the same time, he tried repeatedly to find a way to increase mutation rates by experimental treatments of various kinds. Although these efforts were not successful, they explain the eagerness with which he welcomed the new tool of X-ray induction of mutations discovered in 1927 by Muller. Using Drosophila melanogaster, he studied the frequency and types of changes induced by X-rays a t 22 loci of the X-chromosome. In 1933, in a lecture entitled W h a t Is a Gene?, he described his concept of the gene as a complex organic molecule, within which different kinds of chemical rearrangements were responsible for different allelic states. I n what now seems a remarkable flash of intuition, he used a diagram of the molecular structure of thymus nucleic acid (as it was believed to be in 1933) to illustrate the kind of complex organic molecule a gene might be. During the latter thirties, Dr. Demerec continued to work mainly with Drosophila, correlating X-ray induced lethal mutations with cytologically observable changes in salivary-gland chromosomes, and adding important data to the growing body of information about position

OBITUARY

XXXiX

effects. His work was never far from the most advanced frontier in genetics. His contributions were solid and substantial. I n 1941, Dr. Demerec took over the leadership of the Cold Spring Harbor laboratories. Without slackening the pace of his own work, he began the formidable task of molding the two laboratories into a smoothly functioning unit, which soon became a gathering-place for geneticists from all over the world, and a center from which some of the most exciting discoveries of our time were to emanate. He did not readily delegate authority, even in small matters, and concerned himself as intensely with the clipping of hedges and painting of sheds as with the planning of experiments and organizing of Symposia, shifting gears easily from one kind of problem to another. Partly owing to this devotion to details, the Cold Spring Harbor center bore Dr. Demerec’s personal stamp in countless ways, not the least of which was the atmosphere of friendly informality in which he believed that science could best thrive. During World War 11, Dr. Demerec gave much time and energy to the war effort, supervising (among others) a project aimed a t improving the yield of antibiotics from molds. During this period, too, he played a major part in the birth of bacterial and bacteriophage genetics, starting with his invitation to Max Delbriick to participate in the 1941 Cold Spring Harbor Symposium. With Dr. Demerec’s enthusiastic support, the “phage group” adopted Cold Spring Harbor as unofficial summer headquarters, and as a training center for new recruits. The spectacular progress made by this group, and by others working in microbial genetics, was reported a t the 1946 Cold Spring Harbor Symposium, one of the most historic of the series of memorable meetings organized by Dr. Demerec. B y this time, he had made an active ‘start on his own work with bacteria, having done some of the earliest experiments on radiation-induced mutations in E . coli. For a while, he worked on both Drosophila and E . coli, gradually increasing his involvement in the bacterial work. His contributions include studies of spontaneous and induced mutations to bacteriophage-resistance, to resistance to antibiotics and to prototrophy. In much of this work, he developed basic methods still widely used in bacterial mutation studies. He worked also on chemical mutagenesis, and made important observations on “mutagen-stability.” I n 1954, only six years before his retirement, Dr. Demerec began the work that seems certain to be considered his most valuable scientific contribution. Once again exhibiting the flexibility that characterized his approach to research, he decided to work with Salmonella typhimurium, in order to make use of the newly discovered tool of genetics transduction. In these studies, he provided the first direct evidence for intracis-

XI

OBITUARY

tronic recombination in bacteria, and for the clustering of genes determining functionally related enzymes. Far from slowing down after his retirement, Dr. Demerec continued, literally until the day he died, to pursue with vigor and enthusiasm the implications of these important discoveries. His latest work includes studies of the fine structure of certain genes, of the evolutionary significance of fine-structure differences between homologous genes in different bacterial species, and of the phenomenon of “selfing” in transduction. Dr. Demerec enjoyed gardening, boating on Long Island Sound, and talking shop ; he disliked conventional social gatherings, practical jokes and displays of emotion. He was embarrassed by his many honors, which included the Kimber Genetics Award, election to the National Academy of Sciences and to the Presidency of the Genetics Society of America. He served genetics well, and his life and work are a part of its story forever. Evelyn M . Witkin

With Dr. Bernard D. Davis

MlLlSLAV DEMEREC* (1895-1966) Milislav Demerec was an outstanding geneticist. He was the Director of the world-famous laboratories a t Cold Spring Harbor (The Biological Laboratory and the Department of Genetics of the Carnegie Institution of Washington) during what might easily prove to be their Golden Era. Furthermore, he was a kind and good person. He died on April 12, 1966. Dr. Demerec was a quiet-nay, silent-man. Thus, despite eleven years association with him, I know remarkably little about his early life; the few facts that are known are to be found in his own curriculum vitae or must be learned from his friend of early school days, Alojz TavEar of Zagreb. I n brief, he was born a t Kostajnica, Croatia (Jugoslavia) on January 11, 1895. He attended high school and college in or near Zagreb. Following World War I he attended the College of Agriculture a t Grignon, France and then became a student of R. A. Emerson a t the College of Agriculture, Cornell University. After obtaining his degree in 1923 for work on the genetics of maize, he went to Cold Spring Harbor as a staff member a t the Department of Genetics, Carnegie Institution of Washington; it was this laboratory of which he was Director from 1943 until 1960. Somewhat earlier, in 1941, he had acquired the directorship of the physically adjacent laboratory, The Biological Laboratory-a position he also held until 1960. Following his retirement from the laboratories a t Cold Spring Harbor, he was appointed Senior Geneticist at the Brookhaven National Laboratory for a period of five years. At the age of 71, while organizing a new laboratory a t C. W. Post College, he died. To know these details is not to know Dr. Demerec. H e was, as I said, a quiet man. I n part, he was shy; in part, preoccupied with his own thoughts. During evening parties a t his home, he wanted no more than to have the conversation and activity involve everyone else while he, silent but contented, sat to one side in his favorite chair. What experiments were planned, what sources of funds were identified, what symposia topics were selected, or what committee business was organized during these social evenings only Dr. Demerec knew. Promptly a t ten

* Reprinted through bhe courtmy of Genetics 87:13. xxxiii

xxxiv

OBITUARY

o’clock, if no senior guest had already done so, Dr. Demerec would arise, look a t the clock with a startled exclamation, and announce that the morrow was coming. And so the evening would end. Dr. Demerec was physically a strong man. His day began before six each morning working a t his house-gardening in the spring and summer, otherwise going over data or papers in his study. As Director of two laboratories, he arrived on the grounds a t eight with the first workers, supervised the work schedules, discussed financial matters, listened to and commented on new experimental data described by various colleagues and staff members he happened to encounter, went over carefully the data from his own laboratory staff; and, in addition, carried out the multitude of other tasks that fell on his shoulders either through committee work, editorial duties, or the affairs of various scientific societies and congresses. He was an exceptionally well-organized man but that in itself was not enough, he needed, and possessed, tremendous physical stamina to withstand this routine work load. One of his remarkable abilities was that which enabled Demerec to evaluate a problem, reach a decision, and then to put the matter out of his mind. This ability reduced the strain of supervising the day-byday operations on the laboratory grounds, of supervising the housing and dining of hundreds of summer guests, and of assuming responsibility for the research efforts of numerous post-doctoral fellows and research associates. In each of these matters, he serenely did the best he could do under the circumstances and then refused to fret over what might otherwise have been. The Cold Spring Harbor Symposia on Quantitative Biology were Dr. Demerec’s special joy. With the exception of the periodically recurrent topic, The Gene, suggestions for appropriate subjects came from his many friends and colleagues ; he invariably favored those proposals that were just beyond what a t the moment was fashionable research. His only instruction to the members of each year’s Program Committee was for them to prepare the ideal program as if unlimited funds were at their disposal ; raising funds for these meetings was his responsibility and, should he fail to find enough, it was then that the program would be modified. He had a complete and well-justified faith that funds are easier to find for excellent symposia than for mediocre ones. The only foreign scientist who could not be brought to the meetings a t Cold Spring Harbor despite Demerec’s repeated efforts was J. B. S. Haldane; federal regulations concerning visas effectively barred Haldane’s entry into the country. Ironically, Haldane finally came to the United States to participate in a symposium sponsored by the space agency. The summer courses, including the nature study course for children

OBITUARY

xxxv

of the scientific and neighborhood communities, were also a source of great pride to Dr. Demerec. The Phage Course, first under the direction of Max Delbruck and then for many years under that of the late Mark Adams, was in many respects the birthplace of molecular genetics. The talent that was involved in P. U. (Phage University) a t one time or another during Demerec’s years a t Cold Spring Harbor is nearly unbelievable; four Nobel Laureates come to mind immediately but the list of truly superb research men who passed through this course would be at least twenty times as great. The physical nature of the gene remained throughout Dr. Demerec’s life his main research problem. During the course of his work he passed with deceptive ease from maize t o delphiniums to flies and to microorganisms leaving behind a body of excellent data in the literature on each. The climax of his work was undoubtedly the finding that the linear arrangement of genes (Demerec was reluctant to adopt new terminology such as “cistron”) controlling tryptophan synthesis in Salmonella typhimurium corresponds to the order of biochemical reactions that these genes mediate. “The assembly line has finally been found,” was Haldane’s comment upon hearing of this work. Subsequent studies on S. typhimurium involved analyses of the fine structure of its chromosome (including its large “silent” regions) a comparison of this structure with the corresponding one of Escherichia coli, and a study of the consequences of transferring genes from the chromosome of one of these species to that of the other. In short, Demerec had started a systematic attack on evolutionary genetics a t the molecular level. Dr. Demerec succumbed to a heart attack. His death reminded me of a morning many years before when we walked together from Jones Laboratory (on the waterfront a t Cold Spring Harbor) up the steps toward Bungtown Road. About midway up the rather long slope Dr. Demerec paused, turned toward the harbor and its surrounding hills, and gazed a t the familiar but always beautiful view. Then, quietly as if he were discussing the sea gulls in the mud flats of the inner harbor, he said that his doctor had told him to take it easy and so he would like to rest for a moment. “Anything serious?” I asked as we stood there. “Doughhhh, just my heart, nothing really serious.” Many times during my subsequent years a t Cold Spring Harbor I saw Dr. Demerec pause halfway up those steps and turn to gaze out over the harbor; to the casual observer it would appear that he never tired of his laboratory and its surroundings. And, of course, he never did.

Bruce Wallace

CLASSIFICATION AND MAPPING OF SPONTANEOUS AND INDUCED MUTATIONS IN THE HISTIDINE OPERON OF Sahonella

.

.

Philip E Hartman. Zlata Hartman. Ruth C Stahl Department of Biology. The Johns Hapkinr University. Baltimore. Maryland

Bruce

N. Ames

Department of Biochemistry. University of California. Berkeley. California

I . Introduction . . . . . . I1. Materials and Methods . . A . Isolation of Mutants . . B. Characterization of Mutants I11. Results . . . . . . . I V. Discussion . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Deletion Mutations . . . . . . . . . B . Stable Mutations . . . . . . . . . .

C . Frameshift Mutations . . . . . . . . . D Base Substitutions . . . . . . . . . . E The Ratio of Missense to Nonsense Mutations . . F . Multiple Mutations . . . . . . . . . . G Polarity . . . . . . . . . . . . . H Cross-Reacting Material (CRM) . . . . . . I Spontaneous Mutations . . . . . . . . J . Screening Potential Mutagens . . . . . . . K . Intragenic Complementation . . . . . . . L . Gene-Enzyme Relationships . . . . . . . M . Genetic Mapping . . . . . . . . . . V . Summary . . . . . . . . . . . . . References . . . . . . . . . . . . .

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1 2 2 3 3 9 9 21 21 22 23 24 25 25 26 26 26 27 28 29 30

.

I Introduction

The senior author’s undergraduate class notes in introductory college bacteriology for 1948 carry the notation : “Nucleus-not organized.” The intervening years have seen a drastic reassessment of this idea; now over 250 gene loci have been precisely located on the closed-circular Salmonella typhimurium chromosome (Sanderson. 1970) . I n fact. we 1

2

PHILIP HARTMAN, ZLATA HARTMAN, RUTH STAHL, BRUCE AMES

have devoted considerable effort in elucidating the organization and functioning of about 0.3% of this ‘ h o t organized” entity. Genetic results are reviewed here. Our work on Salmonella grew entirely out of Zinder and Lederberg’s classic report on transduction (1952) and Dr. Milislav Demerec’s recognition that this new technique would allow analysis of genetic fine structure. In what seemed at the time a rash move, Dr. Demerec converted his entire laboratory from studies on Escherichia coli to pursuit of the gene in S. typhimurium. The mutational and recombinational complexity of genes in bacteria was immediately established (Demerec, 1955, 1956; Demerec et al., 1955, 1956; Demerec and Demerec, 1956). Furthermore, these same studies demonstrated that genes with related functions were clustered on the chromosome. One question important to basic genetics had been answered but new questions arose. The strictly genetic work of two of us in Dr. Demerec’s lab was abandoned for two years and then, guided by Dr. Demerec’s inspirations, reinitiated elsewhere (Hantman et al., 1960a,b). Concurrent enzyme analyses promptly established gene-enzyme relationships on a sound basis and demonstrated coordinate control of enzyme production by genes of a cluster (Ames and Garry, 1959; Ames et al., 1960). These lines of evidence were relied upon by Jacob and Monod in their brilliant synthesis outlining the operon theory of gene regulation (1961). I n work since, genetic experiment has paved the way for critical biochemical analysis. II. Materials and Methods

A. ISOLATION OF MUTANTS Almost all mutants were isolated in strain LT2 of 8. typhimurium except for one series isolated as spontaneous mutations in strain LT7 (Tables 1 and 2). The origins and methods of isolation, after penicillin selection, of many mutants with isolation numbers below l 4 l l are given elsewhere (Hartman e t al., 1960b; Loper et aZ., 1964). Also available in the literature are descriptions of the isolation of spontaneous mutations selected for polarity (Fink et al., 1967) and of ICR-induced mutations (Oeschger and Hartman, 1970). Additional mutations not previously described in the literature are included in Table 1 and were isolated by similar procedures. A series of mutations was induced by N-methyl-N‘-nitro-N-nitrosoguanidine (NG) in an arabinose nonutilizing mutant (am-9) of strain LT2. An overnight nutrient broth culture of bacteria was washed and

MUTATIONS IN THE HISTIDINE

OPERON OF

Salmonella

3

suspended for 30 minutes at room temperature in minimal E medium (Vogel and Bonner, 1956) lacking glucose and containing 200 pg/ml NG (Aldrich Chemical) . The suspension was centrifuged, resuspended in E medium, and diluted into a large series of tubes of the same medium containing 20 pg/ml L-histidine. After overnight growth, histidine-requiring mutants were isolated by screening through E medium containing penicillin and an amino acid mixture complete except for the omission of histidine. This screening procedure paralleled that used for isolation of most of the other mutants surveyed here (Hartman e t al., 1960b).

B. CHARACTERIZATION OF MUTANTS Genetic mapping was performed using P22-mediated transduction (Zinder and Lederberg, 1952; Demerec et al., 1956), Hfr crosses (Nikaido e t al., 1967; Sanderson, 1970), or F’ lac-mediated crosses ( A n t h , 1968). Often, preliminary genetic classification was carried out in complementation tests with a series of F’ his of E. coli K12 origin (Garrick-Silversmith and Hartman, 1970; G. R. Fink and J. R. Roth, personal communication). Complementation tests were performed by P22-mediated abortive transduction (Demerec e t al., 1956; Hartman e t al., 1960a; Loper et al., 1964) and additionally in gene hisD, by matings with strains carrying complementing mutations on F’ his of E. coli (Greeb e t al., 1971). Data on the presence and absence of immunologically cross-reacting material have been published: hisB (Loper, 1961), hisC (Whitfield e t al., 1966), hisA (Margolies and Goldberger, 1968), and hisD (Greeb et al., 1971). Procedures for reversion tests follow those outlined previously (Ames, 1971; Whitfield et al., 1966). Identification of nonsense mutations has relied on phenotypic suppression by streptomycin coupled with the detection of nonsense suppressors among revertants (Berkowitz et al., 1968) Strains SB393, SB789, and SB948 were used to test for ochre-amber, amber, and UGA suppressors, respectively. I n addition, some strains were tested by transduction into strains deleted for a large segment of the his operon and carrying amber or UGA suppressors, or by infection with an F’ episome carrying an ochre suppressor (Garrick-Silversmith and Hartman, 1970). Ill. Results

Table 1 lists the pat’terns of reversion of 1020 independently isolated mutations leading to a histidine requirement and mapping in the histidine

TABLE 1 Histidine-Requiring Mutants* NG+ ICR-

NG- ICR-

cp

Deletion (NG- ICR-)

NG- ICR+

NG+ ICR+

Spontaneous LT-2

Bal4m,C15m,Ba20m+, Bad2la,C31a-, A33mi. Db39m-,Bd40m+, F420, C4W, F a , F45m, G46m, Db49m-, C a y , G52m. F58u. Bc59m. BbBlm+, Bad102a-, Dbl3Om-, F1310, Bc136a. Dab139m-, Dbl4lm-. F144, F147, F148m, DablSLlr-, c1516, C163m-, B185m. F186m, BaclWm, Db45lm+, F453a, Db474m+, Da476m+, C5278, A528m+, Dab52%-, A530m+, AS32m+, Db55Sm-

BalZ, B&-, Dab27, H32. Bd47s+, A&. Dab@-. D a m ,ABW, 19% F103. F132a. Dab1378-, Dab142; Dab154, Db477s-, F479, D24418, F2446e

2 2 , F4l,55-, 57,133, 129,134-, 135,152, A478-

C l F , D a b l e - . Bbcd138 Bcdllr, Iab146, C14(T, ~ a b i 4 9 - ~, a b i ~FHS a,

None

Spontaneous LT-7

A30m+, Dab&-, Bd65m+, Db66m. Db68m-. A69m+, G70m. G72m. F73m. Dab74m-. F76m, Db77m+, Dab78m-, Ba79m+. A80m+, Db82m-, DbBm-, Db84m+, C86m, Db88m-. DbWm-, F91. Db92m+. F9fm. F95m. A100n-. C104m, Ibl05m. AlWm-, CllOm. Dblllm+. Dbll3m+,C115m. Bcll6m-, Cll70, G11%, C120m. C121a, Dab123n-, Dablam-, Ia125m, Da126m-, Ealblm, DalGBm-, Bc167m-, Dbl7Om-, Dbl'llm-, 172, 174n. A178m-, A179m+, A181n-, Db182m+, H193m. F212u, Bd213m+, Db214m+, Db215m+, Db216m-. Bd217m+, F218m. F219m, Da220m+, W21, Db223m-, F224, C225m, DbZ26m-, F227. W28, Bcd229m+, Db233m+, Bdd234+, F235m, Db236m+,Db237m-, Db239m-, FHOm, Bcd241n. Ia242m. Bad2438, Db244m-, Db245m+, A247m+, Db248m-, F251, Db254m+, G255m, A263n. EaZMm, Ia266m, A269m-. Db271, Dab274m-, F277m, Bad278u, A279m+, Ea284m. Bd28%+, Db29lm-, F293m, A294m+.Db295m. A300m+, Ia30lm, Db302m+, I177m

A67s-, AH-, Ia998, H107, DblOBe-. F1098, DallZ, F1148. Ball&, Dab1278, A1538, A1578, A1598, Dabl76-, Dabl91-. 81923, C194, Dab23V. -I-. A246-, A253-, Dab273-, Dab4OOn-, AS&

96-, 101-

B d F , F71, Dab85-. C87, C122, Fl68,F180,F232, F256

Cll70

x-ray

Bb53m+, G200u. C201m+, G204m. C202, DS2088,CZW, Jab%. A481h, 203,386,388,399, C207-,C377-, Dab391-, C516-, G205u. Bb206m+. C21Om+. Bb374m. A M , F466~.Dab48%, C493. B482.515, 519.520, C540, B.824 Bcd380a. Db381m+, F382m,Ba39lm, DaMSBs, Dab497, Iab498, A617eT, 533,538,801, D W , Dab.5186, A521-, C5248, 533s. 8Oa,SG9,810,813Db392m+. F3958, Dab396a-. F398m, C483m-, A485n-, C487a+. Dab49om-, F5438, Da54.5-, A547S. AS%-, F557s, F576n, Bd578e, H586n. F59&, C491a, Db492m+, C496m, G499m. F808, A8ll8+. Db81.5+, ABIBs-, A522m-, C5258-. F526u. F534m, A8288, Db8338 A535m+, Dab536a-, C537m. Iab539n. Bd542m, C M a , F548m, A550n-, A551mf, Ia553m. A561m+, Dab563a. F565m.F568m, Ia.570~.Ea571. G5720, Ba-d573a, G575m, G5810, C588m. Bb59om. A593m-, A594m+. E596m. F806m. Bad807a, Bc8128, A820n-, F823a, Ba-d82k, A834-

ZAminopurine G255n, A263n-, F336a. G337m. A338m+, A9678, Da988s+, D2443a F3390, C34Oa, C3418, Ia352m, Bcd353a, C3540, Bc3558, Dab362m-, Db363m-, C3648. C367mC. C368m. Dab4lOa-, Dab4lla-. DWOm-, G4210. DaMZh-. A423m+. F424m, Ba-d425n, C426a-, Ia427m, G4280, Bc429a-. A430m+, A431m-. F432a. C4348-. Dab4368, C439a-, F440. C441, Dab442a-. Dab443a-, A m - , C446a-,A449m+, Da450m-, Db451m+ A4521-, Dab47ln-, F5ooa. CWIa, c5020, Iab50k, C5078, c50&-, A512m+, A513m+, (25140, Ba-d573n, Ba-d647n, F8590,A%&-. A864m+, Bc8658, F8680, C869a. F8720, Dab873a-. A875n-, G8760, C879a. Dab88Oa-, C881a. Dab@%-, AW-, Ea885m. A8866, Dab889n-. C890m, Dab89ln-, Dab892a-, Dab893a-, Dab%-, Dab895m-, Dabs%-, F897m, A916m+, Db924m-, DbMIm-, Dab946n-. F9490,Dab952a-, A955nDab956n-, DabSGOn-, Dab962n-, Da986m-, B9&, Dab992a-, DabNBn-, F1027m, A1031n-, B1032m A1400u. B1408, D24360, A2782m, C2783m, C2785n, C2793o. C2794m, D2812a, A3701m, Dab37100

950

None

None

zz z

Y

3

Ei C W - , DMlZm-. F413o.

C441n

Y

E m

E: ,Y tl

1 %M 9

P

0

3

F 0

5

N

F

(Continued)

TABLE 1 (Continued) NG+ I C R

NG- ICR-

Q,

Deletion (NG- ICB-)

NG- I C P

NG+ ICW

w +I

None

None

None

A2437a, C24388, A2447m. Db2448m. Dab9s48, C2460:E3116*, E2449s. C245On. Dab2452a. Db2462m. Db246b. Db24729.2476s Db2463m. F2464m. I2465m. I247Om. Dab2471m. Db2474m. Db2475m

None

None

None

Dblm, C2m-, A3m+, F b , C8m-, DabW, A3W, BdlSC, F197n. F198, DalGu-, Eabllo, DablZlm-. Eb35m. Bad314, Dab466-. F a % , DbBOW, Dab607 Db36m, Db37m+, C62m. F306n. F308n, F31Om,Eab31la, GWm, Dab461r. F463u, C464m-. Db465m+, Eb48’lm. Bbe470a. Dam-.DaGolm+, Dab603a-, Db604m+

None

None

None

Faat neutrona

Iab333m, GBllu, A613m-, EabBlh, Bd615m. Da619m+. C63Om. Dab&-, Db635m+, Dab639n-

F327,A3W,612, 640.642.644-

Bc328, F330

None

Nitrous acid

F319m. F321m, G3.250. G3260, Bad647a, F654, Bad6568, Bad6578, F6W, Ib648m. F650m. Aalm-. A652m-, C W F661m,.Iab6670, F671a, C683m, F689m. A1450m-

615-,646,658,669, 660.666. 695

CW-,C855, CL756, C857

None

None

None

None

None

None

None

5-Bromouncil

Bd898n. C899m, Fgoom,A901m+, AW3m+, A905m+, C906m, A907m-, G936m. Db1442m

Diethylsulfate

m~vblet light

SZP

Decay

Nitrosoguanidme

Db506m-. Dab5llm-

B1022a.DabllOSm, Bcdl710, A1712u.

None

FGOgS, Bb6248.

Dab626, F63Bs.

Bb6418

None

F1713B3115,’ A1747:B3113m,* Db1714m, Db1715m, C1716a. A1754: B3114,*A17748,A1797: F3118,* Dbl717m. F1718m, D1719a,Bac1721m, A3095 F3119* Db1722m, F17!24m,B1725m,C1726m. Da1727m. F1728m, Iab1729a, F1730m, C1731m. Db1732m, A1733m, 1734111, Db1736m. C1737m. A1738m, F1739m, Ba-c1740m, 1741. Db1742, (374%. C1744m. Da1745m. 1746, Dab1748m. Db1749m, B1750m, Db1751m. C1752m, Db1753m. Db1755m, F1756m.

E w

F e F p zl

s M

4

B

ul

Dbl757m. Ib1758, D1759m. C1760m, C1762m. Db1763m. F1764m. Db1765m. Ba1766m, G1767m. Db1768m, G1769m, Db1770m, Db1772m, G1773m, G1775m. I1776 Dab1777m, Db1778m. A1779m, E1780m, Dab1782m, Dab1783m. I1784n, G1785m, Dab1786m, Dbl787m, Eab1789m. Db1790m, F1791, G1792m, Db1793m. A1794m.

is

2+

2

0

3

Iabl796a. E1798m, Db1799m, G2453u. F2454m, H2459m. 2460. F2767a, E2768n, I2777m. C2784n. Db2788m. Bad279Om, H279lm. E2800m. E2801m, Da2802m, F2807m, Iab2808n. D28110, Db2813m. Db3080u. Db3081m. Da3082m, Iab3083a,A3084m. (33085. Iab3086a. Dab3087m. A3088m. Db3089m, Db309Om. F309ln. Dab3092m, F3094m. F3095m. G3096m. E3097a ICR364-OH

ICR372and ICR191

G3048m, B3055m

None

2

#M

52 El

30508, F3051, C3053, Dab305&, C3064. Da3073, C3077

A3056, A3075,3050 C3049, F3054. C3059. C3062, Dab3052, C3057, C3060, A306; C3063, F3066, C3067, C3069, ‘23065, Dab3068, C3072, c3070, ~ 3 0 7 1 ~. 3 0 7 4 a, 0 7 9 ~ 3 0 7 6~, 3 0 7 s

B2771, (32775, Bad277&, C2781, F2792, A3003, Da3008, Dab3009. F3015S. F3020. A3023a, (2025, A3027. A30348, F30428,Dab3045s, Dab3706

A3000

F2439, C2455, D2456, B2457, F2439. D2769, A2770. A2772, B2458. A2786, (22799, C2803, A2774, G2776, (32779. G2804. F2805. F2806, G2810, Dab2780. D2787, C2789, C2814, (33001,A3005. F3011. C2795. C2798, A3002, C3004, Dab3013, Dab3028. A3030, A3006, C3007, A3010, Dab3012, C3014. A3016, C3035, Dab3036, C3703, A3017, Dab3018, ‘23021, F3704, Dab3708. B3735 A3022. G3024, F3026. Da3029, F3031, C3032, H3033, G3037, F3038,A3039, Dsb3040, F3041, A3043, F3044,F3046. F3047, Dab3702, (3705, D3733, C3737. D3749 (Continued)

3 0

5

0

+4

2

Y 2

N

a

c1

id

TABLE 1 (Continued) Mutagen

NG+ I C R

NG- I C R

Deletion (NG- ICR-)

NG- ICR+

NG+ ICR+

Spont. LT-2 Dab2114n, G2115a. D2116n, Dab2117n. H2142s. H2144s. G2148, BS-d2150, Dab2121, B2224,2225 G2100, G2102 through G2113, G21010, Dab2119n, C2132a. selected for Dab2119n. Dab212On. A212Lh. G2159,H21678. B2178,B218&s. G2192, throngh 2228,B2229, FZ118, Dab2122, C2123, Ba-d2133a,BacdZl35a, growth of Bc2130n. Dab213ln. Bs-d2134n, F22236,C2301. (2302,G2303:C3120.* 2232 through B38, C2124, C2125, C2126,H2129, Bc2147n, B2152a, Bc2153n, mnstitutivea Ba-d2145n, Bc2161n. Bc2177n. 2308, Dab2317, A2322. Dab2369, 2253,2315, B2320, FZ136, (32137, H2138, C2139, Bad216011, G2556 at M C . Bc2179n,Bc21BOn,Bca2183n,Bc2184n. C2499u, Dab2511, Dab2549, DabZ5.50, 2323,2327,2371, H2140, Ba-d2141. H2143, Bc218Sn. Dab2186n. G2187a, FZlSBa, Dab2555, F26Ols. FZW,FZW, 2372,2497.2600,2602, C2146, C2149. C2151, C2154. Bc21W)n. B2272n. F2273n. C2304a, FZ613s,A2615s, F26lb, FZ6176, 2604,2605,2607,2610 G2155 tbmugh G2158, G2162, G2308, C23070, D23090, D2310u. F26208,p2632s. C 2 M F3117; h n g h 2612,2614, C2163, G2164, C2165, C2166, G2168. G2170, G2171. H2172. FZGPBS,26478, C2648S. A264%, D2787, 26%. 2626 tbrougb D2311a, D2312a, D2313s. 02321, Ba-c2442u,F2445n, A2606n, F2636n, F30988 2630,2633,2634,2637, G2173,G2175,G2176,G2181, C2638n 2639,2640,2643,2650 C2182, B2191,(22193,C2194, D2252, F2264 though F2267, througb 2653 F2269, F2270,F2271, F2274, F2275, F2276, D2352, B2498, DZ641 Hmtidime-fequirmg mutanta chilied according to method of induction (left-band mlnmn) and revmion pgtterns (top beadings). hlatinn procedures are described in Section 11. ICR = ICRl9I exuept wbere p i l i e d ; deletion = fails to recombme with two or more mutations that reoombime with each other. In the body of the table, each number refers to an independent mutational event. Capital lettpz prehea denote gene locus designationswhere d i a g n d ; tbis designation ban been omitted from mutations m the “Deletion” mlnmn where more tban one gene locus is affected. Lowex case prehea indica(e the complementation pattern where this has been investigate3 for mutations in tbom four genes (0, B, E . and 0 where intragenic complementation ban been observed to occur. D mutanta are c h i l e d into three mmplementation types: Da, Db, and nonmmplementing (Dab).Mutants of genes I and B exhibit three W c ctuSea. respectively, as in gene D. In gene B there are four h i c mmplementation units, and the entirely nonmmplementing claw is designated Bad. Suffires indicate the following: positive (+)or negative (-) for immunologically -reacting material; a = amber; m = misrenss; n = nomenw, (amba or ochre); o = ochre; s = stable in spontaneous and in mutagen revmion tssts (aU “deletion” mutations also are stable); u = UGA nonsense. Mutations separated by mlow and marked witb asterisks were isolated as double mutations induced in two eeparate gene loci. All sponteneously revertible atrainS that am NG- ICR-. NG- ICR+,and NG+ ICR+ and W i n g let& nuf6xea are masidered to be frameahifta.

N

W

m

2M

MUTATIONS IN THE HISTIDINE OPERON OF

Salmonella

9

operon. No mutations leading to an absolute requirement for histidine have been found to map outside of the operon. Additional properties of the mutations are given in Table 1, as described in the legend. Figure 1 shows the map positions of over 1500 mutations. IV. Discussion

The data of Table 1 are summarized and classified in Table 2. After some comments on classification of Salmonella mutants and actions of mutagens, we point out some special features in our results not directly concerned with these other two aspects. Throughout the discussion we will emphasize points that may have wide applicability in practical experimentation in Salmonella as opposed to speculation on special genetic events. As background on the structure of the his operon and its control we refer the reader to reviews (Brenner and Ames, 1971; Ames et al., 1967). The operon is comprised of nine genes, arranged in the order operator-GDCBHAFIE, and dictates the structures of all of the enzyme proteins specifically involved with biosynthesis of L-histidine. The entire operon is approximately 10,000 nucleotide pairs in length (Brenner and Ames, 1971; Ames et al., 1967). Over 375 recombinationally separable sites of mutation have been discerned in the operon (Fig. 1) , and this number could be drastically increased by further recombination tests between point mutations.

MUTATIONS A. DELETION These mutations (“Deletion” column in Table 1; Column 2 in Table 2) are defined by genetic recombination tests as mutations that fail to recombine with two or more other mutations found to recombine with each other. Some mutations are extended deletions of two or more genes while others are probably deletions of short stretches of nucleotide pairs (Fig. 1 ) . One mutation (hisDCBHAFl52) makes a ‘‘short” messenger RNA (Venetianer et al., 1968) and another (hisBH22) “skips” a period of time in the expression of genes distal to the mutation upon derepression (Marver et al., 1966). Two cases where mutations link two polypeptide chains into one long chain (Yourno et al., 1970; Rechler and Bruni, 1971) also are presumed to be deletions of genetic material including intergenic nucleotides (“intercistronic divide”) . A number of the presumed deletion mutations have been critically tested 8s donors in transduction tests, and no evidence for recombination with centrally located point mutations has been obtained. Such recombi-

I

0611

'fO2

'EL5

E96

186

I

0

D

ob837 b699R b694L ob627L ab625 b585 0459 b457L b345 b344 ob9.C ab34L

b1437L b1436L b1431L b1429L b1423L ob 1416L D 1412L 0ll5lR b941 940

b934L ob883

bI75IRm b1749Rm obI748Rm b1736m b1732Rm 11727Rm b17221 1719Rm b1717Rm blll5Rm ab1709m ob1440L

obl786Lm 2301E 0b1783Rm b2YIOE ob1782Rm 0237% 2378E b1778Rm ob1777Rm 2377E b1772Rm 23130 b1770Rm 2312a b1765m 23110 b1763Rm 23lOU 1759Rm 23390 b1717Rm b1790m bl755Rm b1787m

2433 2432 2431 2430 2429

2428n

2427R 24261 2421 2420 2418 2302E

2473 b2472s ob2471m b2469s b2463m b2462m 24161 0824520 2443. 2441s 24360 2434

b2813m 2788m 27691 26411 obZ5IIRl ob2483E ab2482E ob24elE ab248OE b2479E

k$zT2;

Ob37061 3107 ab3092Rm b3090Lm bMB9Ra ob3087m 03082m b308ILu bMBOLv ab30681 ob3M I ob3032l

03112 3711 ab371h 370% ob37081

3939

3911 24611 2152" I422L

I I52

219%

Ihrwph 2222Y

0 0 4 7 loab41 10-

b2002m

28121

3725"

3633

3635

I

3639

-

b3642

ObyZOn

abS63r ab4OOsob137r

646

2222 -37

I

2906R. 2910R. 2912R.2916R 2225

/2221 63 538

--

- ._

63 5313 2226 2213

7776

2253 0""

3603

3050

\

/

386R.399R.S I~FI.~I~P*Z-

FIG.1. For caption see pp. 18 and 19.

644,3603.3050

b3637 3632 ob6390-

D b 30091

ob95602368 ob8960-

ab895m-

ob893.r Gb8730ob6030ob490mobl54l obI5Omb141m-

C

278M

ob2440m ob2I 1% ob2I 14" b 179% b1793m b I768m b 1442m b1753m ab9960-

3646 3656 b3645 3634 3644 b3629 3630 b3087m ab3058s mb30521 0 b 3 0 I 31

926

417

sw

683-

312

6?U

310

5140 804

577 1744 5 3 7 m 1743 503 140. 5020 1403 496m 1026 160 Ice0 I510 8551

2353P 2 2316 14661 1401

1033

24941 1029 24931 6 580m 0 23021 954

687

36b

2014l 276%

2 I251 1261

307Bl

1731 1410 Iloo7 IS0

22591 2326

980

22541 225661

3540

2354" 1762

595

8441-

3711

30721

0531 653

2769 37270 b460 b1424L 3726u b20I ob92I ObU)45s b173 ob807 ab30281 025 6% eb2106n

673

299 280 298 26 I

249 225m 2071 10E

2794.74

27930-

976

107 871

964 9uI 944 89% 908

860

5401 1210 4 2021 6 h 306M 3724u

Mo 131

I I 5 m 20031

22% I I 30771 2zyI 3721 I 306441 3716" 30071 30631

or91

22571 21661 2 I651 2 I391 I631

2 1320 21371

--

6 2 m 27751

b214m*

IY

461

YI

YB

YA

PII

120m

2091

nm

1170

ne

I

75-4

ab

2630

0129

129

2236 2604

2906R.29lOR. 2912R;

2906R. 2 9 1 0 R . 2912R. 2916R

)6R

6 3 . . . 53u

712

57.2226 2253 644.3603.3050

I

1

2225 152

I

63 152

57.2226

2253 644.3603.3050

I

2650

I

2604

I

I

2605

I

2607

5uL

I

2624

YWVVWV\I

I

-

I

2652

I

712

3 8 6 R . 3 0 0 ~ . 3 9 9 R . 5 I 5 R . 5 1 9 R . 5 2 0 R . 5 3 3 R . 6 5 8 R . 6 6 0 R . 6 0 1 R . 8 0 3 R . 8 0 9 ~ . 1 4 4 8 R . 1 4 8 6 R . 1 ~ 0 7 ~ . 2 3 7 1 R . 2 3 7 2 R . 2 3 7 3 ~ . 2 4 9 7 R . ~ 0 6.ZbWR.Z6IOR.2611R.2612R ~

!I

2628

CZOZ'LWZ

\

SO92 I

b192

LZFZ

I

l

I

I

n /( X

LO92

I

bZ92 I U916Z'Ut 162 '10162 'US062

I I

bO9Z bE9Z

bC92 OZCZ

I

A

I

l

I6950

JI

ZZ

m

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RSZ OZFZ

II

1092

C29Z W l b Z %216Z 'U0162'U9062

to92

5092

0592

m

22 RSZ

m

I I

8

H

ITS%

2329 2330 23u) 2144s 2167s

O b c 17-

oc1721n mr990 c 4ea 6%s429a

3114

24Sm 2351 244%

2142,

2344

I7S4 1147

2341

2336

1407

sw,

C35%

2333 2331

5641

2325

2791~

2347 2143

mz

2609

2349

I

2350

523

I

=E

I IB

I A

2339

21721

2337

2335 3095 1794

21431 z14a 21361 2l29I

644 646

2342 2340 974

.

30331

2346 2345

2336 1 1% %

622 621

1-

I

I

HA IIB

22

I

260s

fPL4R. s7

~

e

24

2253

644.712.26205050.3603 146ZR. 2602R

/

I

3

h

2633 2614

I

2604

2s27

m 1

2634 2633 2614

I

24%R 2-

~

~~

2605 69SR.2651R.2906R.2908R.2910R.2912R.2914R.2916R

2547

I

2227.2323

644.I l2%%30%.36O3

1462R260PR.Z911R.2915R

3101

26261 \ I407R.23711.237ZI).23731.24971.25~R 2 6 O O R . 2 ~ 6 1 1 4 . 2 6 1 2 R

\

26371

a .

FIG.1 (Continued).For caption see pp. 18 and 19.

Ob9

>

m

2il I

BII I

QII I

I

SEl

I

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rfb 4-12 aann Iemtha

17 ~

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6

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l

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.

l

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FIG.1 (Continwd). For caption see pp. 18 and 19.

.

2

3

7

l

~

18

PHILIP HARTMAN, ZLATA HARTMAN, RUTH STAHL, BRUCE AMES

FIa. 1 [pp. 10-181. Map of the histidine operon and adjacent gene region in S. typhimurium. Each gene is designated by a capital letter. The correlation of each gene with an enzyme activity is described elsewhere (Ames et al, 1967; Loper et al., 1964; Martin et al., 1971). The map is not drawn to scale. Each number represents an independently isolated mutation and carries the suffix his except for rfb-806.Mutations indicated below the heavy horizontal line are deletions. A wavy line indicates that the extent of the deletion in the wavy region is unknown and an arrow at the end of the wavy line indicates that the extent of the mutation into the particular gene is unknown. Roman numerals just below the heavy horizontal line designate subregions of each gene as determined by deletion-mapping, except in a few cases where three-point tests were utilized in an earlier map (Loper et al., 1964). Mutational sites are placed above the heavy horizontal line in their most probable map order, determined by deletion mapping and three-point tests. Mutations listed in vertical columns have not been ordered. I n a number of instances more than one column of successively numbered mutations is shown in one gene region. Parentheses indicate that the map order is unknown. Horizontal brackets indicate that the mutation(s) map in a particular region but have not been crossed further. Sets of mutations that fail to recombine with one another are enclosed by vertical brackets. Mutations not mapped in detail but placed only as to gene affected are listed in a cluster, for each gene, at the top of the diagram. Prefixes indicate complementation patterns (for genes D, B, I , and E ) ; consult text for details. Suffix designations follow: C = constitutive (Roth et al., 1966; D. B. Fankhauser, in preparation); F = mutation on E . coli F’ (Garrick-Silversmith and Hartman, 1970; G. R. Fink and B. N. Ames, personal communication; 5. C. Loper, personal communication) ; L = lysogenic stock; M = missing (lost) ; P = polar prototroph (Voll, 1967); R = rough (P22-resistant; in the case of extended deletions this is generally due to inclusion of the rfb operon in the mutation (cf. Nikaido et al., 1967) and in the case of point mutations to a separate mutation in one of the somatic antigen genes) ; a = amber (UAG nonsense) ; c = cold-sensitive, feedback hypersensitive (prototrophic a t 37°C) (St. Pierre, 1968; G. A. O’Donovan, personal communication) ; f = frameshift ; m = missense ; n = nonsense (either amber or ochre) ; o = ochre (UAA nonsense) ; r = feedback-resistant (prototrophic) (Sheppard, 1964; D. N. Ant6n and J. H. Wyche, unpublished) ; s = stable (deletions listed below the heavy horizontal line also are stable to reversion on minimal medium) ; u = UGA nonsense; - = CRM-negative; + = CRM-positive (consult text). Numbers in parentheses following deletion numbers, e.g., 90.9 under hGG, designate constitutive enzyme levels (or absence of detectable enzyme activity) for the remaining intact genes of the operon in mutants lacking the normal his operator-promotor region (Ames et al., 1963; St. Pierre, 1968). One mutation (his-984) located internally in the operon (genetic region DIB) appears to be an absolute polar mutation (S. A. McIntire and J. C. Loper, cited in Greeb et al., 1971). This map supersedes all previous ones, differing in a number of minor details. The map is a compilation of unpublished data and material published in a variety of locations (Loper, 1961; Loper et al., 1964; Whitfield et al., 1966; Fink et al., 1967; Margolies and Goldberger, 1968; Martin and Talal, 1968; Greeb et al., 1971). These publications should be consulted for further details. Description of the regions adjacent to the his operon in S. typhimurium is to be found in Sanderson (1970), Murray and Klopotowski (1968), and Nikaido et al. (1967). Mews. Morris Earl

MUTATIONS IN THE HISTIDINE OPERON OF

Salmonella

19

nation might have been expected were the mutations inversions. Finally, many of the mutations are nonpolar as expected of inframe deletions but not expected of extensive inversion. Therefore, we conclude that inversions are rare or absent in our material while deletions are quite common. The top, horizontal column in Table 2 indicates that deletion mutations are found with fair frequency among spontaneous histidine-requiring mutants of strain LT2, and this frequency is increased when selection is performed for strong polarity (second horizontal column, Table 2). Such mutants are more infrequent among LT7 derivatives since one or two mutator genes that induce base substitutions are present in many LT7 “wild-type” stocks (cf. Kirchner, 1960). Irradiation with X-rays or fast neutrons or treatment with nitrous acid are effective in increasing the relative frequency of deletions (and stable mutations) in bacteria (cf. Schwartz and Beckwith, 1969). The ends of deletion mutations in the histidine operon are located a t over 67 places (Fig. 1). Only a few “repeated” mutations have been detected. A set of five mutations (2228, 2233, 2234, 2235, and 2237) detected by Fink et al. (1967) appears to share one end in common, as do the pairs 1300-1301, 1303-2232 and the trio 2627-264-2643 (in each case the other terminus lies outside of the operon in unmapped territory). Two additional mutations, 2227 and 2323, share one common “end” while in each case the other lies outside of the operon in an unmapped region between the his operon and gnd, some P-12 “genelengths” away. Two ICR-induced mutations appear identical (Oeschger and Hartman, 1970), and hisF41 (spontaneous), and hisFS27 (fast neutrons) appear alike. I n a number of instances, pairs of multisite mutations share one end in common while the other is known to differ in extent, either extending in the opposite direction (63-2605; 2236-2652; 64.5-2315; 640-2547; 2649 and the trio 2627-2640-2643) or in the same direction (649-14.52; 2527-2634; 612-2633; 666-950). This indicates that Harper and David M. Mayer did considerable mapping and mutant characterization in gene B, extended by Miss Ann Boardley under the supervision of Dr. Mary Jane Voll. Credit for other published results incorporated here is due Drs. John C. Loper and John R. Roth. The gene labeled s u p W on the map is an amber suppressor gene and is now designated supD, in keeping with the symbol for a presumably analogous gene (also known as sul and suZ) in E . coli. Strains 203, 644, 1301, 1302, 1303, and 1304 carry a wildtype s u p D gene; other deletions have not been tested. Mutant 3934 is a cold-sensitive mutant that maps in region GV and not in GVI as shown on the map (Rothman-Denes and Martin, 1971); i123 maps in GVI; G mutants 72, 266, 337, and 936 are missense (J. Ferretti, personal communication).

TABLE 2 Summary of Reversion Patterns of 1018 Independently Isolated Histidine-Requiring Mutants and Some Additional Properties

origin SpontaneousLT2 SpontaneousLT2 (Polarity) Sponteoeous LT7

x-ray

Diethylunlhte Nitrous acid Ultraviolet light ICR364-OH ICR372 and ICR191 N-Methyl-N'-nitro-Nnitrosogunidme ZAminopurine

5-Bmmouracil

Total %

83

y

10

199

47

143

2

114

Fast Neutrons

"P b

Total 1

Frame- Frame- Frameshift: shift: shift: Presumed minus minus plus deletion (NG- (NG(NG+ Deletion (stable) I C R 3 ICR+) ICR+) 2 3 4 5 6

23

20 31 37

33 87 119

9 (23 %)

(34%I (10 . %) ..

1 ~~

66

13

11

9

236

(25%)

7 0

16

(44 %)

0

(32%)

3 1

(16 %) (15%) (8%)

0

9

20

(34%)

6

11

9

0

1

2

0

5

0

0

0

6 2

2 4 5

4-6

20

4 0

12

(13%) 3 (13%I 0

0 0

9

6

11

25

44

1

0

0

0

6

(19%) 4 (11%I 26 (79%) 80 (92 %I 0

119

0

10

0

2

0

1020

93 18%

* CRM = presence (+) or abaence (-) t One pmmotor mutation (hisWS2f).

Total additions and deletions 2-6

39

BaaeBaae mbstitnmbsti tions tutions

(NG+

ICR-) 7

44

(NG+

ICR+) 8

0

6

4

3

Total frame shifts

0

0

0

0

95

70

133

54

0 257 25%

of immunologidy orasreacting material.

(47%) 13 (57 %) 5 (25 %)

16 (52 %) 10 (27%) 31 (94%) 87 (100%) 1 (1%) 4 (3%) 0 0 445

44%

(53%) 10

(44%)

14 (70 %I 15

(48%) 27 (73 %) 2 (6%)

0

0 0 0

0

Nonsense Mkneme 9 10 15 (36 %) 45 (98%)t 13 (13%) 24 (41%) 2 (20%) 4 (29 %) 5 (33%) 10 (37 %) 0

36

-

-

29

51

16

30

2

7

0

0

1

1

0

7

0

7

11

0

-

-

-

0

-

0

m

-

-

5

F

11

52

0

3

1

0

0

2

0

0

85

(87%) 34 (59%) 8

(80%) 10 (71%) 10 (67%) 17 (63 %) 2 0

113 (95 %)

0

19 (18%) 78 (70%) 1 (10%)

85 (82%)

553

14 56 %

0

216 (40%)

0

10

0

(97%) 0 10 (100%) 2 0

Double 13

27

0

4

CRM-* 12

(64 %)

0

111

CRM+* 11

34 (30%) 9

(907%) 2 323 (60%)

78 28%

198 72%

8 8%

m 6

1: td

9 9

MUTATIONS IN THE HISTIDINE OPERON OF

Salmonella

21

the identity of termini in the few cases observed may be a consequence of regions of the map that are relatively immune to mutation and thus bare of mutations that would otherwise differentiate deletion mutations from one another. These may be regions that are relatively insusceptible to nonrandomly distributed frameshift mutations (Oeschger and Hartman, 1970) and relatively unessential for catalytic activity and thus also insusceptible to most base substitution mutations. The origin of deletion-type mutations is unknown; their occurrence in E. coli K12 is independent of rec function (Franklin, 1967; Inselberg, 1967; Anderson, 1970) and ,thus probably not due to looping out and unequal crossingover performed by the enzyme complex normally involved in recombination. Extended deletions in the histidine operon of E. coli appear proportionally more frequent and uniform than those observed in Salmonella (Goldschmidt et al., 1970; Garrick-Silversmith and Hartman, 1970).

B. STABLE MUTATIONS Stable mutations (Column 3, Table 2) are presumed deletion mutations as they have not been observed to revert spontaneously or with the mutagens NG or ICR191 (reversion frequency less than 10-lo). Many of these have been extensively crossed with closely allied mutations and not yet found to cover more than one mutational site. Others have not been extensively tested. In either case, it is likely that very extensive tests against a larger number of base substitution mutations would reveal that many of these mutations are short deletions of several nucleotide pairs. Several are nonpolar and some show intragenic complementation; on theoretical grounds these are in-phase deletions of 3 base pairs or a multiple thereof. Other stable mutations are polar and thus presumably shift the reading frame in mRNA. C. FRAMESHIFT MUTATIONS These are spontaneously revertible deletions or additions of bases that cause a shift in the reading frame in mRNA. Recent observations suggest that the vast majority of spontaneous frameshifts as well as those induced by many mutagens involve deletions of base pairs, and the mutations are not suppressed by external suppressors. I n contrast, acridine derivatives frequently cause mutations through basepair additions and are suppressible by external suppressors. Some frameshift mutations fail to respond in reversion tests to the mutagens NG and ICR191, some respond to ICR but not to NG, and a third class responds both to

22

PHILIP HARTMAN, ZLATA HARTMAN, RUTH STAHL, BRUCE AMES

ICR and to NG (Tables 1 and 2 ) . Only one mutation (G.2556) in this third class has been detected among spontaneous mutations and among mutations induced with a number of mutagens. Such NG+ ICR+ mutations are frequently found among mutants originally induced with ICR compounds (Oeschger and Hartman, 1970). Generally, they are more highly revertible in spot tests (Ames and Whitfield, 1966) with ICR191 than they are with NG. We have speculated that many of these mutations are “plus” frameshifts, as actually determined for one of them (Yourno and Heath, 1969). Many, but possibly not all, of these mutations are suppressible by external suppressors (Yourno et al., 1969; Riddle and Roth, 1970). The suppressors restore in-phase translation at an efficiency of 1-15% (Yourno and Tanemura, 1970; Riddle and Roth, 1970). Riddle and Roth (1970) found only one ICR-induced frameshift (F3704) that responded to ICR but not. to NG but is suppressible by external suppressors. None of the mutants in frameshift, stable, or deletion classes has been observed to be phenotypically suppressed by streptomycin (Whitfield et al., 1966; Oeschger and Hartman, 1970).

D. BASESUBSTITUTIONS Mutations that revert spontaneously and whose reversion is increased with NG but not by ICR (NG+ ICR- Column in Table 1 ; Column 7 in Table 2) are considered to be base substitutions. Many of these strains also are reverted by base analogs. For example, about 75% of NG-induced mutants are reverted by 2AP (about a third of these are negative in spot tests but yield positive results after extended growth in broth containing 500 pg/ml 2AP). We have not attempted to separate transition mutations from transversion mutations. Most of the NG-revertible mutations also are revertible with diethylsulfate, and about half are clearly induced to revert with p-propiolactone. Various suppression tests (see p. 3) have allowed subclassification of substitution mutations into missense and various kinds of nonsense (UAA, UAG, UGA triplets). Nonsense mutations also are characterized as polar, as very highly susceptible to NG-induced reversion and to phenotypic reversal with streptomycin, and very often as inactive in intragenic complementation in v i m . Our tests have been extensive enough to lead us to believe that our missense class includes few, if any, missed nonsense. UGA nonsense mutations are rare in our material, presumedly since most such mutants are “leaky” because Salmonella strains translate this codon with low efficiency (Roth, 1970; Ferretti, 1971).

MUTATIONS IN THE HISTIDINE

OPERON OF

Salmonella

23

A small fraction of base-substitution mutations are revertible strongly by NG and weakly by ICR compounds (Whitfield et al., 1966; Fink et al. 1967; Oeschger and Hartman, 1970): (see mutations with suffixes in NG' ICR' Column of Table 1; Column 8 of Table 2). ICR364-OH appears to be more effective than ICR191 in eliciting this type of reversion, although the latter compound is comparatively more effective in intragenic reversion of frameshift mutations (Oeschger and Hartman, 1970). The ICR frameshift mutagens are presumed to act on base-substitution mutants by induction of suppressor mutations, perhaps by adding or deleting bases in a tRNA (Fink et al., 1967). We have checked the purity of these stocks, their mutational responses, and determined for his-dl 33 that the original mutation can indeed be recovered by transduction away from the suppressor. We do not know why reversion of only a few nonsense mutations is elicited by ICR compounds. Evidence has been provided that ICR191 can cause occasional base substitutions in E. coli (Berger et al., 1968) but, based on the observations reported here, its action in this regard can be considered minimal or absent in Salmonella. Table 2 shows that NG, 2AP, and 5BD predominantly or exclusively elicit base substitutions (cf. Eisenstark et al., 1965). The few stable mutants (Column 3 of Table 2; 4 mutants per 249 mutants examined) could be closely allied double mutations, similar to others detected (Column 13 of Table 2 ) ) but within a single gene. Alternatively, the stable mutations could be spontaneous mutations fortuitously selected after mutagen treatment, or they could be a minor mode of mutagenesis by these compounds.

E. THERATIOOF MISSENSETO NONSENSE MUTATIONS Whitfield et al., (1966) noted a great shortage of missense mutations relative to nonsense mutations. They conclude that most missense mutations are not detected since the proteins are partially or completely functional due to substitution of the same or of a related amino acid, or due to substitution of an amino acid in a noncritical portion of the polypeptide chain. This shortage of missense mutations was particularly strong with the transition-inducing mutagen, 2AP. I n our more extensive investigation we find some 2 0 4 0 % of the base substitutions detected are nonsense mutations except those induced by 2AP where approximately 70% are nonsense (Column 9 in Table 2 ) . The surplus of nonsense mutations induced by 2AP and the relative rarity after mutagenesis with NG run counter t o expectations. UGA nonsense mutations are rare in our material (Roth, 1970), while UAA

24 PHILIP HARTMAN,

ZLATA HARTMAN, RUTH STAHL, BRUCE AMES

nonsense can arise by transition only from CAA, and UAG nonsense only from CAG or UGG. All of these transitions involve G/C to A/T changes. Our observed ratio for NG is the opposite of what one would expect if the major means of NG mutagenesis in stationary phase bacteria was the formation of 7-methylguanine (Lawley, 1968) which would be expeoted to lead selectively to G/C to A/T transitions (cf. Drake, 1970). Perhaps NG gives rise to a substantial proportion of transversion mutations. Also, when bacteria are grown in nutrient broth containing 2AP, as was the case in our isolation procedure, the G/C to A/T transition appears to predominate. This is not the behavior expected if the classic DNA polymerase of Salmonella resembles that of E . coli in eliciting incorporation of 2AP preferentially in place of adenine (Rogan and Bessman, 1970) and is involved in DNA replication in vivo.

F. MULTIPLEMUTATIONS Only eight histidine-requiring auxotrophs have been found to contain two mutations, each in a separate gene of the operon (separated by colons in NG- ICR- Column of Table 1 and listed in Column 13 of Table 2). Five of these were obtained in NG-treated cultures, one in a diethylsulfate-treated culture, and two were spontaneous. Although the double mutants were stable to mutagens, both mutations in each of the five NG-induced double mutants were found susceptible to NGinduced reversion when the strains were infected with F’ his episomes functional for one of the two gene loci. Additionally, mutation B3113 was recovered from a double mutant by transduction into a hisD deletion mutant on histidinol-containing plates nonselective for the single and double mutations of the donor strain. Transductional clones were screened for reversion in response to NG, and the missense mutant, B3113, which maps identically to the B mutation in the double mutant, was isolated. Numerous other strains with double mutations were found in the NGtreated bacteria even though (see p. 2) stationary phase cultures were used, NG treatment was not extreme, and selection was made for histidine-requiring bacteria. Among the doubles found were 1 classified by phage typing as rfa, 7 classified as rfb, 3 as rfc, 17 other unclassified rough strains, two p-aminobenzoic acid-requiring mutants, and 1 leaky purine mutant [see Sanderson (1970) for nomenclature]. One strain carried three point mutations, two in the histidine operon (F1713 B3116), and one (rfb-808) in the somatic antigen operon that is closely linked to the his operon (Nikaido et al., 1967). Therefore, one must be ex-

MUTATIONS IN THE HISTIDINE OPERON OF

Salmonella

25

tremely wary of NG as a mutagen; cotransducibility gives no assurance that clusters of mutations have not occurred. G. POLARITY Nonsense and frameshift mutations in the his operon lower enzyme levels of distal genes (polarity) to about 5% to 60% of normal (Whitfield et al., 1966; Fink et al., 1967; Fink and Martin, 1967; Martin, 1967; Martin and Talal, 1968; Oeschger and Hartman, 1970). The degree of polarity is a function of map position of the mutation (Fink and Martin, 1967; Martin and Talal, 1968). Frameshift mutations are polar because they generate nonsense codons (Martin, 1967). Missense mutations are uniformly nonpolar. Out of over 1000 mutations examined, only one absolutely polar mutation that maps within the structural genes of the operon has been found (stable mutation hisD984, S. A. McIntire and J. C. Loper, personal communication); all other mutations with absolute polarity have genetic defects in the operator-promotor region (Ames et al., 1963; Atkins and Loper, 1970; D. B. Fankhauser, personal communication). The rarity of absolute polar mutations in structural genes of the Salmonella his operon contrasts with E. coli K12, where absolute polar insertions of possibly redundant DNA sequences frequently are found in the structural genes of the lac and gal operons (cf. Malamy, 1970; Michaelis et al., 1969).

H. CROSS-REACTING MATERIAL(CRM) Examination of the data in Table 1 shows that only one mutation (Db802) out of 13 multisite mutations allows production of a protein that serologically cross-reacts with wild-type enzyme. About one fifth of strains carrying stable mutations contain CRM. These CRM-positive strains are thought to contain in-phase deletions with mutant protein one or more amino acids shorter than the wildtype. CRM is absent from the 35 revertible frameshift mutants examined (Table 1, NG- ICR- strains without letter suffixes; NG- ICR+ strains). Two out of 62 nonsense mutants examined for CRM contain cross-reacting material. One of these is an amber mutation, hisC487, that maps a t the extreme end of the hisC gene, distal to over 140 other point mutations (Martin and Talal, 1968; Fig. 1 ) . The second, hisB689, is an amber or ochre mutation that maps in the hisB gene, distal to a t least 50 point mutations but, by three-point tests, proximal to a t least 5 other point mutations (Fig. 1 ) . As expected from the high incidence

26

PHILIP HARTMAN, ZLATA HARTMAN, RUTH STAHL, BRUCE AMES

of nonsense mutations among 2AP-induced auxotrophs, CRM-negative strains predominate among 2AP-induced mutants. Approximately half of the missense mutations contain CRM, and the other half are CRMnegative (Loper, 1961; Whitfield et al., 1966; Margolies and Goldberger, 1968; Greeb et al., 1971).

I. SPONTANEOUS MUTATIONS If one disregards nonpolar (missense) mutations, the data in the top two lines of Table 2 indicate that spontaneous his mutations are similar in distribution in strain LT2 whether selected through penicillin or selected directly for polarity. About one fourth of the mutations are nonsense mutations and three fourths are frameshifts and deletions. Extended deletions (multisite mutations) and stable mutations (“short” deletions) together are about as common as revertible frameshift mutations. As pointed out above (see Section IV,C) , spontaneous mutations and mutations induced by all agents tested except the ICR compounds rarely include the type of frameshift mutation that is reverted both with ICR and with NG; such mutations have been interpreted as “plus” frameshifts, perhaps most commonly single-base additions. Thus, it would seem that deletion of genetic material in Salmonella is a relatively common event in mutant production. Spontaneous mutations in strain LT7 are predominantly base substitutions, since many lines of LT7 contain one or two mutator genes which seem to elicit substitutions (Kirchner, 1960). J. SCREENING POTENTIAL MUTAGENS Particular mutants have been chosen from among those reported here for use in rapid, spot-test screening of various compounds for mutagenic activity (Ames, 1971; Smith, 1966; Hartman et al., 1971). The sensitivity of tests for mutagenicity in some of the strains has been increased by the placement of additional mutations that limit repair of genetic lesions (Ames, 1971). Methodology underlying these tests and their utility in the mass-screening of potentially deleterious substances has been reviewed by Ames (1971).

K. INTRAGENIC COMPLEMENTATION Complementation has been observed in vivo among mutants of genes D , B, I , and E, respectively (Hartman et al., 1960a; Loper et al., 1964).

Three of the genes contain two basic complementation groups while the

MUTATIONS IN THE HISTIDINE

OPERON OF

Salmonella

27

fourth gene ( B ) contains four basic groups. Table 1 and Fig. 1 list noncoinplementing mutations as Dab, Ba-d, lab, and Eab ; complementing mutations are designated Da, Db, etc. Noncomplementing mutations are largely of the nonsense, frameshift and deletion classes, but noncomplementing missense mutations also are frequent. Complementing mutations are largely missense but also include a few multisite and some stable mutations (probably all in-phase, nonpolar deletions). Several nonsense and frameshift mutations in the terminal portions of genes D,B , and I also exhibit intragenic complementation in vivo (Fig. 1). All CRM-positive mutations tested in genes B (Loper, 1961) and D (Greeb et al., 1971) complement in vivo; some CRM-negative mutations also complement. The proteins of these latter strains probably undergo transformations to immunologically unrecognizable forms during extraction. Thus, in vivo complementation appears to be a more sensitive test for protein with minor alterations than does detection of CRM. On the other hand, detection of CRM may be equally sensitive to in vitro complementation or the assay of “secondary” residual enzyme function in cases where each of these activities occurs (Loper, 1961). Gene D codes an enzyme with two identical subunits (Loper, 1968; Yourno, 1968; Lew and Roth, 1971) and the gene B product shows evidence of subunit structure (Vasington and LeBeau, 1967). The relationship between the genetic map (Fig. 1 ) and the complementation map (Loper et al., 1964) appears complex for gene B. No intragenic complementation has been observed among mutants of genes G, C, A , and F , respectively (Loper et al., 1964). The tests include missense mutations in each case. While gene A specifies a protein functional as a monomer (Margolies and Goldberger, 1967), multimeric proteins built of identical polypeptide chains are specified by genes G (hexamer-Voll et al., 1967) and C (dimer-Yourno et al., 1970; Rechler and Bruni, 1971). Therefore, while the observation of intragenic complementation is an indicator of multimeric enzyme structure, lack of observed intragenic complementation does not ensure that the “native” gene product is a monomeric enzyme.

L. GENE-ENZYMERELATIONSHIPS The enzymology of histidine biosynthesis has been reviewed (Martin

et al., 1971), as have gene-enzyme relationships in Salmonella (Loper et al., 1964; Ames et al., 1967), and aspects of regulation (Ames et al., 1967; Brenner and Ames, 1971). There still remains uncertainty as to the number of polypeptide chains involved in the structures of the enzymes coded for by genes B, H , F , I, and E. Complementation

28

PHILIP HARTMAN, ZLATA HARTMAN, RUTH STAHL, BRTJBE AMES

behavior among B mutants was mentioned above. I n addition, we have noted in abortive transduction tests relatively weak complementation between a class of B mutants and a class of H mutants that we now recognize as nonsense and frameshift mutants. Whether these results are due to protein-protein interactions of gene products or to termination of transcription and consequent polarity (or to both) remains uncertain. The structure(s) of the I and E enzyme(s) also awaits elucidation. The sedimentation profiles of these two activities are very similar (Whitfield et al., 1964). The complementation patterns and map positions of mutations (Fig. 1) do not allow definite interpretation. Nonsense and frameshift mutations in gene I fail to complement E mutants, but this could be due to polarity rather than to dictation of a unit polypeptide chain by the entire E-I region. In addition, a mutation that has been thoroughly examined and classified as missense (hisI333) fails to complement either E or I mutants. Purely genetic tests cannot differentiate between several possibilities of enzyme structure for the product of this gene region. M. GENETICMAPPING Much of the genetic mapping has relied upon the all-or-none presence of wild-type recombinants in crosses involving stable deletion strains as recipients. Three-point tests have utilized double his- strains, in which case prototrophic (His+)recombinants are scored, or hishis0 double mutants, in which case the his0 serves as an unselected marker whose segregation in the cross is followed. Mapping in the operator region has relied exclusively upon three-point tests (D. B. Fankhauser, unpublished). With numerous and carefully performed three-point tests, the order of mutant sites often can be determined unambiguously, but cases do occur in which an unambiguous order cannot be established by this method (cf. Loper et al., 1964; Martin and Talal, 1968). The behavior in recombination tests appears to be an inherent property of the alleles involved in the cross. This was tested by transducing hisBl2 and hisB24, two frameshifts that fail to recombine with each other, into a standard genetic background. Even in isogenic stocks, 24 recombines severalfold more freely (i.e., produces an excess of wild-type recombinants) than does 1.2 in crosses with mutations located to either side of the mutant pair. In spite of the above difficulties, accurate two-point transduction tests do indicate that the histidine genes shown in Fig. 1 lie closely adjacent and do not bear lengthy “spacers” or other genes between them (J. Bagshaw and P. E. Hartman, unpublished, 1965). The experiments in-

MUTATIONS IN THE HISTIDINE OPERON OF

Salmonella

29

volve reciprocal crossing of several point-mutations mapping in one end of a gene with several mutations mapping in che nearest portion of the adjacent gene. These recombination figures ar2 then contrasted to those obtained in similar crosses that, for example, involve mutations a t opposite ends of the same gene. The data are expressed in terms of p, the average probability of a crossover separathg the two markers to yield wildtype recombinants (Hartman, et al., 1960b; Loper et al., 1964). Values obtained are the following: across D , 0.182; D-C, 0.024; across C 0.092; beginning of D to beginning of C, 0.221; end of D to end of C , 0.153; C-B, 0.031; across F , 0.205; F-I, 0.038; across I , 0.114; I to middle of E , 0.066. These are overestimates of the sizes of possible intergenic spaces since we now have mutations located closer to the ends of some genes than those utilized and, as pointed out below, there is evidence that mutations are not distributed randomly and that a t least the proximal portions of some genes are relatively insusceptible to mutation. Also, negative interference would maximize the smaller map distances. Rechler and Martin (1970) have begun an analysis of the D-C intercistronic divide, and a similar approach is being used to obtain information pertaining to the G-D intercistronic divide (J. Yourno, I. Ino, and P. E. Hartman, unpublished, 1970). Frameshift mutations are not randomly distributed in the histidine operon; they are clustered in particular regions (Oeschger and Hartman, 1970; and Fig. 1). In addition, nonsense and frameshift mutations (chain-terminating mutations) are frequent and missense mutations are infrequent in the proximal regions of several genes. The ratio of nonsense and frameshift mutations to missense mutations for proximal regions are (hisG, 7 : l ; hisD, 1 5 : l ; hisB, 9 : l ; hisC, 11:5; h i d , 8:6; hisF, 5:14; and hisl, 11:l (7 nonsense and 4 frameshifts). I n contrast, as the extreme example, mutations in the remaining (distal) portion of the hid gene are 10 missense, 1 nonsense, and no frameshifts. Mutations in the most distal portion of the operon, hisE, comprise 15 missense, 4 nonsense, and no frameshifts. Thus, no frameshifts have been observed in the terminal segment of the operon. I n region CIV, the distal end of the hisC gene, there is clustering of mutations of similar type: 1 missense-8 frameshifts-6 missense-8 amber mutations (Martin and Talal, 1968). V. Summary

Over 1500 histidine-requiring mutants of independent origin have been mapped in the histidine operon of S. typhimurium. About 1000 mutants have been characterized as to type of mutation. The type

30

PHILIP HARTMAN, ZLATA HARTMAN, RUTH STAHL, BRUCE AMES

of mutation (extended deletions; frameshifts of the plus and minus types; base pair substitutions causing missense or nonsense) has been determined for each mutant by the collective results of recombination tests, analysis of the pattern of spontaneous, N-methyl-N’-nitro-N-nitrosoguanidine, and acridine half-mustard induced reversion, and the presence of suppressors among revertants. The type of mutation is correlated with polarity in the operon, presence of immunologically detectable cross-reacting protein, complementation, and genetic map position. The spectrum of spontaneously occurring histidine-requiring mutants is compared with that found after treatment of bacteria with a number of widely used mutagens. ACKNOWLEDGMENTS We owe ever so much to Dr. Milislav Demerec. This work also has depended upon fine collaboration and stimulating discussions with Drs. R. F. Goldberger, J. C. Loper, and R. G. Martin as well as with Drs. G. R. Fink, J. R. Roth, H. J. Whitfield, Jr., and J. Yourno, and many other associates through the years. It was supported, in part, by Research Grant A141650 of the National Institute of Allergy and Infectious Diseases, US. Public Health Service to PEH, and AEC Grant (11-1)34 Agreement 156 to BNA. Dr. Hugh J. Creech, Institute for Cancer Research, Philadelphia, Pennsylvania, kindly supplied each of the ICR compounds used in our tests. Mrs. Margaret W. Nolley brought illustrative order to Fig. 1, and Mrs. Kathryn Levine assisted in proofreading. Contribution number 621 of the Department of Biology.

REFERENCES Ames, B. N., 1971. The detection of chemical mutagens with enteric bacteria. In “Chemical Mutagens : Principles and Methods for Their Detection” (A. Hollaender, ed.), Vol. 1, pp. 267-282. Plenum, New York. Ames, B. N., and Garry, B. 1959. Coordinate repression of the synthesis of four histidine biosynthetic enzymes by histidine. Proc. Nut. Acad. Sci. U.S. 45, 1453-1461.

Ames, B. N., and Whitfield, H. J., Jr. 1966. Frameshift mutagenesis in Salmonella. Cold Spring Harbor Symp. Quant. Biol. 31,221-225. Ames, B. N., Garry, B., and Herzenberg, L. A. 1960. The genetic control of the enzymes of histidine biosynthesis in Salmonella typhimurium. J. Gen. Microbiol. 28, 369-378.

Ames, B. N., Hartman, P. E., and Jacob F. 1963. Chromosomal alterations affecting the regulation of histidine biosynthetic enzymes in Salmonella. J . Mol. Biol. 7, 23-42. Ames, B. N., Goldberger, R. F., Hartman, P. E., Martin, R. G., and Roth, J. R. 1967. The histidine operon. In “Regulation of Nucleic Acid and Protein

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Biosynthesis” (V. V. Konigsberger and L. Bosch, ed.), pp. 272-287, Elsevier, Amsterdam. Anderson, C. W. 1970. Spontaneous deletion formation in several classes of E. coli mutants deficient in recombination ability. Mutat. Res. 9, 155-165. A n t h , D. N. 1968. Histidine regulatory mutants in Salmonella typhimurium. V. Two new classes of histidine regulatory mutants. J. Mol. Biol. 33, 533-546. Atkins, J. F., and Loper, J. C. 1970. Transcription initiation in the histidine operon of Salmonella typhimurium. Proc. Nut. Acad. Sci. U.S. 6.5, 925-932. Berger, H., Brammar, W. J., and Yanofsky, C. 1968. Spontaneous and ICR191-Ainduced frameshift mutations in the A gene of Escherichia coli tryptophan synthetase. J . Bacteriol. 96, 167Z1679. Berkowitz, D., Hushon, J. M., Whitfield, H. J., Jr., Roth, J., and Ames, B. N. 1968. Procedure for identifying nonsense mutations. J . Bacteriol. 96,215-220. Brenner, M., and Ames, B. N. 1971. The histidine operon and its regulation. In “Metabolic Regulation” (H. J. Vogel, ed.) [Vol. 5 of “Metabolic Pathways”-D. Greenberg, ed.1, pp. 349-387. Academic Press, New York. Demerec, M. 1955. What is a gene?-Twenty years later. Amer. Natur. 89, 5-20. Demerec, M. 1956. A comparative study of certain gene loci in Salmonella. Cold Spring Harbor Symp. Quant. Bwl. 21, 113-121. Demerec, M., and Demerec, Z. E. 1956. Analysis of linkage relationships in Salmonella by transduction techniques. Brookhaven Symp. Biol. 8, 75-84. Demerec, M., Blomstrand, I., and Demerec, Z. E. 1955. Evidence of complex loci in Salmonella. Proc. Nut. Acad. Sci. U.S. 41, 359-364. Demerec, M., Hartman, Z., Hartman, P. E., Yura, T., Gots, J. S., Ozeki, H., and Glover, S. W. 1956. Genetic Studies with Bacteria, Carnegie Inst. Wash. Publ. 612, 136 pp. Drake, J. W. 1970. “The Molecular Basis of Mutation.” Holden-Day, San Francisco, California. Eisenstark, A,, Eisenstark, R., and Van Sickle, R. 1965. Mutation of Salmonella typhimurium by nitrosoguanidine. Mutat. Res. 2, 1-10. Ferretti, J. J. 1971. Low-level reading of the UGA triplet in SaZmonella typhimurium. J . Bacteriol. 106, 691-693. Fink, G. R., and Martin, R. G. 1967. Translation and polarity in the histidine operon. 11. Polarity in the histidine operon. J. Mol. Biol. 30, 97-107. Fink, G. R., Klopotowski, T., and Ames, B. N. 1967. Histidine regulatory mutants of Salmonella typhimurium. IV. A positive selection for polar histidine-requiring mutants from histidine operator constitutive mutants. J. Mol. Biol. 30, 81-95. Franklin, N. C. 1967. Extraordinary recombinational events in Escherichia coli. Their independence of the rec+ function. Genetics 55, 699-707. Garrick-Silversmith, L., and Hartman, P. E. 1970. Histidine-requiring mutants of Escherichia coli K12, Genetics 66, 231-244. Goldschmidt, E. P., Cater, M. S., Matney, T. S., Butler, M. A,, and Greene, A. 1970. Genetic analysis of the histidine operon in Escherichia coli K12. Genetics, 66, 219-229. Greeb, J., Atkins, J. F., and Loper, J. C. 1971. Histidinol dehydrogenase (hisD) mutants of Salmonella typhimurium. J. Bacteriol. 106, 421431. Hartman, P. E., Hartman, Z., and Serman, D. 1960a. Complementation mapping by abortive transduction of histidine-requiring Salmonella mutants. J. Gen. MicTobiol. 22, 354-368. Hartman, P. E., Loper, J. C., and Serman, D. 1960b. Fine structure mapping by

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complete transduction between histidine-requiring Salmonella mutants. J . Gen. Microbiol. 22, 323-353. Hartman, P. E., Levine, K., Hartman, Z., and Berger, H. 1971. Hycanthone: a fremeshift mutagen. Science 172, 1058-1060. Inselberg, J. 1967. Formation of deletion mutations in recombination-deficient mutants of Escherichia coli. J . Bacteriol. 94, 12661267. Jacob, F., and Monod, J. 1961. Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3, 318-356. Kirchner, C. E. J. 1960. The effects of the mutator gene on molecular changes and mutation in Salmonella typhimurium. J . Mol. Biol. 2, 331-338. Lawley, P. D. 1968. Methylation of DNA by N-methyl-N-nitrosourethane and Nmethyl-N-nitroso-N’-nitroguanidine.Nature 218, 580-581. Lew, K. K., and Roth, J. R. 1971. Genetic approaches to determination of enzyme quaternary structure. Biochemistry 10, 204-207. Loper, J. C. 1961. Enzyme complementation in mixed extracts of mutants from the Salmonella histidine B locus. Proc. Nat. Acad. Sci. U.S. 47, 1440-1450. Loper, J. C. 1968. Histidinol dehydrogenase from Salmonella typhimurium. J . Biol. Chem. 243, 3264-3272. Loper, J. C., Grabnar, M., Stahl, R. C., Hartman, Z., and Hartman, P. E. 1964. Genes and proteins involved in histidine biosynthesis in Salmonella. Brookhaven Symp. Biol. 17, 15-50. Malamy, M. 1970. Some properties of insertion mutations in the lac operon. I n “The Lactose Operon” (J. R. Beckwith and D. Zipser, eds.), pp. 359-373. Cold Spring Harbor Laboratory, New York. Margolies, M. N., and Goldberger, R. F. 1967. Physical and chemical characterization of the isomerase of histidine biosynthesis in Salmonella typhimurium. J . Biol. Chem. 242, 256-264. Margolies, M. N., and Goldberger, R. F. 1968. Correlation between mutation type and the production of cross-reacting material in mutants of the A gene of the histidine operon in Salmonella typhimurium. J. Bacteriol. 95, 507-519. Martin, R. G. 1967. Frameshift mutants in the histidine operon of Salmonella typhimurium. J. MoZ. Biol. 26, 311328. Martin, R. G., and Talal, N. 1968. Translation and polarity in the histidine operon. IV. Relation of polarity to map position in hisC. J. Mol. Biol. 36, 219-229. Martin, R. G., Berberich, M. A., Ames, B. N., Davis, W. W., Goldberger, R. F., and Yourno, J. 1971. In “Methods in Enzymology,” Vol. 17B (C. Tabor and H. Tabor, eds.), pp. 3-44. Academic Press, New York. Marver, D., Berberich, M. A., and Goldberger, R. F. 1966. Correlation between location and time of expression for genes in a single operon. Science 153, 1655-1656. Michaelis, G., Saedler, H., Venkov, P., and Starlinger, P. 1969. Two insertions in the galactose operon having different sizes but homologous DNA sequences. Mol. Gen. Genet. 104, 371-377. Murray, M. L., and Klopotowski, T. 1968. Genetic map position of the gluconate-6phosphate dehydrogenase gene in Salmonella typhimurium. J. Bacteriol. 95, 1279-1282. Nikaido, H., Levinthal, M., Nikaido, K., and Nakane, K. 1967. Extended deletions in the histidine-rough-B region of the Salmonella chromosome. Proc. Nat. Acad. Sci. U.S. 57, 1825-1832.

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Salmonella

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Oeschger, N. S., and Hartman, P. E. 1970. ICR-induced frameshift mutations in the histidine operon of Salmonella. J . Bacteriol. 101, 490-504. Rechler, M. M., and Bruni, C. B. 1971. Properties of a fused protein formed by genetic manipulation. J . Biol. Chem. 246, 1806-1813. Rechler, M. M., and Martin, R. G. 1970. The intercistronic divide: Translation of an intercistronic region in the histidine operon of Salmonella typhimurium. Nature 226, 908-911. Riddle, D. L., and Roth, J. 1970. Suppressors of frameshift mutations in Salmonella typhimurium. J. Mol. Biol. 54, 131-144. Rogan, E. G., and Bessman, M. J. 1970. Studies on the pathway of incorporation of 2-aminopurine into the deoxyribonucleic acid of Escherichia coli. J. Bacteriol. 103, 622-633. Roth, J. 1970. UGA nonsense mutations in Salmonella typhimurium. J . Bacteriol. 102, 467475. Rothman-Denes, L., and Martin, R. G. 1971. Two mutations in the first gene of the histidine operon of Salmonella typhimurium affecting control. J. Bacteriol. 106, 227-237. St. Pierre, M. 1968. Mutations creating a new initiation point for expression of the histidine operon in XalmoneEla typhimurium. J. Mol. Biol. 35, 71-82. Sanderson, K. E. 1970. A current linkage map of Salmonella typhimurium. Bacteriol. Rev. 34, 176-193. Schwartz, D. O., and Beckwith, J. R. 1969. Mutagens which cause deletions in Escherichia coli. Genetics 61, 371-376. Sheppard, D. E. 1964. Mutants of Salmonella typhimurium resistant to feedback inhibition by L-histidine. Genetics 50, 611-623. Smith, D. W. E. 1966. Mutagenicity of cycasin aglycone (methylazomethanol), a naturally occurring carcinogen. Science 152, 1273-1274. Vasington, F. D., and LeBeau, P. 1967. Sedimentation properties of the enzymes of the histidine B gene. Biochem. Biophys. Res. Commun. 26, 153-161. Venetianer, P., Berberich, M. A., and Goldberger, R. F. 1968. Studies on the size of the messenger-RNA transcribed from the histidine operon during simultaneous and sequential derepression. Biochim. Biophys. Acta 166, 124-133. Vogel, H. J., and Bonner, D. M. 1956. Acetylornithinase of Escherichia coli: partial purification and some properties. J . Biol. Chem. 218, 97-106. Voll, M. J. 1967. Translation and polarity in the histidine operon. 111. The isolation of prototrophic polar mutations. J. Mol. Biol. 30, 109-124. Voll, M. J., Appella, E., and Martin, R. G. 1967. Purification and composition studies of phosphoribosyl-adenosine triphosphate: pyrophosphate phosphoribosyltransferase, the first enzyme of histidine biosynthesis. J. Biol. Chem. 242, 1760-1767. Whitfield, H. J., Jr., Smith, D. W. E., and Martin, R. G. 1964. Sedimentation properties of the enzymes of the histidine operon. J . Biol. Chem. 239, 32883291. Whitfield, H. J., Jr., Martin, R. G., and Ames, B. N. 1966. Classification of aminotransferase (C gene) mutants in the histidine operon. J. Mol. Biol. 21, 335-355. Yourno, J. 1968. Composition and subunit structure of histidinol dehydrogenase from Salmonella typhimurium. J. Biol. Chem. 243,3277-3288. Yourno, J., and Heath, S. 1969. Nature of the hisDSO18 frameshift mutation in Salmonella typhimurium. J. Bacteriol. 100, 460-168.

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Yourno, J. and Tanemura, S. 1970. Restoration of in-phase translation by an unlinked suppressor of a frameshift mutation in SalmoneEla typhimurium. Nature (London) 225, 422426. Yourno, J., Barr, D., and Tanemura, S. 1969. Externally suppressible frameshift mutant of Salmonella typhimurium. J . Bacteriol. 100,453-459. Yourno, J., Kohno, T., and Roth, J. R. 1970. Enzyme evolution: Generation of a bifunctional enzyme by fusion of adjacent genes. Nature 228, 82M24. Zinder, N. D.,and Lederberg, J. 1952. Genetic exchange in Salmonella. J . Bacteriol. 64, 679-699.

Genetics of the Enterobacteriaceae A. GENETIC HOMOLOGY IN THE ENTEROBACTERIACEAE Kenneth E. Sanderson* Department of Biology, The University of Calgary, Calgary, Alberta

Introduction. . . . . . . . Glossary of Abbreviations. . . . Concepts of Microbial Taxonomy. . Use of Genetic Data in Systematics . A. Genetic Transfer . . . . . B. Genetic Recombination. . . . C. Comparisons of the Linkage Maps V. Use of Data on Protein Structure. . VI. Summary. . . . . . . . . References . . . . . . . .

I. 11. 111. IV.

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. . 3 5 . . 36 . .37 . .39 . . 40 . . 41 . .43 . .47 . .48 . .49

I. Introduction

The main thrust of the work of Dr. M. Demerec was toward a clearer view of the structure of the gene, of mutation, and of the nature of gene action. His work on maize, on Drosophila, and on Delphinium, through the 1920s and 1930s, was concerned mainly with gene structure and mutation. I n the 1930s and 1940s he analyzed the effect of environmental agents, first ionizing and non-ionizing radiation, and later chemicals, on the genetic properties of Drosophila, of bacteria, and of bacteriophages. The discovery of transduction provided a means of genetic exchange in bacteria, with which he began an analysis of the fine structure of genetic material which led to his detailed observations of clusters of genes for related functions. A bibliography of Dr. Demerec’s papers is found a t the end of this volume (p. 349).

* The author acknowledges support from the National Research Council of Canada through an Operating Grant, and from the National Science Foundation of the United States through Grant GB-20464. 35

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Through his entire life, Dr. Demerec’s interest in organisms as experimental subjects was primarily in terms of their suitability for the solution of particular problems of genetics. However, in the 1950s and 1960s, while Dr. Demerec’s interest in gene structure, function, and mutation did not diminish, he also began to see the possibilities of genetic analysis to give information on taxonomic relationships in the Enterobacteriaceae. This analysis was presented in several papers (Demerec and Ohta, 1964; Demerec and New, 1965; Demerec, 1965; Ino and Demerec, 1968; St. Pierre and Demerec, 1968). This present group of three papers by Middleton and Mojica-a (this volume), by Brenner and Falkow (this volume), and by myself, reviews the work of Dr. Demerec, and others, and presents new data on the genetic and molecular relationships in the Enterobacteriaceae. It is the purpose of this paper to summarize the present data, which indicate considerable similarities of linkage maps in different members of the Enterobacteriaceae, and to try to evaluate this comparison in terms of systematics. The paper by Middleton and Mojica-a (this volume) reviews data, collected by Demerec and his associates, as well as by others, on genetic intercrosses between members of the Enterobacteriaceae ; these data reveal that the frequency of recombination in crosses between different genera, e.g., Escherichia coli and Salmonella typhimurium, is usually quite low. In addition, Brenner and Falkow (this volume) discuss the molecular relationships among the Enterobacteriaceae based on methods of DNA-DNA and DNA-RNA hybridization. The purpose of these three contributions is to evaluate the present state of knowledge in these areas in which Dr. Demerec’s work was important.

II. Glossary of Abbreviations aromatic amino acid lac lactose nonutilization requirement nic nicotinamide requirement biotin requirement bio Pro proline requirement chl PYr pyrimidine requirement chlorate resistance cysteine requirement thi thiamine requirement CYS galactose nonutilisation thr threonine requirement gal hag, H I , H 2 H-antigen defective thy thymine requirement host specificity tna tryptophanase hap histidine requirement hut trP tryptophan requirement inositol fermentation in1 uvr ultraviolet light resistance ZY 1 xylose nonutilization aro

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Ill. Concepts of Microbial Taxonomy

According to Dobzhansky’s concept of the genetic nature of species, ‘(Species are formed when a once actually or potentially interbreeding array of Mendelian populations becomes segregated in two or more reproductively isolated arrays. Species are, accordingly, groups of populations the gene exchange between which is limited or prevented in nature by one, or by a combination of several, reproductive isolating mechanisms.” These reproductive isolating mechanisms may be of two general types. Firstly, geographic isolation prevents two populations from interbreeding due t o spatial separation. Secondly, reproductive isolation, which prevents populations sharing the same habitat from interbreeding, may function by ecological or behavioral factors, or due to an inability of intercrosses to form fertile hybrids. The continuous process of interbreeding between organisms of the same species makes the species of sexually cross-breeding organisms generally recognizable by different investigators, regardless of the traits these investigators are using to differentiate their species. Agreement about classification to lower ranks, such as subspecies and races, or to higher ranks, such as families and orders, is much more difficult to achieve. Dobzhansky excluded the asexual species from his genetic definition of the species. He pointed out that those plant groups most difficult to classify into species were usually those with mainly asexual or selffertilization mechanisms. In these species, while variation is not completely continuous, the range of variation of each species is often greater than in sexual species, and the phenotypes are clustered around certain “adaptive peaks.’’ However, these species are not real entities, unified through exchange of genes, but only clusters of separate clones of similar phenotypes. Dobzhansky considered the bacteria to be examples of asexual species. In the past few years, three new factors have entered the field of bacterial taxonomy. Genetic exchange has been discovered in several new groups of organisms, including the bacteria. This has enabled a new look at the systematics of these groups, and such examinations have already been undertaken (Marmur et al., 1963; Mandel, 1969; Jones and Sneath, 1970). At about the same time, the concept of separation of bacterial species by individual characteristics in the manner dictated by the use of a key was not always satisfactory as more information became available, and this has generated the field of numerical taxonomy. I n numerical taxonomy, all the information comparing two

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KENNETH E. SANDERSON

groups is used to decide on their relationship, and characters are often weighted equally (Davis and Heywood, 1963). The large amount of data used necessitates mathematical treatment and the use of high-speed computers. Over the same period, the field of molecular biology has enabled the measurement of a number of characteristics which may turn out to be of crucial importance in making valid decisions on taxonomic relations. Direct comparison of the genetic material of two organisms is possible from DNA-DNA or DNA-RNA hybridization methods, and direct comparison of proteins, a product which closely reflects the sequence of nucleotides of the genetic material, is possible through studies of amino acid sequence. In the remainder of this section, the theoretical basis for the use in bacterial systematics of data derived from the first of these new approaches, the use of intercrosses, will be considered. In the bacteria, a large number of groups has been demonstrated to be capable of genetic exchange. However, the fact that genetic exchange is possible or even proven to occur in nature cannot be taken to indicate that bacteria exist as Mendelian populations. True Mendelian populations can exist only in those species which have an obligate sexual cycle in each generation, and in which cross-fertilization rather than self-fertilization is normally or always used. In the bacteria, reproduction is normally the product of asexual division, and genetic exchange operates independently of reproduction, for genetic exchange is not necessary for reproduction. As indicated earlier, one of the isolating mechanisms which separate populations of sexually reproducing organ'isms and create species is the inability of individuals of the two populations to intercross to produce fertile hybrids. In nature, such inability may be due to ecological, seasonal, or behavioral characteristics which can be bypassed in the laboratory so that fertile hybrids may be technically possible between species which do not normally hybridize in nature. However, formation of fertile hybrids between separate Mendelian populations is often impossible, even in laboratory conditions, and such inability may indicate separate species. Genetic crossing in bacteria can be used to produce a type of taxonomic analysis similar to that in higher organisms. Crosses in bacteria differ from those in higher plants and animals in several respects: the parents are usually haploid, rather than diploid or of higher ploidy; the donor parent usually transfers only part of its genetic material to form a merozygote, rather than a holozygote as in the higher organisms; no meiosis intervenes in the life cycle; and segregation to re-form the haploid often occurs immediately after transfer. To enable an analogy

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to crossing in higher organisms, the entire process in bacteria may be considered as two stages. First, genetic transfer is the transmission of genetic material from one cell to another; this genetic material may or may not replicate, and may or may not integrate into the recipient chromosome. Second, genetic recombination will be used in this artiple to refer to the process of integration of genetic material of the donor into the recipient chromosome, with subsequent replication. Transfer must precede recombination, but recombination need not occur following transfer. Genetic transfer is considered analogous to the formation of a hybrid in crosses between diploid, sexual organisms. Such hybrids may be formed in higher organisms in crosses between quite unrelated organisms, in which case the hybrid will usually be sterile. Sterility of hybrids is due to two major reasons, genic and chromosomal. Genic sterility results in failure of the hybrid to produce functional meiotic cells, or to produce gametes after meiosis. Chromosomal sterility results from a failure of homologous chromosomes of the hybrid to pair normally, to form bivalents, and to disjoin normally at anaphase (Dobzhansky, 1951). The corollary is that the formation of a fertile hybrid indicates sufficient genomic relationship to permit synapsis and crossing-over. Thus we may draw an analogy between the formation of a sterile hybrid due to chromosomal sterility in higher organisms, where the chromosomes can function but are not sufficiently homologous to pair, and formation of a merozygote between bacterial species where the hybrid is formed but recombination does not occur as measured by a failure of integration of the donor genes into the chromosome of the recipient. This may indicate about the same degree of relationship. The formation of a fertile hybrid in higher organisms indicates a greater degree of relationship, though it does not prove that the parents belong to the same species. In the bacteria, the observation of a high frequency of integration of genetic material of the donor into the chromosome of the recipient may be taken to indicate a similar level of relationship. To summarize, genetic transfer without recombination in bacteria, and formation of a sterile hybrid in higher organisms, suggests that the parents have a low order of relationship. Genetic transfer and recombination, as detected by integration in bacteria, is analogous t o the formation of fertile hybrids in higher organisms, and indicates a closer degree of relationship.

IV.

Use of Genetic Data in Systematics

There are two types of genetic data of value to the systematics of the Enterobacteriaceae. First, there are data on transfer and recombina-

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KENNETH E. SANDERSON

tion from genetic crosses; the theoretical basis for the use of these data was described in the previous section. Second, the similarity of the linkage map in terms of locations of genes for identical functions can be compared among the members of this group. The value of the genetic map as a taxonomic criterion has not been clear up to now, and an attempt will be made to evaluate it.

A. GENETICTRANSFER Data on intercrosses between species and genera have been derived primarily by phage-mediated transduction and by episome-mediated conjugation. Transduction has two disadvantages, the narrow host ranges of the few transducing phages available, and the fact that only small pieces of chromosome can be transmitted. Use of the process of conjugation has produced more data, since transfer between genera is not limited by phage sensitivity. Jones and Sneath (1970) have summarized the data from many authors who have studied the transfer of episomes such as R-factors, F-prime factors, and col-factors, and they conclude that intercrosses between the following genera are frequently or invariably successful in the transfer of the episome from the donor to the recipient: Escherichia, Shigella, Salmonella, Klebsiella, Serratia, Citrobacter, and Proteus. Other groups to which transfer from the above genera was less frequent are Providence, Enterobacter, Hafnia, Aerobacter, Arizona, Vibrio, Yersinia (a part of the Pasteurella group) , and Alcaligenes. It is clear that genetic transfer is possible over a very broad range of genera, to all or almost all of the genera normally placed in the family Enterobacteriaceae (Edwards and Ewing, 1962) , and even to some usually placed in other families, such as Vibrio, Pasteurella, and Alcaligenes (Breed, Murray, and Smith, 1957). The frequencies of transfer obtained in crosses between different genera show a wide range of variability. I n fact, intercrosses involving different strains of one genus with different strains of another genus have also shown considerable variability. A number of factors which may be of only peripheral systematic importance affect the success of genetic transfer, factors such as host restriction and modification, and success in the formation of conjugation bridges between cells of different species. However, as pointed out in the previous section, genetic transfer does not prove close relationship, and so we must examine data on genetic recombination (integration) .

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B. GENETICRECOMBINATION Intercrosses between different strains of E. coli, such as E. coli K12 Hfr X E . coli B F- (Boyer, 1964) and E. coli K12 Hfr X E. coli C F(Lieb et al., 1955) have shown a high level of acceptance and integration of the chromosomal genes of the donor by the recipient, though the frequencies of recombination are in some cases reduced by host restriction. Apparently, all the strains of E . coli so far tested belong to a single species by a genetic definition, which is the ability to give stable, integrated recombinants on intercrossing. Luria and Burrous (1957) crossed E . coli K12 Hfr X Shigella dysenteriae, and found that the frequency of recombinants was reduced by a factor of to from the frequency found with E . coli Hfr X E . coli F- crosses, but most of the hybrids were stable, indicating that the donor genes had integrated in the recipient chromosome. Falkow et al. ( 1963) and Schneider and Falkow (1964) found a reduction of only 10-1 to in crosses of E . coli K12 Hfr X Shigella flezneri F-, as compared to homologous E. coli crosses, with over 90% of the recombinants stable. Crosses of E . coli Hfr strains to other genera of the Enterobacteriaceae have revealed a different pattern, for the frequency of recombinants isolated is much lower, or undetectable, and in a high proportion of these recombinants the donor fragment does not integrate, but remains as a stable partial diploid. Crosses of E. coli Hfr to S. typhimurium F- (Baron et al., 1959; Miyake and Demerec, 1959; Zinder, 1960; Falkow et al., 1962) gave a low frequency of recombination for chromosomal genes (lo-* to though this frequency could be increased by lo4 by selection of F- lines which have enhanced recipient ability, apparently due to loss of their capacity for restriction of E. coli DNA (Okada et al., 1968; Colson et al., 1969). Baron et al. (1968) reviewed the results of crosses of E . coli Hfr to Salmonella typhosa and S. typhimurium, and concluded that the formation of recombinants is reduced to to 10-3 from homologous E. coli x E . coli crosses, and that in very few if any of the recombinants are the E . coli genes integrated into the chromosome with the elimination of the Salmonella genes to form a stable haploid. The initial conclusions were based on the observation that when the donor gene is dominant, as is usually the case, segregation of the hybrid with recovery of the recessive recipient phenotype was usually observed. However, the use of recessive genes from the donor, such as auxotrophy or streptomycin resistance, showed a low frequency of integration. In contrast, Demerec and Ohta (1964), Demerec and

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KENNETH E. SANDERSON

New (1965) , Demerec (1965), Ino and Demerec (1968) , and St. Pierre and Demerec (1968) reported on transduction studies using E. coli H f r X S. typhimurium hybrids, and concluded that a substantial proportion of the hybrids were stable haploids. This conclusion was based on failure to obtain segregation, and on the results of P22-mediated transduction experiments with hybrids as donors, and S. typhimurium mutants as recipients. Demerec and Ohta (1964) found that the frequency of transduction for certain unselected Salmonella genes which map close to the gene selected from E. coli was reduced to as low as 1% of the frequency of transduction for genes in other regions of the chromosome. It was interpreted that the wild-type Salmonella gene was replaced by a wild-type E . coli gene. If these lines had been partial diploids, one would expect only a small decrease, if any, in the frequency of transduction, since the Salmonella chromosome would be available for transduction. Middleton and Mojica-a (this volume), review the literature and present new data on this topic. The explanation for the differing results apparently resides in the use of different strains. Gemski et al. (1967) and Baron et al. (1968) conclude that transfer of chromosomal genes from E . coli Hfr to Proteus F- occurs a t a low frequency of to lo-* recombinants per donor cell, and that all the recombinants isolated were partial diploids with no evidence that integration ever occurred. These authors demonstrated that DNA of the guanine plus cytosine ratio characteristic of E. coli (51%) was present in the hybrids in the proportion expected for the number of E. coli genes they carried. Clarke (1961) and Stouthamer and Pietersma (1970) both report failure to obtain recombinant formation in crosses of E. coli Hfr x Klebsiella species, though numerous authors have reported ready transfer of episomes into Klebsiella species (Jones and Sneath, 1970). Reports on attempts to transfer chromosomal genes from E. coli Hfr to other members of the Enterobacteriaceae were not found. It is possible that they have been attempted, but have failed and have not been reported. The data above indicate that the Enterobacteriaceae and related genera are a single group in terms of ability to accept, episomes and chromosome fragments carried on episomes from other members. However, tests for genetic recombination, involving crosses with selection for chromosomal genes using E. coli Hfr strains as donors and several genera as recipients, divide these genera into four groups: E. coli, high transfer and high integration; Shigella, transfer reduced by a factor high integration; Salmonella, transfer reduced to of lo-' to to low integration; Proteus, transfer reduced to to no integration detectable ; other genera like Klebsiella, to which transfer

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of chromosomal genes has been undetectable, or remains untested. These data give a similar pattern to data from molecular hybridization (Brenner and Falkow, this volume), which show Shigella clmely related to E . coli, with all other species much less related. However, the data have the same shortcomings in that genera like Salmonella and Proteus can be compared only with E. coli, not with each other, for most intercrosses and studies on molecular hybridization have used only E . coli as a reference point. Studies using genera other than Escherichia as the reference point are needed. I n similar studies using Salmonella montevideo Hfr X S. typhimurium hybrids the S. montevideo genes from the hybrid were integrated into S. typhimurium in only 5 to 30% of the frequency for homologous S. typhimuriuva crosses (Atkins and Armstrong, 1969) . I n the above study the electrophoretic analysis of the reductoisomerase enzymes of the isoleucinc-valine pathway, which are distinguishable in S. typhimurium and S. montevideo, confirmed the prediction of the genetic analysis as to which parent contributed the reductoisomerase gene to each recombinant. C. COMPARISONS OF

THE

LINKAGE MAPS

A second criterion of genetic relatedness of two species is similarity of the linkage map. This comparison can be done only in those few cases where related species of genera have been genetically studied so as to yield a detailed overall linkage map, or where sections of the maps can be compared based on deletion analyses or other local analysis. I n the following discussion, the linkage maps of members of the Enterobacteriaceae will be compared, in the light of other measures of relatedness of species. Overall linkage maps of the following species of the Enterobacteriaceae have been published (the accompanying number indicates the number of genes mapped) : E . coli K12 (Taylor, 1970), 310 genes; E . coli B (Boyer, 1966), 13 genes; E . coli C (Wiman et al., 1970), 39 genes; 8. typhimurium (Sanderson and Demerec, 1965; Sanderson, 1970), 251 genes; Salmonella abony (Makela, 1963), 18 genes. I n all cases the overall linkage map is circular and was established from F-mediated conjugation studies, frequently studied with interrupted conjugation. Fine structure mapping was usually done by generalized or specialized transduction. The most detailed comparisons can be made between E . coli K12 (Taylor, 1970) and S. typhimurium (Sanderson, 1970). A total of 116 genes which are mapped in both genera appear to determine a product which has the same function in both organisms. For example, in a clearly

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KENNETH E. SANDERSON

identified case, the structural gene for tryptophan synthetase (trp) is found in the same general location on the linkage map of E. coli (Signer et al., 1965) and S. typhimurium (Blume and Balbinder, 1966) (Fig. 1 ) . Data which illustrate that the enzyme formed by the trp genes of the two genera is similar but not identical (Creighton et al., 1966) will

FIQ.1. Linkage maps of Escherichia coli (Taylor, 1970), shown on the outside of the circle, and of Salmonella typhimurium (Sanderson, 19701, on the inside of the circle. The numbers indicate the time required for chromosome transfer, in minutes, from interrupted conjugation experiments. Only a small number of the 310 genes mapped in E . coli and of the 251 genes mapped in S. typhimurium are shown. A few genes with similar map locations in each genus are shown as reference points (thr, his, thy, zyl), though differences in the time of entry for the entire chromosome places these at time intervals which do not correspond exactly in the two genera. Several other genes which differ in map location are shown on the maps, and discussed in the text. The abbreviations used in the table are explained in Section 11. This table draws on material published in Bacteriological Review.

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be discussed below. In other cases, the basis for assuming homology of genes is not as clear. For example, mutants which result in an inability to utilize D-xylose as the sole carbon source map in a single and similar region of the linkage map of both genera, but there are as yet no enzymatic data to indicate if the genes are really homologous in both. However, for the purposes of comparison the xyl genes and numerous other pairs like them are considered to produce homologous gene products. Of the total of 116 genes mapped in both genera, almost all occur in identical map locations so far as the data permit a comparison. A comparison in any region may show apparent differences usually resulting from uncertainties in map location in one genus or the other. However, there are a number of differences. Firstly, there is a case of a chromosomal rearrangement. The genes trpOABEDC-cysB-pyrF are all arranged in the same order in E. coli and S . typhimurium. (Different locus designations are used in the two genera, and the Salmonella designations are used here, but homologous genes are in the same order.) But this group of genes is inverted in the two genera with respect to the remainder of the linkage map (Sanderson and Hall, 1970). There are, in addition, several cases in which genes, or gene activities, or sections of genetic material are missing from one genus or the other. I n E. coli, proA, proB, and proC are rather widely separated, proA and proB by more than 2 minutes on the linkage map, while several genes including the lac operon are between proB and proC. I n S. typhimurium proA and proB appear to be contiguous by mutation and recombination studies, and are jointly transduced with proC by P22 phage (Miyake and Demerec, 1960). The inability to ferment lactose suggests the absence of the lac operon from S . typhimurium and may indicate a deletion of genetic material between proA and proC, and a similar explanation seems to be necessary to explain the close proximity of proA and proB as well. Mutant strains which can ferment lactose slowly have been isolated from Salmonella lines which were unable to ferment lactose (Schafler et al., 1960) though there is no evidence that the genetic material involved in production of the relevant enzymes is homologous with the lac operon of E. coli. T,he observation of Brenner and Falkow (this volume) that the binding of E . coli lactose messenger RNA was higher to Salmonella DNA than was the binding of E . coli bulk RNA suggests the presence of genetic material in Salmonella homologous to the lactose operon of E . coli, but further tests will be needed. Further, S. typhimurium uses histidine as a sole carbon source, while E . coli does not, and several genes and enzymes have been observed for this pathway which are absent from E. coli (Brill and Magasanik, 1969; Meiss et al., 1969). The enzyme tryptophanase, determined by the gene tna, for

46

KENNETH E. SANDERSON

breakdown of tryptophan to indole, is found in E. coli but not in Salmonella (Gartner and Riley, 1964). The gene for phase 1 flagellar antigen of S. abony is allelic to H, the gene for the only flagellar antigen of E. coli, whereas the 8. abony gene H2 has no counterpart in E. coli (Makelii, 1964). Thus the genetic maps of the two genera are strikingly similar, with few major differences. The linkage maps of E. coli C, E. coli B, and S. abony have no proven differences from those of E. coli and S. typhimurium except those differences between the genera which were already discussed. The above analysis of the linkage maps is mostly based on genetic recombination using conjugation or transduction, though deletion analysis with or without genetic crosses was used in some cases. Deletion mapping of chlorate-resistance mutants, in E. coli (Puig and Azoulay, 1967; Adhya et al., 1968), in 8. typhimurium (Stouthamer, 1969; M. Alper and B. Ames, personal communication), and in Klebsiella aerogenes (Stouthamer and Pietersma, 1970) reveals a striking similarity of gene orders in two map regions of these three genera. I n the first region, the order of genes in Klebsiella is nicA-aroG-gal-chlD-hut-biouvrB-chlA. The order in E . coli is the same, except that hut is missing since E. coli cannot utilize histidine, but Stouthamer and Pietersma (1970) point out that there seems to be an inversion of the bio-chZD region between S. typhimurium and Klebsiella. I n the second region, mutants in the chlG region have been shown to have the same order for nicB-thiB and inlB in K. aerogenes and S. typhimurium. There are striking similarities of the linkage maps of all the Enterobacteriaceae examined. This suggests the possibility that all bacteria may have related linkage maps. An examination of the linkage maps of Bacillus subtilis (Dubnau et al., 1967; Goldthwaite et al., 1970) reveals a striking similarity in that the clusters of genes for related functions so commonly found in the enteric bacteria (Demerec, 1964) are also present in the genes for synthesis of methionine, tryptophan, isoleucine-valine, and histidine, as well as other groups. However, the location of these clusters of genes one to another in B. subtilis bears no apparent relation to that in the Enterobacteriaceae. Thus it is likely that the linkage maps are basically unrelated, with clusters of genes for related functions present in each group because of a selective advantage of this arrangement (Demerec, 1964). Pseudomonas, by contrast, shows little clustering, for the histidine genes appear in five unlinked groups by transduction studies in Pseudomonas aeruginosa (Mee and Lee, 1969) instead of in one group as in the enteric bacteria, and the arginine genes are in seven independent groups instead of being partially grouped as in the enterics (Feary et

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al., 1969). Numerous other groups show the same pattern, with genes of the isoleucine-valine pathway (Pearce and Loutit, 1965) an exception in showing clustering in P. aeruginosa as well as the enterics. I n addition, a preliminary linkage map based on conjugation (Stanisich and Holloway, 1969) shows no similarity to that of the enterics. V. Use of Data on Protein Structure

Demerec (1965) noted that E. coli and S. typhimurium have very similar gross linkage maps, but that the genetic homology of the two genera is very low as measured by intercrossing by conjugation or transduction, followed by integration of chromosomal genes, or as measured by molecular hybridization. This suggested to him that the explanation for this divergence might be that the linkage maps are concerned with whole gene loci, whereas the studies on genetic recombination or molecular hybridization processes occur a t the subgenic, molecular level. Thus the genes might be homologous in terms of function yet differ in their molecular structure. This interpretation was based, in part, on the observations made a t this time that homologous proteins for the same function in different organisms frequently differ in their amino acid sequence (Matsubara et al., 1968). Such differences had been widely studied in other organisms by 1965, but homologous proteins had not been sequenced in the Enterobacteriaceae. Subsequently, variation in a homologous protein in different genera was confirmed by Creighton et al. (1966). They compared the structure of the tryptophan synthetase a-subunits in several members of the enteric bacteria by comparing tryptic-chymotryptic peptides. Their results indicate that the proteins in E. coli, 8. dysenteriae, 8. typhimurium and Aerobacter aerogenes appear to be homologous, with similar size based on sedimentation data, and similar numbers of peptides after digestion. The peptide patterns on two-dimensional paper chromatography indicate identity between E. coli K12 and E. coli B, whereas the pattern with S. dysenteriae differs from E . coli in about three peptide spots. The S. typhinturium and A . aerogenes subunits, though clearly related to E. coli, differed markedly from E . coli and from each other. Of 38 to 41 major peptides detected, the peptide pattern of S. typhimurium has 21 peptide spots in common with E. coli, while A . aerogenes has 18 peptides in common with E. coli. However, since the amino acid content of each peptide was not analyzed, the number of amino acid changes could only be estimated. The complete amino acid sequence of the tryptophan synthetase a-subunit of E . coli K12 was later determined

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KENNETH E. SANDERSON

(Yanofsky et al., 1967), and recently C. Yanofsky (personal communication) has extended these data by determining the sequence of the first 50 amino acids of this protein in S. typhimurium. There are 6 amino acid differences between the two genera in these 50 residues, each of which is explainable by a ,single nucleotide change per codon. Therefore the minimum base changes per nucleotide between E . coli and S. typhimurium based on this very small sample of the total number of nucleotides (150 of a total of approximately 5 )( lo6) is 6/3 X 50 (100) = 4%. Such differences between the proteins of the two genera are not always distinguishable. For example, Neu and Winshell (1970) , studying penicillinases produced in R-factor negative strains of S. typhimurium and E . coli, were not able to demonstrate any difference in the enzymes by chromotography on DEAE cellulose and Sephadex G100, and by measures of activity, but such differences might have been discovered by more detailed tests such as fingerprint analysis. An estimate of the percentage of altered bases between the DNA of E . coli and S. typhimurium can be made by molecular hybridization methods as well. Brenner and Cowie (1968) assayed the binding of S. typhimurium DNA to E . coli DNA ; reassociation between the heterologous DNA was 35.5% at 6OoC and 27% at 66OC, where reassociation between homologous E . coli DNA is normalized to 100%. The reassociation product of E . coli-S. typhimurium DNA a t 6OoC has a reduction in melting temperature (AT,) of 12.5OC compared with similarly treated E . coli DNA, while the 66OC product has a AT, of 10.5OC. Laird et al. (1969), in studies with B. subtilis, demonstrated that hybrids between nucleic acids chemically altered from one another in 1.5% of their bases had a reduction of l 0 C in the T, compared with the homologous hybrid. Therefore the observed A T , of 10.5OC for E. coli-S. typhimurium hybrids indicates altered base pairs of 10.5 X 1.5 = 15% to 16%. VI. Summary

Four separate measures of genetic relatedness among the members of the Enterobacteriaceae and related genera have been considered. First, transfer of genetic elements can occur in intercrosses within the entire group. However, integration of chromosomal genes from Escherichia coli occurs with high frequency into the chromosome of Shigella, with much reduced frequency into Salmonella typhimurium, and is generally undetectable into other genera of the group. Second, DNA-DNA or DNARNA hybridization methods (Brenner and Falkow, this volume) indicate

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that E. coli and Shigella are closely related, while other genera are much less related to E. coli. Third, the linkage map is a conservative character, which is relatively unaltered in the genera Escherichia, Salmonella, and Klebsiella, and may be similar in the entire family, while there is no evidence of similarity to the linkage maps of unrelated groups such as Bacillus and Pseudomonas, to which transfer does not occur. Fourth, amino acid sequences of tryptophan synthetase reveal that these proteins in E . coli and S. typhimurium are related, but with a significant number of amino acid differences.

REFERENCES Adhya, S., Cleary, P., and Campbell, A. 1968. A deletion analysis of prophage lambda and adjacent genetic regions. Proc. Nut. Acad. Sci. U.S. 61, 956-962. Atkins, C. G., and Armstrong, F. B. 1969. Electrophoretic study of Salmonella typhimurium-Salmonella montevideo hybrids. Genetics 63, 775-779. Baron, L. S., Carey, W. K., and Spilman, W. M. 1959. Hybridization of Salmonella species by mating with Escherichia coli. Science 130, 566-567. Baron, L. S., Gemski, P., Jr., Johnson, E. M., and Wohlhieter, J. A. 1968. Intergeneric bacterial matings. Bacteriol. Rev. 32, 362-369. Blume, A. J., and Balbinder, E. 1966. The tryptophan operon of Salmonella typhimurium. Fine structure analysis by deletion mapping and abortive transduction. Genetics 53, 577492. Boyer, H. 1964. Genetic control of restriction and modification in Escherichia coli. J . Bacteriol. 88, 1652-1660. Boyer, H. 1966. Conjugation in Escherichia coli. J. Bacteriol. 91, 1767-1774. Breed, R. S., Murray, E. G. D., and Smith, N. F. 1957. I n “Bergey’s Manual of Determininative Bacteriology,” 7th ed. Williams & Wilkins, Baltimore, Maryland. Brenner, D. J., and Cowie, D. B. 1968. Thermal stability of Escherichiu coli-Salmonella typhimurium deoxyribonucleic acid duplexes. J. Bacteriol. 95, 2258-2262. Brill, W. J., and Magasanik, B. 1969. Genetic and metabolic control of histidase and urocanase in Salmonella typhimurium, strain 15-59. J . Biol. Chem. 244, 5392-5402.

Clarke, C. H. 1961. Genetic studies with Klebsiella pneumoniae. Nature 190, 194. Colson, A. M., Colson, C., and Van Pel, A. 1969. Host-controlled restriction mutants in SalmoneEla typhimurium. J. Gen. Microbiol. 58, 57-64. Creighton, T. E., Helinski, D. R., Somerville, R. L., and Yanofsky, C. 1966. Comparison of the tryptophan synthetase a-subunits of several species of Enterobactsriaceae. J. Bactem’ol. 91, 1819-1826. Davis, P. H., and Heywood, V. H. 1963. “Principles of Angiosperm Taxonomy.” Van Nostrand-Reinhold, Princeton, New Jersey. Demerec, M. 1964. Clustering of functionally related genes in Salmonella typhimurium. Proc. Nut. Acad. Sci. U.S. 51, 1057-1060. Demerec, M. 1965. Homology and divergence in genetic material of Salmonella typhimurium and Escherichia coli. In “Evolving Genes and Proteins” (V. Bryson and H. J. Vogel ed.), pp. 505510. Academic Press, New York. Demerec, M., and New, K. 1965. Genetic divergence in Salmonella typhimurium,

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S. montevideo, and Escherichia coli. Biochem. Bbphys. Res. Commun. 18, 652-655. Demerec, M., and Ohta, N. 1964. Genetic analysis of Salmonella typhimurium x Escherichia coli hybrids. Proc. Nut. Acad. Sci. U.S. 52, 317-323. Dobzhansky, T. 1951. “Genetics and the Origin of Species,” 3rd ed. Columbia Univer. Press, New York. Dubnau, D., Goldthwaite, C., Smith, I., and Marmur, J. 1967. Genetic mapping in Bacillus subtilis, J . Mol. Biol. 27, 163-185. Edwards, P. R., and Ewing, W. H. 1962. “Identification of Enterobacteriaceae.” Burgess, Minneapolis, Minnesota. Falkow, S., Rownd, R., and Baron, L. S. 1962. Genetic homology between Escherichia coli K12 and Salmonella. J. Bacteriol. 84, 1303-1312. Falkow, S., Schneider, H., Baron, L. S., and Formal, S. B. 1963. Virulence of Escherichia-Shigella genetic hybrids for the guinea pig. J. Bacteriol. 88, 12511258. Feary, T. W., Williams, B., Calhoun, D. H., and Walker, T. A. 1969. An analysis of arginine requiring mutants in Pseudomonas aeruginosa. Genetics 82, 673-686. Gartner, T. K.,and Riley, M. 1964. Genetic studies on tryptophanase mutants of Escherichia coli K12. Bacterial. Proc. p. 18. Gemski, P., Jr., Wohlhieter, J. A., and Baron, L. S. 1967. Chromosome transfer between Escherichia coli Hfr strains and Proteus mirabilis. Proc. Nut. Acad. Sci. US. 58, 1461-1467. Goldthwaite, C., Dubnau, D., and Smith, I. 1970. Genetic mapping of antibiotic resistance in markers of Bacillus subtilis. Proc. Nut. Acad. Sci. U.S. 85, 96-103. Ino, I., and Demerec, M. 1968. Enteric hybrids. 11. Salmonella typhimurium-E. coli hybrids for the trp-cysB-pyrF region. Genetics 59, 167-176. Jones, D., and Sneath, P. H. A. 1970. Genetic transfer and bacterial taxonomy. Bacteriol. Rev, 34, 40-81. Laird, C. D., McConaughy, B. L., and McCarthy, B. J. 1969. Rate of fixation of nucleotide substitutions in evolution. Nature 224, 149-154. Lieb, M., Weigle, J. J., and Kellenberger, E. 1955. A study of hybrids between two strains of Escherichia Cali. J. Bacteriol. 69, 468-471. Luria, S. E., and Burrous, J. W. 1957. Hybridization between Escherichia coli and Shigella. J. Bacterial. 74, 461476. Makela, P. H. 1963. Hfr males in Salmonella abony. Genetics 48, 423-429. Makela, P. H. 1964. Genetic homologies between flagellar antigens of Escherichia coli and Salmonella abony. J . Gen. Microbiol. 35, 503-510. Mandel, M. 1969. New approaches to bacterial taxonomy: perspective and prospects. Annu. Rev. Microbiol. 23, 239-274. Marmur, J., Falkow, S., and Mandel, M. 1963. New approaches to bacterial taxonomy. Annu. Rev. Microbiol. 17, 329-372. Matsubara, H., Jukes, T. H., and Cantor, C. R. 1968. Structural and evolutionary relationships of ferredoxins. Brookhaven Symp. Biol. 21, 201-216. Mee, B. J., and Lee, B. T. 0. 1969 A map order for his I , one of the genetic regions controlling histidine biosynthesis in Pseudomonas aeruginosa, using the transducing phage F116. Genetics 62, 687-696. Meiss, H. K.,Brill, W. J., and Magasanik, B. 1969. Genetic control of histidine degradation in Salmonella typhimurium strain LT2. J . Bwl. Chem. 244, 538% 5391. Miyake, T.,and Demerec, M. 1959. Salmonella-Escherichia hybrids. Nature 183, 1586.

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Miyake, T., and Demerec, M. 1960. Proline mutants of Salmonella typhimurium. Genetics 45, 755-762. Neu, H. C., and Winshell, E. B. 1970. Purification and characterization of penicillinases from Salmonella typhimurium and Escherichia coli. Arch. Biochem. Biophys. 139, 278-290. Okada, M., Watanabe, T., and Miyake, T. 1968. On the nature of the recipient ability of Salmonella typhimurium for foreign deoxyribonucleic acids. J. Gen. Microbiol. 50, 241-252. Pearce, L. E., and Loutit, J. S.1965. Biochemical and genetic grouping of isoleucinevaline mutants in Pseudomonas aeruginosa. J. Bacteriol. 89, 58-63. Puig, J., and Azoulay, E. 1967. Etude gCnCtique et biochemique des mutants rksistant au C103- (genes chlA, chlB, chlC). C . R . Acad. Sci. 264, 1916-1918. Sanderson, K. E. 1970. Current linkage map of Salmonella typhimurium. Bacteriol. Rev. 34, 176-193. Sanderson, K. E., and Demerec, M. 1965. The linkage map of Salmonella typhimurium. Genetics 51, 897-913. Sanderson, K. E., and Hall, C. A. 1970. F-prime factors of Salmonella typhimurium and an inversion between S. typhimurium and Escherichiu coli. Genetics 64, 215-228.

Schafler, S., Mintzer, L., and Schafler, C. 1960. Acquisition of lactose-fermenting properties by salmonellae. J. Bactem'ol. 79, 203-212. Schneider, H., and Falkow, S. 1964. Characterization of an Hfr strain of Shigella flexneri. J. Bacteriol. 88, 682-689. Signer, E. R., Beckwith, J. R., and Brenner, S. 1965. Mapping of suppressor loci in Escherichia coli. J. Mol. Biol. 14, 153-166. Stanisich, V., and Holloway, B. W. 1969. Conjugation in Pseudomonas aeruginosa. Genetics 61, 327-339. Stouthamer, A. H. 1969. A genetical and biochemical study of chlorate-resistant mutants of Salmonella typhimurium. Antonie van Leeuwenhoek J . Microbiol. Serol. 35, 505-521. Stouthamer, A. H., and Pietersma, K. 1970. Deletion-mapping of resistance against chlorate in Klebsiella aerogenes. Mol. Gen. Genet. 106, 174-179. St. Pierre, M. L., and Demerec, M. 1968. Hybrids of enteric bacteria. I. Salmonella typhimurium-Salmonella montevideo hybrids for the histidine region. Genetics 59, 1-9. Taylor, A. L. 1970. Current linkage map of Escherichia coli. Bacteriol. Rev. 34, 155-175.

Wiman, M., Bertani, G., Kelly, B., and Sasaki, I. 1970. Genetic map of Escherichia coli strain C. Mol. Gen. Genet. 107, 1-31. Yanofsky, C., Drapeau, G. R., Guest, J. R., and Carleton, B. C. 1967. The complete amino acid sequence of the tryptophan synthetase A protein (a subunit) and its colinear relationship with the genetic map of the A gene. Proc. Nut. Acad. Sci. U.S. 57, 296298. Zinder, N. D. 1960. Hybrids of Escherichia and Salmonella. Science 131, 813-815.

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B HOMOLOGY IN THE ENTEROBACTERIACEAE BASED ON INTERCROSSES BETWEEN SPECIES Richard B. Middleton* and Tobias Mojica-at Department of Biology. McGill University. Montreal. Quebec

I. Introduction . . . . . . . . . . . . . . . A . Nomenclature . . . . . . . . . . . . . B. Definitions and Abbreviations . . . . . . . . I1. Production of Hybrids . . . . . . . . . . . . A. Differences in Cell Surfaces . . . . . . . . . B. Differences in Genetic Material . . . . . . . . C. Effects of Restriction . . . . . . . . . . . D . Alteration of Restriction . . . . . . . . . . I11. Stability of the Hybrids . . . . . . . . . . . A. Male DNA Associated with the Chromosome . . . . B. Male DNA Not Associated with the Chromosome. . . I V . Characterization of the Hybrids . . . . . . . . . A. Homology . . . . . . . . . . . . . . B. Normalization . . . . . . . . . . . . . V. Genetic Homologies between Species. . . . . . . . A. Summary of Data . . . . . . . . . . . . B. Reliability of Homology Values . . . . . . . . VI . Use of Hybrids for Mapping . . . . . . . . . . A . Ordering of Nearby Genes Which Are Not Cotransduced . B. Orientation of the ilvE-metA Region, 122-129 Minutes . C. Locating Certain Wild-Type Genes . . . . . . . VII Summary . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . .

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53 54 55 55 55 56 56 58 59 59 61 62 62 63 65 65 65 72 72 73 75 76 76

I. Introduction

Mandel (1969) among others has pointed to the sexual versatility of the enteric bacteria and the resulting potential of this group for studies of genetic relatedness . A special advantage in constructing viable interspecific hybrid recombinants results from the constancy of the genetic

* Present address : Faculty of Medicine. Memorial University. W . John’s. Newfoundland. t Present address: Polish Academy of Sciences. Institute of Biochemistry and Biophysics. 36 Rakowiecka St., Warszawa 12. Poland . 53

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RICHARD B. MIDDLETON AND TOBIAS MOJICA-A

map throughout the Enterobacteriaceae (Sanderson, 1970, 1971; Taylor, 1970). Integration of male genetic material after conjugation can, due to synapsis of similar regions of the genetic material, result in the formation of recombinant prototrophic hybrids. Early reports of genetic hybridization between different genera include studies by Luria and Burrous (1957) for Escherichia coli and Shigella, which are both included in the host range of the generalized transducing coliphage P1, and Baron et al. (1959a,b) for E. coli and various Salmonella species. Of particular interest for a quantitative description of genetic homology are subsequent studies, by means of transduction analysis, of hybrids between E. coli and Salmonella typhimurium (Zinder, 1960; Miyake, 1962; Demerec 1964, 1965a,b; Demerec and Ohta, 1964; Demerec and New, 1965; Eisenstark, 1965a,b; Middleton and Demerec, 1966; Ino and Demerec, 1968; Mojica-a and Middleton, 1970a), and between Salmonella species (Glatzer et al., 1966; Armstrong, 1967; LaBrie and Armstrong, 1968; St. Pierre and Demerec, 1968). Transduction crosses with hybrids as donors and various strains of the female parental species as recipients allow homologous genetic material of the two parental species to be involved in recombinational events. Comparison of the frequency of such interspecific recombinations provides a measure of the similarity of the genetic material in the region under consideration (Demerec and New, 1965). Other approaches to the study of the genetic relatedness of species of enteric bacteria are comparison of the genetic maps (Sanderson, 1971) and DNA-DNA and DNA-RNA associations (Brenner and Falkow, 1971). This review concentrates on the genetic homology of the enteric bacteria as indicated by transduction analysis of hybrids which result from intercrosses between species. Generally excluded is consideration of episomic transfer between species (see Falkow et al., 1961) as well as of the intensive studies, largely of Baron and his co-workers, on hybrid heterogenotes of varying stability. Much of the latter work was the subject of a recent review (Baron et al., 1968). The survey of literature for the present review was concluded in November 1970. A. NOMENCLATURE The nomenclature of Demerec et al. (1966) is followed. The designation of the five structural genes of the tryptophan operon, trpA-E, follows the E. coli convention (Taylor, 1970). Since S. typhimurium is the female parent of most hybrids discussed, all other gene symbols, locus designations, and map positions (minutes) of genes, in general, follow S. typhimurium (Sanderson, 1970) with certain exceptions, as noted.

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B. DEFINITIONS AND ABBREVIATIONS Throughout this paper female refers to a strain used in a conjugation cross with another species and recipient is a strain, of the female species, used in transduction crosses with hybrids as donors. Unless otherwise specified, female and recipient refer to S. typhimurium. Similarly males participate in interspecific conjugation crosses whereas donors refer to phage preparations of hybrids which are utilized in transduction crosses with recipients of the female species. Unless specified, males are E. coli Hfr or F’ strains. Hybrids are recombinants which result from conjugation crosses between species, and unless specified, result from matings between E. coli males and S. typhimurium females. Hybrids susceptible to genetic analysis by transduction have the male genetic material integrated into recombinant chromsomes from which the corresponding female segments are displaced. The distinctions between such hybrids and heterogenotes (partial diploids) are discussed in Section 111, below. P22, a generalized transducing phage of S. typhimurium, is most commonly used for the genetic analysis of hybrids. Abbreviations are DNA (deoxyribonucleic acid) , e.0.p. (efficiency of plating), F (sex factor) , F’ (F-prime, sex factor which carries a segment of the bacterial chromosome) , fer (fertile mutation, after Miyake, 1959), NG (N-methyl-” nitro-N-nitrosoguanidine) , RNA (ribonucleic acid) , R T F (resistance transfer factor) , and UV (ultraviolet irradiation). CI. Production of Hybrids

A characteristic feature of matings between E. coli Hfr’s and Salmonella female strains is the low recovery of prototrophic recombinants. A typical frequency of recombination in crosses of E . coli Hfr’s and (Mojica-a, S. typhimurium LT2 female strains is of the order of 1971; Mojica-a and Middleton, 1970a). Strain LT7 of S. typhimurium is somewhat more fertile: 10-3-10-~(Baron et al., 1959a; Miyake, 1962; R. B. Middleton, unpublished observations). Three possible explanations for the low fertility of interspecific crosses are discussed in the light of the methods employed to overcome infertility.

A. DIFFERENCES IN CELLSURFACES Differences in the cell surfaces of mating cells may introduce a barrier to the conjugal transfer of genetic material. Female strains of two species

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RICHARD B. MIDDLETON AND TOBIAS MOJICA-A

might be expected t o differ in the receptor sites presumed to interact with the sex pili of the male strain. Recently it has been shown that S. typhimurium LT2 male and female strains may have their conjugal fertilities affected by known changes in cell wall polysaccharide (Watanabe et al., 1970); in particular, fertility of female strains is markedly increased by the loss of the 0 side-chain (abequose-mannoserhamnose-galactose) %. A NG-induced mutant of S. typhimurium LT2 (J. M. Somers, onpublished observation) that has an increased fertility for E. coli RTF’s with respect to the parent strain and is sensitive to coliphage P1, appears to retain its ability to restrict E. coli-modified DNA and to impart Salmonella-modification (Middleton and Mojica-a, 1970; Mojica-a and Middleton, 1970b). It seems most likely that a cell-surface alteration is responsible for the increased fertility rather than an impairment of the restriction mechanism, but the mutant has not yet been fully characterized. IN GENETICMATERIAL B. DIFFERENCES

Differences in the DNAs of the mating cells would decrease the frequency of transfer during conjugation as well as reduce the frequency of integration of the male genetic material into the female chromosome. It has been shown that the presence in a Salmonella typhosa hybrid of the leading region of an E. coli Hfr increases the frequency with which subsequent E. coli genes are recovered upon a further mating (Johnson et al., 1964). This effect of the resident E. coli genetic material fits very well in the current model of conjugation (Curtiss et al., 1968; Curtiss, 1969) which requires early pairing of DNA molecules.

C. EFFECTS OF RESTRICTION The restriction mechanisms of the female strains may render genetically inactive the male genetic material after transfer; recent reviews of DNA restriction and modification are by Arber (1965) and Arber and Linn (1969). It was proposed (Baron et al., 1959a) that the low frequency of recombination represented the selection of rare, high-fertility female cells in an otherwise infertile culture. As expected the recombinants were found to have higher fertility with E. coli than did the female parent strain. Salmonella typhimurium LT7 mut which carries a mutator gene (Miyake, 1959, 1960) gave characteristically low frequencies of recombinants for a rather wide range of male markers in conjugational crosses with E . coli Hfr strains. By indirect selection, high

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57

fertility strains (LT7 f e r for fertile) have been isolated without previous participation in conjugal matings (Miyake, 1962). It was therefore concluded that S. typhimurium LT7 mut populations are heterogeneous with respect to the ability to act as fertile females in conjugation with E. coli Hfr’s. Presumably the mut phenotype induced fertility ( f e r ) in infertile (fer+) LT7 mut strains of S. typhimurium to yield fertile female strains, LT7 mut fer. These observations are consistent with the operation of restriction (Grasso and Paigen, 1968a,b). Salmonella typhimurium LT7 fer strains have in addition the following properties (Okada and Watanabe, 1968; Okada et al., 1968): (1) the frequency of transfer of R T F and F’ from E. coli K12 to LT7 f e r is considerably higher than to S. typhimurium fer+ strains-LT2 mut+, LT7 mut+, or LT7 mut; (2) the conjugal transfer of the R T F and F’ episomes from LT2, LT7 mut+,and LT7 mut to derivatives of these fer+ strains occurs at frequencies almost equal to the frequency of transfer between E. coli K12 and LT7 fer; (3) S. typhimurium phage P22 grown and titered on LT7 fer (e.0.p. = 1.0) gives a much reduced e.0.p. on any other derivatives of S. typhimurium: e.0.p. on LT2 6.0 X The e.0.p. on LT7 mut+3.0 X lo+, on LT7 mut 1.0 X of phage P22 grown on LT7 mut is only slightly lower on LT2 mut+, LT7 mut+,and LT7 mut than on LT7 fer. Radioisotope studies show that the DNA of P22 prepared on LT7 fer is rapidly degraded in LT2, LT7 mut+,and LT7 mut: in 10 minutes 3040% of the DNA is degraded to acid-soluble material. Okada et al. (1968) concluded that the results are due to host-controlled restriction and modification of the type defined by Arber (1965). Two alternative explanations were offered: (a) LT7 f e r is a restrictionless and modificationless mutant or (b) LT7 mut+ and LT2 mut+ are modificationless whereas LT7 fer and LT7 mut are able to carry out modification and LT7 fer is restrictionless. The results of Okada et al. (1968), however, favor the first interpretation. The second interpretation is less probable since a strain that is able to restrict and is modificationless has not been found in S. typhimurium (Colson et al., 1969) or in E. coli and would presumably be lethal (Boyer and Roulland-Dussoix, 1969). Eisenstark (1965a) reported induction of hybridization for crosses between E. coli Hfr and S. typhimurium LT2 females by plating mating mixtures in the presence of NG. He concluded that the action of NG in inducing fertility is only on female cells and that the selected phenotypes produced resulted from hybridization and not from reverse mutation. A small number of recipient strains and two E. coli Hfrs (CS101 and P4X6) were used. It has also recently been shown that UV treatment of female LT2 strains increases fertility to a similar extent (Mojica-a

58

RICHARD B. MIDDLETON AND TOBIAS MOJICA-A

and Middleton, 1970a, and unpublished results) . These observations are also consistent with the operation of restriction (Bertani and Weigle, 1953; Luria, 1953).

D. ALTERATION OF RESTRICTION If it is possible to induce fertile mutations and/or select for rare fertile clones in a generally infertile population of cells, i t may also be possible to find appropriate physiological conditions which will permit infertile cell populations to behave temporarily as fertile females. It has been shown that by growing the S. typhimurium LT2 female cells in minimal salts supplemented with appropriate nutritional requirements rather than in nutrient broth the frequency of intergeneric recombinations is increased approximately 100-fold. The additional treatment of preincubating the females at 5OoC for 2 0 4 0 minutes immediately before mating with the E. coli Hfr strains increases the frequency of recombination approximately 100-fold (Mojica-a, 1971; Mojica-a and Middleton, 1970a). The final frequencies of recombination observed are similar to those for S. typhimurium LT7 fer females. These effects are presumably generalized although most tests were made with various 8. typhimurium LT2 females with deletions in the tryptophan operon, and the successful E. coli Hfr strains injected the selected genes fairly early (Mojica-a, 1971). This protocol appears to have three advantages for the production of hybrids: (1) nearly all genetically studied strains of S. typhimurium are derived from LT2, rather than LT7, and these are now routinely available as female parents of hybrids; (2) uncontrolled side effects of mutagens (W and NG) are avoided; (3) the hybrid recombinants produce P22 which is not restricted by S. typhimurium fer+ recipients, in contrast with hybrids produced by fer strains, which simplifies the genetic analysis of the hybrids. Both growth medium (Grass0 and Paigen, 1968a,b) and high temperature (Gwinn and Lawton, 1968) affect host-restriction of phage DNA. The pretreatments of the female therefore appear to reduce the effectiveness of the restriction system of the female in contributing to the low fertility of Salmonella strains with E . coli males. It is concluded that there are a t least three factors responsible for the low recovery of recombinants in conjugal crosses between E . coli Hfr’s and Salmonella strains: (1) differences in the cell surface that affect genetic transfer, (2) differences in the DNA sequences of the parent strains that affect both transfer and integration of male genetic material, and (3) the restriction system of the female that inactivates

ENTEROBACTERIACEAE : HOMOLOGY FROM INTERCROSSES

59

male genetic material after transfer. It is not possible to determine the relative importance of these three factors for the fertility of interspecific matings. The apparent importance of female restriction may be due to the present ability to alter its effectiveness by experimental means. 111. Stability of the Hybrids

When S. typhimurium-E. coli hybrids are used as donors in transduction crosses with S. typhimurium recipients, few wild-type recombinants are generally observed for donor genes which are derived from the E. coli parent. Is this depression in the recombination frequency due solely to inhomology of the genetic material, or might the physical state of the male DNA affect the donor efficiency of the hybrids? Various observations indicate that the physical state of the male DNA in the hybrids probably has a detectable effect on the depression of the frequency of recombination, although the mechanism by which this effect is achieved is by no means clear. There appear to be two possible physical states of the male DNA in the hybrids: it can either be closely associated (integrated) or not closely associated with the female genome. Both possibilities are discussed in the light of the effects observed. WITH A. MALEDNA ASSOCIATED

THE

CHROMOSOME

Eisenstark (1965b) has shown that P22 is able to transduce E. coli genetic material which is integrated into the S. typhimurium genome. In addition, hybrids have frequently been identified in which E. coli DNA has been stably integrated into the Salmonella chromosome (for example Ino and Demerec, 1968). I n contrast, Baron et al. (1968) have stated that if stable integration occurs at all, it is an extremely rare event. Both classes of recombinants have however been observed within the same set of hybrids (T. Mojica-a and R. B. Middleton, unpublished observations). The criteria used to establish stable integration are the following: (1) inability to segregate clones with a phenotype like the female parent strain (usually auxotrophic clones) ; (2) ability of the male genes to be transduced ; (3) demonstration, by cotransduction with a closely linked marker, of the absence of a cryptic female gene for which the integrated male gene should be substituted ; (4) absence of .extrachromosoma1 DNA. None of the above criteria is completely conclusive and the uncer-

60

RICHARD B. MIDDLETON AND TOBIAS MOJICA-A

tainties for each include the following: (1) phenotypic stability of some hybrids may reflect a very close relationship other than integration of the male genetic segment with the corresponding female region; (2) the absence of donor ability of a male gene may result either from the possible inaccessibility of a nonintegrated male segment or from very low homology of the genes of the two species; (3) the presence of the cryptic female gene cannot distinguish between integration of the male gene in a novel region or the lack of integration; (4) the presence of a satellite DNA is difficult to establish if the physical properties of the two DNAs are similar (e.g., E. coli and Salmonella) or if the satellite DNA is too small to detect. Of Trp+ hybrids generated from E. coli trp’ males X 8. typhimurium trp females, 60-750/0 are stable and “integrated” by the first three criteria outlined above (R. B. Middleton, unpublished observations; Mojica-a, 1971) : no Trp- clones are segregated, Trp’ transductants are produced, and no Trp- cotransductants are found after selection for cysB’ recombinants. Similar results were obtained by Ino and Demerec (1968) for other Trp’ hybrids. St. Pierre and Demerec (1968), however, were unable to select from conjugation crosses stable S. typhimurium-E. coli hybrids in the histidine region and conclude that the absence of integration of E. coli material may be due to the presence of the somatic antigen loci of E. coli mapping very near the histidine region. Another possibility is that the male genetic material is not efficiently recognized by enzymes necessary for recombination. Garrick-Silversmith and Hartman (1970) found that F’ his’ grid' of E. coli is stable in S. typhimurium; the episome remains intact, or is lost entirely, and yields no integrated hybrids. Similarly, a S. typhimurium F’ rfb’ his+ gnd+ episome does not appear to interact with the E. coli chromosome to give stable hybrids (M. J. Voll and P. E. Hartman, personal communication). There are two alternatives for the location of the selected, integrated genetic material: 1. The male genetic material displaces homologous female genetic material, resulting in a patchwork hybrid chromosome similar to the original female chromosome in the continuous linear arrangement of nucleotides. In this case it is possible to estimate the size of the integrated segment of male DNA by its donor efficiency in transduction. It is assumed that the replacement prototrophic alleles are regulated normally by the female regulatory machinery. 2. The male DNA is tandemly inserted into the host chromosome (not necessarily in the region of functional homology) producing a duplicated region. A genetic estimate of the size of the insertion will be difficult since the homologous female genes are not displaced and are efficiently

ENTEROBACTERIACEAE : HOMOLOGY FROM INTERCROSSES

61

transmitted in transduction. There is some evidewe that tandem insertions may occur near, but not displacing, the hc,mologous female genes: about half of a group of Trp+ hybrids which sail1 harbor a cryptic trpB female allele show a significant decrease in t h z frequency of cotransduction between trpB and cysB. The lengthering of the genetic distance suggests that the Trp' male genetic material may be integrated between trpB and cysB (R. B. Middleton, unpublished observations). There are two approaches toward locating the inserted male genetic material in the chromosomes of hybrids: 1. If gene products of the corresponding genes of the two species can be differentiated electrophoretically (Atkins and Armstrong, 1969 ; Lew and Roth, 1971), the enzymes of stable hybrids can be characterized. If the male gene displaces the female gene, only the male enzyme is found (Atkins and Armstrong, 1969). If only the female enzyme is found, such a small part of male gene is integrated as not to affect the characteristic electrophoretic mobility of the female enzyme (F. B. Armstrong, L. Glatzer, and C. G . Atkins, unpublished observations). The presence of both male and female enzymes would indicate the insertion of the male gene into a chromosome which still contains the female gene. 2. If the marker selected against in the female is a mutation in a structural gene in an operon with an operator-constitutive mutation, a tandem insertion of the male gene into the female operon would result in constitutive enzyme levels for some male genes. Some female genes would at the same time become inducible or repressible due to interruption of the female operon. On the other hand, an insertion of the selected male gene into a novel region of the female chromosome, distant from the corresponding female operon, would leave the enzyme levels of all functional female genes at constitutive levels.

B. MALEDNA NOT ASSOCIATED WITH

THE

CHROMOSOME

The male DNA in a hybrid conceivably might not be associated with the chromosome. The male genetic segment would probably replicate independently of the chromosome, and its size would be measurable only by physical rather than by genetic means. A strain carrying such an exogenote would be expected to segregate clones lacking the functions coded for by this male DNA. Baron et al. (1968) reported hybrid strains that behave as if the male DNA were not integrated into the female chromosome. This is perhaps not unexpected since the functions of the markers selected, namely lac+, ara+, rha+, xyl', fuc+,tna+, r c p f , and T,rcp+ of the E. coli male, are all lacking from the wild-type female, Salmonella typhosa.

62

RICHARD B. MIDDLETON AND TOBIAS MOJICA-A

The absence of female genetic material homologous with the selected male alleles may greatly decrease the frequency of integration of the selected genes into a hybrid chromosome. I n contrast the stable integration was reported of markers carried by the wild type of both species, namely met+ and str+. I n some cases the reported losses of male material were possibly mutations to loss of function; for example, the frequency of segregation of Lac- clones was not stated and, if infrequent, might represent lac+ to lac mutations rather than loss of male genetic material from the cells. Leavitt et al. (1970) have recently reported the characterization of E. coli DNA in S. typhosa-E. coli Xyl+ hybrids by sucrose density gradient centrifugation and electron microscopy. They found circular molecules with a molecular weight of 66 X lo6 daltons and a length of 34 mp. This finding may answer the question of physical state of the male DNA in the cases of unstable diploid hybrids, and may provide an approach to the understanding of other questions, such as replication, stability, regulation, and interaction with the host chromosome of pieces of male DNA which are not integrated into the chromosome. Thus male DNA may exist in each hybrid in one of two different forms: associated with the chromosome or as an exogenote. Although both types of hybrids have been reported, it is not yet possible to make detailed statements on the interactions of the male DNA with the female chromosome or on the replication, stability, or regulation of male DNA in hybrids. IV. Characterization of the Hybrids

Transduction crosses mediated by phage P22 are carried out with hybrids and wild-type 8. typhimurium LT2 as donors. Titers are ordinarily determined on LT2. Recipients are 8. typhimurium auxotrophs representative of the chromosomal region under study, usually that region selected for in conjugation. At least one additional recipient with a marker outside the region under study should be included; the choice of this recipient depends on the conditions of the conjugation matings, particularly the Hfr used and duration of mating. It is assumed that the hybrids do not carry any male genetic material in this outside region. The additional recipient, as will be seen in Section IV, B below, is used to normalize for donor phage efficiency. A. HOMOLOGY Homology is defined as the ratio between the frequency of recombination (in phage-mediated transduction) in which an E. coli-S. typhimur-

ENTEROBACTERIACEAE : HOMOLOGY FROM INTERCROSSES

63

ium (or other) recombinant is used as a donor with an S. typhimurium recipient and the frequency in which the donor and the recipient are of the same species (for example, S. typhimurium X S. typhimurium). Homology, usually expressed as a percentage, appears to relect similarities in the genetic material of related organisms. It should be noted that homology is assessed as a consequence of a recombinational event and could perhaps be better termed “recombinational homology.” Of course the relationship between “recombinational homology” and the similarity of the nucleotide sequences is not known, although small differences in DNA sequence may be reflected as great differences when recombinational events are scored since transduction analysis has high resolution. Approaches to the description of the relationship between the homology values generated by transduction analysis of hybrids and the “microhomology” of the DNA sequences of two species may be (1) DNA-RNA hybridization studies of genetically identified regions, and (2) comparison of the amino acid sequences of homologous enzymes.

B. NORMALIZATION P22-mediated transductions with wild-type S. typhimurium LT2 as donor with a series of S. typhimurium recipients permit normalization for recipient efficiencies. Donor efficiencies are calculated from the inclusion of a recipient outside the region(s) in which male genetic material may be integrated. The procedure is outlined in St. Pierre and Demerec (1968) and in Ino and Demerec (1968) ; see Table 4 in Section VI for data which have been normalized. The most common case is the analysis of a particular region of the chromosome; an example of a hypothetical case is shown in Table 1. Normalization is useful for quantitative studies of particular regions of the bacterial chromosome and is valid if the following assumptions and observations are made: 1. The outside marker does not contain male DNA. 2. The hybrid strains contain male DNA for a least the sites selected for in the conjugation cross; that is, a recombination between the female and the male has resulted in a hybrid chromosome with a linear arrangement of nucleotides functionally indistinguishable from the wild type of the female in the selected region. Cases in which the latter assumption does not appear to be valid have been described (St. Pierre and Demerec, 1968; Baron et al., 1968) and are discussed in Section I11 above. 3. The vector phage used in transduction is able to transfer male DNA from hybrids. For P22 this assumption appears to be valid. Eisenstark (1965b) made thymineless mutations in hybrids in which the

64

RICHARD B. MIDDLETON A N D TOBIAS MOJICA-A

thymine gene was of E . coli origin and used these mutations as recipients in transduction in which the donor was the prototrophic hybrid thy+ gene from E . coli parent. The high frequency of Thy+ transductants shows that P22 can efficiently transmit E . coli DNA. In addition the frequent appearance of abortive transductants in crosses a t hybrid regions indicates that P22 does carry male genetic material (Demerec 1964; Glatzer e t al., 1966; Mojica-a, 1971). 4. The number of transductants observed is greater than zero as would be expected unless (a) the homology is so low as to be not measurable TABLE 1 Determination of Percentage Homology (Hypothetical Example) Numbers of wild-type recombinants with various recipients Donors (phage preparations)

his-1

trp-1

trp-9

S . typhimurium LT2 Hybrid X*

1000 900

2000 400

1500 50

* Trp+ recombinant isolated from a cross between E. coli Hfr X S. typhimurium trp-d under conditions were the his+ region is most likely not transmitted from the E. coli parent. Normalization for recipient efficiencies: 1000 2000

-

1000 1500

-

for trp-1;

for trp-2

Normalization for donor phage efficiency:

1000 900

Normalized colony counts: 1000 1000 400 X -X - = 222

2000

1000 50X---X-= 1500

900

1000 900

37

for trp-1 for trp-8

Normalized percentage homology values : 222 X

100 = 22.2%

for trp-1

-X 100 = 3.7% 1000

for trp-d

1000

37

ENTEROBACTERIACEAE HOMOLOGY FROM INTERCROSSES

65

in transduction crosses or (b) the host specificity locus [hsp', 89 minutes in the E . coli map (Taylor, 1970)] of the male displaces the female locus, presumed to occupy a similar position, with the result that transducing fragments would be susceptible to degradation in recipient strains of the female species. The only apparent case of complete lack of transductants due to very low homology is for the lactose region (Zinder, 1960). Lac+ hybrids yield no recombinants with S. typhimurium recipients which may be a result of the characteristic lack of the lactose operon in S. typhimurium. Evidence for the effects of E . coli hsp' locus has been found in hybrids which have male genetic material incorporated near the top of the map, 0-5 min (H. Itikawa and M. Demerec, unpublished results). The low homologies found in this region and an apparently generalized lowering of donor efficiency in transduction may be consequences of E . coli hsp+ modification of the hybrid DNA. V. Genetic Homologies between Species

A. SUMMARY OF DATA Homology values calculated from the data of various workers and shown as percentages, normalized when possible, are summarized in Table 2: gene symbols and map positions (minutes) are those of S. typhimurium (Sanderson, 1970), which is the female parent of hybrids with E. coli, Salmonella nrontevideo, and Salmonella abony males. Figure 1 summarizes the homology values between S. typhimurium and E. coli.

B. RELIABILITY OF HOMOLOGY VALUES The high resolving power and sensitivity of transduction analysis yield quantitative estimates of the decrease in integration of male genetic markers due to lack of homology of the region of genetic material under consideration. To facilitate interpretation and comparison of different experiments the normalized numbers of transductants are presented as percent homologies. 1. Direct Selection for Male Genes

The most reliable values appear to be those generated from transduction analysis of male genes for which the hybrids have been directly selected. When the female markers used are stable, preferably short deletions, the hybrids are certain to contain the desired male gene. Heterogenotes are unlikely if the hybrids are phenotypically stable, and

66

RICHARD B. MIDDLETON AND TOBIAS MOJICA-A

TABLE 2 Summary of Percentage Homology Values of S. typhimurium with E . coli, S. montevideo, and S . abony Gene symbol* ara argA

H

C F AHCF B D

E

aroA B

C

aac cysA B C CD

H Z

3 gal

dt

BlyA guaA his ilV

A C D

E

leu lysA metA C

E F nkcA Pan

Ph

pheA PPC

Map position* (min) 4 128 128 128 128 128 91 8 102 45 108 108 23 76 52 90 90 90 90 90 33 33 80 79 65 122 122 122 122 122 4

91 129 103 123 128 33 9 120 88 128

Percentage homologyt with

E . coli 0h,5k,

7s'

<1b,2d-e,lgh 0.6h < lb,4d 7h 8h 7h

S. montevideo

S. abony

1l b 26',318 17b,328 27" 31' 17O

3h

17h, 0 . 1 ~(Deletion in pyrF), 0.6d 0.3d, 0.5d 0.2d 0.5d 65" 16" 21h 15h, 20b, 1000, 16h 14h 1k,40J', <0.58,
ll-16b 70b 10-150,

ll-16b 2oj 220 9' 16' 110

23',24' 10-15b 23' 24'

568

lOOb

ENTEROBACTERIACEAE

67

: HOMOLOGY FROM INTERCROSSES

TABLE 2 (Continued) Gene symbol* pwB ZlC G PYrA

C

D E F serA B thT

B

thyA tTP B

C

E ABCDE tyrA

Map position* (min) 43 79 81 2 42 41 116 51 95 1 1 1 91 52 52 52 52 52

a4

Percentage homologyt with

E. coli 10h 12h, 4d1 390,

0.7' (Deletion in F, long insertion); 12b, 66g (Deletion in F, short insertion),
S. montevideo

S . abony

ll-16b 10-15b >1000 61b 5l b 35b 81b

1000 1000

700,

1OOb

* Gene symbols and map positions are those of 8.typhimurium (Sanderson, 1970), except for locus designations of trp which follow the E. coli convention (Taylor, 1970). t a, Armstrong (1967); b, Demerec, M. (unpublished observations forwarded by Dr. P. E. Hartman); c, Demerec and New (1965); d, Demerec and Ohta (1964), and Demerec (1965b) and see Table 4; el Eisenatark (1965b); f, Glatzer et al. (1966); g, Ino and Demerec (1968); h, Middleton, R. B. (unpublished results); i, Mojica-a and Middleton (1970a), j, St. Pierre and Demerec (1968); k, Zinder (1960). are excluded if cotransduction fails to reveal a cryptic female allele as discussed in Section II1,A above. If genetic analysis of a group of hybrids is limited to a single transducing fragment, unselected male genetic material which may have been incorporated in other regions of the chromosome can be eliminated by transferring the interesting transducing fragment of a group of hybrids into a recipient strain of the female species. Examples of well-studied homologies of directly selected male genes in S. tgphimurium females are the E. coli cysC (Demerec, 1964, 1965b; Demerec and Ohta, 1964) and trp regions (In0 and Demerec, 1968; Mojica-a, 1971; Mojica-a and Middleton, 1970a) and the S. montevideo

68

RICHARD B . MIDDLETON AND TOBIAS MOJICA-A u)

mdov

0 _.

Fro. 1. Percentage homology between S. typhimurium and E. coli, from column 3 of Table 2. Inside the circle are values based on selection for the locus, on an integrated segment which includes a selected locus, or on the identification of a phenotype derived from the male parents. Outside the circle are values inferred from low donor ability of hybrids. Map positions, 1-138 minutes, are indicated at 5-minute intervals.

ilv region (Atkins and Armstrong, 1969). The recipient strains used

for transduction analysis should have stable single-site or short multisite mutations in order to assume that the homology values reflect the behavior of the gene under study rather than that of neighboring genetic regions.

W. Regions of Low Hom#ology Regions of the genetic map which have low genetic homology are more easily distinguished than regions of higher homology. I n addition to greater precision in the determination of homology values for selected markers, the ends of the incorporated male segment of genetic material can be identified. This latter characteristic permits the study of reciprocal effects of male and female segments on markers near the junction of the two types of genetic material. Demerec and Ohta (1964) reported

ENTEROBACTERIACEAE : HOMOLOGY FROM INTERCROSSES

69

that the S. typhimurium genes of composite transducing fragments were incorporated into transductants with full efficiency (100%), whereas E . coli genes were incorporated a t 40%. Since homology in the cysC transducing fragment is less than 1% when all genes are E . coli, they concluded that the synaptic forces acting between homologous portions of a composite transducing fragment and recipient chromosome can increase the donor efficiency of E.coli genes about 40-fold. Glatzer et al. (1966) reported the opposite effect in S. montevideo-8. typhimurium hybrids in the ilv region, namely, proximity of a male region caused a decrease in the donor efficiency of the female ilvC+ gene of two hybrids. In addition, the genetic identification of a hybrid ilv gene cluster (Atkins and Armstrong, 1969) and hybrid trpA+ gene (Middleton and Mojica-a, 1970) makes possible the correlation of genetic analysis and biochemical studies of the gene products. The electrophoretic mobility of the product (reductoisomerase) of the ilvC+ gene for any hybrid studied was characteristic of either the male (S. montevideo) or female (S. typhimurium) enzyme and agreed with the origin of the gene as assigned to male or female by transduction analysis. Selection of a hybrid trpA+ gene resulted from a cross between an E . coli male deleted for the operator-distal portion of the trpA gene and a S. typhimurium female deleted for the operator-proximal portion of the trpA gene. Stable Trp+ recombinants with a secondary trpE mutation produce high levels of the trpA+ gene product (G. R. Drapeau, personal communication) , the A protein of tryptophan synthetase, which can be analyzed for characteristics of the parental enzymes which differ in physical properties (Creighton et al., 1966). This approach may lead to a means of associating quantitative biological homology with actual differences in the nucleotide sequence of the two genes, since the A protein of tryptophan synthetase of E . coli has been sequenced (Yanofsky et al., 1967). There are three conspicuous regions of low homology between E . coli and S. typhimurium, and hybrids in these regions indicate the lengths of male genetic material which may be integrated into the chromosome: The cysC region. Demerec and Ohta (1964) selected Cys+ hybrids from crosses between E . coli and two S. typhimurium females: with a short deletion and with two single-site mutations within the cysC gene cluster. Rather long segments of male DNA were incorporated (Table 3 ) . All 12 hybrids carried E . coli genes for the 5 genes tested within the cysC transducing fragment, and in 10 hybrids the E. coli segment extended clockwise, or both clockwise and counterclockwise. The largest E . coli segment included purG and argE, 80-102 min, and

70

RICHARD B . MIDDLETON A N D TOBIAS MOJICA-A

TABLE 3 Lengths of E . coli Genetic Material Incorporated into Hybrids (Calculated from Demerec, 196513)*

No. of hybrids

Inclusive E. coli genes

Estimated percentage of Map position total genome in (min) E . coli segment

~

2 2 2 1 1 1 1 1 1

CYSJ+-C~SC+ C ~ J+-lysA S cysJ+-serA+ cysJ+-argE+ cysJ+-metC+ purG+-serA + pheA +-met C+ tyrA+-metC+ purG+-argE+ +

90-90 90-91 90-95 90-102 90-103 80-95 84-103 84-103 80-102

1 2 4 8.7 9.4 11 14 14 16

* Derived fromTable 4; based on S. typhimuriummap, total 138 minutes (Sanderson, 1970). is estimated to comprise about 16% of the total genome (Table 3) based on the total map length, 138 min. Homology is low, 0.2-7% throughout the region, purG to metC, 80-103 min. The serB-pan region. A second region of generally low homology is serB-pan, 1-9 min, with homologies, 0-1276 except for pyrA, 39% (Table 2 ) . A study of two groups of hybrids generated by E . coli Hfr AB261 and two S. typhinzurium LT7 strains again indicates a tendency for the incorporation of long E. coli segments in this region (R. B. Middleton, unpublished results). Such incorporations occur whether the selection is made within the region (thrB, 1 min) as in one group of 12 hybrids, or outside the region (ilv and trpB, 122 and 52 min) as in the other group of 31 hybrids. The largest male segments in the serB-pan region were metrl-pan, 129-9 min, 14% of the total genome, and serBpan, 1-9 min, 7% of the genome. As noted by Glatzer et al. (1966), one might account for long incorporations of male genetic material in regions of low homology as a consequence of fewer locations of identical homology. I n such regions there would be a considerable distance from one crossover to the next. They found maximum S. montevideo segments of 4% of the S. typhimurium chromosome in the ilv region, presumably a consequence of higher homology in the ilv region than in serB-pan or cysC regions and of the closer relationship between two Salmonella species than between S. typhimurium and E . coli.

ENTEROBACTERIACEAE

:

HOMOLOQY FROM INTERCROSSES

71

The trp region. A third region of low homology between E . coli and S. typhimurium is the tryptophan region; the trp transducing fragment contains the five structural genes, A-E, of the trp operon, as well as cysB and pyrF. The orientation of this group of seven genes is reversed in the two species, although the order of the genes is the same (Sanderson, 1970; Taylor 1970) : pyrF-cysB-trpOEDCBA. From the left, these genes are oriented clockwise in S. typhimurium and counterclockwise in E . coli (Sanderson and Hall, 1970). A female with a deletion for most of the trp operon yielded three clases of hybrids as determined by increasing size of the inserted male segment: trp operon only, trp operon plus most of the region between trpE and cysB, and trp operon plus cysB. A female with a short deletion of pyrF also yielded three hybrid classes: E . coli for only a part of pyrF locus, for pyrF plus cysB, and for pyrF plus cysB plus trp operon. The last class, where the entire transducing fragment appears to be E . coli in origin, generated the percentage homology values reported: pyrF, 0.7; cysB, 0.1; trp, <0.03 (In0 and Demerec, 1968). The hybrids with E . coli inserted in part of the pyrF gene were peculiar in two respects: they exhibited a partial requirement for uracil (slow growth on minimal medium) and were high-efficiency donors in crosses with pyrF recipients (apparent homology 66%). In the latter respect these hybrids resemble those generated from a female with a stable trpB point mutation; the hybrids were similarly efficient donors with a trpB recipient, 80% homology (R. B. Middleton, unpublished results). Three groups of hybrids, generated from females with deletions in trpA, C , and E , were inefficient donors in crosses with the same deletions as recipients: homology <0.05% for all groups. Higher efficiency for neighbouring genes as well as an ascending gradient of efficiency with distance from the selected gene indicate that the E . coli insertions are limited to the selected genes (Mojica-a and Middleton, 1970a). These results agree with Ino and Demerec (1968) in two respects: (1) when deletion strains are used to generate and test hybrids the homology is generally low throughout the trp transducing fragment; (2) in contrast to Demerec and Ohta (1964), and in agreement with Glatzer et al. (1966), an insertion of male genetic material can depress the donor efficiency of nearby female genes. The low homology of the trp region, and perhaps the inversion of this region between the two parental species, result in hindrance of genetic exchange which has observable consequences. A symptom of the rarity of recombination exchange in this region may be the identification of classes of hybrids (In0 and Demerec, 1968).Hybrids with very short male insertions may be of intermediate efficiency, perhaps one third

72

RICHARD B . MIDDLETON AND TOBIAS MOJICA-A

less efficient than a wholly female donor gene but nevertheless a t least 50 times more efficient than an E. coli gene (In0 and Demerec, 1968). 3. Regions of Intermediate Homology Intermediate homology values, 31-69% for example, become difficult to ascertain as the values increase since high donor efficiency of a wildtype male gene resembles the genetic behavior of a wild-type female gene. Additional difficulties arise from attempts to infer the origin of genes which are not selected in the interspecific cross or which &re not phenotypically distinguishable between the parents. The appearance in a hybrid of significantly lowered donor efficiency for an unselected wild type gene is, of course, evidence that the male gene has been integrated into the hybrid chromosome or has displaced the corresponding wild-type female gene. Hybrids diploid for the gene should permit transductions at full efficiency. Inferred homology values usually agree fairly well with the values produced from studies on selected genes (Fig. 1 ) . Wherever the inserted male genetic material is less than one transducing fragment long, however, as may be the case for unselected genes in particular, the homology values are not reliable. Such inferred values are therefore presented separately (Fig. 1, outside the circle) from the values for directly selected loci (inside the circle). The identification of regions of varying homology is of interest in considering the extent of divergence among enteric species of nucleotide sequences in the various parts of the genetic map. VI. Use of Hybrids for Mapping

A. ORDERING OF NEARBY GENESWHICHARE NOTCOTRANSDUCED The mapping of loci in enteric bacteria is usually accomplished by two methods : by interrupted matings and by three-point reciprocal crosses of markers which share a transducing fragment. Interrupted matings have a usuable lower limit of a few minutes when results from different crosses must be compared. Except in crosses with a special arrangement of mutant genes in the female strain (Sanderson, 1965), the order of genes a few minutes apart cannot be reliably determined. On the other hand, the ordering of genes, or sites, on a transducing fragment is limited by the size of each transducing fragment, about 1% of the bacterial genome or 1 min of the total map for 8. typhimurium with phage P22. As a consequence, genes which are slightly too far apart to share a transducing fragment are difficult t o order on the map. Biological hybrids provide an approach to mapping a t this intermediate level of

ENTEROBACTERIACEAE : HOMOLOGY FROM INTERCROSSES

73

resolution. I n regions of fairly low homology, the ends of an integrated length of male genetic material can be identified, since wild-type genes carried in the donor region will be reflected in low transduction frequencies in crosses with recipient strains. The selection of a group of hybrids which carry a prototrophic (male) allele can result in the incorporation of various lengths of male material in the hybrids. The ordering of genes can then be made by a process analogous to that followed in mapping by means of overlapping deletions. An example (Table 4) is that of Demerec (1965b) who selected a number of hybrids with cys+ E. coli alleles in the cysC gene cluster of an S. typhiinuiium genome. Table 4 shows the transduction frequencies and normalized percentage homology values ; the genes are arranged in an order consistent with the incorporation of E. coli pieces of various lengths which include cysC+. As can be seen in Table 4, the homology between E. coli and S. typhimurium genetic material in the cysC region is low, the ends of the integrated E. coli pieces of each hybrid can be easily identified, and an unambiguous map order can be constructed of all genes which lie adjacent to the ends of the E. coli pieces. In Table 4, lysA-argB and pheA-tyrA are pairs of nearby genes for which the order could not be inferred from the group of hybrids studied although the lysA-argB order is now known from other evidence (Sanderson, 1970). I n addition, since all E . coli segments include all markers tested on the cysC transducing fragment, the orientation of the cysC fragment relative to the genetic map is not known.

B. ORIENTATIONOF

THE

ILVE-META REGION, 122-129 MINUTES

Glatzer et al. (1966) and Armstrong (1967) employed transduction analysis of S. montevideo F+-S. typhimurium hybrids and cotransduction tests to infer the order of a number of genes on the chromosome map and the orientation of genes within three transducing fragments, respectively. The segments of S. montevideo male chromosome integrated into hybrids selected for markers in the ilvE-metA region varied from 1-4% of the total map, and the patterns of incorporation led to the following proposed map order, reading clockwise from the left: Map position (min)

ilvE ilvD ilvA ilvC metE metB metF p p c argA argH argC argF purD metA 122 123 128 129

This map order agrees with that produced independently by time-ofentry conjugation crosses (Sanderson, 1970).

TABLE 4

Transductants* with 8.typhimurium Mutants as Recipients and S.'typhimurium X E. coli Hybrids as Donors Map poaition (min):

108

Recipients: cy&S82 Donors S. fuphimurium LT2 100 Hybrids A48 B33 135 B-20 A-57 45.3 B-24 113 A45 144 B-11 128 A40 A-I4 A-19 B-51 107 0-74 146 Number of hybrids with E. coli loci (normalized homology values 134%. in heavy type): 0 Percentage homology laveraze) : .

-

* Normalied (from data of

105 dCS2

104 argE116 100

95

91

serAlS

lyd8

arpB69

100

100 19.9

dGC697 @lOf!l

dGCD619 ddH76

100 2 3 0.9 0.3 0 1 0 0.6 0.1

100 1.4 0.4 0.1 0.1 0 0 0.4 0 0.2 0.8

0 0.2

1 4

40 0 0 0 0

0.3 0. I 0.9 0.3 3 0 0

0.1 0.9 0.4 1 2.5 0 5.1 1 1

0 0 0 0 0.3 0

100 0 2.2 0.5 0 0 0 0 0.6 0 0 0 0.6

4

5

8

10

10

12

9

4.9

3.4

0.32

0.94

100 18

-

100 33.1

375

-

429

-

131

-

335

101

3.5

20 85.1

80.0 105

115 479 44.0

1490 48.1

6.6

59

4.

21.4 183

74.8

89

90 cysc cluster

117 57.0 0

2.0

100 1.2

&I68

M5S8

pheAS

:WAS

100 0.2

100 72.9 65.4

100 100

100 -

100

138.3 100 136 100 136

311 119 107 108 127

100

0 0.3 -

-

0.8 0.2

0 0.4 0

100 1.3 1 0.6 0 0.2 0.2 0 2.1 0.2 0 0.2 0.2

10

7

8

12

0.34

0.47

0.18

0.5

-

0.8

-

0

-

-

80

-

0.6

-

0.1

-

0 0.2

88 119 87.5 119 0 100 84.8 0.96 9.18 14.3

0.2

-

237 1.3 16.2

purGS03

2.9

-

-

5.5

115

49.8 129

5

3

2

5.0

5.9

4.2

Demerec, 1965b) for recipient (LT2 values set a t 100 %) and for donor efficiencies (oluAl, @AS, or &AS values set at 100 %).

&/A1

-

-

100

-

100

1W

-

I00 100 100

100

0

-

ENTEROBACTERIACEAE

: HOMOLOGY FROM INTERCROSSES

75

C. LOCATING CERTAINWILD-TYPEGENES ( E . coli GENESAND MAP POSITIONS) Recombinants selected from E . coli Hfr X S. typhosa matings were used to locate the genes for 30 S ribosomal proteins on the genetic map (O’Neil et al., 1969). A hybrid, diploid for about one-third of the genome, clockwise from strA to proB (64-9 min), yielded 30 S ribosomal protein bands in gel electrophoresis which contain both E. coli (exogenote) and S. typhosa (endogenote) components. A second hybrid carried somewhat less E . coli genetic material, either integrated or as an exogenote, clockwise from malP to lac (66-10 min) ; apparently the E . coli 30 S ribosomal protein cistrons were not included since the electrophoretic pattern is characteristic of S. typhosa. The third hybrid had an integrated E . coli segment from strA to zyl ( W 7 1 min) and produced only the E . coli 30 S ribosomal protein pattern. It therefore appears that the cistrons for 30 S ribosomal proteins of both E. coli and S. typhosa are clustered in a small region, probably 64-66 min (of the E . coli map, Taylor 1970). By similar comparison of three 50 S proteins which differ in E . coli and S. typhosn, the same three hybrids indicate that the cistrons for these 50 S proteins are also located near strA, 64 min (Sypherd et al., 1969). A difference between the 23 S RNAs of these two species was found. Differentially labeled 23 S RNA was isolated from E. coli and 8.typhosa, mixed and digested with pancreatic ribonuclease. The resulting oligonucleotides were resolved on a DEAE-urea column and followed by high-voltage electrophoresis of the various size groups. A hexanucleotide unique to each species was identified: A,U from E . coli 23 S RNA, A,GC from S. typhosa. The first hybrid contains the hexanucleotides characteristic of both parental species, whereas the third hybrid has only the hexanucleotide from the S. typhosa cistron. It was concluded that the cistrons for 23 S RNA lie in the broad region from xyl to proB, 71-9 min, and that these rRNA cistrons are not closely linked to the cistrons for ribosomal proteins, near 64 min. It therefore appears unlikely that rRNA acts as message for ribosomal proteins (Sypherd et al., 1969). These examples illustrate the special use of hybrids (1) for mapping the order of genes near one another on the chromosome map but not sharing a transducing fragment, (2) for orienting a transducing fragment on the map, and (3) for placing on the map genes, common to enteric species, which are phenotypically distinguishable.

76

RICHARD B. MIDDLETON AND TOBIAS MOJICA-A

VII. Summary

The genetic behavior of interspecific hybrids in transduction analysis provides a quantitative measure of the microhomology of two species for the region considered. Whereas the similarity of the genetic maps of the enteric bacteria facilitates the formation of viable hybrids, divergence in the nucleotide sequences in the chromosomes of two species hinders efficient recombination in transduction crosses. The percentage homology values in general confirm the traditional assignments of biological relatedness: for any gene or region, closely related species have a higher homology value than do less related species. Hybrids permit an approach to the study of a number of interesting problems which include (1) the response of genes from the male species to regulatory genes of the female, (2) the elucidation of the structure of ribosomes and enzymes whose constituent parts are distinguishable between two species, (3) the properties of hybrid genes and gene products, and (4) the relative divergence of various regions of the chromosome map among enteric species.

ACKNOWLEDGMENTS The authors’ work was supported in part by the U.S.Atomic Energy Commission, Research Corporation, Medical (MA21601 and National (A3452) Research Councils of Canada, and Food and Drug Directorate of Canada. The authors appreciate the critical reading of the manuscript by a number of colleagues, and valuable comments by P. E. Hartman, K. E. Sanderson, F. B. Armstrong, Marie L. Godfrey, C. G. Atkins, and I. Takahashi.

REFERENCES Arber, W. 1965. Host-controlled modification of bacteriophage. Annu. Rev. Microbiol. 19, 365-378. Arber, W., and Linn, S. 1969. DNA modification and restriction. Annu. Rev. Biochem. 38, 467400. Armstrong, F. B. 1967. Orientation ,and order of loci of the met-arg region in the Salmonella typhimurium linkage map. Genetics 56, 463-466. Atkins, C. G., and Armstrong, F. B. 1969. Electrophoretic study of Salmonella typhimurium-Salmonella montevddeo hybrids. Genetics 63, 775-779. Baron, L. S., Carey, W. K., and Spilman, W. M. 1959a. Genetic recombination between Escherichia coli and Salmonella typhimurium. Proc. Nat. Acad. Sci. U.S. 45, 976-984. Baron, L. S., Carey, W. F., and Spilman, W. M. 1959b. Hybridisation of Salmonella species by mating with Escherichia coli. Science 130, 566-567.

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Baron, L. S.,Gemski, P., Jr., Johnson, E. M., and Wohlhieter, J. A. 1968. Intergeneric bacterial matings. Bacteriol. Rev. 32, 362369. Bertani, G., and Weigle, J. J. 1953. Host-controlled variation in bacterial viruses. J . Bacteriol. 65, 113-121. Boyer, H. W., and Roulland-Dussoix, D. 1969. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J . Mol. Bwl. 41, 459472. Brenner, D. J., and Falkow, S. 1971. Molecular relationships among members of the Enterobacteriaceae. Advan. Genet. 16, 81-118. Colson, A. M., Colson, C., and Van Pel, A. 1969. Host-controlled restriction mutants of Salmonella typhimurium. J . Gen. Microbiol. 58, 57-64. Creighton, T. E., Helinski, D. R., Somerville, R. L., and Yanofsky, C. 1966. Comparison of the tryptophan synthetase a subunits of several species of Enterobacteriaceae. J . Bacteriol. 91, 1819-1826. Curtiss, R. 1969. Bacterial conjugation. Annu. Rev. Microbiol. 23, 69-136. Curtiss, R., Charamella, L. J., Stallions, D. R., and Mays, J. A. 1968. Parental functions during conjugation in Escherichia coli K-12. Bacteriol. Rev. 32, 320-348. Demerec, M. 1964. Organization of genetic material in Salmonella. In “Struktur und Funktion des Genetischen Materials: Erwin-Bauer-Gedachtnisvorlesungen 111,” pp. 51-56. Akademie-Verlag, Berlin. Demerec, M. 1965a. Gene differentiation. Nut. Cancer Inst. Monogr. 18, 15-20. Demerec, M. 196513. Homology and divergence in genetic material in Salmonella typhimurium and Escherichia coli. In “Evolving Genes and Proteins” (V. Bryson and H. J. Vogel, eds.), pp. 505-510. Academic Press, New York. Demerec, M., and New, K. 1965. Genetic divergence in Salmonella typhimurium, S. montevideo, and Escherichia coli. Biochem. Biophys. Res. Commun. 18, 652455. Demerec, M., and Ohta, N. 1964. Genetic analysis of Salmonella typhimurium x Escherichia coli hybrids. Proc. Nat. Acad. Sci. US. 52, 317-323. Demerec, M., Adelberg, E. A., Clark, A. J., and Hartman, P. E. 1966. A proposal for a uniform nomenclature in bacterial genetics. Genetics 54, 61-76 [reprinted in J. Gen. Microbiol. 50, 1-14 (1968)l. Eisenstark, A. 1965a. Mutagen-induced hybridization of Salmonella typhimurium LT2 x Escherichia coli K-12 Hfr. Proc. Nat. Acad. Sci. U.S. 54, 117-120. Eisenstark, A. 1965b. Transduction of Escherichia coli genetic material by phage P22 in Salmonella typhimurium x E . coli hybrids. Proc. Nut. Acad. Sci. U.S. 54, 1557-1560. Falkow, S.,Marmur, J., Carey, W. F., Spilman, W. M., and Baron, L. S. 1961. Episomic transfer between Salmonella typhosa and Serratia marcescens. Genetics 46, 703-706. GarrickSilversmith, L., and Hartman, P. E. 1970. Histidine-requiring mutants of Escherichia coli K-12. Genetics 66, 231-244. Glatzer, L., LaBrie, D. A., and Armstrong, F. B. 1966. Transduction of Salmonella typhimurium-Xalmonella montevideo hybrids. Genetics 54, 423-432. Grasso, R. J., and Paigen, K. 1968a. The effect of amino acids on host-controlled restriction of lambda phage. Virology 36, 1-8. Grasso, R. J., and Paigen, K. 1968b. Loss of host-controlled restriction of h bacteriophage in Escherichia coli following methionine deprivation. J. Virol. 2, 1368-1373.

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Gwinn, D. D., and Lawton, W. D. 1968. Alteration of host specificity in Bacillus subtilis. Bacteriol. Rev. 32, 297-301. Ino, I., and Demerec, M. 1968. Enteric hybrids. 11. S. typhimurium-E. coli hybrids for the trp-cysB-pyrF region. Genetics 59, 167-176. Johnson, E. M., Falkow, S., and Baron, L. S. 1964. Recipient ability of Salmonella typhosa in genetic crosses with Escherichia coli. J. Bacteriol. 87, 54-60. LaBrie, D. A., and Armstrong, F. B. 1968. Transduction using ilv mutants of Salmonella typhimurium-Salmonella montevideo hybrids. J. Bacteriol. 95, 1193-1194. Leavitt, R. W.,Wohlhieter, J. A., Johnson, E. M., Ladda, R. L., and Baron, L. S. 1970. Isolation of circular DNA from Salmonella typhosa hybrids obtained from matings with Escherichia coli. Bacteriol. Proc. p. 55. Lew, K. K., and Roth, J. R. 1970. Genetic approaches to determination of enzyme quaternary structure. Biochemistry 10, 204-207. Luria, S. E. 1953. Host-induced modification of viruses. Cold Spring Harbor Symp. Quant. Biol. 18, 237-244. Luria, S. E., and Burrous, J. W. 1957. Hybridization between Escherichia coli and Shigella. J. Bacteriol. 74, 461476. Mandel, M. 1969. New approaches to bacterial taxonomy : perspectives and prospects. Annu. Rev. Microbiol. 23, 239-274. Middleton, R. B., and Demerec, M. 1966. Genetic homology of Salmonella typhimurium and Escherichia coli. Genetics 54,347-348. Middleton, R. B., and Mojica-a, T. 1970. Homology of Salmonella typhimurium and Escherichia coli: selection of hybrids and restriction of coliphage P1. Proc. 10th Znt. Congr. Microbiol. p. 52. Miyake, T. 1959. Fertility factor in Salmonella typhimurium. Nature (London) 184, 657-658. Miyake, T. 1960. Mutator factor in Salmonella typhimurium. Genetics 45, 11-14. Miyake, T. 1962. Exchange of genetic material between Salmonella typhimurium and Escherichia coli K-12. Genetics 47, 1043-1052. Mojica-a, T. 1971. Genetic behavior of male genetic material in enteric hybrids. M.Sc. thesis, McGill University. Mojica-a, T., and Middleton, R. B. 1970a. Genetic homology of Salmonella typhb murium and Escherichia coli: production of hybrids and analysis of the tryptophan operon. Bacteriol. Proc. p. 22. Mojica-a, T., and Middleton, R. B. 1970b. Host restriction and modification of coliphage P1 by Salmonella typhimurium. Bacteriol. Proc. p. 35. Okada, M., and Watanabe, T. 1968. Isolation of Salmonella typhimurium mutants with increased recipient ability by use of R factor. Nature (London) 217, 854-866. Okada, M., Watanabe, T., and Miyake, T. 1968. On the nature of the recipient ability of Salmonella typhimurium for foreign deoxyribonucleic acids. J . Gen. Microbiol. 50, 241-252. O’Neil, D. M., Baron, L. S., and Sypherd, P. S. 1969. Chromosomal location of ribosomal protein cistrons determined by intergeneric bacterial matings. J. Bacteriol. 99, 242247. St. Pierre, M. L., and Demerec, M. 1968. Hybrids of enteric bacteria. I. S. typhimurium-S. montevideo hybrids for the histidine loci. Genetics 59, 1-9. Sanderson, K.E. 1965. Information transfer in Salmonella typhimurium. Proc. Nat. Acad. Sci. U.S. 53, 1335-1340.

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Sanderson, K. E. 1970. Current linkage map of Salmonella typhimurium. Bacteriol. Rev. 34, 176-193. Sanderson, K. E. 1971. Genetic homology in the Enterobacteriaceae. Advan. Genet. 16, 35-51.

Sanderson, K. E., and Hall, C. A. 1970. F-prime factors of Salmonella typhimurium and an inversion between S. typhimurium and Escherichia coli. Genetics 64, 215-228.

Sypherd, P. S., O’Neil, D. M., and Taylor, M. M. 1969. The chemical and genetic structure of bacterial ribosomes. Cold Spring Harbor Symp. Quant. Biol. 34, 77-84.

Taylor, A. L. 1970. Current linkage map of Escherichia coli. Bacteriol. Rev. 34, 155-175.

Watanabe, T., Arai, T., and Hattori, T. 1970. Effects of cell wall polysaccharide on the mating ability of Salmonella typhimurium. Nature (London) 225, 70-71. Yanofsky, C., Drapeau, G. R., Guest, J. R., and Carlton, B. C. 1967. The complete amino acid sequence of the tryptophan synthetase A protein (a subunit) and its colinear relationship with the genetic map of the A gene. Proc. Nat. Acad. Sci. U.S. 57, 296-298. Zinder, N. D. 1960. Hybrids of Escherichia and Sulmonella. Science 131, 813-815.

.

C MOLECULAR RELATIONSHIPS A M O N G MEMBERS OF THE ENTEROBACTERIACEAE

.

Don J Brenner and Stanley Falkow Division of Biochemistry. Walter Reed Army Institute of Research. Washington. D.C., and the Department of Microbiology. Schools of Medicine ond Dentistry. Georgetown University. Washington. D.C.

I. Introduction . . . . . . . . . . . . . . . . . . 81 I1. Techniques Used in Nucleic Acid Reassociation . . . . . . . . 82 A . Density Gradient Methods . . . . . . . . . . . . . 83 B . Nitrocellulose Filter and DNA-Agar Methods . . . . . . . 83 C . Hydroxyapatite Method . . . . . . . . . . . . . . 85 D . Measurement of DNA Reassociation from Renaturation Rate . . . 86 I11. Factors Affecting Nucleic Acid Reassociation . . . . . . . . . 86 A . Purity of Nucleic Acid Preparations . . . . . . . . . . 86 B . Base Composition . . . . . . . . . . . . . . . . 87 C. DNA Fragment Size . . . . . . . . . . . . . . . 87 D . Ionic Strength . . . . . . . . . . . . . . . . . 87 E. Incubation Temperature . . . . . . . . . . . . . . 87 F. Nucleic Acid Concentration and Time of Incubation . . . . . . 88 I V. Interpretation of Reassociation Data . . . . . . . . . . . 90 V . Taxonomy and Nomenclature . . . . . . . . . . . . . 95 V I . Nucleic Acid Relationships among Enterobacteria . . . . . . . 96 A . Divergence among Different Strains of E. coli . . . . . . . . 97 B . Relatedness between E. coli K12, Strains of the Alkalescens-Dispar Group and Shigella Species . . . . . . . 99 C . Relative Relatedness between E coli and Other Enterobacteria . . 101 V I I . Divergencein Specific Portions of the Genome . . . . . . . . 105 A . Detection of E. coli-Specific DNA in the Genomes of Heterologous Organisms . . . . . . . . . . . . . 105 B . Divergence of the Lactose Operon among Enterobacteria . . . . 108 C . Conservation of Ribosomal RNA Genes . . . . . . . . . 110 D . Conservation of Transfer RNA Genes . . . . . . . . . 111 VIII . Relationships between Extrachromosomal Elements . . . . . . 112 References . . . . . . . . . . . . . . . . . . 116

.

I. Introduction

The family Enterobacteriaceae is a large and diverse group of microorganisms which are often found as commensal members of the intestinal 81

82

DON J. BRENNER AND STANLEY FALKOW

tract and are sometimes pathogenic for man, animals, and even plants. The medical and agricultural importance of the enterobacteria has stimulated intense interest in their serology, biochemistry, and genetics. These organisms exhibit a wide range of differences in distribution, host specificity, virulence, phage susceptibility, antigens, and biochemical capacity. This variability is not surprising in view of the fact that their guanine cytosine content in deoxyribonucleic acid (DNA) varies from 37% in Proteus vulgaris to about 58% in strains of Serratia marcescens and Aerobacter aerogenes (Marmur and Doty, 1962). I n the past decade advances in nucleic acid technology and in microbial genetics have allowed a preliminary assessment of genetic relatedness among many of these organisms. Comparative recombination studies among many enterobacteria are plagued by several factors, including restriction and modification of DNA, surface incompatibility, different fertility groups, different specificities of recombinational enzymes, and dissimilarity in DNA base composition. These genetic difficulties may be circumvented by directly comparing the ability of DNA from one organism to specifically reassociate or reanneal with DNA from another organism. Nucleic acid reassociation studies have also been used for the purpose of establishing a phyletic or natural classification. This type of study attempts to establish the lines of evolutionary divergence within the Enterobacteriaceae. Polynucleotide sequence relatedness between members of the enterobacteria and enterophages has also been investigated. These investigations have been extended to assess relationships among several plasmids found in enterobacteria and between these plasmids and Escherichia coli. Nucleic acid reassociation studies have also been extended to determine relatedness among specific portions of the genome. Thus far the lactose operon, the ribosomal ribonucleic acid (rRNA) cistrons, and the transfer RNA (tRNA) cistrons have been investigated. We shall attempt to summarize results obtained from all of these studies in order to present current views regarding genetic relationships among the Enterobacteriaceae, their viruses and their plasmids. We begin with a discussion of the types of methodology employed in these studies and the parameters that affect nucleic acid reassociation studies.

+

II. Techniques Used in Nucleic Acid Reassociation

Native bacterial DNA is composed of two chains of covalently linked purine and pyrimidine bases. The two strands are held together largely by hydrogen bonds formed between the complementary bases adenine

ENTEROBACTERIACEAE : MOLECULAR RELATIONSHIPS

83

and thymine, and guanine and cytosine. With the exception of rRNA cistrons, which are present in 6 5 copies (Kohne, 1968), the bacterial genome does not have significantly repetitious DNA. In other words, there is but one copy of each gene. DNA strands can be separated by heating them at a temperature high enough to break the hydrogen bonds and overcome base-stacking energy. If the DNA is then quickly cooled, the strands will remain separated. If, after thermal denaturation, the separated strands are incubated at a suitable temperature, they will specifically reassociate or reanneal with their complementary strands to form a double-stranded molecule. Single-stranded DNA will reassociate with a complementary DNA strand or a complementary RNA strand from the same or a different organism. This ability to dissociate DNA strands and allow them to reanneal specifically with nucleic acids from a homologous or heterologous source is the basis for all nucleic acid relationship studies. All of the methods discussed below rely on the specificity of DNA-DNA or DNA-RNA reassociation and the ability to separate reassociated nucleic acid products from any unreacted molecules.

A. DENSITY GRADIENTMETHODS These techniques involve mixing two DNAs or DNA and RNA, allowing them to reassociate, and then detecting reassociated duplexes in CsCl density gradients. In one application, DNA from one organism was uniformly labeled with a “heavy” isotope and allowed to reassociate with unlabeled DNA from another source. The presence of reassociated duplexes composed of one “light” strand and one “heavy” strand was the criterion employed to determine DNA similarity (Schildkraut et al., 1961). This technique is impractical for routine work as it is quite time consuming and also has the serious disadvantage of only detecting very closely related duplexes. CsCl has also been used to detect or confirm the presence of DNA-tRNA hybrids (Goodman and Rich, 1962; Brenner, et al., 1970). In this case one nucleic acid was labeled with 32Pand the gradient was assayed for radioactivity at the density equivalent to a DNA-RNA hybrid.

FILTER AND DNA-AGAR METHODS B. NITROCELLULOSE The DNA-agar technique was the first generally applicable method for measuring nucleic acid relatedness (Bolton and McCarthy, 1962). In this method, high-molecular-weight DNA is denatured and added

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DON J. BRENNER AND STANLEY FALKOW

to a molten agar solution which is then quickly cooled. The DNA-agar is then extensively washed to remove any DNA not bound firmly to the agar. Radiolabeled RNA or radiolabeled, sheared, denatured DNA is incubated with the DNA-agar to allow reassociation between labeled nucleic acids and agar-immobilized DNA. DNA is sheared in order to allow free diffusion through the agar matrix so that label present in the DNA-agar after incubation is the result of nucleic acid reassociation and not physical trapping of labeled DNA in the DNA-agar. Shearing is accomplished in different laboratories by sonication or mechanical shearing in a pressure cell or pressure pump. The average DNA fragment is 125,000 or 250,000 daltons per single strand depending upon the conditions employed. Incubation is carried out for a period sufficient to insure maximum reaction between the labeled nucleic acid and the unlabeled, immobilized DNA. The agar is then washed thoroughly to remove all labeled DNA that has not reacted with the DNA-agar. Reassociated, labeled DNA or RNA is removed from the agar either by increasing the temperature and dropping the salt concentration to a point where the molecules are dissociated, or by removing the reacted labeled nucleic acid in a series of thermal elutions. It is difficult to reproducibly weigh out agar samples because each preparation contains different amounts of moisture and the moisture content varies with storage. It is also difficult to prevent or control leaching of DNA from the agar a t high incubation or elution temperatures. The nitrocellulose filter method has largely replaced the DNA-agar method because samples need not be weighed out, lower background values are obtained, and more samples can be conveniently handled. In principle the nitrocellulose filter method is similar to the DNA-agar method. High-molecular-weight, denatured DNA in dilute solution is slowly impinged on a nitrocellulose filter. The procedure for binding DNA to filters differs depending upon whether DNA-DNA (Denhardt, 1966) or DNA-RNA (Gillespie and Spiegelman, 1965) reactions are of interest. Filters are dried and then washed to remove unbound or loosely bound DNA. Sheared, denatured, labeled DNA (or labeled RNA) is added to a filter containing DNA and the reaction mixture is incubated as in the agar procedure. Elution procedures are similar to those employed in the agar method. Problems encountered with the filter method include variability in binding of DNA by different batches of filters and leaching of DNA a t high temperatures. Many laboratories are now using denaturants, such as formamide, which suppress the thermal stability of DNA and allow reactions and elutions to be carried out a t significantly lower temperatures (McConaughy et al., 1969). In the above methods, one labeled nucleic acid preparation is reacted

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with a series of different unlabeled DNA preparations in agar or on filters. It is sometimes preferable to carry out competition experiments. In a competition experiment both the labeled DNA and the immobilized DNA are from the same source. This homologous reaction is carried out and the degree of binding is obtained. The homologous reaction is also carried out with a series of tubes containing increasing amounts of unlabeled competitor DNA from the same or different organisms to generate a series of competition curves. Homologous competitor DNA reacts with most of the labeled DNA before it can react with complementary sequences on the filter or in the agar, and the amount of binding is markedly reduced. The decrease in binding (if any) caused by each heterologous competitor DNA is compared to the decrease in binding caused by the homologous competitor DNA and an index of relatedness is obtained. Nygaard and Hall (1963) developed a method in which labeled RNA is incubated free in solution with a large excess of unlabeled DNA. This incubation mixture is then passed through a nitrocellulose filter which retains DNA-RNA hybrids and single-stranded DNA, but does not bind unreacted RNA.

C. HYDROXYAPATITE METHOD Hydroxyapatite (HA) is a modified calcium phosphate gel that is extremely effective in fractionating single- and double-stranded DNA (Bernardi, 1965; Miyazawa and Thomas, 1965; Britten and Kohne, 1966). Labeled, sheared DNA fragments (or labeled RNA molecules) are mixed with unlabeled, sheared DNA fragments and the mixture is thermally denatured. A mixture of labeled, sheared DNA fragments and unlabeled, sheared DNA fragments (or RNA molecules) is thermally denatured and incubated a t the desired temperature until maximal reassociation has occurred. The mixture is then passed through HA contained in a water jacketed column or by a batch procedure (Brenner et al., 1969a). The HA is equilibrated in 0.12 or 0.14 M phosphate buffer a t the temperature employed during incubation (usually 60 or 75OC). Under these conditions single-stranded DNA does not adsorb to HA, while reassociated DNA-DNA duplexes and DNA-RNA hybrids are adsorbed. Nucleic acids are eluted by raising the ionic strength t o a point where double-stranded DNA and DNA-RNA hybrids are not bound to HA (0.4 M phosphate buffer is usually employed) or by raising the temperature to a point where the duplexes or hybrids are dissociated and elute from HA. Reassociation of labeled DNA fragments with one another is not a

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DON J. BRENNER AND STANLEY FALKOW

problem in the agar or filter systems as these duplexes will be washed out along with unreacted labeled fragments. I n the HA system, however, it is impossible to discriminate between the desired labeled-unlabeled DNA duplex and the undesired labeled-labeled DNA duplex. DNA reassociation is an essentially typical collision dependent, second-order reaction (Britten and Kohne, 1966). Therefore the concentration of unlabeled fragments is made the rate-determining factor (Britten and Kohne, 1966) by employing a tremendous excess of unlabeled fragments in the incubation mixture. This allows virtually complete reassociation of unlabeled fragments while virtually precluding reassociation of labeled fragments with one another. Thermal stability profiles of reassociated nucleic acids bound to HA are generated by washing a t a series of increasing temperatures. When the temperature exceeds the denaturation temperature of a duplex it will elute as single-stranded DNA.

D. MEASUREMENT OF DNA REASSOCIATION FROM RENATURATION RATE A new method based on renaturation rate determinations of DNA from each of two organisms and from their mixture has recently been reported by De Ley and his collegues (De Ley et al., 1970). The method compares optical rates of reassociation of homologous and heterologous DNA a t various temperatures. It appears to be sensitive at a 6OoC criterion with organisms showing moderate or greater relatedness. The obvious advantages of such a procedure are that no radiolabeled preparations are necessary and data are obtained in a short period of time. 111. Factors Affecting Nucleic Acid Reassociation

In a reassociation experiment, nucleic acids are incubated under a set of conditions such tha,t single-stranded DNA fragments (or RNA molecules) may collide with unlabeled DNA (or RNA) in free solution (HA and Nygaard-Hall methods), immobilized in agar, or on filters. Double-stranded DNA duplexes (or DNA-RNA hybrids) are formed, as bases in one strand form hydrogen bonds with their complementary bases in the other strand. The extent and specificity of nucleic acid reassociation in any method is affected by a number of parameters, some of which are summarized below. OF NUCLEICACID PREPARATIONS A. PURITY The extent of reassociation obtained with a nucleic acid preparation, especially a labeled preparation, is dependent upon the purity of that

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preparation. The stability of reassociated DNA decreases when contaminating RNA or protein are present. It is particularly essential to remove contaminating nucleic acid and protein when looking a t a specific region of the genome or a specific population of RNA molecules. €3. BASE COMPOSITION

Guanine and cytosine base pairs exhibit greater thermal stability than adenine and thymine base pairs; therefore the greater the amount of guanine cytosine base pairs within a given DNA duplex, the higher is the thermal stability of that duplex (Marmur and Doty, 1962). In addition, the rate of reassociation increases slightly with increased guanine cytosine content (Wetmur and Davidson, 1968).

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C. DNA FRAGMENT SIZE Any short sequence of bases will be present in all DNA molecules. Therefore, no specificity is to be expected in reactions between very small DNA fragments. In bacteria, the minimal specific DNA fragment size is about 15 nucleotides (about 4500 daltons) (McCarthy, 1967; McConaughy and McCarthy, 1967). The fragment size employed in most investigations is either about 125,000 daltons (300-400 nucleotides/single strand) or 250,000 daltons. The larger fragments reassociate 25% faster than the smaller ones (Britten and Kohne, 1966).

D. IONIC STRENGTH The rate of DNA reassociation increases as the ionic strength of the incubation medium is increased (Britten and Kohne, 1966). Thermal stability of reassociated DNA also increases (Marmur and Doty, 1962). One can easily shift the midpoint temperature of strand separation by 2OoC or more and increase the rate of reassociation by 10-fold or more by changing ionic strength. Most investigators tend to keep ionic strength constant during a given experiment. It has been more convenient in our hands (Brenner et al., 1969b) to vary the experimental criterion by changing incubation temperature rather than shifting salt concentration.

E. INCUBATION TEMPERATURE The temperature a t which optimal reassociation occurs is some 25 to 3OoC below the temperature a t which a given DNA is denatured

88

DON J. BRENNER AND STANLEY FALKOW

(Marmur and Doty, 1961; Marmur et al., 1963). The temperature a t which DNA dissociates is determined by its guanine cytosine content (Marmur and Doty, 1962) and the reaction conditions, especially salt concentration (Britten and Kohne, 1966). Incubation temperatures significantly below the optimum allow distantly related and unrelated sequences to reassociate (McCarthy, 1967; Johnson and Ordal, 1968). I n heterologous reactions between moderately or distantly related bacteria the amount of reaction can be shifted up to 10-fold by varying the temperature of incubation (Brenner et aZ., 1967; Johnson and Ordal, 1968). Thermal stability of interspecies DNA duplexes formed between distantly or moderately related bacteria is also significantly changed depending upon the temperature of incubation (Brenner and Cowie, 1968; Johnson and Ordal, 1968). These points will be considered in detail in a later section.

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F. NUCLEICACID CONCENTRATION AND TIME OF INCUBATION The concentration of labeled DNA (or RNA) and unlabeled nucleic acid, as well as the time of incubation, must be carefully chosen in order to obtain meaningful data on reassociation and relatedness among bacteria. Generally, in agar and filter reactions, the ratio of unlabeled to labeled nucleic acid is kept a t 1OO:l or higher. This provides an excess of available sites with which any related labeled nucleic acid sequence can reassociate. Incubation times are chosen so that reassociation between labeled DNA (or RNA) and immobilized DNA is maximal. DNA concentration and incubation time are particularly critical in free-solution reactions. As previously mentioned, reassociation of labeled fragments with one another cannot be distinguished from the desired reassociation product of labeled with unlabeled DNA fragments. Therefore the labeled DNA concentration is kept small enough to insure little or no label-label reassociation during the course of reaction. Britten and Kohne (1966) introduced the term ‘‘Cot” in order to present measurements of the time course of reassociation. When temperature, salt concentration, and DNA fragment size are held constant, reassociation is determined by the concentration of DNA and the time of incubation. The term Cot is conveniently defined as the product of nucleic acid concentration (C,) and the time ( t ) of incubation. The Cot value is calculated as the product of nucleic acid concentration in optical density units a t 260 nm (a 1 pg/ml solution of single-stranded DNA has an adsorbance of 0.024 a t 260 nm) divided by 2 and multiplied by the time of incubation expressed in hours. For example, 83 pg/ml of DNA corresponds to an optical density a t 260

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nm of about 2.0, and will give a Cot of 1 when incubated for one hour [ (83) (0.024)/2 x 1 = 11. An example of a time course reassociation reaction for E. coli DNA is shown in Fig. 1. The reassociation follows second order kinetics and is indicative of a reaction in which all segments of the DNA are re-

0.0I

0. I

I.o

Cot

10

I00

FIQ.1. Time course reassociation of E. coli DNA. Labeled and unlabeled sheared, denatured E. coli DNA were incubated to various Cot values at 60°C in 0.12 M phosphate buffer and then passed through HA to determine the degree of reassociation.

annealing a t the same rate. This is true in bacterial DNA reactions because (with few exceptions) there is only one copy of each gene: Eucaryotic DNA preparations show biphasic to multiphasic reassociation curves because these organisms contain repetitious DNA, that is, “families” of related genes present as many copies per cell (Britten and Kohne, 1966). For the E. coli reaction the midpoint of reassociation

90

DON J. BRENNER AND BTANLEY FALKOW

or C0t/2 (that Cot value a t which 50% of a given DNA has reassociated) is between 5 and 6. This value will differ with ionic strength, DNA fragment size, and incubation temperature. A comparable reaction assayed spectrophotometrically will give a C,t/2 of about 9. This reflects that, early in the reaction, DNA molecules containing double-stranded regions (and therefore adsorbing to HA) express only about half of their total hypochromicity (Britten and Kohne, 1966). A further examination of the E. coli Cot curve indicates that reassociation is essentially complete a t a Cot of 100 and that little reassociation occurs before a Cot of 0.2 is reached. With this knowledge regarding the kinetics of reassociation one can design conditions so that unlabeled DNA is completely reassociated while the possibility of labeled DNA fragments reassociating with one another is virtually precluded. The standard conditions in our laboratory are as follows (Brenner et al., 1969b): 400 pg/ml of unlabeled DNA are incubated with 0.1 pg/ml of labeled DNA for 21 hr. The kinetics of reassociation are governed by the unlabeled DNA. The optical density of 400 pg a t 260 nm is 9.6. Then 9.6/2 times 21 yields a Cot of 100.8, enough to insure complete reassociation of unlabeled DNA and of labeled DNA complementary to the unlabeled DNA. The Cot for labeled DNA under these conditions is 0.025; small enough to rule out significant label-label reassociation. The ability to carry out reactions a t these concentrations depends upon having a sufficiently concentrated unlabeled preparation and a labeled preparation with a specific activity high enough to use a t about 0.1 pg/ml. When necessary one can alter these conditions and still obtain the desired Cot. For instance, if the specific activity of the labeled preparation is too low to use a t 0.1 pg/ml, the concentration of unlabeled DNA can be increased and the incubation period can be shortened. On the other hand, if the concentration of unlabeled DNA is too low the incubation period can be lengthened in order to obtain the desired Cot. IV. Interpretation of Reassociation Data

Reassociation experiments can be used to determine the extent of relatedness between organisms, the stability of related polynucleotide sequences, the genome size and to select for specific portions of the genome. A large body of data has accumulated that attempts to define relationships in bacteria and in certain specific regions of the bacterial genome by studying heteroduplex DNA molecules. There has been a good deal of confusion in interpreting heteroduplex reassociation data. One reason

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for this is lack of specificity in some early studies. By specific binding we mean binding in excess of that obtained in negative control reactions. Lack of specificity becomes a problem when one incubates a t temperatures much below 3OoC under the T, (temperature a t which 50% of a given DNA is denatured) (McCarthy, 1967; Johnson and Ordal, 1968). Furthermore, there has been a tendency to equate the degree of reassociation of DNA fragments from one species with DNA from another species ( = DNA binding) with the degree of homology between these organisms. I n other words it was assumed that percentage DNA binding meant the existence of identical stretches of nucleotide sequence between the organisms in question. It will be seen below that this is certainly not the case among most members of the Enterobacteriaceae. Moreover, it is often meaningless to assign a numerical value to relatedness among organisms as this value will change depending upon the experimental criteria. These latter points are illustrated by reactions involving labeled E. coli DNA and unlabeled DNA from Salmonella typhimurium and Shigella flexneri. These organisms were first tested for similarity by allowing a mixture of DNAs, one of which was uniformly substituted with a “heavy’) isotope such as 15N, to reassociate. After treatment with E . coli phosphodiesterase to remove unrenatured DNA, the presence of reassociated duplexes composed of one “heavy” strand and one “light” strand was determined in CsCl density gradients. I n this system, hybrids were detected between S. flexneri and E. coli, but not between S. typhimurium and E. coli (Schildkraut et al., 1961). These conditions only detect very closely related duplexes. hlore recent studies have detected between 70 and 9% binding between E. coli and S. typhimurium, and 80 to 85% binding between E. coli and S. flexneri (Bolton and McCarthy, 1962; Brenner et al., 1967, 1969b; Brenner and Cowie, 1968). What then can be implied from a DNA reassociation experiment and what is the “correct” degree of relationship between these organisms? These results are not as disappointing and irreproducible as they may appear. It turns out that each of these values is “correct” for the criterion employed; it is simply that the percentage of DNA fragments bound in any experiment does not by itself yield enough information concerning the amount of paired bases present in the reassociated nucleotide sequences. As mentioned previously, most laboratories choose to vary the stringency of reassociation conditions by varying the incubation temperature. The effect of incubation temperature on binding of labeled E . coli DNA fragments to DNA from representative enterobacterial species is shown in Table 1. The amount of E . coli DNA bound to an Alkalescens-Dispar

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DON J. BRENNER AND STANLEY FALROW

TABLE 1 Effect of Incubation Temperature on DNA Duplex Formation* Relative binding Reaction

50°C

60°C

66°C

75°C

E. colit/E. coli E . coZit/Alkalescens-Dispar strain 08 E . colit/Shigella boydii etousa E . colit/Salmonella typhimurium E. colit/Enterobac!er hafniae

100 97

100 95 89 45 21

100

100 93 85 11 4

-

-

87 28 11

* The reactions shown were incubated at the indicated temperature in 0.14 M phosphate buffer and assayed on HA (Brenner and Fanning, unpublished data). t Indicates labeled DNA. strain and to a strain of S. flexneri is minimally affected by increasing the incubation temperature. These results are typical of reactions between closely related organisms. Increasing the incubation temperature results in a marked decrease in the amount of E. coli DNA that binds to DNA from Enterobacter and S. typhimurium. This result is typical of organisms that show 50% or less reaction with E . coli. An investigation into the thermal stability of interspecies DNA duplexes revealed that at a higher incubation temperature, decreased binding is accompanied by a significant increase in thermal stability. Thermal elution profiles illustrating this point are shown in Fig. 2. The reactions shown are representative of thermal stability profiles obtained from homologous E. coli reactions, and interspecies reactions involving organisms that are closely and distantly related to E. coli. The thermal elution profiles obtained from a homologous E. coli reaction a t both 60 and 75OC exhibit a normal distribution pattern and an elution midpoint at about 90°C. The heteroduplexes formed between E . coli and a closely related Shigella dysenteriae strain are somewhat less stable than the homologous duplexes at 6OoC, but retain an essentially normal distribution. At 75OC the E . coli-S. dysenteriae duplexes exhibit a slightly higher thermal stability. The 6OoC reaction product of E . coli and Bethesda 1A DNAs is obviously significantly less stable than the other reactions and has a very disperse elution profile. At 75OC, less than 10% of E . coli DNA can reassociate with DNA from Bethesda 1A (as compared to 45% at 6OOC). Those duplexes that do form a t the higher temperature, although still significantly less stable than homologous duplexes, show markedly increased stability and tend towards a normal distribution.

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-

-20 C 3

0

E.coli/ Betherda I A

U

7 E

a 0 m

10

36

0 60

70

a0 Temperature, "C

90

100

!,?q 0.4M

PB

FIQ.2. Thermal elution profiles of enterobacterial DNA duplexes. Labeled E . coli DNA was incubated with unlabeled DNA from E. coli, S. dysenteriae, and Bethesda strain 1A. Incubations were carried out in 0.14 M phosphate buffer and held a t the temperature employed for incubation. Reassociated duplexes were eluted from HA in a series of elutions with 0.14 M phosphate buffer at increasing 2.5"C temperature increments to 100°C. The HA was then washed with 0.4 M phosphate buffer to insure that all DNA has been eluted. Part A = 60°C incubation. Part B = 75°C incubation.

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DON J. BRENNER AND STANLEY FALKOW

Increased stability of duplexes formed a t 75OC, coupled with decreased reassociation, is due to a population of fragments that can form stable reassociation products a t 60°C, but which are not stable a t the more stringent 75OC criterion. The diminished thermal stability observed especially in 6OoC reactions may be due to unpaired bases within the duplexes. Diminished stability might also be due to .preferential relatedness among the adenine- thymine-rich sequences in enterobacteria, since adenine thymine base pairs are less stable than guanine cytosine base pairs. Experiments using E. coli and S. typhimurium DNA (Brenner and Cowie, 1968) showed that at least half of the instability of this interspecies duplex was due to unpaired bases. Unpublished experiments in this laboratory could not demonstrate preferential binding of adenine- thymine-rich E . coli DNA fractions to DNA from S. typhimurium, Enterobacter aerogenes, or S. flexneri. It is assumed, therefore, that all instability observed in interspecies enterobacterial DNA duplexes is due to the presence of unpaired bases. If decreased thermal stability is, in fact, due to unpaired bases within reassociated nucleotide strands, one would like to correlate T m ( e()= temperature at which 50% of reassociated DNA has eluted from HA) and percentage unpaired bases. Bautz and Bautz (1964) , using thermal stability of synthetic copolymers containing varying amounts of noncomplementary bases, and Laird et al. (1969) , using DNA treated with nitrous acid to deaminate bases and alter their pairing with previously complementary bases, have attacked this problem. Their data indicate that an approximate l.O°C decrease in thermal stability results from 1% unpaired bases within a DNA duplex. With this relationship between percent unpaired bases and AT^^^, , as well as quantitative reassociation data a t different criteria, an attempt can be made to categorize enterobacterial relationships from the standpoint of divergence. If it is assumed that the enterobacteria evolved from a common ancestor one can determine how much DNA has diverged to a point where it is no longer related to E. coli DNA a t the criteria employed. Furthermore, one can approximate the degree of divergence present in DNA held in common between two enterobacteria. It must again be emphasized that both percentage binding and thermal stability are dependent upon the conditions chosen for reassociation. There is, therefore, no one number to describe relatedness between two organisms. If DNA from two organisms does not reassociate these organisms are not now related. On the other hand, two organisms showing almost complete relatedness will give only minimally different values at different reassociation criteria. Since there are no formal guidelines available with which to correlate genetic and molecular information to taxonomic group-

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ings, any attempt to equate the extent and stability of interspecies duplexes with lines of speciation would be somewhat premature and certainly arbitrary. A rule of thumb used in our laboratory is to employ less stringent, but specific, reaction conditions to detect overall relatedness between species. Highly related or specific interspecies DNA duplexes should be studied under stringent reaction conditions. For the Enterobacteriaceae we test for overall relatedness in 0.14 M phosphate buffer a t 6OoC, which is close to optimal for reassociation of enterobacterial DNA. Our stringent criterion for reassociation is 75OC in 0.14 M phosphate buffer. V. Taxonomy and Nomenclature

A detailed discussion of taxonomy and nomenclature of enterobacteria is, of course, beyond the scope of this report. A few comments, however, will serve to clarify both the nomenclature used here and the established taxonomic relationships among enterobacteria. For in-depth treatment of these topics the reader is referred to “Identification of Enterobacteriaceae” (Edwards and Ewing, 1966) and to the International Bulletin of Taxonomy and Nomenclature. On the basis of biochemical and serological reactions the enterobacteria are usually divided into four groups: 1. The Escherichia-Shigella group includes the genus Shigella and all strains of E. coli. Escherichia coli is the only species currently recognized, although the Alkalescens-Dispar group are usually included as strains of E. coli. 2. The Salmonella-Arizona-Citrobacter group includes the vast number of Salmonella species and serotypes and the closely related Arizona organisms. Also included is the genus Edwardsiella and the Citrobacter group (Citrobacter is the name currently used for strains formerly known as Escherichia freundii). The Bethesda organisms are identical to Citrobacter species except that they ferment lactose slowly. These organisms are usually included as members of Citrobacter. 3. The Klebsiella-Aerobacter-Serratia group. Klebsiella and Aerobacter species have very similar biochemical capabilities. The genus Enterobacter is often used to include organisms formally contained in the genera Aerobacter and Hafnia. The genuls Serratia and the phytopathogenic Erwinia are also included in this group. 4. The Proteus-Providence group includes the diverse species of Proteus and Providence strains (formerly called Proteus inconstans). A simplistic scheme depicting biochemical relationships among enterobacteria is presented in Table 2.

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DON J. BRENNER AND GTANLEY FALKOW

TABLE 2 The Principal Taxonomic Groups of Enterobacteriaceae* Serrafia

\

Kle bsiella

"\

\/

Bethesda Cifrobacter

Enferobacfer

\ .

Escherichia coli

/

Shiiella

Proreus Providence

* This schematic representation is adapted from Edwards and Ewing (1966). It attempts to portray relationshipsof the main groups of enteric bacteria to E . coli, based, in the main, on biochemical data.

In general we have retained the names of cultures as we have received them; both for historical reasons and to test the new names and groupings from the point of view of nucleic acid relatedness. Therefore, we distinguish, for example, between Bethesda and Citrobacter and between Escherichia and Alkalescens-Dispar.

VI. Nucleic Acid Relationships among Enterobacteria

The first attempt at establishing genetic relationships among enterobacteria using nucleic acid reassociation was carried out by Marmur and his colleagues (Schildkraut et al., 1961). They were successful in showing heteroduplex formation between E. coli and S. flexneri, but were unable to demonstrate duplex formation between DNA from E . coli and S. typhirnurium, Erwinia caratovora, or E . freundii. Their

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method of reassociating density labeled and unlabeled DNA and looking for hybrid bands in CsCl detected only essentially identical duplexes. Using DNA-agar, McCarthy and Bolton ( 1963) surveyed heterologous reactions in about a dozen enterobacteria and a few representatives of other bacterial families. Their experiments showed that reproducible results were obtainable using both DNA-DNA and DNA-RNA reassociation. The incubations were apparently carried out a t conditions optimal for reassociation and they obtained a range of different degrees of relatedness among enterobacteria. Subsequent studies have f ailed to confirm several of their quantitative results ; however, the qualitative relationships they obtained remain valid. These investigators realized the application of nucleic acid reassociation experiments in determining evolutionary relationships between organisms. The more recent studies on enterobacterial relatedness utilize either the nitrocellulose filter technique with labeled RNA or free-solution reactions assayed on HA (Brenner and Cowie, 1968; Brenner et al., 1969b; Brenner e t al., 1971 ; Brenner and Fanning, unpublished observations). Thus far most studies utilized E. coli strains as reference organisms. It is necessary to carry out reciprocal reactions using labeled DNA from other groups of enterobacteria and also to examine relationships among members of various enterobacterial genera and groups. Implicit in all bacterial relationship studies is that the genomes of the organisms under test have similar molecular weights. This assumption appears to be generally true, as reciprocal reactions in several groups of bacteria yield comparable binding percentages (reciprocal binding percentages are only obtained when the genome sizes are essentially equal) , however, careful determinations of reciprocal binding percentages (Brenner et al., 1971) indicate that E. coli strain BB is some 6% larger than E . coli K12. A similar finding was obtained (Gillis et al., 1970) by measuring genome size in these organisms using reassociation kinetics. These investigators find that most enterobacteria tested have a genome size within &lo% that of E . coli K12; however, certain species tested have as much as genomes 40% larger than E. coli. These data must be kept in mind when interpreting relatedness. A. DIVERGENCE AMONG DIFFERENT STRAINS OF E. coli

It was assumed that various E. coli strains differed largely by singlebase changes and that reassociation techniques would not differentiate a t the strain level. This assumption has recently been tested (Brenner et al., 1971) and it is incorrect. Reactions representative of some 30 strains of E . coli tested are shown in Table 3. Escherichia coli strain

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DON J. BRENNER AND STANLEY FALKOW

K12 is the source of labeled DNA fragments in the reactions shown; however, comparable results are obtained when E . coli BB or E. coli 02A are employed as reference strains. Strains C600, 1485 T-,and W3110 are all derivatives of strain K12 and show virtually complete reassociation with K12 at the optimal 6OoC incubation temperature. The near identity of these strains is also evident from the minimal decrease in duplex stability (AT,,,, values are only 0.1 to 0.3OC below that of the homologous reaction). The B strains tested with K12 behave identically, exhibiting about 94% reassociation at 6OoC and 90% reaction a t 75OC. These reaction products are only slightly less stable than the homologous K12 reaction product. The remainder of the reactions shown is indicative of results obtained with some two dozen strains of diverse clinical origin. Certain of these strains (strain 128, for example) are almost identical with K12 a t either incubation criterion. I n several strains binding to K12 DNA a t 6OoC is less than 90% (190, 04) and the decrease in stability is 3OC or greater (025, 07, 04, 01A). At the higher 75OC criterion, as much as 20% of the DNA of these strains does not form stable duplexes with K12 DNA. It is also evident that the thermal stability of these reactions increases at the higher incubation temperature. Relatedness among E . coli strains may be summarized as follows: (1) at optimal reassociation conditions as much as 10-15% of the DNA of some strains has diverged to a point where it no longer reacts with DNA from K12; (2) reaction products from optimal 6OoC incubations contain as much as 4% unpaired bases (l.O°C drop in thermal stability per 1.0% unpaired bases; Laird et al., 1969) ; (3) a t incubation conditions designed to preclude formation of all but highly complementary polynucleotide sequences, up to 20% of the DNA in many E. coli strains is unable to form duplexes with K12 DNA. Those duplexes that form still exhibit as much as 3% divergence; (4) the thermal binding index (TBI; last column, Table 3) is the ratio of relative reassociation a t 75 and 6OoC (Brenner et al., 1969b). The TBI is useful in detecting the presence or absence of highly related genetic material in heterologous reassociation reactions. A low TBI indicates that most of the duplexes formed at optimal conditions are not stable, and therefore not highly complementary a t stringent reassociations. The high TBI values observed in reactions between strains of E . coli indicate that most of the polynucleotide sequences contain only minimal amounts of unpaired bases. In terms of polynucleotide sequence relatedness, it appears that the species E. coli may be described as a group of strains whose DNAs are at least 85% related. The related sequences in these strains have diverged up to about 4%.

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ENTEROBACTERIACEAE : MOLECULAR RELATIONSHIPS

TABLE 3 Divergence among Strains of E. coli

Reaction K 12$/K12-1 K12$/C600 K12$/1485T K12$/W3110(21) K12$/B K12 $/B/r Kl2$/BB K12#/128 K12$/0128A K12$/190 K12 $/025 K12$/07 K12#/04 K12$/01A

Relative Relative percent percent binding AT,+)* binding AT,,.) at 60°C a t 60°C a t 75°C a t 75°C 100 98 99 97 94 94 92 98 97 87 92 91 85 94

0.1 0.3 0.1 0.8 0.8 0.7 0.4 1.o 2.5 3.0 3.6 3.1 3.2

100 96 100 96 88 90 89 98 94 82 88 82 78 80

0.3 0.3 0.1 1.0 1.1 0.6 0.4

0.5 2.0 2.2 2.6 2.0 2.8

TBIt

0.98 1.o 0.99 0.94 0.96 0.97 1.o 0.97 0.94 0.95 0.90 0.92 0.85

* AT,,,(,) is the decrease in !l'+ between ) heterologous reactions and the homologous K12-1 reaction. t TBI = relative binding a t 75°C divided by relative binding at 60°C. 3 = Source of labeled DNA fragments. B. RELATEDNESS BETWEEN E. coli K12 STRAINS OF THE ALKALESCENS-DISPAR GROUPAND Shigella SPECIES Data obtained from K12-Alkalescens-Dispar group and K12-Shigella spp. reassociation reactions are presented in Table 4 and Table 5. Under. optimal reassociation conditions, DNA from members of the Alkalescens-Dispar group is 88-95% related to E. coli K12 DNA. The relative stabilities of these reaction products indicates divergence of 1 to 2.5% in the E . coli-Alkalescens-Dispar heteroduplexes. With the possible exception of AD-03, there is little decrease in binding a t more stringent incubation conditions. On the basis of these data Alkalescens-Dispar strains are as closely related to E. coli K12 as most E. coli strains. These group relationships, at 6OoC, are shown in Fig. 3. It is clear from the data and this diagrammatic representation that the Alkalescens-Dispar group is virtually indistinguishable from E. coli strains and probably should be included as members of the species E. coli as suggested by Ewing on the basis of serologic and biochemical data (Edwards and Ewing, 1966).

100

DON J. BRENNER AND STANLEY FALKOW

TABLE 4 DNA Reassociation Reactions between E. coli K12 and Members of the Alkalescens-Dispar Group

Reaction

Relative Relative percent percent binding ATm(e) binding ATm(i) at 60°C a t 60°C at 75°C at 75°C

K12*/AD-O1 K12*/AD-02 K12*/AD-03 K12*/AD-03 (Ceylonensis) K12*/AD-04 K12 */AD-05 K 12* /AD-06 K12 */AD-07 K12*/AD-08

*

89 91 88

0.9

90 93 95 88 90 94

1.8 0.5 1.5

-

2.2

-

0.5

82 86 78

1.2

88 94 89 84 89 92

1.2 0.6 1.3

-

1.7

-

0.5

TBI 0.92 0.95 0.89 0.98 1.0 0.94 0.95 0.99 0.98

= Source of labeled DNA fragments.

Those Shigellae tested form a close group with respect to relatedness to E. coli. Under optimal conditions 8049% reaction is obtained with Tmc,,values indicating 1 to 2% divergence. From 71 to 85% of E . coli DNA forms highly stable heteroduplexes with Shigella DNAs at stringent conditions. The range of binding percentages obtained is TABLE 5 DNA Reassociation Reactions between E. coli K12 and Shigellu spp.

Reaction K12*/Shigellu sonnei (a) K12*/S. sonnei (b) K12*/S. sonnei (c) K12*/8. jZexneri (Newcastle) K12*/S. jZexneri K12*/S. boydii K12*/S. boydii (etousu) K12*/S. dysenteriue I K12*/S. dysenteriue I1 K12*/S. dysentwiue I11

*

Relative Relative percent percent binding ATm(e) binding AT,(#) at 60°C at 60°C at 75°C at 75°C 87 84 87 85 84 80 89 82 89 80

= Source of labeled DNA fragments.

0.7 0.9 0.5

1.o

1.0

-

1.3 1.3

1.7

-

85 83 79 83 79 71 85 78 85 76

0.8 0.7 0.7

1.o

0.8

-

1.0 0.9 0.5

-

TBI 0.98 0.99 0.91 0.98 0.94 0.88 0.96 0.95 0.98 0.95

ENTEROBACTERIACEAE

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101

slightly lower than that obtained in reactions involving E. coli strains or members of the Alkalescens-Dispar group. These data support the bulk of taxonomic observations which assign Shigella and E. coli to different genera, while acknowledging that these organisms are highly related.

C. RELATIVERELATEDNESS BETWEEN E . coli OTHERENTEROBACTERIA

AND

Except for other E. coli strains, the Alkalescens-Dispar group, and members of the genus Shigella, all enterobacteria tested exhibit 50% or less reaction with E. coli DNA. Under optimal conditions we have not observed any organism reacting with E. coli in the range 50-80%. At the stringent 75OC criterion for reassociation, no evidence of relatedness between 20 and 70% has been observed. All of the organisms to be discussed (the few exceptions are noted), form extremely unstable products at 6OOC. At 75OC the degree of relative reassociation is markedly decreased with a concomitant increase in thermal stability of those polynucleotide sequences that are able to form duplexes. TBI values for these reactions rarely exceed 0.25, in contrast to E . coli-Alkalescens-Dispar and E. coli-Shigella reactions, where the TBI is rarely below 0.9. Representative reactions are shown in Table 6 and the range of reactions for each group is given in Fig. 3. Citrobacter strains show about 50% relatedness to E. coli. The related sequences have diverged some 12-13% (heteroduplexes contain between 12-13% unpaired bases a t 6OOC). When only highly complementary sequences are allowed to reassociate, one sees about 12% relatedness and these heteroduplexes still contain 5-6% unpaired bases. Citrobacter strain 3796, which behaves similarly to the other Citrobacter strains at 60°C, a t 75OC exhibits almost twice the relatedness t o E . coli as do other Citrobacter strains. It will be of interest to isolate these sequences and determine their relatedness to E. coli and to other Citrobacter strains. Reassociation a t 6OoC of DNAs from Bethesda strains and E. coli is usually between 42 and 50%; somewhat less than that seen with Citrobacter strains. The related sequences appear t o have diverged by about 13-1676. Under stringent reassociation conditions 7-10% reaction occurs and divergence appears to be about 6-796. These reactions again appear to be slightly lower than those obtained with Citrobacter strains. The question of whether Bethesda and Citrobacter strains fall into the same group, with respect to nucleic acid similarity, remains open. These organisms must be investigated in detail. Bethesda strain 5A appears

102

DON J. BRENNER AND STANLEY FALKOW

5 t

1

C ITROBACTER

5 60W

LL

r 5 W

tY

40-

ENTEROBACTE NI I

20P.MIRABILIS

AEROBACTER ENTEROBACT~ STRAINS

FIO.3. Polynucleotide sequence relatedness among enterobacteria. This figure presents a diagrammatic representation of nucleotide sequence relatedness of enterobacteria to E . coli K12. Each box represents the range of binding percentages and AT,,,(.) values obtained from DNA reassociation reactions between a group of enteric bacteria and E . coli K12.The width of the box is the range of relative binding percentages and the length of the box is the range in AT,,,(,,) values.

to be an exception in that it exhibits only 22% reaction with E . coli a t 6OoC and only 3% reaction a t 75OC. The stability of reacted E . coli-Bethesda 5A duplexes is only slightly less than those formed between E. coli and other Bethesda strains. Only two Salmonella species have been tested. These DNAs show 45% relatedness to E. coli DNA at 6OoC and the duplexes contain about 20% unpaired bases. At 75OC 6 to 11% reassociation is observed. Salmonella species and members of the Arizona group must be examined in detail, both for relatedness to E. coli and for intrageneric relationships. Enterobacter-Klebsiella species comprise three classes based on their reactions with E . coli. At optimal conditions, an aberrent strain of Enterobacter cloacae is 48% related to E . coli. Two other E . cloacae strains and two E . aerogenes strains, as well as six strains of four Klebsiella species, show between 35 and 40% reaction with E . coli. A third class within the Enterobacter-Klebsiella group contains E . liquifaciens and E. hafniae which show 15-21% reaction with E . coli. Reassociated heteroduplexes from all 3 classes show 12-17% divergence. At 75OC binding drops significantly, as does the percentage of unpaired bases in the heteroduplexes. As is the case with other groups, these data do not answer

ENTEROBACTERIACEAE

:

103

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TABLE 6 Relative Relatedness between E. coli and Distantly Related Enterobacteria

Reaction

RelaPertive centage percent diverbinding gence at at 60°C 60°C

Relative percent binding

at

75°C

Kl2lCitrobacter 3796 Kl2/Citrobacter P11-C Kl2/Citrobacter “Zurich”

54 50 48

12 13 13

21 11 12

Kl2/Bethesda K12/Bethesda K12/Bethesda K12/Bethesda

8A 1A 2A 5A

50 44 42 22

13 14 15

10 7

Kl2/Salmonella typhi 643 Kl2/Salmmella typhimurium LT2

44 45

Kl2/Enterobacter cloacae Kl2lEnterobacter cloacae(aberrant) K12/Enterobacler aerogenes K 12/ Klebsiella ozaenae Kl2/Klebsiella pneumoniae I1 Kl2/Enterobacter aluei K12/Enterobacter hafniae

Percentage divergence at 75’C*

TBI

5 6 6

0.39 0.22 0.25

-

7

3

6 7

0.20 0.16 0.17 0.12

15 12

6 11

6 3

0.14 0.24

35 48 37 38 38 17 21

13 13 14 14 14 15 16

5 11 5

5 6

8 7 4 4

4 5 -

-

0.15 0.23 0.14 0.21 0.18 0.24 0.18

K12/Serratia marcescens K 12/Serratia kiliensis

24 24

14 13

3

5 5

0.11 0.18

Kl2/Proteus mirabilis Kl2lPrdteus morganii

7 17

17 17

1.5 2.5

-

0.23 0.15

K12IErwinia amyliwora K 12lErwinia caratiwora Kl2lErwinia dissoluens

30 19 35

14 15 13

6 6 8

4 2 6

0.21 0.29 0.23

4

-

* Percentage divergence is calculated by multiplying the ATm(e)by the percentage of unpaired bases (1.0%) thought to cause a 1°C drop in thermal stability (Laird el al., 1969). questions pertaining to the relationship between these three classes of organisms. All of these groups must be tested for intrageneric relatedness. It is especially interesting to determine the relatedness between E . aerogenes and K . pneuinoniae strains. Two strains of Serratia marcescens and a Serratia kiliensis strain were identical in reactions with E . coli, with 24% binding a t 6OoC and 3 4 % binding a t 75OC.

104

DON J. BRENNER AND STANLEY FALKOW

The genus Erwinia, containing plant pathogens, is significantly related to E. coli. The strains thus far examined (Brenner and Fanning, unpublished) show 15-20% and 30-3576 reaction a t 6OOC. At 75OC the reactions fall to 6-876. The thermal stability of these reactions is comparable to that seen with other groups. It is interesting to note that the two extents of relatedness to E. coli seen in Erwinia spp. do not follow the classification of these organisms into the so-called true Erwinia spp. and the soft-rot-causing Erwinia species. Neither does there appear to be EL correlation between the relatedness of these organisms to E . coli and the closeness of their guanine+ cytosine contents to that of E . coli. Both of these implications need confirmation, and, of course, these organisms must be tested for intrageneric relatedness. King and Adler (1964) reported a previously undescribed group of Enterobacteriaceae which they named the Bartholomew group. Sakazaki (1965) and Ewing and collegues (Ewing et al., 1965) had collected and studied similar cultures dating from 1959. Sakazaki called these organisms the Asakusa group. Ewing assigned them to the new genus, Edwardsiella, and suggested the species name, Edwardsiella tarda. We obtained some 20 cultures of E. tarda from W. J. Martin, including isolations from human infections and from reptiles. These strains originated in the United States, Ecuador, Japan, Canada, Israel, and the Congo. We have preliminary data as to their relatedness with E. coli under optimal reassociation conditions. Most strains show about 2Q% reaction with E . coli DNA. The reaction products, in most cases, have AT,(,, values of between 12 and 14OC. The highest binding observed was about 30% with a AT,^^, of only 7.5OC. These results must be confirmed and extended. Proteus species showed the least relatedness to E. coli. Proteus mirabilis DNA binds about 7% and Proteus morganii DNA binds about 17% to E. coli DNA a t 6OOC. At 75OC these reactions fall to 1.5 and 2.5% respectively. Reacted DNA from both of these organisms contains about 18% unpaired bases a t 60OC. The twofold difference in reactivity of P. morganii and P. mirabilis is probably attributable to the fact that P. mirabilis DNA contains only about 38% guanine cytosine, while P . morganii DNA contains 50% guanine cytosine, as does E. coli DNA. Intrageneric studies have been carried out in the genus Proteus. The data obtained indicate that the protei are a diverse group. P. mirabilis and P. vulgaris are highly related (90%), however, P . mirabilis shares only about 15% nucleotide sequences with P. inconstans and about 5% with Proteus rettgeri. Proteus mirabilis-labeled DNA incubated a t 60°C shows between 4 and 7% reaction with E. coli, S . typhimurium, Salmonella typhi, and E . aerogenes. At 75OC these reactions are all 1%or lower.

+

+

ENTEROBACTERIACEAE : MOLECULAR RELATIONSHIPS

105

VII. Divergence in Specific Portions of the Genome

The degree of nucleic acid binding is a measure of overall relatedness between organisms; however, reactions involving unfractionated nucleic acids give no information as to the relative rates of divergence in specific portions of the genome. One may ask questions such as: (1) Are the genes involved in transcription and translation preferentially conserved in order to maintain universality of the genetic code? (2) Are essential metabolic enzyme systems conserved to a greater degree than enzymes concerned, for example, with fermentation of a carbohydrate not essential for cell growth? (3) Are rates of divergence different from enzymes that are normally constituitive as opposed to inducible enzymes? (4) Does the physical position of the gene on the chromosome have an effect on divergence? Answers to these and other questions concerning divergence in specific genetic or physical regions of the chromosome have been slow in coming because only a handful of genes have been prepared in pure form. Thus far only the genes that specify rRNA, tRNA, and the lactose operon have been tested for divergence in the enterobacteria. OF E. COli-SPECIFIC DNA IN THE GENOMES A. DETECTION OF HETEROLOGOUS ORGANISMS

One approach to isolating specific genetic regions, particularly with respect to their physical location on the chromosome, is t o detect and isolate a group of E . coli genes in the genome of a heterologous organism. This type of experiment has been carried out using S. typhi-E. coli hybrids and P . inirabilis strains with diploid regions that contain E . coli DNA. I n one series of experiments, labeled E . coli DNA fragments were allowed to reassociate at 60 and 75OC with unlabeled DNA fragments from E . coli, S. typhi, and three S . typhi-E. coli hybrid strains: S. typhi 643 lac3, which had substituted 7-13% of the E. coli chromosome for its own, and S. typhi 643 X30T and S. typhi 643 X30W, which had substituted 20-28% and 39-44%, respectively, of the E. coli chromosome (Brenner et al., 1969b). The reaction mixtures were passed through HA and the bound DNA was thermally eluted. The thermal elution profile obtained after reassociation of E. coli DNA fragments a t 6OoC tends toward a Gaussian distribution with a T m ( e ) close to 90°C. In contrast, a profile obtained from an E . coli-S. typhi reaction at 6OoC is very broad and has a Tm(e,some 13OC below that of the E. coli reaction, The presence of increasing amounts of E . coli-

106

DON J. BRENNER AND STANLEY FALKOW

TABLE 7 DNA Duplex Formation between E . coli, S. typhi, and P. mirabilis Strains

Reaction

AT,(,) at 60°C

E . eoli/E. coli E . coli/S. typhi 643 1. coli/S. t y p h i 643 lacs 1. coli/S. typhi 643 X30T E. coli/S. typhi 643 X30W E . coli/P. mirabilie E . coli/P. mirabilis F-lact

12.5 11.5 7.5 4.5 14.0 7.5

-

Relative percent binding AT,,,(#) at 60°C at 75°C 100 43 52 59 58 6 8

-

Relative percent binding at 75°C

4.5 3.5 0.5 0.0

100 9 16 32 38

0.5

3

-*

1

TBI

0.21 0.31 0.54 0.66 0.17 0.38

* Too little reassociation to allow accurate assay. specific DNA in S. typhi strains lac3, X30T, and X30W is evident from the increased relative binding percentages and the increased Tmc,)(Table 7 ) . Thermal elution profiles from E. coli-8. typhi hybrid reactions at 6OoC are biphasic in nature, with the appearance of a thermally stable E. coli-like peak (Fig. 4A). Elution profiles obtained from all interspecies reactions at 75OC show increased thermal stability (Table 7 ) . These increases are due to the loss of reassociated sequences which are stable a t 6OoC, but could not withstand the more stringent incubation temperature. The loss of these sequences results in sharper, more stable elution profiles as seen in Fig. 4B. The results obtained with 8. typhi-E. coli hybrid strains are summarized in Table 8. The data indicate that the degree of reassociation a t 75OC may be used to obtain a reasonable estimate as to the degree of specific E . coli genetic substitution in these strains, and to isolate this material. Approximately 9% relative reassociation occurs between DNA from E . coli and S. typhi at 75OC. This “background” problem was largely avoided by using a strain of P. mirabilis that carries the extrachromosoma1 element F-lac‘. The F-lac+ element is equivalent to about 2.5% of the E. coli genome and is present as an addition to the Proteus genetic material. The presence of F-lac+ does indeed increase the relative reassociation of E. coli DNA and P. mirabilis DNA by about 2% and concurrently significantly increases the thermal stability of this reaction product (Table 7 ) . The increased amounts of E. coli material in P . mirabilis F-lac+ and in the E . coli-S. typhi hybrids is readily apparent from the increased TBI values (Table 7).

ENTEROBACTERIACEAE : MOLECULAR RELATIONSHIPS

107

E.col i - " X 3 0 T " 60%

Temperature , O C

,,I E.col i --

E.coli-"X3P W " 75% --

-'I

X 3 0 T...... " 75'c

........

Temperature , O C

FIG.4. Detection of E . coli-specific DNA in the genome of S. typhi genetic hybrids. "P-Labeled E . coli DNA fragments were incubated with unlabeled DNA from the indicated organisms. The reaction mixtures were passed through HA and bound material was eluted in a series of washes with 0.14 M phosphate buffer at increasing 2.5"C temperature increments. Part A = profiles obtained from 60°C reactions. Part B = profiles obtained from 75°C reactions.

108

DON J. BRENNER AND STANLEY FALKOW

TABLE 8 Detection of E. coli-Specific DNA in the Genomes of S. typhi Hybrid Strains

Source of unlabeled DNA

S. typhi 643 8.typhi 643 lacJ S. typhi 643 X30T

S. typhi 643 X30W

Estimated E . coli genetic substitution* 0 7-13 20-28 39-44

Relative binding of E . coli DNA 60°C 43 52 59 58

f4 f2 f1 f5

75°C 9 16 32 38

f 3

f1 f3 f2

Estimated E. coli DNA substitution t 0 7 25 33

* All data expressed in percentages. The estimate of the degree of E. coli substitution was calculated on the basis of E. coli characters stably present in E. coli X S. typhi hybrids assuming uniform substitution of E . coli material into the genome of 8. typhi. It was assumed that the chromosomes of E. coli K12 and S. typhi 643 are both 2.5 X 100 daltons. The genetic analysis of these hybrids is presented in detail by Falkow et al. (1962). t The estimate of the degree of E . coli-specific nucleic acid substitution into the S. typhi chromosome was calculated by subtracting the “background” reaction between E . coli and S. typhi at 75°C from the percentage of E. coli DNA bound by the S. typhi hybrid strains at this temperature. It was also assumed that the “background” binding is distributed at random along the S. typhi chromosome and tha t the genetic substitution replaces a proportional amount of this “background” reaction. E. coli/ E . coli reactions a t 60°C and 75°C were arbitrarily designated 100%; all binding percentages are relative to these homologous reactions. This type of experiment is sensitive enough to detect a few percent of specific DNA in the genome of a heterologous organism, especially when the reaction between the parental organisms is minimal, as is the case with E. coli and P. mirabilis. With the proper selection of material from small, well-defined regions around the genetic map, divergence of genetic material from the standpoint of physical location can be assayed. B. DIVERGENCE OF THE LACTOSE OPERONAMONG ENTEROBACTERIA Messenger RNA highly enriched for lactose operons was obtained by pulse-labeling RNA in E. coli under conditions where p-galactosidase was preferentially synthesized (Brenner et al., 1969b). A portion of the pulse-labeled RNA was incubated with filter-immobilized DNA from several enterobacteria for measurement of the overall relative binding of the RNA preparation. Another portion of the RNA preparation was repeatedly reacted with the DNA of an E. coli strain with a genetic deletion that included the entire lactose operon. The RNA that remained

ENTEROBACTERIACEAE : MOLECULAR RELATIONSHIPS

109

after reaction with the deletion strain (about 3% of the original radioactivity) is presumed to be relatively enriched for lactose operon messenger RNA. This lactose messenger RNA was tested for its binding to heterologous DNA species. With most enterobacteria, the percentage relative binding with lactose messenger RNA was lower than the binding of bulk messenger RNA (Table 9 ) . With S. flezneri DNA, the binding TABLE 9 Binding of Pulse-Labeled E . coli Lactose Messenger RNA to Enterobacterial DNA Bulk messenger RNA RNaseresistant cpm bound

Source of unlabeled DNA on filter

E. coli K12 lac+ E. coli K12 lac A P. mirabilis P. morganii A . aerogenes S. typhi S. typhimurium S. jlezneri S. marcescens

39,267* 39 , 156 1,149 2 464 7,945 8,599 8, 864 32,149 3,215 8 ,864 8,334 9,868 130

Bethesda 10 Salmonella sp. (Her.) ballerup Salmonella sp. (ser.) arizonae None ~

~

Lactose messenger RNA

Relative RNaseRelative percent resistant percent binding cpm bound binding 100 99 3 6 20 22 22 83 8 22 21 25 1

1,320 27 28 26 190 220 460 910 29 340 1 ,230 270 28

100 2 2 2 14 17 34 69 2 26 93 20 2

~~

*Hybridization reactions were carried out by the procedure of Gillespie and Spiegelman (1965). Many of these bulk reactions are lower than those reported for DNA-DNA reactions. The reason is that these experiments were carried out at 66"C1 a more stringent incubation temperature than the 60°C employed in the DNA-DNA reactions.

decreased by 13%. A decrease was expected, since it has been reported that the y region of the lactose operon is deleted in strains of Shigella species (Luria et al., 1960). No measurable reaction was obtained with DNA from P . mirabilis, P. morganii, or S. marcescens. An unexpected finding was that the reaction with the lactose messenger RNA fraction was about 70% higher with Salmonella sp. (ser) ballerup and about 10% higher with S. typhimurium than was the bulk messenger RNA reaction with DNA from these organisms. It must be emphasized that these are only preliminary data and that

110

DON J. BRENNER AND STANLEY FALKOW

the lactose operon was only part of the genetic deletion in the strain employed in these experiments. Portions of the genome adjacent to the lactose operon may account, therefore, for part of the observed reaction. The deletion, however, is not more than 2%. At any rate, it appears that, assuming a common ancestor, particular regions of the genome of enteric organisms have diverged a t quite different rates. Perhaps material obtained from isolation procedures such as that used by Beckwith and his colleagues to isolate the lactose operon (Shapiro et al., 1969) will facilitate the study of divergence of specific genes. OF RIBOSOMAL RNA GENES C. CONSERVATION

Several laboratories have investigated rRNA relatedness among members of thc Enterobacteriaceae. McCarthy (Moore and McCarthy, 1967) and ourselves (Brenner et al., 196913) used labeled 23 S rRNA and reacted it with filter-immobilized DNA according to the Gillespie and Spiegelman (1965) technique. Kohne (1968) isolated and purified rRNA cistrons from E. coli and P. mirabilis and used this DNA in reassociation reactions followed by binding assays and thermal elution profiles on HA. Canter (personal communication) is presently extending Kohne’s observations. All of these results are summarized in Table 10. TABLE 10 Reaction of 23s rRNA or rRNA Cistrons from E. coli with Unfractionated Enterobacterial DNA Relative percent binding Source of unlabeled, unfractionated DNA

E . coli P . mirabilia P . morganii P . rettgeri P . vulgaris S. marcescens A . cloacae A . aerogenes Paracolobactrum aerogenoidea S . typhi S . typhimurium S . flexneri

* Moore and McCarthy

t Kohne (1968).

(1967).

$ Brenner el al., (1969b). $ Canter, personal communication.

23 S rRNA 100 84*, 76% 44 *, 52 $ 79 *

105*

77*, 741 89 * 63*, 80$ 83 * 79 $

-

92 t

rRNA cistrons 100 93t, 938 -

-

93 8 95 8

-

97ti 978

-

ENTEROBACTERIACEAE : MOLECULAR RELATIONSHIPS

111

It is immediately apparent that the rRNA genes are highly conserved throughout the Enterobacterih'ceae. It appears that the 23 S rRNA genes have diverged to a greater extent than bulk rRNA. Thermal stability profiles carried out by Kohne (1967) and by Canter (personal communication) show very little decrease in stability in heteroduplexes formed between E . coli rRNA cistrons and DNA from other enterobacteria. The maximum is about 4% in P . mirabilis. Conservation o f rRNA sequences is not surprising in view of the evidence of a universal genetic code. D. CONSERVATION OF TRANSFER RNA GENES Goodman and Rich (1962) first assessed divergence of tRNA genes in enterobacteria by assaying tRNA-DNA hybrids in CsCl gradients. Their data are summarized in Table 11. It is again evident that the tRNA cistrons are highly conserved throughout the enterobacteria tested. The method used by these investigators requires essentially complete complementarity in order to observe hybrid formation. It is possible that the values obtained are low. Recent experiments using isolated tRNA cistrons from E . coli (Brenner et al., 1970) show 85% relative reassociation between tRNA cistrons and P . mirabilis DNA. The stability of this reaction product is only 4OC less than that seen in an intraspecies E . coli tRNA cistron reaction. The results with rRNA, as well as with tRNA, need to be expanded. TABLE 11 Hybridization of tRNA with DNA from Enterobacteria*

Source of DNA

Approximate relative percent binding of tRNA

E . coli B E . coli K12 E . freundii S. marcescens P . morganii S . typhimurium A . aerogenes K . pneumoniae P . aerogenoides P . vulgaris

100 88 68 65 60 55 52 52 47 32

* Data are approximate values taken from Goodman and Rich

(1962).

112

DON J. BRENNER AND STANLEY FALKOW

It is, however, quite clear that the genes that specify both of these classes of molecules are highly conserved with respect to the genetic pool as a whole. VIII. Relationships between Extrachromosomal E1emen.h

Extrachromosomal elements carrying a variety of functions are transmissible among many species of enteric organisms (Falkow et al., 1967; Meynell et al., 1968; Novick, 1969). These genetic elements have the advantage that they ordinarily replicate independently of the host chromosome, are usually infectious, and are not essential to the host. They provide enteric species with a gene pool which may often give a marked selective advantage without the necessity of any permanent chromosomal change. The full extent of the pool of extrachromosomal elements found in enteric bacteria is still unknown, although it is clear that the incidence is increasing. We can make this unequivocal statement because it is clear that over the last decade a significant proportion of commensal and pathogenic enterobacteria have been shown to have acquired transmissible multiple drug resistance factors (R-factors) (Watanabe, 1963). The increased use (and misuse) of antibiotics in man and his domestic animals is likely responsible for this. One can appreciate the full impact of this increase by noting that prior to 1960, few Shigella strains isolated from dysentery patients in Japan harbored R-factors; at present about 85% of the strains isolated from patients harbor these elements (Mitsuhashi, 1969). Similarly E. S. Anderson (1968, 1969) reports from England that S. typhimurium isolates harboring R-factors have increased dramatically since 1961. Furthermore, there has been an associated progressive expansion of the genes carried on R-factors. In the S. typhimurium example, starting with resistance to streptomycin and sulfonamides in 1963, successive resistance appeared to tetracycline, ampicillin, neomycin-kanamycin, furazolidine, and chloramphenicol. Presumably this successive appearance reflects a sequential buildup of the genetic complexity of the genetic element by recombination in response to the increased use of each of the respective antibiotics. We can add to R-factors a growing list of extrachromosomal elements in enteric species. They include transmissible and nontransmissible lac+ elements and colicin factors (Falkow e t al., 1967). More recently two transmissible elements have been described which appear to be associated with bacterial virulence (Smith, 1969). These two, H l y and Ent, confer upon certain E. coli hosts the ability to synthesize an alpha-hemolysin and endotoxin respectively. A common extrachromosomal element found

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113

along with Ent and Hly is one which carries a determinant for a surface antigen (K88). Finally, we point out that lysogeny is common to bacteria; temperate phages abound in enteric organisms. Clearly then, the extrachromosomal agents are an important consideration when describing the evolution of enteric species. The recent wide dissemination of R-factors seems especially significant. Since R-factors, like most transmissible agents, promote not only their own transfer but also transfer of the bacterial chromosome, it is interesting to wonder what will be the eventual consequences to the evolution of enteric species. The presence of a transmissible plasmid in a bacterial cell is manifested by two effects. The first, obviously, is the ability of the cell to act as a genetic donor. The second major effect is that the cell produces a specific cell appendage which appears in the electron microscope as a fine filament. These proteinacious filaments are called sex pili. Recent studies divide sex pili into two types: F-like and I-like, referring to whether they resemble the pili seen in cells harboring the classical F factor of E. coli K12 or the colicin I determinant (Meynell et al., 1968; Meynell and Datta, 1969). The division into F-like or I-like pili is made with respect to both morphological and phage adsorption characteristics. It seems highly significant that approximately 85% of the transmissible plasmids studied thus far (about 65 independent isolates) fall unequivocally into one or the other of these two major classes (Meynell et al., 1968). Table 12 presents the nucleotide sequence relationships that have been observed (Guerry and Falkow, unpublished observations) between a n F-like R-factor and several other plasmids. The data indicate that all of the F-like transmissible extrachromosomal elements show a significant degree of relatedness which is precise in terms of their TBI. There is a relatively low degree of relatedness between the F-like element and any I-like element studied in our laboratory. Preliminary studies indicate that the reciprocal relationship is also true; i.e., I-like elements are significantly related to each other, but not to F-like elements. Clearly, extrachromosomal elements belonging to the sex pili type are by no means necessarily identical, but can differ in genetic and physicochemical organization in important ways. Yet, the data (still limited, to be sure) suggest that the members of each group are almost certainly related phylogenetically and that there are only two major ancestral types of transmissible extrachromosomal elements in enteric bacteria. The latter point is of interest since transmissible extrachromosomal elements can be freely transmitted in the laboratory among all enteric genera tested and, indeed, are found readily (R-factors for instance) in clinical isolates representing all enteric genera.

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TABLE 12 Polynucleotide Sequence Complementarity among Enterobacterial Plasmids

Source of unlabeled DNA Rl(R, Su, Sm, Cm,S Km, Am) RTF (R) 222(R, Su, Sm, To, Cm) F+ N3(R, Su, Sm, Tc) R-l44(R, Km) col I E. coli K12

* F-like t I-like

F-like*

I-liket

+ ++ +-

-

-

+ + + -

Mol. wt. x 10' daltons

65 50 67 60 46 42 75 2500

Relative percent binding 60°C

75°C

TBI

100 67 80 36 14

100 70 74 35 8 14 16 4

1.04 0.92 0.97 0.61 0.76 0.89 0.29

18

18 15

-

= transfer agent shows sex pili characteristic of the E . coli K12 F agent. = transfer agent shows sex pili characteristic of the col I agent. Reactions

were carried out on hydroxyapatite after incubations a t 60 or 75°C sufficient t o achieve essentially complete reassociation of the unlabeled DNA preparation. R-1 DNA incubated alone showed 1-3 % reassociation under these conditions. This background level was used to correct the binding level obtained in these reactions. $ R refers to the transfer agent of the R-factor; resistance to: Su = sulfonamide, Sm = streptomycin, Tc = tetracycline, Cm = chloramphenicol, Km = kanamycin, Am = ampicillin.

A major property of transmissible elements which is often cited is their ability to occasionally acquire genes from their host. One of the first examples of this phenomenon was the demonstration that the F agent could acquire the lac+ genes of E. coli K12. That such an event can occur in nature is suggested by the isolation of transmissible lac+ elements from clinical isolates (Falkow et al., 1967, 1969). It would be interesting to examine in detail the relationships between extrachromosomal elements and their hosts. Unfortunately there are only two examples which can be cited. One is between an F-like R-factor and E . coli K12 and the other is between F and E. coli K12. I n both instances, as reported in Table 12, there was significant relatedness although the F agent possessed significantly more nucleotide sequence relationship with host DNA than did the R-factor. It may be significant that F (and a few F-like colicine factors) is the only element thus far studied which shows a high degree of association with its host chromosome. Significantly, the nucleotide relationship between the host and the extrachromosomal elements is not very precise and the degree of binding decreases sharply under stringent conditions.

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A recent report (Jenkins and Drabble, 1969) indicates hybridization between an R-factor carrying resistance to tetracycline, streptomycin, sulfonamides, chloramphenicol, and kanamycin, and DNA from S. typhimurium. Neither the thermal stability of the duplexes formed nor the relative percentage of relatedness can be calculated from the preliminary data shown. It is clearly necessary to test relatedness between a variety of extrachromosomal elements and a spectrum of bacterial hosts. The relationships between temperate phages and between temperate phages and their hosts has been reviewed recently (Falkow et al., 1969) and will not be repeated here. The studies of phage-phage relationships suggest that the lambdoid phages and Salmonella phage P22 may have a common ancestor. All of the phages interact with their (nominal) host DNA to between 20 and 50% at 60°C, but a full spectrum of reassociation conditions has yet to be carried out. The significance of the relatedness between extrachromosomal elements and their hosts with respect to evolution of the element (and the host) remains to be elucidated. We should point out an example of a potential pitfall in determining nucleotide relationships between enteric species infected with extrachromosomal elements. Ordinarily a transfer factor or phage represents only a small proportion of DNA extracted from a cell. On the average, the molecular size of the element is in the range of 25-100 x lo6 daltons (equivalent to 1-9% of the E . coli K12 chromosome) , and there is usually only a single copy of the extrachromosomal element per chromosome. This is not always the case, however. I n some instances, extrachromosomal elements, particularly R-factors, may be present as multiple copies in an enteric species. Indeed, this is documented for R-factors in Proteus, Rlebsiella, and E . coli strains (Rownd et al., 1966; Falkow et al., 1969; Clowes, personal communication). I n these instances the extrachromosomal DNA may represent from 10% to more than 40% of the total DNA extracted from a cell. Clearly if such a strain were employed in relationship studies, falsely high or falsely low results would be obtained, depending upon the presence or absence of this episome in other strains. It is, therefore, useful to ascertain wherever possible the extrachromosomal DNA complement of bacterial strains selected for study. We also emphasize that the nucleotide relationships determined between different bacterial species reflect all of the DNA present without regard to whether the DNA is chromosomal or extrachromosomal. ACKNOWLEDGMENTS The authors should like to thank G. R. Fanning, K. E. Johnson, and Patricia Guerry for their help in the preparation of this manuscript. We also thank D. E.

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Kohne, Dorothy Canter, and B. P. Doctor for permitting us to cite their unpublished observations. We are indebted to A. Riggsby for preparing the figures. The work of one of us (S. F.) was supported by a grant (13035) from the National Science Foundation and by grant FR5360 from the U.S.P.H.S.

REFERENCES Anderson, E. S. 1968. The ecology of transferable drug resistance in the Enterobacteriaceae. Annu. Rev. Microbial. 22, 131-180. Anderson, E. S. 1969. Ecology and epidemiology of transferable drug resistance. In “Bacterial Episomes and Plasmids” (G. E. W. Wolstenholme and M. O’Connor, eds.), pp. 102-119. Churchill, London. Bautz, E. K. F., and Bautz, F. A. 1964. The influence of noncomplementary bases on the stability of ordered polynucleotides. Proc. Nat. Acad. Sci. US. 52, 14761481. Bernardi, G., 1965. Chromatography of nucleic acids on hydroxyapatite. Nature (London) 206, 779-783. Bolton, E. T., and McCarthy, B. J. 1962. A general method for the isolation of RNA complementary to DNA. Proc. Nat. h a d . Sci. U.S.48, 1390-1397. Brenner, D. J., and Cowie, D. B. 1968. Thermal stability of Escherichia colGSa1monella typhimurium deoxyribonucleic acid duplexes. J . Bacterwl. 95, 2258-2262. Brenner, D. J., Martin, M. A., and Hoyer, B. H. 1967. Deoxyribonucleic acid homologies among some bacteria. J . Bacteriol. 94, 48M87. Brenner, D. J., Fanning, G. R., Rake, A., and Johnson, K. E. 1969a. A batch procedure for thermal elution of DNA from hydroxyapatite. Anal. Bwchem. 28, 447459. Brenner, D. J., Fanning, G. R., Johnson, K. E., Citarella, R. V., and Falkow, S. 1969b. Polynucleotide sequence relationships among members of the Enterobacteriaceae. J . Bacteriol. 98, 637-650. Brenner, D. J., Fournier, M. J., and Doctor, B. P. 1970. Isolation and partial characterization of the transfer ribonucleic acid cistrons from Escherichia coli. Nature (London) 227, 448-451. Brenner, D. J., Fanning, G. R., Skerman, F. J., and Falkow, 8. 1971. J . Bacteriol. in press. Britten, R. J., and Kohne, D. E. 1966. Nucleotide sequence repetition in DNA. Carnegie Znst. Washington Year. 65, 78-106. De Ley, J., Cattoir, H., and Reynaerts, A. 1970. The quantitative measurement of DNA hybridization from renaturation rates. Eur. J . Biochem. 12, 133-142. Denhardt, D. T. 1966. A membrane filter technique for the detection of complementary DNA. Bwchem. Biophys. Res. Commun. 23, 641-646. Edwards, P. R., and Ewing, W. H. 1966. “Identification of the Enterobacteriaceae,” pp. 83-89. Burgess, Minneapolis, Minnesota. Ewing, W. H., McWhorter, A. C., Escobar, M. R., and Lubin, A. H. 1965. Edwardsiella, a new genus of Enterobacteriaceae based on a new species, E . tarda. Znt. Bull. Bacteriol. Nomenclat. Taxon. 15, 33-38. Falkow, S., Rownd, R., and Baron, L. S. 1962.Genetic homology between Escherichia coli K-12 and Salmonella. J . Bacteriol. 84, 1303-1312. Falkow, S., Johnson, E. M., and Baron, L. S. 1967. Bacterial conjugation and extrachromosomal elements. Annu. Rev. Genet. 1, 87-116.

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Falkow, S.,Haapala, D. K., and Silver, R. P. 1969. Relationships between extrachromosomal elements. I n “Bacterial Episomes and Plasmids” (G. E. W. Wolstenholme and M. O’Connor, eds.), pp. 136-158. Churchill, London. Gillespie, D., and Spiegelman, S. 1965. A quantitative assay for DNA/RNA hybrids with DNA immobilized on a membrane. J. Mol. Biol. 12, 829-842. Gillis, M., De Ley, J., and De Cleene, M. 1970. The determination of molecular weight of bacterial genome DNA from renaturation rates. Eur. J. Biochem. 12, 143-153.

Goodman, H. M., and Rich, A. 1962. Formation of a DNA-soluble RNA hybrid and its relation to the origin, evolution and degeneracy of soluble RNA. Proc. Nut. Acad. Sci. U.S. 48, 2101-2109. Jenkins, P. G., and Drabble, W. T. 1969. RNA from Salmonella typhimurium hybridizible with R-factor DNA. Nature (London) 223, 296-297. Johnson, J. L., and Ordal, E. J., 1968. Deoxyribonucleic acid homology in bacterial taxonomy : effect of incubation temperature on reaction specificity. J. Bacteriol. 95, 893-900.

King, B. M., and Adler, D. L. 1964. A previously undescribed group of Enterobacteriaceae. Amer. J. Clin. Pathol. 41, 230-232. Kohne, D. E., 1968. Isolation and characterization of bacterial ribosomal RNA cistrons. Biophys. J. 8, 1104-1118. Laird, C. D., McConaughy, B. L., and McCarthy, B. J. 1969. On the rate of fixation of nucleotide substitutions in evolution. Nature (London) 224, 149-154. Luria, S. E., Adams, M. J., and Ting, R. C., 1960. Transduction of lactose-utilizing ability among strains of E . coli and S. dysenteriae and the properties of the transducing phage particles. Virology 12, 348-390. McCarthy, B. J. 1967. Arrangement of base sequences in deoxyribonucleic acid. Bacteriol. Rev. 31, 215-229. McCarthy, B. J., and Bolton, E. T. 1963. An approach to the measurement of genetic relatedness among organisms. Proc. Nat. Acad. Sci. U.S.50, 156-164. McConaughy, B. L., and McCarthy, B. J. 1967. The interaction of oligodeoxyribonucleotides with denatured DNA. Biochim. Biophys. Acta 149, 18&189. McConaughy, B. L., Laird, C. D., and McCarthy, B. J. 1969. Nucleic acid reassociation in formamide. Biochemistry 8, 3289-3295. Marmur, J., and Doty, P. 1961. Thermal renaturation of DNA. J. Mot. BWZ. 3, 585-594.

Marmur, J., and Doty, P. 1962. Determination of the base composition of deoxyribonucleic acid from its thermal denaturation point. J. Mol. Bid. 5, 109-118. Marmur, J., Rownd, R., and Schildkraut, C. L. 1963. Denaturation and renaturation of DNA. Progr. NucZ. Acid Res. Mol. Biol. 1, 231-300. Meynell, E., and Datta, N. 1969. Sex factor activity of drug-resistance factors. I n “Bacterial Episomes and Plasmids” (G. E. W. Wolstenholme and M. O’Connor, eds.), pp. 120-133. Churchill, London. Meynell, E., Meynell, G. G., and Datta, N. 1968. Phylogenetic relationships of drug-resistance factors and other transmissible bacterial plasmids. Bacteriol. Rev. 32, 55-83. Mitsuhashi, S. 1969. Review: The R-factors. J. Infec. Dk. 119, 89-100. Miyazawa, Y., and Thomas, C. A. 1965. Composition of short segments of DNA molecules. J . Mol. Biol. 11, 223-237. Moore, R. L., and McCarthy, B. J. 1967. Comparative study of ribosomal ribonucleic acid cistrons in enterobacteria and myxobacteria. J. Bacteriol. 94, 1066-1074.

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Novick, R. P. 1969. Extrachromo.soma1 inheritance in bacteria. Bacte~ol.Rev. 33, 210-235.

Nygaard, A. P., and Hall, B. D. 1963. A method for the detection of RNA-DNA complexes. Biochem. Bwphys. Res. Commun. 12, 98-104. Rownd, R., Nakaya, R., and Nakamura, A. 1966. Molecular nature of the drug resistance facbrs of the Enterobacteriaceae. J . Mol. Biol. 17, 376-393. Sakazaki, R., 1965. A proposed group of the family Enterobacteriaceae, the Asakusa group. Znt. Bull. Bacteriol. Nomenclat. Taxon. 15, 4547. Schildkraut, C. L., Marmur, J., and Doty, P. 1961. The formation of hybrid DNA molecules and their use in studies of DNA homologies. J . Mol. Biol. 3, 595-617. Shapiro, J., Machattie, L., Eron, L., Ihler, G., Ippen, K., and Beckwith, J. 1969. Isolation of pure lac+ operon. Nature (London) 224, 768-774. Smith, H. W. 1969. Vetinary implications of transfer activity. I n “Bacterial Episomes and Plasmids” (G. E. W. Wolstenholme and M. O’Connor, eds.), pp. 213-223. Churchill, London. Watanabe, T. 1963. Infective heredity of multiple drug resistance in bacteria. Bacteriol. Rev. 27, 87-115. Wetmur, J. G., and Davidson, N. 1968. Kinetics of renaturation of DNA, J . Mol. Bwl. 31, 349-370.

METABOLITE ANALOGS AS GENETIC AND BIOCHEMICAL PROBES H. E. Umbarger Department of Biological Sciences, Purdue University, Lofayette, Indiana

I. 11. 111. IV. V. VI. VII.

Introduction . . . . . . . . . Tryptophan Analogs . . . . . . . Tyrosine Analogs . . . . . . . Phenylalanine Analogs . . . . . . Histidine Analogs. . . . . . . . Analogs of Isoleucine, Valiie, and Leucine Outlook and Recommendations. . . . References . . . . . . . . .

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

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

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

. . . . . . . . .

119 122 126 127 128 131 135 136

I. Introduction

I n the course of their fine-structure studies of the genetic material in Salmonella typhimurium, Demerec and colleagues were greatly intrigued by their discovery that genes controlling related biochemical steps were often linked. At the time, Demerec (1955) suggested that this nonrandom gene distribution, which had not been noted with fungi, was not a matter of chance but a result of some strong evolutionary advantage, perhaps peculiar to bacteria. What that advantage was probably escaped him and it cannot yet be fully explained. On the basis of some of the studies that stemmed from his pioneering experiments, however, we can cite one very distinct advantage that the clustering of related genes provided. That advantage was recognized when it was discovered that the expression of gene clusters could be regulated together, presumably through another genetic element, the operator (Ames and Garry, 1959; Jacob and Monod, 1961). Clearly, the role of a regulatory element that controls the expression of a gene cluster is to “recognize” some specific signal generated within the cytoplasm that allows the gene cluster to function a t varying rates. In the case of a cluster of genes involved in amino acid biosynthesis, a signal to “turn off” or repress gene function is generated when the amino acid is supplied in the medium. Since it is quite unlikely that the DNA of the postulated regulatory element or operator could directly 119

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recognize an amino acid, some additional regulatory element must be postulated to mediate the interaction between excess amino acid and the DNA of the operator. In the model of Jacob and Monod (1961), the repressor has been postulated to play this mediating role. Proposing this second element leads to the assumption that a second regulatory gene must exist. Since regulatory elements involved in repression studies will not give the phenotype of growth factor requirement upon loss of function, genetic techniques different from those employed in the isolation of auxotrophic or carbohydrate nonfermenting mutants had to be devised. To date, the most successful probe for the analysis of these gene-gene interactions leading to control of gene functions is the use of metabolite analogs that are inhibitory to cell growth. Many examples serve to illustrate the way in which this approach has been successful, but it should be noted that the approach is far from being fully exploited. One of the striking facts to emerge from this kind of an approach is that the generation of repression signals can be far more complex than the two-element model originally postulated by Jacob and Monod (1961).In other words, there can be several elements involved in end product recognition, suggesting that a series of reactions might be involved. It might be of some interest to review some of the patterns that appear to be emerging from these ongoing attempts in several laboratories to ana1yz;e the mechanisms of gene expression. Before doing so, however, it might be well t o define some terms and to outline what this writer looks upon as the 1970 version of the Jacob-Monod model in its pure form. As postulated in 1961, expression of a cluster of structural genes, and presumably that of a single structural gene as well, is controlled by a repression recognition element, the operator. Transcription of the structural genes occurs only if the operator region is not bound to the repressor, which is itself the product of a regulatory gene. Since the regulatory gene functions via a cytoplasmic product, the location with respect to the cluster is left unspecified. While the original model did not specify the nature of the repressor, the two repressors that have been isolated are, in fact, protein (Gilbert and Muller-Hill, 1966; Ptashne, 1967). The general properties of these repressors will be taken as the criteria for a repressor in the strict sense of the word: a protein that binds specifically to a fragment of doublestranded DNA containing a specific repression recognition (operator) region (Gilbert and Muller-Hill, 1967). Thus, genetic evidence for an element required for generation of a repression signs1 is no longer sufficient evidence to postulate a repressor. The gene cluster with its adjacent operator was originally considered

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to be the operon. A feature of the operon was that, even though it might have been composed of several structural genes, it functioned as a single unit of expression with presumably transcription of the genetic information beginning at the first gene in the cluster and continuing through the last. Unsettled even today is the question whether the operator is transcribed and, if transcribed, whether it is translated. The failure to find a fragment of the z gene of the lac operon in Escherichia coli corresponding to the operator region (Bhorjee et al., 1969) does not appear to be relevant since it is inferred that a t least a portion of the N-terminal region has been removed (e.g., p-galactosidase does not contain an N-formylmethionine group). That the operator might indeed be transcribed can be concluded from the demonstration of still another element, the promoter, which appears to be the point at which RNA polymerase begins its transcription. I n the case of the lac operon, for which the evidence for a promoter is strongest, the promoter is also now believed to be an element of control in that it includes the recognition region for catabolite repression or perhaps more precisely for the cyclic 2’,3’-adenosine monophosphate (CAMP)-dependent protein. Whereas the repressor is a negative control element, the CAMP-dependent protein is a positive control element (Emmer et al., 1970; Beckwith, 1970). The lac promoter thus has two postulated functions that have not been separated structurally: a point of transcription initiation and a recognition site for a positive control element. Whether these two functions will be necessary properties of all promoter regions remains to be seen. That two kinds of recognition element have evolved in the case of the lac operon can be interpreted in terms of the physiological advantage that was achieved. Although the original Jacob-Monod model did not postulate either a promoter or a positive control element, it does seem fair to retain both of these elements in the 1970 version of the model. Therefore, in this paper, the model will be considered to involve the operon consisting of the promoter, operator, and structural gene(s), in that order, and presumably, but not necessarily, two regulatory genes that are themselves structural genes for a positive and a negative control element. A minor departure, such as a series of reactions required to convert some small molecule to the metabolite that binds to the repressor or to the positive control element, would also be in accord with the model. At this point, the question of what regions are transcribed in the mRNA or translated into polypeptide can remain unspecified. Also permissible in the interpretation of the model being followed here would be an operon without an operator. It would not be difficult to imagine a system in which a repression recognition region was not

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present and the entire control of the operon resided in the kind of specificity exhibited by a positive control element. (The CAMPdependent protein appears to have a very broad specificity affecting a variety of, if not most, catabolic enzymes.) Thus, a highly specific positive control element analogous, if not homologous, to the CAMPdependent protein might eliminate the need for a repressor. Conversely, a “low-level” promoter, which recognized either no protein analogous to the CAMP-dependent protein or one in which its activity did not vary with the environment, might account for formation of proteins that are always found in fixed levels, if any such do exist (Pardee and Beckwith, 1963). For the purposes of this paper, it will be assumed that, none of the systems that will be considered here have yet been shown to fulfill the necessary features of the Jacob-Monod model. Specifically, the missing evidence in each case is failure to demonstrate a repressor, i.e., a protein binding to the operator. Therefore, general terms, noncommittal with respect to mechanism, will be preferred to the terms operator and repressor which now refer to very precisely defined physical entities. Thus, until binding to a specific repressor is demonstrated, the terms “repression recognition element” or “induction recognition element,” will be preferred to the term “operator.” (The genetic designation for such elements with the symbol 0 as in his 0 and leu 0 is heartily endorsed, however.) Similarly, “repression signal” or “induction signal” will be preferred to “repressor.” (The apparent complexity of some of the systems generating such signals is a strong justification for this operational terminology.) The model employed here is thus not intended as an alternative to the Jacob-Monod model but as a more general scheme that encompasses alternatives to the model. Furthermore, no restrictions will be made with respect to control at the transcriptional or translational level. II. Tryptophan Analogs

The technique of using an analog as a genetic and biochemical probe in bacteria had its origin with the isolation by Cohen and Jacob (1959) of 5-methyltryptophan-resistantstrains of E. coli. These workers demonstrated that the mutated genetic locus ( t r p R ) was distant from the tryptophan structural genes on the E. coli chromosome (Cohen and Jacob, 1959). It will be recalled that the trp R lesions resulted in nonrepressible synthesis of the tryptophan biosynthetic enzymes. The existence of such mutants was readily interpreted by a version of the

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Jacob-Monod model in which the repressor (in this case the trp R gene product) was active only when excess endproduct was present (in this case tryptophan). Today, the trp R gene is usually referred to as the structural gene for the tryptophan repressor (Taylor, 1970). In the more general terminology recommended here, the trp R gene should be looked upon for the present merely as an element required for generating the signal, “tryptophan excess.” One does not know whether it is the only element that is required or not.” The 5-methyl analog of tryptophan was also employed by Moyed (1960) who found an entirely different kind of 5-methyltryptophanresistant mutant. This mutant was shown to have an anthranilate synthetase and a phosphoribosyl anthranilate transferase that were resistant to inhibition by tryptophan or by 5-methyltryptophan. Surprisingly, these mutants were also derepressed with respect to the tryptophan biosynthetic enzymes. Mutants of this type have subsequently been studied in both E. coli and X. typhirnurium and have thus far been only partially explained (Somerville and Yanofsky, 1965; Cordaro and Balbinder, 1967). [That two enzymatic steps were sensitive t o end product inhibition is explained by the fact that the synthetase and the transferase exist as a complex and that the inhibition of the transferase activity is due to binding of tryptophan to the synthetase (It0 and Yanofsky, 1966, 1969). It should be noted, however, that the transferase was also sensitive to tryptophan when complexed with a tryptophaninsensitive anthranilate synthetase. Clearly, the tryptophan site on the anthranilate synthetase had not been destroyed but only its interaction with the catalytic site.] The genetic study of these same mutants demonstrated that most had lesions in the “initial” region (i.e. near the beginning of the gene cluster which, in fact, begins with the anthranilate synthetase structural gene) (Somerville and Yanofsky, 1965; Cordaro and Balbinder, 1967). The simultaneous development of derepression and loss of end product sensitivity exhibited by these mutants thus raised two questions: ( a ) Was the repression recognition site (operator) part of the first structural gene in the operon? and ( b ) were end product sensitivity and repression inextricably linked in function? An affirmative answer to the first question would have been rather readily accommodated by the Jacob-Monod *It is ironical that the tryptophan biosynthetic system is one of the few in which a test for a repressor is possible. Preparations of trp DNA with and without the repression recognition region ( t r p 0) are readily available and the trp R gene has been shown to be the structural gene for a protein (Matsushiro e t ~ l . , 1965; Ito e t al., 1969). However, it is this writer’s impression that attempts to find a trp 0-binding protein in t r p R+ cells have been unsuccessful to date.

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model, whereas an affirmative answer to the second would have been strong support for an alternative that involved control at the level of translation and more specifically the binding of end product and the first enzyme in the sequence. Indeed, a very specific model to account for translational control of gene expression was proposed several years ago by Cline and Bock (1966) based on the assumption that end product inhibition and repression were linked and on the occurrence of several other observations that were not immediately predicted by the Jacob-Monod model. For the most part, however, that model was not uniquely supported by the observations that were cited. While the model proposed by Cline and Bock (1966) was perhaps much more explicit than the data would justify, there are a number of observations made on apparent relationships of end product-sensitive enzymes to repression that are difficult to account for at present by a repressor-operator model. Nevertheless, in no case do these examples point so strongly to alternative models that an ad hoc accommodation with the repressor-operator model is ruled out. Two quite different classes of 5-methyltryptophan-resistant mutants of E . coli have more recently been identified by Hiraga and co-workers (Hiraga, 1969; Hiraga et al., 1968). One of these classes was particularly important for it provided the first genetic evidence in this organism for a repression recognition region ( t r p 0). An operator region had been inferred to exist but, as well as this reviewer can determine, the inference was from analogy with the Jacob-Monod model and from the direction of polarity of the genetic information (Yanofsky and Ito, 1966). There were, however, defective @80 phages that carried varying portions of the trp gene cluster in their genomes (Matsushiro e t al., 1965). Two of these that carried all five trp cistrons differed from each other in that one was repressible and the other was not. The repression recognition element was therefore assumed to have been severed in the one case. This kind of observation, however, does not provide as rigorous evidence for a repression recognition element as does the O++ 0" type of mutation first demonstrated for the lac operon (Jacob and Monod, 1959). The other class of 5-methyltryptophan-resistant mutants has not as yet been explained biochemically. They affect a region of the chromosome (designated mtr) that is remote from t r p R , the t r p structural gene cluster, and trp S, the tryptophanyl-tRNA synthetase structural gene (Hiraga et al., 1968). At the present time there is no evidence that these mutations in any way affect expression of the trp operon. It is of interest that the genetic studies of Hiraga (1969) with the trp Oc mutant suggested that the trp 0 region was about one fourth as long as the entire t r p operon. If the t r p 0 region were transcribed

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(as might be inferred from the current idea of the role of the promoter as found in the lac system), it would constitute a significant fraction of the mRNA hybridizable with trp DNA after derepression of the trp operon. However, Imamoto (1969) has shown that a mutant bearing a deletion of all but about one eighth of the operator-proximal portion of the trp operon has retained the repression recognition site. Measurement of trp mRNA formed by this mutant indicates that if trp 0 is transcribed, it cannot account for more than 3% of the trp operon. It may be, of course, that the size estimate of the trp 0 region made by Hiraga was in error because some unknown factor perturbed the recombination frequency or UV-inactivation measurements. Evidence for a repression recognition region preceding the trp structural genes has also been obtained for X. typhi1nurium by Callahan et a2. (1970) who isolated derepressed mutants with lesions in the postulated trp 0 region that were resistant to 5-methyltryptophan and t o 6-fluorotryptophan. They also marshalled evidence that the promoter element deleted in some tryptophan auxotrophs described by Margolin and Bauerle (1966) was adjacent to the repression recognition region and distal to the structural genes similar to the arrangement in the lac system of E . coli. Held and Smith (1970a)b) have analyzed the effect of another analog of tryptophan, 7-methyltryptophan, as well as two analogs of tryptophan precursors, 3-methylanthranilatc and 7-methylindole1 which can be converted to 7-methyltrytophan. They showed that all three compounds caused a derepression of the trp operon in wild type E. coli which they attributed to tryptophan starvation resulting from inhibition of anthranilate synthetase by 7-methyl-tryptophan. Two classes of 3-methylanthranilate-resistant mutants were found-one class with lesions closely linked to the trp gene cluster, and a second class with lesions close to aroG, the structural gene for the phenylalanine-sensitive DAHP synthetase. The former contained anthranilate synthetases that were not sensitive to end product inhibition. These mutants were also resistant to derepression of the t r p genes by 7-methyltryptophan. It was concluded on this basis that the primary cause of derepression by 3-methylanthranilate was the inhibition of anthranilate synthetase by the endogenously formed 7-methyltyptophan. As will be discussed later, however, it may not always be possible to identify the target site of an analog as the site of analog resistance. The 3-methylanthranilate-resistant mutant of the second class was interesting not because it pointed to an unrecognized genetic region but because it revealed a previously unrecognized metabolic relationship. This mutant had a DAHP synthetase that was resistant to inhibition

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by phenylalanine and was shown to overproduce chorismate (Held and Smith, 1970a). Since the latter is the substrate that competitively overcomes 7-methyltryptophan inhibition of anthranilate synthetase, it would appear that it was overproduction of substrate that was responsible for resistance. While overproduction of a competitive substrate had been anticipated as a mechanism of analog resistance (Davis, 19571, one cannot a priori assume that a sufficiently elevated pool of the substrate can, in fact, be achieved. The mutant in this case suggests that the chorismate pool can be so elevated. The second class of mutant is, of course, one that would account for resistance if the postulated target site were the actual one. However, it would also account for resistance if what was really necessary was increased metabolite flow over the tryptophan biosynthetic pathway and an increased internal tryptophan pool. In part, the use of analogs has been used to support the argument that the activation of tryptophan is not necessary for the generation of the repression signal recognized by the trp 0 region. That there might be some role of tryptophanyl-tRNA synthetase in repression had been indicated by the demonstration that a mutant with an altered synthetase exhibited an altered pattern of repression and derepression of the t r p operon (Kano et al., 1968; Hiraga et al., 1967). That this involvement did not include the charging of tRNA with tryptophan, however, was suggested by the observations on three analogs of tryptophan: 7-azatryptophan, 4-methyltryptophan1 and 6-methyltryptophan (Mosteller and Yanafsky, 1970). The first two could be incorporated into protein, whereas the third could not. In contrast, the first was unable to repress the formation of specifically hybridizable t r p mRNA, whereas the second and third could. 111. Tyrosine Analogs

Quite a number of tyrosine analogues have been tested in microorganisms, but only a few have been subjected to the kind of genetic and biochemical analysis that is being considered here. Among these is 4-fluorophenyla1aninel an analog shown years ago by Munier and Cohen (1956) to be incorporated into E. coli protein. More recently, 4-fluorophenylalanine-resistantmutants of S. typhimurium have been examined in which the lesions are closely linked to structural genes for two enzymes that are coordinately derepressed in these mutants, the tyrosine-repressed DAHP synthetase and prephenate dehydrogenase (Gollub and Sprinson, 1969). While other, unlinked tyrosine repressible gene products have not been examined, it may be that, in this case,

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a specific repression recognition region has undergone an 0' + 0"type mutation. Another kind of mutation has been found in E. coli mutants resistant to 4-aminophenylalanine (Wallace and Pitt.ard, 1969). I n these, the mutated gene, tyr R, also causes derepression of the aroF or tyr A genes but is unlinked to them. In addition, transaminase A, an enzyme for which the structural gene has not been located, is derepressed. At present, in the operational terms employed here, tyr R is the gene for some element (not necessarily a repressor) required for generating the tyrosine excess signal. Still another analog of tyrosine, 2-aminotyrosine, has been employed with E. coli strain 9723 as a biochemical probe but not as a genetic probe. Sloane and Smith (1968), in an attempt to explain the reversal of the inhibitory effect of this tyrosine analog by p-hydroxymethylphenylalanine but not by phenylalanine, obtained isotopic evidence that p-hydroxymethylphenylalaninecould be converted to both phenylalanine and tyrosine. Further analysis yielded evidence that some phenylalanine could be converted to p-hydroxymethylphenylalanine which, in turn, could be an intermediate in the low-level conversion of phenylalanine to tyrosine that these workers observed. Whether this route is unique to certain strains of E. coli and what its relationship is to the well-known route to tyrosine via prephenate remains an open question. Interestingly, however, it is a route that, on the basis of mutant methodology with the more commonly used strains of E. coli and S. typhimurium, has been eliminated as a significant one. Finally, D-tyrosine has been employed as an analog of L-tyrosine in Bacillus subtilis by Champney and Jensen (1969). A resistant mutant was isolated in which the gene lesion was in the structural gene for prephenate dehydrogenase, which, in the mutant, differed from that in the parent in being resistant to end product inhibition by tyrosine. Such findings are extremely important, for the parallel observation that such mutants excrete L-tyrosine provides evidence that the end product sensitivity observed in vitro is indeed physiologically significant. The studies did not reveal, as these workers correctly interpreted, whether inhibition of prephenate dehydrogenase or some other enzyme by D-tyrosine was the primary cause of the growth inhibition. This principle is sometimes forgotten in the interpretation of analog-resistance studies. IV. Phenylalanine Analogs

An often used analog of phenylalanine is p-2-thienylalanine. One of the first uses of it as a biochemical probe was reported by Ezekiel (1965)

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who showed that it inhibited the phenylalanine-repressible DAHP synthetase as well as phenylalanyl-tRNA synthetase. A resistant mutant exhibited what was very probably an altered phenylalanine repressible DAHP synthetase. The genetic analysis of this strain does not appear to have been completed, however. In B. subtilis, the site of end product inhibition by phenylalanine is prephenate dehydratase. Coates and Nester (1967) have isolated two types of p12-thienylalanine-resistantmutants in which the properties of this enzyme are altered. I n one type, the enzyme is insensitive to phenylalanine and in the other the enzyme is actually stimulated by phenylalanine. Genetic analysis verified that the structural gene for the enzyme was modified. Phenylalanine and tyrosine provide two very striking examples of amino acids for which analogs have certainly been underexploited. There is such a wide variety of analogs for these amino acids (for a list of some, see Shive and Skinner, 1963) that have not a t all been subjected to a combined biochemical and genetic analysis. V. Histidine Analogs

Probably the best example of the utility of amino acid analogs as biochemical and genetic probes is to be found in the studies conducted by Hartman and Ames and their colleagues. That the genetic analysis has been so successful is due to a great extent to the very extensive genetic mapping studies that have been conducted with the histidine auxotrophs of S. typhimurium. The introduction of histidine analogs into serious microbial studies is due to Moyed (1961), who employed 2-thiazolealanine as an inhibitor of E . coli. Moyed discovered that this compound was an inhibitor of the first enzyme in the pathway to histidine. At low levels of thiazolealanine, growth inhibition was, however, only transient, since the limitation of histidine caused the derepression of all the histidine biosynthetic enzymes including the sensitive one. This mechanism, termed by Moyed “induced phenotypic resistance,” has been found many times since with analogs. It will not occur with all analogs, however, because some will prevent protein synthesis (and hence the derepression necessary to overcome the inhibition) or may, in fact, be incorporated into protein but yield inactive or “false” proteins. Another possible reason for the absence of induced phenotypic resistance is that derepression does not occur because the analog mimics the effect of the natural amino acid as a repressor. This mechanism has often been postulated but is difficult

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to distinguish from “false protein” formation. I n only a few cases does it appear that this distinction has been achieved. One of the most strongly supported cases for an analog mimicking the effect of an end product as a repressor is that of 1,2,4-triazolealanine1 studied by Levin and Hartman (1963). Triazolealanine is a histidine analog that is incorporated into protein to yield, in at least some cases, biologically inactive protein. These workers examined several histidine biosynthetic enzymes formed in the presence of triazolealanine by a mutant that had completely lost the repression recognition region as well as part of the gene for the first enzyme in the pathway but could express the other his genes, (i.e., a his Oc G mutant). It was possible to demonstrate that active histidinol phosphate phosphatase and imidszole acetolphosphate transaminase were formed in the presence of triazolealanine, whereas an active histidinol dehydrogenase was not formed. However, when a leaky mutant that, in the absence of histidine, formed both enzymes a t a derepressed rate was given either triazolealanine or histidine, both enzymes were repressed. Unless, by some peculiar mechanism, these two enzymes were formed in the deletion mutant because histidine released by protein turnover was incorporated into these two enzymes in preference to the analog, one must conclude that triazolealanine can indeed be used to mimic a “histidine excess” signal. The first extensive genetic analysis of mutants resistant to a histidine analog was that performed by Sheppard (1964) with S. typhimurium mutants resistant to triazolealanine, the analog shown by Moyed to act as a “false feedback” inhibitor. Sheppard noted that most of the mutants had lesions in a particular region of the his G gene which specified the structure of the first enzyme in the pathway to histidine. In such mutants, the enzyme was refractory to inhibition by both histidine and thiazolealanine. While this region is not necessarily that which specifies the histidine binding site, it does apparently specify a sequence in the protein that is a t least important in end-product sensitivity. The region is also of special interest since mutations occurred here that appeared to result in the generation of new transcription initiation points (promoters?) in a strain that has lost by deletion both the repression recognition region as well as the postulated promoter (St. Pierre, 1968; Ames et al., 1963). Furthermore, some of the reinitiation mutations in this region, when transferred to a strain in which the rest of the his operon was intact, led t o cold-sensitive strains that were super-sensitive to inhibition by histidine similar to the E. coli mutants described by O’Donovan and Ingraham (1965). The genetic analysis of S. typhiinurium mutants in which regulation of formation of the histidine biosynthetic enzymes was altered, has been

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more spectacular than the analysis of the mutants with altered end product sensitivity. These studies have revealed that regulation in this system is indeed more complex than was a t first realized. Because triazolealanine prevents derepression, it will prevent the growth of organisms that depend upon derepression of the histidine gene cluster for growth. Examples would be bradytrophs (“leaky” mutants in which one enzyme in the pathway was only partially active) and cells growing in the presence of 1,2,4-amino triazole, an inhibitor of imidazoleglycerol phosphate dehydrase. In such cases, derepression was required for an adequate rate of histidine biosynthesis. The first gene to be recognized by lesions leading to derepression was designated his R , undoubtedly in anticlipation that it was the gene for the repressor of the Jacob-Monod model (Roth and Hartman, 1965). Subsequently, it was found that the derepressed mutants contained lesions in any of several additional loci, which were designated his S, T , U , and W (Roth et al., 1966, Anthn, 1968). Examination of these revealed that the his S mutants had lesions in the structural gene for histidyl-tRNA synthetase, while his U , his W ,and the earlier recognized his R mutants had reduced levels of histidine acceptor tRNA (An&, 1968; Roth and Ames, 1966; Silbert et al., 1966). While there might be reason to suspect that his R, W , and U are structural genes for histidine acceptor tRNA, there is no evidence that there are three distinct species. It is not a t all clear how these genes might affect the quantity of a single species of tRNA. The possibility might be considered that they are multiple copies of a gene that does not function very effectively so that all three are required for the full quota of histidine acceptor tRNA.” The his T gene is not yet explained but it does appear to be the structural gene for a protein (B. N. Ames, personal communication). What can be concluded at this point is that activation of histidine by the histidyl-tRNA synthetase and an intact his T gene are essential for the generation of the excess histidine signal. That a deficiency of the corresponding tRNA itself also prevents the generation of the signal suggests that histidyl-tRNA itself is directly on the pathway to the repression signal. Indeed, a direct involvement of these recognized elements would still be compatible with the Jacob-Monod repressor-operator model. Alternatives are possible, however, including some in which histidine acceptor tRNA plays an indirect role. One alternative is based on the observations of Goldberger and his associates (Kovach et al., 1969) that, in certain mutants with an initial enzyme in the pathway

* More-recent observations indicate that the levels of histidine acceptor tRNA in his U and his W strains are normal. Only his R mutants exhibit reduced levels of histidine acceptor tRNA.

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that is histidine-insensitive, triazolealanine cannot repress the histidine biosynthetic enzymes, and that, with histidine itself, the pattern of repression is markedly altered. Their further observations that the initial enzyme binds histidyl-tRNA (though not completely specifically) had induced them to invoke some not-yet clear role for the first enzyme in the generation of the repression signal (Kovach et al., 1970). Certainly, the studies currently in progress with this system are worthy of our continued attention. Another analog of histidine, D-a-hydrazinoimidazolepropionic acid (HIPA) has been useful in the study of the active transport systems for histidine in 8. typhimurium (Ames and Roth, 1968). HIPA is an inhibitor of growth and, while the primary site of its inhibitory effect is presumably not active transport, apparently the most effective way for the organism to develop resistance is to prevent the uptake of the analog. The analysis with HIPA and a variety of other analogs indicated that histidine was concentrated in the cell by means of two uptake systems, one highly specific for histidine and histidine analogs and another for aromatic amino acids or histidine. More recently, studies involving utilization of D-histidine in place of L-histidine have revealed genetic evidence for two histidine-binding proteins in addition to the component earlier referred to as a “permease” (Ames and Lever, 1970). This example is only one of many that could be cited in which analogs have been used to select for mutants with altered uptake of the analog as well as the natural product. Clearly, analogs should be useful in helping to unravel the complex system that the word “permease” conceals. VI. Analogs of Isoleucine, Valine, and Leucine

As might be expected from the similarity in structure of isoleucine, valine, and leucine, these amino acids have sometimes functioned as analogs of each other. Indeed, one of the earliest examples of “amino acid imbalance” was observed with these amino acids by Gladstone (1939) using B . anthracis. The well-known inhibition of the growth of the K-12 strain of E. coli by valine, and its reversal by isoleucine, provided the subject of the studies conducted by Ramakrishnan and Adelberg (1965a), whose analysis provided us with our present-day picture of the arrangement of the five structural genes for the isoleucine and valine biosynthetic enzymes. Their work demonstrated that, unlike the genes in the tryptophan cluster in E . coli and S. typhimurium, there was more than one repression recognition element and, therefore, presumably more than one operon or functional grouping.

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Very briefly, they presented evidence that the structural genes were arranged in the order B , C, A, D, and E corresponding to enzymes 2, 3, 1, 4, and 5 in the sequence (Ramakrishnan and Adelberg, 1965a). One class of valine-resistant mutant was that described biochemically several years earlier by Leavitt (Leavitt and Umbarger, 1962), in which the normally valine-sensitive enzyme (enzyme 2) was resistant. Such mutants, in which the ilv B gene was altered, were useful to locate the position of that gene on the chromosome since E . coli mutants lacking this enzyme have probably not yet been found, though probably mutations leading to abnormal enzyme have been observed (Ramakrishnan and Adelberg, 1965a,b). Another class of valine-resistant mutant was one in which the enzymes coded for by the ilv genes A , D , and E were derepressed (Ramakrishnan and Adelberg, 1965b). While the basis for valine-resistance in these mutants cannot yet be explained (Umbarger, 1969), they did provide a means of identifying the repression recognition element, designated ilv 0. Another class was found to have a derepressed ilv B gene, thus resulting in high levels of the valine-inhibited enzyme (Rarnakrishnan and Adelberg, 196513). Interestingly, these mutants had only the valine-sensitive enzyme derepressed indicating that the affected repression recognition region ilv P controlled only the adjacent ilv B gene. No mutations were found to affect regulation of the ilv C gene which is now known to be controlled in E . coli and S. typhimurium by substrate induction rather than by repression (Arfin et al., 1969). The first nonnatural analog of a branched-chain amino acid employed systematically as a biochemical and genetic probe was 5’,5’,5’-trifluoroleucine, a compound shown by Rennert and Anker (1963) to be incorporated into protein of E . coli and under suitable conditions, as yet not fully understood, to replace fully the leucine of proteins in that organism. This study, initiated by Calvo with S. typhimurium, led to the recognition of four classes of trifluoroleucine-resistant mutants. One very rare class contained an isopropyl malate synthetase (the end product-sensitive enzyme) that was resistant to inhibition by both leucine and trifluoroleucine (Calvo et al., 1969a). Another class was characterized as having normal leucine biosynthetic enzymes, but derepressed levels of all three (Calvo et al., 1969b). I n these, the lesion has affected the repression recognition site (designated leu 0) adjacent to the structural genes for the leucine biosynthetic enzymes. Another kind of trifluoroleucine-resistant mutant was one in which the lesion was not linked to the leu gene cluster. I n these, not only were the leucine biosynthetic enzymes derepressed, but also five isoleucine and valine biosynthetic enzymes. This property was of particular interest because it was known that for repression of the isoleucine and valine biosynthetic enzymes

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all three branched-chain amino acids had to be in excess (Freundlich

et al., 1962). The affected gene (leu S ) in these mutants was thus one that was required for generation of the “leucine excess” signal not only for interaction with the leu 0 region but with the presumed ilv 0 and ilv P regions as well. (Although ilv 0 and ilv P have not been demonstrated in S. typhimurium, the near-universal homology between gene order and function in E. coli and that in S. typhimurium serve to justify the presumption.) The leu S lesions have since been shown to result in leucyl-tRNA synthetases that have a reduced affinity for leucine. That difficulty in generation of the repression signal for multivalent repression of the ilv gene cluster accompanied the difficulty in activating leucine was not surprising in view of the findings of Neidhardt and his colleagues (1966) that under conditions of restricted valyl-tRNA synthesis the “excess valine” signal required for multivalent repression was not generated. That the “excess isoleucine” signal also required a normal activating enzyme was indicated by a study of E. coli thiaisoleucine resistant mutants (Szentirmai e t al., 1968). These mutants exhibited reduced levels of isoleucyl-tRNA synthetase. Subsequent genetic analysis of these has demonstrated that for the thiaisoleucine resistance and derepression observed in the original isolates, two mutational steps were required (Coker and Umbarger, 1970). One of these affected the structural gene, ilv S, of isoleucyl-tRNA synthetase, resulting in an enzyme with reduced activity and reduced affinity for isoleucine. The second gene, ilv U , appears to be a regulatory gene that leads to derepression of isoleucyl-tRNA synthetase when isoleucine is limiting. Cells containing only the ilv S lesion, although partially resistant to thiaisoleucine, appear to have a derepressed level of a poor synthetase. The derepression would appear to compensate at least in part for the reduced efficiency of the enzyme (resulting in about 50% of the wild-type activity) so that no depression of the isoleucine and valine biosynthetic enzymes results. When the ilv U mutation was introduced into an ilv S mutant, derepression of the poor synthetase was no longer possible, and only about 5% of the wildtype synthetase activity was observed in extracts. This introduction of ilv U was accompanied by an increase in thiaisoleucine resistance and derepression of the ilv gene cluster. [Actually in E. coli strain K-12, only the ilv ADE operon is derepressed when isoleucine is limiting, whereas in S. typhimurium all five enzymes are elevated (Dwyer and Umbarger, 1968) .] The introduction of ilv U into a wild-type organism led to levels of thiaisoleucine resistance and derepression less than those observed when ilv U and ilv S were both present. While an ilv U mutant exhibits

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a normal level of isoleucyl-tRNA synthetase when grown in minimal medium, it does not exhibit derepression of the synthetase when the isoleucine supply is restricted, as observed by Nass and Neidhardt (1967). It is of interest that the regulation of isoleucyl-tRNA synthetase is not multivalent but specifically responsive, via the ilv U gene, to isoleucine. I n some, but not all, thiaisoleucine-resistant mutants a third gene has been affected. This gene, ilv T, leads to a low level of derepression but not to thiaisoleucine resistance. It, too, may act synergistically with ilv S but its role is more obscure than that of ilv U . Thiaisoleucine has also been employed as a probe with S. typhimurium, but all the resistant mutants were those with an end product-insensitive threonine deaminase (Blatt, unpublished observations). This difference in pattern of analog resistance in two such similar organisms illustrates well the utility of examining the same analog in a variety of organisms that can be studied genetically. While the analog itself may not behave significantly differently in the two organisms, the permissible and feastble mutations leading to resistance in the two organisms may differ markedly. I n S. typhimurium, ilv S mutants were isolated as isoleucine auxotrophs with isoleucyl-tRNA synthetase with only 1/1000th the wild-type affinity for isoleucine (Blatt and Umbarger, 1970). In these organisms, the ilv S lesion itself led to derepression of all five isoleucine and valine biosynthetic enzymes. Another example of the advantage of utilizing a variety of analogs in a variety of organisms was noted when mutants comparable to those found by Calvo in S. typhimurium were sought in E . coli. However, trifluoroleucine was not a particularly effective inhibitor of E . coli, so that 4-azaleucine was employed an analog not particularly effective in S. typhimurium. Mutants of E . coli resistant to azaleucine were isolated that had, like leu S mutants of S. typhimurium, derepressed leucine biosynthetic enzymes and derepressed isoleucine and valine biosynthetic enzymes (except, of course, the inducible ilv C gene product that was not well induced because of the effectiveness of end product control on the valine-sensitive enzyme). However, this mutation was in an entirely different gene, and neither leucyl-tRNA synthetase, nor any leucine acceptor tRNA, were any different from those in wild-type E . coli. At the present time, the responsible gene, designated ad, is viewed as a necessary element along with normal leu 0, and leu S gene functions and excess leucine to generate the signal “excess leucine” and along with normal ilv 0, val S, ilv S, and ilv P gene functions and excess valine and isoleucine to generate the multivalent repression signal. Although one could formulate a model involving these elements it would

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seem premature to do so. Indeed, it might be anticipated that continued use of analog probes will reveal more elements with a role in regulation of these enzymes. From the results obtained with the branched-chain amino acids, one might doubt that the genetic analysis of the regulation of any amino acid biosynthetic pathway will be complete when evidence for a repression recognition region and a single unlinked regulatory element (as postulated by the Jacob-Monod model) has been found. The generation of repression signals may well be far more complex than we have anticipated and a combined genetic and biochemical probing may be almost essential. It is also quite possible that different analogs will result in different kinds of resistant mutants. Similarly, a comparative study of 8. typhimurium and E . coli with respect to the spectrum of regulatory elements that can be recognized may yield much information that will be complementary. This prediction is based upon the great extent of genetic homology that is found between the two species until one reaches the base-pair level of resolution. It is a t this latter level that the kind of effects that can be obtained by permissible changes in a base pair is determined. Such differences may account for the observation that a given analog may select for end product insensitive enzymes in one species and derepressed operons in another. VII. Outlook and Recommendations

The theme that I have tried to develop here, is that there is yet much to be learned by using metabolite analogs to select resistant mutants. The mutants so found may not only reveal biochemical relationships not earlier recognized, but also gene functions that are not recognized until altered. One might ask, cannot these interacting elements also be discovered by sufficiently detailed biochemical analysis? The answer is usually yes, but it does appear that some biological interactions are so tightly coupled that, without genetically controlled modification, resolution of the components involved would be difficult indeed. An example might be the proteins of the ribosomes that are tightly bound and then function as a single unit. An example a t another level might be the processes of transcription and translation of genetic information which, while apparently biochemically distinct, are so difficult to uncouple that it is not yet possible to find convincing evidence that control of gene expression is a t one level or the other. (Perhaps we should keep in mind that the goal in evolution was to achieve control of gene function and perhaps the means to achieve the end has not always been the same in each case.)

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While only a few examples have been chosen for discussion here, specifically analogs of the aromatic and branched-chain amino acids and histidine, it has been possible to show that their study has had considerable impact on the attempts to understand the mechanisms of regulation of gene expression in bacteria. The results have been quite enlightening to those of us who, following the introduction in 1961 of the very clear, concise and esthetically pleasing repressor-operator model, went off to our own biosynthetic and catabolic systems in bacteria, fungi, and even in developing embryos to invoke operators, repressors, and, more recently, promoters with complete abandon, only because we had no evidence to countraindicate them. This zeal on the part of the disciples was exactly opposite to the mode of operation used by Jacob and Monod (1961), who invoked only regulatory elements for which genetic and biochemical evidence was available. The more carefully examined picture currently seems quite complex. Indeed, the differences thus far revealed, even in such similar systems as these amino acid biosynthetic pathways, cause us to hesitate before concluding (paraphasing the Philistinism of a contemporary political revertant) that to have seen one operon is to have seen them all. One might wonder whether the regulation of gene expression, which must have evolved after the appearance of the function itself, might not in each case have developed along unique lines. While we might easily recognize analogous elements involved in repression and induction of certain gene functions, whether these elements are homologous in function and structure may require more detailed study. Metabolite analogs should continue to prove valuable in this area, which is one of the many that are extensions of what Demerec began at Cold Spring Harbor.

REFERENCES Ames, B. N., and Gamy, B. 1959. Coordinate repression of the synthesis of four histidine biosynthetic enzymes by histidine. Proc. Nat. Acad. Sci. U.S. 45, 1453-1461.

Ames, G. F., and Lever, J. 1970. Components of histidine transport : histidine-binding protein and h i P protein. Proc. Nat. Acad. Sci. U.S. 66, 10961103. Ames, G. F., and Roth, J. R. 1908. Histidine and aromatic permeases of Salmonella typhimurium. J . Bacteriol. 96, 1742-1749. Ames, B. N., Hartman, P. E., and Jacob, F. 1963. Chromosomal alterations decting the regulation of histidine biosynthetic enzymes in Salmonella. J. Mol. Biol.

7,2342.

Anttin, D. N. 1968. Histidine regulatory mutants in Salmonella typhimurium. V. Two new classes of histidine regulatory mutants. J . Mol. Biol. 33, 633-546. Arfin, S. M., Ratzkin, B., and Umbarger, H. E. 1969. The metabolism of valine

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and isoleucine in Escherichia coli. XVII. The role of induction in the derepression of acetohydroxy acid isomeroreductase. Biochem. Biophys. Res. Commun. 37, 902-908. Beckwith, J. 1970. Gene expression in bacteria and some concerns about the misuse of science. Bacteriol. Rev. 34, 222-227. Bhorjee, J. S., Fowler, A. V., and Zabin, I. 1969. Biochemical evidence.that the operator locus is distinct from the z gene in the lac operon of Escherichia coli. J . Mol. Biol. 43, 219-222. Blatt, J. M.1970. Unpublished observations. Blatt, J. M., and Umbarger, H. E. 1970. Relation of isoleucyl-tRNA synthetase to repression of branched-chain amino acid biosynthetic enzymes in Salmonella typhimurium. Bacteriol. Proc. p. 136. Callahan, R., 111, Blume, A. J., and Balbinder, E. 1970. Evidence for the order promoter-operator-first structural gene in the tryptophan operon of Salmonella. J . Mol. Biol. 51, 709-714. Calvo, J. M., Freundlich, M., and Umbarger, H. E. 1969a. Regulation of branched chain amino acid biosynthesis in Salmonella typhimurium: isolation of regulatory mutants. J . Bacteriol. 97, 1272-1282. Calvo, J. M., Margolin, P., and Umbarger, H. E. 1969b. Operator constitutive mutations in the leucine operon of Salmonella typhimurium. Genetics 61, 777-787. Champney, W. S., and Jensen, R. A. 1969. n-Tyrosine as a metabolic inhibitor of Bacillus subtilis. J . Bacteriol. 98, 205-214. Cline, A. L., and Bock, R. M. 1966. Translational control of gene expression. Cold Spring Harbor Symp. Quant. Biol. 31, 321-333. Coates, J. H., and Nester, E. W. 1967. Regulation reversal mutation: characterization of endproduct-activated mutants of Bacillus subti1i.s. J . Bwl. Chem. 242, 49484955. Cohen, G. N., and Jacob, F. 1959. Sur la rbpression de la synth6s des enzymes intervenant dans la formation du tryptophane chez E. coli. C.R. Acad. Sci. 248, 3490-3492. Coker, M., and Umbarger, H. E. 1970. Genetic lesions leading to thiaisoleucine resistance in Escherichia coli. Bacteriol. Proc. p. 135. Cordaro, C., and Balbinder, E. 1967. An operator constitutive mutation in a mutant for the first structural gene of the tryptophan operon. Bacteriol. Proc. p. 51. Davis, B. D. 1957. Physiological (phenotypic) mechanisms responsible for drug resistance. Ciba Found. Symp. Drug Resist. Micro-Organisms pp. 165-179. Demerec, M. 1955. Annual Report of the Director of the Department of Genetics, Carnegie Institution of Washington, No. 55, 213, Washington. Dwyer, S. B., and Umbarger, H. E. 1968. Isoleucine and valine metabolism of Escherichia coli. XVI. Pattern of multivalent repression in strain K-12. J . Bacteriol. 95, 1680-1684. Emmer, M., de Crombrugghe, B., Pastan, I., and Perlman, R. 1970. Cyclic AMP receptor protein of E . coli: its role in the synthesis of inducible enzymes. Proc. Nat. Acad. Sci. US.66, 480-487. Ezekiel, D. H. 1965. False feedback inhibition of aromatic amino acid biosynthesis by p-2-thienylalanine. Biochim. Biophys. Acta 95, 54-62. Freundlich, M., Burns, R. O., and Umbarger, H. E. 1962. Control of isoleucine, d i n e and leucine biosynthesis. I. Multivalent repression. Proc. Nat. Acad. Sci. U.S. 48, 1804-1808.

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Gilbert, W. and Muller-Hill, B. 1966. Isolation of the lac repressor. Proc. N u t . Acad. Sci. U.S. 56, 1891-1898. Gilbert, W., and Muller-Hill, B. 1967. The lac operator is DNA. Proc. N a t . Acad. Sci. U.S. 58, 2415-2421. Gladstone, G. P. 1939. Inter-relationships between amino-acids in the nutrition of Bacillus anthracis. Biit. J . Exp. Pathol. 20, 189-200. Gollub, E. and Sprinson, 0. B. 1969. A regulatory mutation in tyrosine biosynthesis. Biochem. Biophys. Res. Commun. 35, 389-395. Held, W. A., and Smith, 0. H. 1970a. Regulation of the Escherichia coli tryptophan operon by early reactions in the aromatic pathway. J. Bacteriol. 101, 202-208. Held, W. A., and Smith, 0. H. 1970b. Mechanism of 3-methylanthranilic acid derepression of the tryptophan operon in Escherichia coli. J. Bacteriol. 101, 209-217. Hiraga, S. 1969. Operator mutants of the tryptophan operon in Escherichia coli. J . Mol. Biol. 39, 159-179. Hiraga, S., Ito, K., Hamada, K., and Yura, T. 1967. A new regulatory gene for the tryptophan operon of E . coli. Biochem. Biophys. Res. Commun. 26, 522-527. Hiraga, S., Ito, D., Matsuyama, T., Ozaki, H., and Yura, T. 1968. 5-Methyltryptophan-resistant mutations linked with the arginine G marker in Escherichia coli. J . Bacteriol. 96, 1880-1881. Imamoto, F. 1969. Unimpaired repressibility of transcription of the operator-proxima1 eighth of the first (E)gene of the tryptophan operon in Escherichia coli. Mol. Gen. Genet., 105, 298-305. Ito, J., and Yanofsky, C. 1966. The nature of the anthranilic synthetase complex of Escherichia coli. J. Biol. Chem. 241,4112-4114. Ito, J., and Yanofsky, C. 1969. Anthranilate synthetase, an enzyme specified by the tryptophan operon of Escherichia coli: comparative studies on the complex and the subunits. J. Bacteriol. 97, 734-742. Ito, K., Hiraga, S., and Yura, T. 1969. Temperature-sensitive repression of the tryptophan operon in Escherichia coli. J. Bacteriol. 99, 279-286. Jacob, F., and Monod, J. 1959. GBnes de structure et gknes de r6gulation dans la biosynthhse des prot6ines. CJZ. Acad. Sci. Ser. D 249, 1282-1284. Jacob, F., and Monod, J. 1961. On the regulation of gene activity. Cold Spring Harbor Symp. Quant. Biol. 26, 389-401. Kano, Y., Matsushiro, A,, and Shimura, Y. 1968. Isolation of the novel regulatory mutants of the tryptophan biosynthesis system in Escherichia coli. Mol. Gen. Genet. 102, 15-26. Kovach, J. S., Phang, J. M., Blaai, F., Barton, R. W., Ballesteros-Olmo, A., and Goldberger, R. F. 1970. Interaction between histidyl tRNA and the first enzyme for histidine biosynthesis of Salmonella typhimurium. J . Bacteriol. 104, 787-792. Kovach, J. S., Phang, J. M., Ference, M., and Goldberger, R. F. 1969. Studies on repression of the histidine operon, 11. The role of the first enzyme in control of the histidine system. Proc. N a t . Acad. Sci. U.S. 63, 481-488. Leavitt, R. I., and Umbarger, H. E. 1962. .Isoleucine and valine metabolism in Escherichia coli. XI. Valine inhibition of the growth of Escherichia coli strain K-12. J . Bacteriol. 83, 624-630. Levin, A. P., and Hartman, P. E. 1963. Action of a histidine analogue, 1,2,4-triazole3-alanine, in Salmonella typhimurium. J. Bacteriol. 86, 820-828. Mmgolin, P., and Bauerle, R. H. 1966. Determinants for regulation and initiation

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of expression of tryptophan genes. Cold Spring Harbor Symp. Quant. Biol. 31, 311-320.

Matsushiro, A,, Sato, K., Ito, J., Kida, S., and Imamoto, F. 1965. On the transcription of the tryptophan operon in Eschem’chia coli. I. The tryptophan operator. J . Mol. Biol. 11, 54-63. Mosteller, R. D., and Yanofsky, C. 1970. Repression of the tryptophan operon of E . coli. Fed. Proc., Fed. Amer. SOC. Exp. Biol. 29, 598. Moyed, H. S. 1960. False feedback inhibition : inhibition of tryptophan biosynthesis by 5-methyltryptophan. J. Biol. Chem. 235, 1098-1102. Moyed, H. S. 1961. Interference with the feedback control of histidine biosynthesis. J. Biol. Chem. 236, 2261-2267. Munier, R., and Cohen, G. N. 1956. Incorporation d’analogues structuraux d’amino acides dans les protCines bactbriennes. Biochim. Biophys. Acta 21, 592-593. Nass, G., and Neidhardt, F. C. 1967. Regulation of formation of amino acyl-ribonucleic acid synthetases in Escheiichin coli. Biochim. Biophys. Acta 134, 347-359. Neidhardt, F. C. 1966. Roles of amino acid activating enzymes in cellular physiology. Bacteriol. Rev. 30, 701-719. O’Donovan, G. A., and Ingraham, J. L. 1965. Cold-sensitive mutants of Escherichk coli resulting from increased feedback inhibition. Proc. Nut. Acad. Sci. U.S. 54, 451457. Pardee, A. B., and Beckwith, J. R. 1963. Control of constitutive enzyme synthesis. I n “Informational Macromolecules” (H. J. Vogel, V. Bryson, and J. 0. Lampen, eds.), pp. 255-269. Academic Press, New York. Ptashne, M. 1967. Isolation of the phage repressor. R o c . Nut. Acad. Sci. U.S. 57, 306-313. Ramakrishnan, T., and Adelberg, E. A. 1965a. Regulatory mechanisms in the biosynthesis of isoleucine and valine. 111. Map order of the structural and operator genes. J . Bacteriol. 89, 661-664. Ramakrishnan, T., and Adelberg, E. A. 196513. Regulatory mechanisms in the biosynthesis of isoleucine and valine. 11. Identification of two operator genes. J. Bacteriol. 89, 654-660. Rennert, 0 M., and Anker, H. S. 1963. On the incorporation of 5’5’5’-trifluoroleucine into proteins of E. coli. Biochemistry 2, 471476. Roth, J. R., and Ames, B. N. 1966. Histidine regulatory mutants in Salmonella typhimurium. 11. Histidine regulatory mutants having altered histidyl-tRNA synthetase. J. Mol. Biol. 22, 325-334. Roth, J. R., and Hartman, P. E. 1965. Heterogeneity in P22 transducing particles. Virology 27, 297-307. Roth, J. R., Antbn, D. N., and Hartman, P. E. 1966. Histidine regulatory mutants in Salmonella typhimurium. I. Isolation and general properties. J . Mol. Biol. 22, 305-323. St. Pierre, M. L. 1968. Mutations creating a new initiation point for expression of the histidine operon in Salmonella typhimurium. J . Mol. Biol. 35, 71-82. Sheppard, D. E. 1964. Mutants of Salmonella typhimurium resistant to feedback inhibition by L-histidine. Genetics 53, 445-459. Shive, W., and Skinner, C. G. 1963. Amino acid analogs. In “Metabolic Inhibitors” (R.M. Hochster and J. H. Quastel, eds.), Vol. I, pp. 1-73. Academic Press, New York. Silbert, D. F., Fink, G. R., and Ames, B. N. 1966. Histidine regulatory mutants

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in Salmonella typhimurium. 111. A class of regulatory mutants deficient in tRNA for histidine. J. Mol. Biol. 22, 335-342. Sloan, N. H., and Smith, S. C. 1968. Hydroxymethylation of the benzene ring. IV. p-Hydroxylmethyl-L-phenylalanine, a naturally occurring amino acid in Escherichia coli: identification and biochemical properties. Biochim. Biophys. Acta 158, 394401. Somerville, R. L., and Yanofsky, C. 1965. Studies on the regulation of tryptophan synthesis in Escherichia coli. J . Mol. Biol. 11, 747-759. Szentimai, A., Szentirmai, M., and Umbarger, H. E. 1968. Isoleucine and valine metabolism in Escherichia coli. XV. Biochemical properties of mutants resistant to thiaisoleucine. J. Bacteriol. 95, 1672-1679. Taylor, A. L. 1970. Current linkage map of Escherichia coli. Bacteriol. Rev. 34, 155-175.

Umbarger, H. E. 1969. Regulation of the branched-chain amino acids. Curr. Top. Cell. Regul. 1, 57-76. Wallace, B. J., and Pittard, J. 1969. Regulator gene controlling enzymes concerned in tyrosine biosynthesis in Escherichia coli. J. Bacteriol. 97, 1234-1241. Yanofsky, C., and Ito, J. 1966. Nonsense codons and polarity in the tryptophan operon. J. Mol. Biol.21, 313-334.

S-AMINO ACID METABOLISM AND ITS REGULATION IN Escherichia coli AND Salmonella fyphimurium

D. A. Smith Genetics Department, University of Birmingham, England

I. The Enzymes and Genes Involved in the Synthesis of Methionine and Cysteine. . . . . . . . . . . . A. The Synthesis of 0-Succinylhomoserine from Aspartate . . B. The Synthesis of Cysteine from Sulfate . . . . . . C. The Synthesis of Homocysteine . . . . . . . . . D. The Methylation of Homocysteine . . . . . . . . E. Uptake of Methionine . . . . . . . . . . . F. The Fate of Cysteine and Methionine . . . . . . . 11. The Genetic Map Location and Nature of the Structural Genes 111. Regulation . . . . . . . . . . . . . . . . A. Cysteine Pathway . . . . . . . . . . . . B. Methionine Pathway . . . . . . . . . . . . C. Possible Cysteine and Methionine Regulatory Relationships IV. Conclusions . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . .

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142 144 144 145 146 147 147 147 151 151 152

157 158 161

It is almost unnecessary to say that many lines of work which are now culminating in increasingly deeper understanding of the regulation of anabolic pathways in bacteria were either initiated by Dr. Demerec himself or fostered in his laboratories at Cold Spring Harbor and Brookhaven during the last 15 years of his life. The work on cysteine and methionine synthesis is no exception, and it is a privilege and pleasure to contribute to a memorial volume to one whose influence in many areas of genetics has been so profound. I shall always be proud to acknowledge my personal contact with Dr. Demerec as one of the many who shared with him the exciting experience of working a t Cold Spring Harbor in the mid-l950s, a contact which was sustained by somewhat succinct, pithy, but always helpful occasional correspondence until his death in 1966. The justification for the joint consideration of cysteine and methionine synthesis in bacteria is that convergent rather than divergent pathways and scattered genes rather than neatly defined operons are involved, 141

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a situation much more akin to that in higher organisms. We restrict our consideration to work with Escherichia coli and Salmonella typhimurium because both the biochemical and the genetical information relevant to regulation is more complete for these microorganisms than for others. I n spite of the obvious attempt to make this review as up-to-date as possible, it really describes only the early and, perhaps, some of the middle stages of this work. The layout of the review is conventional, with biochemical and genetical considerations of the various parts of the pathways initially dealt with separately and then, in terms of regulation, with closer integration. There is inevitably unevenness because as one small area of the overall regulation of S-amino acid synthesis is explored, the paucity of information in other areas becomes only too apparent. A number of related reviews appeared recently. A general account of cysteine synthesis is included in the comprehensive article by Trudinger (1969) ; aspects of the methylation of homocysteine are covered by Weissbach and Taylor (1966) in an account of the role of cobalamin in methionine synthesis. A number of excellent papers on various aspects of regulation appeared in the 1966 volume (33) of the Cold Spring Harbor Symposia on Quuntitative Biology, and Umbarger (1969) has more recently dealt specifically with the regulation of amino acid metabolism. In addition, although an article by de Robichon-Szulmajster (1967) is concerned mainly with the regulation of the two separate but related pathways of synthesis of threonine and methionine and of isoleucine and valine in Saccharomyces cerevisiae, many comparisons with the equivalent bacterial systems are made.

1. The Enzymes and Genes Involved in the Synthesis of Methionine and Cysteine

The known steps in the pathways of synthesis of these two amino acids in 8. typhimurium and E . coli and their utilization are summarized in Fig. 1. Mutants deficient in most of these steps have been isolated and the nature of their defects deduced from the results of enzyme assays and from their growth responses and behavior in cross-feeding experiments, thus permitting the identification of many of the genes involved. Where enzymes have been partially or completely purified, their relevant properties will be described. Biochemical homology between the two organisms is assumed unless an explicit distinction is made. As the pathways of synthesis of O-succinylhomoserine and cysteine

Aspartate

I-l,

P-Aspartyl phosphate

Enzyme

H,R<

Enzyme

T&F Glycine

cysA

* Met1

Serine

tRNA-Protein

cysD

cySC

?

A

cysH

cysllG

................. .......................................... "I

metE

-

t OAS cysE j

Serine

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

I

z

I

met met tRNA

FIG.1. An ouhline of the metabolism of S-amino acids in Escherichia coli and Salmonella typhimurium. A list of genes, plus their nomenclature, full and abbreviated, follows. cysA, sulfate permease ; cysC ATP-adenylplsulfate 5'-phosphotransferase-APS kinase or PAPS synthetase; cysD, ATP sulfate adenylyl transferase-ATP sulfurylase or PAPS synthetase ; cysE, serine transacetylase ; cysH, adenosine 3'-phosphate 5'-sulfatophosphate reductme-PAPS reductme ; cysGIJ, hydrogen sulfide NADP oxidoreductase (cysGPQ in E . col)-suliite reductase ; metA, homoserine-0-transsuccinylase ; metB, cystathionine-y-synthehse ; metC, p-cystathionase; metE, N5-methyltetrahydropteroyltriglutamttite-homocysteine transmethylase-cobalamin-independent methylase; metF, N6N'O-methy1tetrahydrofolate reductase-tetrahydrofolate reductase; metG, methionyl-tRNA synthetase; metH, N'-methyltetrahydrofolate homocysteine transmethylase-cobalamin-dependent methylase; metP, methionine permease. APS, adenosine-5sulfatophosphate ; PAPS, adenmine-3-phosphate 5-sulfatosphospha~te; OAS, 0-acetylserine ; H,PteGlu, tetrahydropteroyl monoestablished repression; or triglutamate; SAM, S-adenosylmethionine; ---- +, feedback inhibition; ..... -, induction; x-X-D g ~ g r assumed , repression.

.

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D. A. SMITH

converge in the formation of cystathionine, the synthesis of these two compounds will be dealt with first. Two further convenient blocks of reactions are those involved in the synthesis of homocysteine from O-succinylhomoserine and cysteine and the alternate routes for the methylation of homocysteine. Although the uptake of methionine is not strictly a step in its synthesis in the sense of the uptake of sulfate in relation to cysteine synthesis, the recent recognition of a methionine-specific transport system in S. typhiinuriurn and one of its structural genes is reported if only for the relevance of this work to the regulation of methionine synthesis. The fate of methionine and cysteine in bacteria while not part of their syntheses is similarly relevant to regulation and so this is discussed as well. OF 0-SUCCINYLHOMOSERINE FROM ASPARTATE A. THESYNTHESIS

p-Aspartokinase catalyzes the phosphorylation of aspartate to yield p-aspartyl phosphate, which is then reduced by aspartate semialdehyde dehydrogenase to give aspartate semialdehyde, the common precursor of lysine, threonine, and isoleucine as well as methionine. The next stage in methionine synthesis is also common to that of threonine and isoleucine synthesis. It is the reduction of aspartate semialdehyde by homoserine dehydrogenase to give homoserine (Stadtman, 1963; Cohen, 1968). I n E. coli K12, p-aspartokinase comprises three (I, 11, and 111) and homoserine dehydrogenase two (I and 11) isofunctional enzymes, each exhibiting different end-product inhibition and repression control. Protein complexes are formed between aspartokinase I and homoserine dehydrogenase I (mol. wt. 300,000) and aspartokinase I1 and homoserine dehydrogenase I1 (mol. wt. 150,000). Complex I1 is absent from E . coli B (Cohen, 1968; Pattk e t al., 1967). Mutants have been isolated with deficiencies for the different components of complex I (one or other or both the aspartokinase and the homoserine dehydrogenase) and also for both components of complexes I and 11, and both components of complex I1 alone or in combination with a defective aspartokinase I11 (Janin e t al., 1967; Patt6 et al., 1967; Richaud and Cohen, 1968). The first specific precursor of methionine, 0-succinylhomoserine, is formed by acylation of homoserine with succinyl coenzyme A by homoserine-0-transsuccinylase (rnet.4) (Rowbury, 1964; Rowbury and Woods, 1964a ; Smith and Childs, 1966).

B. THESYNTHESIS OF CYSTEINE FROM SULFATE Uptake of sulfate involves a specific permease (cys.4) (Dreyfuss, 1964; Ellis, 1966; Howarth, 1958; Mizobuchi et al., 1962), and its subse-

E . coli

AND

S. typhimurium S-AMINO ACID

SYNTHESIS

145

quent activation by ATP is catalyzed by ATP sulfate adenylyl transferase ( c y s D ), resulting in the formation of adenosine 5‘-sulfatophosphate (APS) . A specific kinase, ATP adenylylsulfate 5’-phosphotransferase ( c y s C ), produces a phosphorylated derivative, adenosine 3’-phosphate 5’-sulfatophosphate (PAPS) (Dreyfuss and Monty, 1963; Pasternak, 1962). This is reduced first by PAPS reductase ( c y s H ) to sdfite (Demerec et al., 1963; Dreyfuss and Monty, 1963; Pasternak et al., 1965) and then by hydrogen sulfide NADP oxidoreductase (sulfite reductase) to sulfide. Sulfite reductase involves several gene productsthose of the cysG, I , and J genes in S. typhimurium (Demerec et al., 1963; Dreyfuss and Monty, 1963; Mizobuchi et al., 1962) and the cysG, P , and Q genes in E. coli (Jones-Mortimer, 1968a; Mager, 1960). This complexity is reflected in the structure of the enzyme. It is a large flavin and iron-containing NADPH-dependent complex with a molecular weight of 700,000, capable not only of, catalyzing the reduction of sulfite to hydrogen sulfide, but also that of nitrite and hydroxylamine to ammonia (Kamin et al., 1967; Lazzarini and Atkinson, 1961). The final reaction to give cysteine is that between sulfide and O-acetylserine (OAS) . This convergent step is catalyzed by O-acetylserine sulfhydrylase. No mutants deficient in this enzyme are available. O-Acetylserine is synthesized from acetyl coenzyme A and serine by the cysE enzyme serine transacetylase (Kredich and Tomkins, 1966; Jones-Mortimer et al., 1968). O-Acetylserine sulfhydrylase of S. typhimurium is a dimer of two identical polypeptides of molecular weight 34,000.The molecular weight of serine transacetylase is 160,000; it may consist of two subunits. Cysteine synthase consists of two molecules of sulfhydrylase and one molecule of transacetylase and has a molecular weight of 309,000 (Becker et al., 1969; Kredich et al., 1969).

C. THESYNTHESIS OF HOMOCYSTEINE Homocysteine is the immediate precursor of methionine and it is synthesized in two stages. The succinyl group of O-succinylhomoserine is replaced by cysteine in a reaction catalyzed by cystathionine-y-synthetase (metB) to give the thioether cystathionine (Kaplan and Flavin, 1966a; Rowbury and Woods, 1964b; Smith and Childs, 1966). I n S. typhimurium this enzyme is oligomeric and comprises four identical subunits each of molecular weight 40,000 (Kaplan and Flavin, 1966b). Cystathionine is then hydrolyzed by p-cystathionase (metC) to homocysteine, pyruvate, and ammonia; this enzyme has been only partially purified (Delavier-Klutchko and Flavin, 1965a,b; Rowbury and Woods, 1964b; Smith, 1961; Wijesundera and Woods, 1962).

146

D. A. SMITH

D. THEMETHYLATION OF HOMOCYSTEINE Two sources of methyl groups for this reaction are the methylated derivatives of the folic acid cofactors tetrahydropteroyl monoglutamate (H,PteGlul) or triglutamate (H,PteGlu,). These compounds are synthesized in two steps. First, iV5,,Nt0-methyleneH,PteGlu, or Glu, is formed by the transfer of the hydroxymethyl group from serine by the action of serine hydroxymethyl transferasc (glyA) , glycine being formed as a by-product. The methylene derivatives are then reduced by N5N10methyltetrahydrofolate reductase (metF) to give N5-methyl H,PteGlu, or Glu3 (Cauthen e t al., 1966). There are alternative pathways for the actual methylation of homocysteine (Woods et al., 1964). In one, the enzyme involved, N5-methyltetrahydrofolate homocysteine transmethylase ( m e t H ), is cobalamin-dependent, and either N5-methyl H,PteGlul or Glu3 can serve as a methyl donor (Childs and Smith, 1969; Guest et al., 1964; Katzen and Buchanan, 1965; Taylor and Wcissbach, 1967a,b). It should be mentioned that in E . coli B, Taylor and Weissbach (1967a,b) have shown that S-adenosylmethionine and methyl cobalamin can also act as methyl donors in reactions catalyzed by the same enzyme, a large molecule with a molecular weight of 140,000. The reactions involving N5-methylfolate and S-adenosylmethionine require a reducing system and a methionine synthetase [purified from E . coli K12 by Galivan and Huennekens (1970) ] with a similar requirement consists of two components, designated M and S, with molecular weights of 125,000 and 3000, respectively. I n the other main pathway of methylation, which is independent of cobalamin, only N5-methyl H,PteGlu3 is the methyl donor in a reaction catalyzed by N5-methyltetrahydropteroyltriglutamate-homocysteine transmethylase ( m e t E ) (Cauthen et al., 1966). Using E . coli K12, Whitfield and Weissbach (1968) demonstrated the binding of folate substrate to a purified preparation of this enzyme. Physical studies of a preparation of 94% purity from a derepressed mutant indicated a molecular weight of 80,000 for the enzyme; it could be denatured into subunits of molecular weight 50,000 (Whitfield et al., 1970). M e t E mutants require cobalamin for growth in the absence of methionine (Davis and Mingioli, 1950; Smith, 1961) presumably because they are unable to synthesize enough of it to facilitate function of the cobalamin-dependent metH enzyme. A third way of methylation of homocysteine has been demonstrated in E . coli Texas M by Balish and Shapiro (1967). Either methylmethionine or adenosylmethionine can act as methyl donors ; presumably this methyltransferase requires that either the cobalamin-dependent or independent methylation pathways function normally.

E. coli

AND

S . typhimurium S-AMINOACID

SYNTHESIS

147

E. UPTAKEOF METHIONINE Although a methionine-specific transport system in E. coli K12 has been described by Piperno and Oxender (1968), it has only recently been studied in S. typhimurium (Ayling and Bridgeland, unpublished). Wild-type S. typhimurium grown in the presence of chloramphenicol and methylmethionine-IT at 25OC were sampled a t 15-second intervals over periods of one minute using a membrane filtration technique, and the amount of isotope in whole cells and TCA-precipitable material determined. The results obtained suggested involvement of two permease systems, one with an affinity for methionine 50 times that of the other. Uptake of methionine was significantly reduced by the presence of the analogs a-methylmethionine, ethionine, norleucine, and methionine sulfoximine, at a concentration 1000 times that of methionine, but not by any other amino acids normally occurring in protein at concentrations 100 times that of methionine. Mutants resistant to inhibition by a-methylmethionine and others resistant to both a-methylmethionine and to methionine sulfoximine each possessed a defective high-affinity permease system, the gene for which is designated metP.

F. THBFATE OF CYSTEINEAND METHIONINE Cysteinyl-tRNA and both species of methionyl-tRNA (Bruton and Hartley, 1968) are formed by their respective tRNA synthetases in normal protein synthesis, and the metG gene of S. typhimurium is the structural gene for methionyl-tRNA synthetase as mutants of i t possess a methionyl-tRNA synthetase with a K, for methionine 100-1000 times greater than that of wild-type (Gross and Rowbury, 1969,1970). Cysteine is a component of the tripeptide glutathione and is the source of the sulfhydryl group of such substances as tRNA and biotin. X-Adenosylmethionine (SAM), which is involved in all transmethylation reactions (Cantoni, 1965), is synthesized from methionine by SAM synthetase, and although this reaction has been well studied (Mudd, 1965) it is only recently that mutants of E. coli with low levels of the enzyme have been reported (Green et al., 1970). Methionine is also involved more directly in the synthesis of other substances; for example, it is the source of both the methyl group and the sulfur atom of the thiazole component of thiamin (Johnson et al., 1966). II. The Genetic Map location and Nature of the Structural Genes

On the basis of conjugation and transduction analysis the positions of most of the cys and met genes on the linkage maps of 8.typhimurium

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D. A. SMITH

and E . coli (Fig. 2) are the same (Sanderson, 1970; Taylor, 1970). The study of both cysteine and methionine mutants of S. typhimurium has been more extensive than in the case of E . coli. Genetical analyses by complete and abortive transduction of large numbers of S. typhimurium mutants have been carried out, resulting not only in more precise mapping but also in shedding light on the structure of some of the enzymes.

FIQ. 2. Part of the linkage map of Salmonella typhimurium. Map positions, 1-138 minutes, are indicated at 20-minute intervals. { = Transduction fragment. { I = Transduction fragment, orientation unknown. ( ) = Precise location unknown. *Equivalent to cysP and Q translocated in E . coli. Gene abbreviations: arg, arginine ; aro, aromatic ; cys, cysteine ; his, histidine, leu, leucine ; met, melthionine; pur, purine; pyr, pyrimidine; rfa, rough; ser, serine; thi, thiamin; trp, tryptophan; trz, triazole resistance.

I n mapping experiments some clustering of related genes in both the cysteine and methionine systems is apparent, but there is no evidence of extensive contiguity as for the histidine (Ames et al., 1967) and tryptophan (Bauerle and Margolin, 1966) genes (Fig. 2). The genes

E. coli

AND

S. typhimurium S-AMINO ACID

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for APS kinase (cysC) and ATP sulfurylase (cysD) are contiguous, as are two of the sulfite reductase genes ( c y s l and J ) , but although these and the cysH (PAPS reductase) gene are all cotransducible, there are gaps of unknown function, the so-called “silent sections,” between cysCD and H , and cysH and I J . (Clowes, 1958; Demerec et al., 1963; Itikawa and Demerec, 1967; Mizobuchi et al., 1962; Qureshi, personal communication). The cysA (permease), cysE (transacetylase), and another sulfite reductase gene (cysG) are each located in quite different regions of the map. The six methionine structural genes are, if anything, more scattered. M e t A and H , the genes for homoserine-0-transsuccinylase and the cobalamin-dependent transmethylase are cotransducible (Ayling and Chater, 1968; Childs and Smith, 1969), as are metB and F (Smith, 19611, the genes for cystathionine synthetase and tetrahydrofoloate reductase, but in neither case are the pairs of genes likely t o be adjacent. The metC (cystathionase) , metE (cobalamin-independent transmethylase), and metG (methionyl-tRNA synthetase) genes are located separately, and recently the site of mutation of an additional mutant deficient in the cobalamin-dependent methylation of homocysteine has been located in the serA region of the map (Whitehouse, personal communication). The glyA (serine hydroxymethyl transferase) gene is unlinked to any methionine genes (Dalal and Gots, 1965). Complete homology of cys and met gene location in S. typhimurium and E . coli is disturbed in two regions. The linked cys gene sequence is cysCDHIJ in S. typhimuriurn. The cysI and J genes of this organism presumably correspond to cysP and Q in E . col?:,and the sequence most compatible with the results of recent transduction analysis is cysC ( P Q )H , (Jones-Mortimer, personal communication). Also the CYSB gene is located counterclockwise to the trp cluster in S. typhimurium and clockwise in E . coli. M e t P (permease) mutants have been studied only in S. typhimurium; their sites of mutation are located in the leu-purE region of the map (Ayling and Bridgeland, unpublished ; Chater, 1969). It is of interest to consider the results of abortive transduction analysis (Ozeki, 1956) of cysteine (Clowes, 1958; Demerec et al., 1963; Howarth, 1958) and methionine (Childs and Smith, 1969; Smith and Childs, 1966) mutants of S. typhimuriurn in relation to the structure and function of relevant enzymes. C y s A mutants, which comprise three complementation groups, are unable to take up sulfate but they possess normal sulfate-binding properties (N. Ohta an1 A. B. Pardee, personal communication to Jones-Mortimer). A protein able to bind sulfate and of molecular weight 32,000 has been

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purified by Pardee (1966) ; no sulfate binder negative mutants have been reported and there is no information on the sulfate transport system. Further studies of genetical and biochemical aspects of sulfate uptake could reveal more of an interesting permeability system. The cysC, D, and H enzymes have not been purified; the cysC mutants form two cistrons (Qureshi, personal communication) and the cysD and H mutants one each. Mutants in three different cistrons (cysl, J, and G ) are unable to reduce sulfite to sulfide; cysI and J are adjacent, but cysG is located well away from them. This situation is compatible with the existence of the large sulfite reductase enzyme complex (Kamin et al., 1967). Similarly, the cysE mutants fall into two complementation groups and the cysE enzyme, serine transacetylase, comprises two subunits (Kredich et al., 1969). The purified metB enzyme (cystathionine-y-synthetase) is an oligomer of four identical subunits (Kaplan and Flavin, 196613). This is reflected in the complex pattern of complementation between metB mutants. The complementation map of the metC gene is also complex, suggesting that P-cystathionase may be similer. Another oligomer whose structure is supported by complementation data is the methionyl-tRNA synthetase, although the enzyme, a tetramer with a molecular weight of about 173,000 was purified from E . coli K12 (Lemoine et al., 1968) and the complex pattern of abortive transduction was obtained between metG mutants of S. typhimurium which possess this enzyme in an altered form. A puzzling situation exists with respect to the metE mutants which are unable to carry out the cobalamin-independent methylation of homocysteine. Mutants of each of the two complementation groups I and I1 into which they fall have the same enzyme deficiency (Cauthen et al., 1966). No in witro complementation between mutants of different groups has been detected, but the tetrahydrofolate reductase (metF) activity of group I mutants was twice that of group 11, suggesting a possible regulatory role for some metE mutants. Three complementation groups have been recognized among cobalamin-dependent transmethylase mutants ; two cistrons (met H ) may be adjacent but the site of mutation of the single representative of the other group is probably located a t least 30 minutes from them in the serA region (Fig. 2). As the cobalamin-dependent methylation of homocysteine is catalyzed by an enzyme of high molecular weight (Taylor and Weissbach, 1967a,b), and as there is already evidence that the enzyme required for two of the reactions involved comprises two subunits (Galivan and Huennekens, 1970), further biochemical studies of the different metH mutants should shed more light on the nature and function of what could be an interesting enzyme complex.

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111. Regulation

Against the background of apparently identical pathways of cysteine and methionine synthesis involving similar structural genes in E . coli and S. typhimurium, a unified consideration of regulation of the pathways in both organisms is probably justified, although in some cases different aspects have been studied with different organisms. Regulation will be considered in relation to the cysteine and methionine pathways separately and then in terms of their possible inter-relationship. An indication of established feedback inhibition, repression, and induction is incorporated in Fig. 1.

A. CYSTEINEPATHWAY The activity of the sulfate transport system of E. coli (Ellis, 1966) and of serine transacetylase, the cysE enzyme of both E . coli and S. typhimurium (Kredich and Tomkins, 1966), are subject to inhibition by cysteine. I n addition to this fine control, the syntheses of all the other cysteine enzymes are repressed by cysteine although this repression is not coordinate (Dreyfuss and Monty, 1963; Kredich and Tomkins, 1966; Mager, 1960; Pasternak, 1962; Pasternak et al., 1965; Wheldrake and Pasternak, 1965). On the other hand, OAS is required for the cysA (sulfate permease) and cysD (ATP sulfurylase) enzymes and the sulfite reductase complex (Jones-Mortimer et al., 1968). Pleiotropic cysB and cysE mutants of E. coli K12 which do not synthesize any of the cysteine-repressible enzymes have been identified and studied by Jones-Mortimer (1968a,b). CysB+is dominant to a pleiotropic negative mutant allele in heterozygotes, thus strongly suggesting positive control of sulfate activation and reduction in cysteine synthesis. The product of the cysE gene (serine transacetylase), is necessary for the synthesis of OAS, which induces the cysteine enzymes, so it would seem that both the cysBCgene product and OAS are necessary for induction. Essentially similar results have been reported by Spencer et al. (1967) from work with cysteine mutants of S. typhimurium. Another approach to the regulation of cysteine synthesis may be developing through study of mutants resistant to the inhibitory effects of selenate, which prevents sulfate activation in E. coli (Pasternak, 1962). Although the sites of mutation of these mutants are 90% contransduced with cysA, the permease gene, they are not deficient in this structural gene but are derepressed at least for sulfite reductase (D. Hulanicka, personal communication). Studies of the nature of the inhibitory effects of triazole on wild-type

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S. typhimurium and the properties of mutants resistant to this inhibition could shed further light on the regulation of cysteine synthesis (Hulanicka, unpublished). She found that triazole does not inhibit the activity of any of the cysteine enzymes in vitro but that extracts of wild-type organisms grown in medium supplem.ented with triazole and OAS show very low cysteine enzyme activities as compared with similar extracts from organisms grown in the presence of OAS alone. It is suggested that triazole interferes with the induction of cysteine enzymes by OAS. Neither the sulfate permease nor the PAPS synthetase of triazole resistant mutants (trzA) is derepressed, whereas induction of these enzymes by OAS is unaffected by triazole. Cotransduction (50%) of trzA mutants with the cysA (permease) gene has been obtained, and the wild-type trzA+ allele is dominant to trzA in partial diploids. It could be that uptake of triazole is defective in trzA mutants, thus eliminating an assumed intracellular competition between triazole and OAS for some site of interaction with the cysB+ gene product. On the other hand triazole may still enter trzA mutants but fail to interact with a cysB+ gene product whose configuration had been altered as a result of the trzA mutation, i.e., perhaps the key activating molecule is a polymer comprising trzA+and cysB+components. B. METHIONINEPATHWAY 1. Feedback Inhibition

The activity of the metA enzyme homoserine-0-transsuccinylase is subject to feedback inhibition by methionone and its derivative, S-adenosylmethionine, either separately or, more effectively, in combination (Rowbury, 1964; D. A. Lawrence, unpublished; Lee et al., 1966). False feedback inhibition is achieved by the analog a-methylmethionine which is unable to replace methionine in protein synthesis and whose addition to cultures of both E . coli and S. typhimurium rapidly inhibits their growth (Lawrence et al., 1968; Rowbury, 1968; Schlesinger, 1967; Smith, 1968). Mutants of S. typhimurium resistant to inhibition by a-methylmethionine were repressible by methionine but overproduced it. They were originally designated metI mutants (Lawrence et al., 1968). These authors found that metI sites of mutation were greater than 95% cotransducible with metA. A complem.entation test in appropriate partial diploids showed that metI was dominant to metI+ only when coupled in the cis-position with metA+ (Chater and Rowbury, 1970). The same authors also showed that a careful deletion analysis of eight metI mutants indicated that their sites of mutation were located in a t least three

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different regions within the metA gene. In addition, D. A. Lawrence (personal communication) has shown that the homoserine-0-transsuccinylase activity in cell-free extracts of four metI mutants was insensitive to inhibition by either a-methylmethionine or methionine; finally, Smith and Childs (1966) failed to detect complementation in paired combinations of 38 metA mutants. The overall conclusion is that metA is another example of a gene which codes for a single polypeptide species (homoserine-O-transsuccinylase) which includes both catalytic and regulatory sites. With this in mind, the metI designation has now been dropped; all feedback-resistant mutants have been given a metA number. It should be noted that the hisG feedback-resistant mutant situation is similar but that the sites of mutation are clustered in particular regions of the hisG gene (Sheppard, 1964). d. Repression

The information of relevance to the control of the synthesis of the methionine enzymes concerned with the direct pathway from aspartate to homocyteine is much more precise than that relating to regulation of the subsequent complex alternative pathways of methylation of homocysteine (see Fig. 1 ) . One of the reasons for this is that in the published genetical regulation work on this system (Lawrence et al., 1968) the enzymes of this last stage have been assayed as a unit “methylase complex” in contrast to the individual assays carried out for the other enzymes. Despite this reservation, some clear deductions about methionine regulation are permissible. These will be presented first and then the early stages of an analysis of the interplay and regulation of the various enzymes concerned with the methylation of homocysteine in relation to their substrates and products will be discussed separately. Regulation of the methionine enzymes is apparently similar in wildtype E. coli and S. typhimurium. The syntheses of the methionine-specific p-aspartokinase and homoserine dehydrogenase of complex I1 (Patt6 et al., 1967; Pattk et al., 1963; Rowbury et al., 1968), and homoserineO-transsuccinylase, cystathionine-7-synthetase,p-cystathionase, and the homocysteine methylase complex (Rowbury, 1964; Rowbury and Woods, 1961, 196413, 1966), are all repressed by methionine, although, as would be anticipated in a scattered gene system, this repression is not ccjordinate (Lawrence et al., 1968). MetJ and metK mutants of S. typhimurium described by Lawrence et al., (1968) possess regulatory defects. They were originally isolated as resistant to inhibition by methionine analogs; metJ mutants are resistant only to ethionine, and metK mutants to ethionine, a-methyl-

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methionine, and norleucine. All nietJ mutants grow slightly more slowly than wild-type organisms in minimal medium, overproduce methionine, and have derepressed and nonrepressible levels of methionine enzymes. MetK mutants fall into two groups. Some overproduce methionine and are very similar to metJ mutants, differing only in their very slow growth rate in normal medium. Others do not excrete methionine and their regulatory abnormalities are less well marked. (All enzymes except homoserine-O-transsuccinylase have been assayed.) The sites of mutation of all metJ and metK mutants are each located in separate clusters, those of metJ being close to the metB gene and those of metK loosely linked to the serA gene (Ayling and Chater, 1968; Lawrence et al., 1968). Suppression of metG mutants which possess a methionyl-tRR’A synthetase with a high K, for methionine by metJ and metK mutations (but, curiously, not by feedback-insensitive metA mutations) is thought to reflect a high intracellular methionine concentration in these double mutants (Chater et al., 1970). Partial diploids established by the transfer of F’ factors carrying metJ+ and metK+ from E. coli to metJ and metK mutants, respectively, of 8. typhimurium were sensitive to the relevant analogs. Cystathionase (metC) activity was then assayed in these merodiploids and showed normal methionine repressibility, i.e., the wild-type alleles were dominant (Chater, 1970). From these results it can be concluded that the overall regulation of methionine synthesis is by negative (repression) control involving the metJ and K gene products. The recent isolation of metJ and metK mutants revertible by nonsense suppressors (Minson, personal communication), is evidence that these products are likely to be proteins. Do either or both of these genes code for the aporepressor itself or components of it, and what is the most likely corepressor? Chater (1970) cites three observations in support of the metJ protein being the aporepressor. First, one mutant metJ allele was not completely recessive to metJ+ whereas all three metK alleles tested were recessive to metK+. This suggests that the metJ protein may be oligomeric and that the incompletely recessive allele may produce a subunit capable of interacting with and thus inactivating wild-type subunits, a situation similar to that of the lac aporepressor of E. coli (Miiller-Hill et al., 1968). Second, a metJ mutant obtained by D. A. Lawrence (unpublished) was found by Chater (1969) to possess a partially derepressed cystathionase activity, whether grown in the presence or absence of methionine, suggesting the possession by this strain of a methionine aporepressor whose conformation is unaffected by the corepressor but which retains some regulatory activity. Third, ethionine inhibits growth by replacing methionine in

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protein synthesis (Spizek and Janecek, 1969) ; metJ mutants are resistant only to ethionine and all overproduce methionine so, presumably, their possession of a defective aporepressor results in a high intracellular concentration of methionine which competes successfully with ethionine for incorporation into protein. In contrast, all metK mutants are resistant to a-methylmethionine as well as ethionine and they do not all overproduce methionine, indicating a different regulatory defect (Lawrence, et al., 1968; Chater, 1970). What of the role of the metK protein? The resistance of metK mutants to a-methylmethionine could result from altered feedback sensitivity, Le., an alteration in a component of homoserine-0-transsuccinylase, but if that was so, resistance would have been dominant to sensitivity (unless the original mutation had been an amber mutation) but in any case it has been shown that this enzyme is sensitive to feedback inhibition in cell suspensions of a metK mutant (Chater and Rowbury, 1970). On the basis of an apparent defect in the uptake of ethionine-I4C by metK in contrast to metJ organisms, Lawrence et al. (1968) suspected a permeability defect, but the rate of uptake of methionine-14C is unaffected by a metK mutation and the metP gene for the high affinity permease is located far from metK (Ayling and Bridgeland, unpublished). Finally, the suggestion by Lawrence et al., (1968) that metK was involved in the synthesis of methionyl-tRNA is eliminated because the metG gene for methionyl-tRNA synthetase has been identified and is also located away from the metK gene, and metG mutants possess normal methionine repressibility (Gross and Rowbury, 1969, 1970). Perhaps as Chater (1970) suggests, the fate of methionine in, for example, the synthesis of X-adenosylmethionine (SAM), is relevant in considering the metK gene. SAM can act cooperatively with methionine as a feedback inhibitor of homoserine-0-transsuccinylase (Lee et al., 1966) and if SAM is also involved in repression control, mutants with defects in SAM synthetase could be resistant to both a-methylmethionine and ethionine, and might well grow more slowly than wild-type organisms. If metK mutants do possess a SAM synthetase deficiency it is difficult although not impossible to accommodate the feedback sensitivity of their homoserine-0-transsuccinylase (Chater and Rowbury, 1970), but the existence of SAM synthetase deficient mutants of E . coli K12 which are ethionine resistant and possess high levels of cystathionine synthetase and cystathionase and of intracellular methionine (Green et al., 1970) strengthens the case for S. typhimurium metK mutants possessing a similar deficiency. On the other hand, in preliminary experiments, Gross and Rowbury (personal communication) found no evidence of SAM synthetase deficiency in metJ or K regulatory mutants.

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Very recently Su et al. (1970) reported the isolation of mutants of E. coli K12 similar to the metJ mutants of S. typhimurium. They were constitutive for SAM synthetase as well as methionine enzymes, their sites of mutation were cotransduced at high frequency with metB, and the mutant alleles were recessive in diploids, thus underlining the implication of SAM in methionine regulation and keeping open a possibility that metJ may be the SAM synthetase gene. Concerning the nature of the corepressor, the wild-type repressibility of methionine enzymes of metG mutants with an altered methionyltRNA synthetase (Gross and Rowbury, 1969, 1970) and the normal methionyl-tRNA synthetase and methionyl-tRNA of metJ and metK mutants (Gross and Rowbury, unpublished) eliminate serious consideration of methionyl-tRNA as the corepressor. Does this only leave methionine as the most likely corepressor? If the properties of the SAM synthetase deficient mutants of E. coEi K12 (Greene et al., 1970) are taken into account some doubt about this must be retained because these mutants with their high levels of intracellular methionine nevertheless have elevated rather than repressed levels of methionine enzymes ; this could implicate SAM as the corepressor. The further work with the SAM synthetase constitutive mutants (Su et al., 1970) is also compatible with this hypothesis. Thus, in the negative repression control of methionine synthesis, the metJ protein appears to be the aporepressor but the nature of the corepressor is unresolved; it may be methionine or SAM. If it is SAM, the metK or possibly the metJ protein may facilitate the regulatory function of this compound. Concerning the methylation of homocysteine, some uncertainty exists as to which enzyme or enzymes were being assayed in the regulation work described so far, as the overall conversion of homocysteine to methionine was measured. A candidate for a rate-limiting role is the tetrahydrafolate reductase (metF) enzyme which controls the formation of the methyl donors for both the cobalamin-dependent and independent pathways. Certainly this enzyme was earlier reported to be more sensitive to methionine repression than the cobalamin-independent transmethylase (metE) enzyme. (Foster et al., 1963). However, some involvement of cobalamin itself in methylase activity seemed likely from the observation of Lawrence et al. (1968) that extracts of wild-type and regulatory mutants of S. typhimurium gave lower levels of methylase activity assayed in the presence of cobalamin than in its absence. I n further studies of the rate-limiting steps in methylation, J. Dawes and M. A. Foster (personal communication) have shown that cobalamin represses both the metF and the metE enEymes, and also that in the presence

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of cobalamin the metF enzyme is rate limiting but in its absence the metE enzyme assumes this role. Also Milner et al. (1969) have evidence that the synthesis of the metE enzyme is repressed and that the synthesis of the metH enzyme, the remaining component of the methylase complex, is activated by cobalamin. Although the tetrahydrofolate reductase levels differ in mutants defective in the cobalamin-dependent transmethylase, they are in general lower than in wild-type (Whitehouse, personal communication), which could indicate a reductase regulatory abnormality. All this evidence could indicate that cobalamin is involved directly in the regulation of the methylation of homocysteine. The situation is further complicated by observations of Cauthen et al. (1966) that extracts of cobalamin-independent transmethylase mutants of one of the two metE complementation groups possessed very low levels of metF reductase; the possible regulatory significance of this has not been explored. Also Whitehouse (personal communication) has carried out enzyme assays on extracts of a recently recognized mutant with an otherwise wild-type phenotype whose growth is stimulated by serine. This is almost certainly due to a deficiency of C, units necessary for the formation of N”N’O-methylene H,PteGlu, (Fig. 1). She found that this mutant also has a reduced level of tetrahydrofolate reductase (metF) bringing out the importance in regulation of the intracellular concentration of these C, units. Finally, the discovery of S. typhimurium mutants physiologically and genetically similar to metJ mutants which can suppress a deficiency in the metH enzyme (Smith, unpublished) may indicate a firm relationship between the overall regulation of the methionine pathway and the cobalamin-dependent transmethylation of homocysteine. There could be interesting similarities between these mutants and those of E. coli K12 which are derepressed for SAM synthetase (Su et al., 1970).

C. POSSIBLE CYSTEINEI AND METHIONINE REGULATORY RELATIONSHIPS There is no evidence that cysteine represses the synthesis of methionine enzymes although it can inhibit some of them (Rowbury, personal communication ; Rowbury et al., 1968 Wijesundera and Woods, 1962). Ellis et al. (1964) showed that methionine does not repress the cysteine enzymes of sulfate activation except in the presence of sulfate, and JonesMortimer ( 1968 ~ )found that this repression did not occur in mutants unable to reduce sulfite, suggesting that endogenous cysteine (rather than exogenous methionine) is responsible for this control. This view was confirmed by the observation of Wheldrake (1967) that the intracellular cysteine concentration is greater in wild-type cells grown in the

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presence of both sulfate and methionine than in the presence of sulfate alone. The observation of Hulanicka (personal communication) that methionine enhances the inhibitory effect of selenate in both wild-type and selenate-resistant mutants is also compatible with this hypothesis. Another line of work which sustains interest in the effect of these two related amino acids on each other’s synthesis and activity is being carried out by Qureshi (personal communication). He is studying a number of mutants (CYM) of S. typhimurium able to grow equally well in the presence of cysteine or methionine as their sole source of sulfur. The protein of these CYM mutants after growth on r n e t h i ~ n i n e - ~con~S tains no c y ~ t e i n e - ~ ~thus S , eliminating direct conversion of methionine into cysteine. Inhibition of the growth of CYM mutants by selenate is overcome by cysteine but not methionine, indicating that the cysteine enzymes are functioning during the growth of these mutants on methionine. On the results of complete and abortive transduction experiments with CYM mutants they fell into 7 distinct groups. Single groups were each located within the cysCD region and the cysH, I, and J genes, respectively; two groups were contransduced with the cysA gene, and another is as yet unmapped. CYM mutants whose sites of mutation were located within the cysCD region lacked the relevant cysteine enzyme, PAPS synthetase, and, similarly, those in the cysl and J regions lacked sulfite reductase. Does the growth response of CYM mutants to methionine result from a sparing effect on their intracellular cysteine requirements (see Fig. 1) ? If this was so they should possess leaky cysteine enzyme deficiencies, but the PAPS synthetase or sulfite reductase levels in CYM mutants were similar to those of non-leaky cysC and D or cysI and J mutants, respectively. Also, in titration experiments, the minimum amount of methionine to support maximum growth both of a CYM mutant and wild-type organisms in sulfur-free minimal medium was the same. For the moment the conclusion is that these mutants possess neither normal structural nor regulatory properties ; perhaps mutation at certain sites in cysteine genes results in an enzyme requiring activation by methionine. IV. Conclusions

I n broad summary the pathways of synthesis of cysteine and methionine in E . coli and S. typhimurium and the way in which they relate to each other are well established. Large numbers of mutants with clearly identified structural gene defects and also some with regulatory defects are available. Although some groups of related structural genes are lo-

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cated close enough to be cotransduced there is limited clustering, only two pairs of the 17 known structural genes being actually contiguous. Some understanding of the regulation of the methionine and cysteine parts of the pathway has been obtained. I n addition to feedback controls, the synthesis of cysteine enzymes is under positive control and for the methionine enzymes as a whole, negative control has been demonstrated. I n neither case is this control coordinate. One cysteine and two methionine regulatory genes have been recognized ; the probable methionine aporepressor gene is adjacent to one of the methionine structural genes. There is no evidence that cysteine affects the synthesis of methionine enzymes and the effect of methionine on cysteine enzymes is probably an indirect biochemical one. The existence of mutants with cysteine structural gene deficiencies but able to utilize methionine as their sole source of sulfur and the effect of methionine on the specific inhibition of cysteine enzymes could indicate some other form of interrelationship. Future study of the systems as a whole requires that many deficiencies are made good. Although mutants of E. coli which lack components of the p-aspartokinase and homoserine dehydrogenase exist (Janin et al., 1967; Patt6 et al., 1967; Richaud and Cohen, 1968) no genetical analysis of them has been reported. Thus the relationship between the p-aspartokinase and homoserine dehydrogenase genes and those concerned with methionine (and, for that matter, threonine and isoleucine) synthesis has not been worked out. Sulfate-binder negative, O-acetylserine sulfhydrylase, and cysteinyl-tRNA synthetase mutants are required to make the collection of cysteine mutants fully comprehensive, but OAS sulfhydryalse mutants might well accumulate intracellular sulfide a t a toxic level so that they would be inviable (Jones-Mortimer, personal communication). I n relation to the later stages of methionine synthesis (and perhaps its overall regulation) S-adenosylmethionine (SAM) synthetase-deficient mutants of S. typhimurium should be identified or sought; they should be ethionine-resistant (Greene et al., 1970) a phenotype which would permit genetical analysis. As SAM is a methyl donor in the cobalamin-dependent methylation of homocysteine, assays of SAM synthetase in each of the different types of metH mutants might be profitable. With respect to the uptake of methionine, the isolation of mutants defective in the low affinity permease could be achieved by isolation of a frameshift, nonsense, or deletion metP (high-affinity permease) mutant, coupling it with a metE mutation so that the double mutant will respond to either methionine or cobalamin, and then isolating from this a mutant unable to utilize methionine (Ayling and Bridgeland, personal communication). At the purely genetical level the precise location of the metH site

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in the serA region of the S. typhimurium map and the metP gene in the leu-purE region is desirable and so is the position of the unmapped CYM sites of mutation. It should be noted that mapping work with cys mutants of E . coli and S. typhimurium has revealed further differences between them in gene sequence (Sanderson, 1970; Taylor, 1970). The relative positions of the cysP and Q genes in E . coli (equivalent to cysI and J in S. typhimurium) with respect to cysC appear to result from a translocation with or without inversion. With regard to regulation, the unequivocal recognition and study of mutants derepressed for cysteine synthesis seems a high priority, and a preliminary report of such mutants has appeared (Spencer et al., 1967). Further information may come from work with selenate- and triazole(trzA)-resistant mutants. A search for suitable analogs of cysteine to permit a similar approach to that made in the methionine system has not yet been successful (Jones-Mortimer, personal communication) . The role of the metJ and K proteins in methionine regulation needs to be clarified, especially against the background of apparently conflicting results concerning their relationship to SAM ; in particular, assays of SAM synthetase measuring K,,, and V,,,, should be carried out. An interesting extension of the work with metJ and K mutants could be the isolation of superrepressed mutants which would be more sensitive to inhibition by methionine analogs than wild-type organisms if, as seems to be the case, resistance to inhibition by the analogs is related to the intracellular concentration of methionine. Detailed knowledge of the regulation of the methylation of homocysteine is minimal. The part played by cobalamin needs further exploration, preferably with mutants altered in their response to this compound. Other potentially interesting approaches could be through further studies of the regulatory abnormalities of some metE and H mutants and a better understanding of the nature of the mutation resulting in stimulation of growth by serine and the accompanying effects of the intracellular concentration of C, units vital for methylation. Genetical studies of the homocysteine transmethylation in which methylmethionine and adenosylmethionine act as methyl donors and which is inducible by homocysteine (Balish and Shapiro, 1967) could add an extra dimension to this work. Perhaps the most outstanding requirement is for operator constitutive cysteine and methionine mutants but their isolation will require unique techniques as yet undevised although seriously contemplated. The recent successful isolation of such mutants in the arginine scattered gene system of E . coli is an encouragement and stimulus (Jacoby and Gorini, 1969; Karlstrom and Gorini, 1969). The CYM mutants are a puzzle and the results of current experiments

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designed to test methionine activation of altered cysteine enzymes to confirm or deny this hypothesis are awaited. In the context of the relationship between control of cysteine and methionine synthesis, it could be of interest to study the effect of coupling a methionine regulatory and a CYM mutation in the same organism (Jones-Mortimer, personal communication).

ACKNOWLEDGMENTS This review could not have been put together without the willing communication of unpublished results and comments on the manuscript by Drs. P. D. Ayling, E. Bridgeland, M. C. Jones-Mortimer, and Mr. M. A. Qureshi and Mrs. Julie Whitehouse of the Genetics Department, Birmingham University; Dr. A. C. Minson of the Virology Department, Birmingham University; Dr. K . F. Chater of the John Innes Institute, Norwich; Dr. R. J. Rowbury of the Botany Department, University College, London; and Dr. T. S. Gross, of the Genetics Department, Sheffield University; Dr. M. A. Foster and Mrs. Joan Dawes of the Microbiology Unit, Department of Biochemistry, Oxford University; and finally, Dr. D. Hulanicka, of the Institute of Biochemistry and Biophysics, Warsaw. I would like to thank all of them and hope that they feel that an appropriate commentary on enjoyable and fruitful collaboration has been achieved. Financial support by the Science Research Council, both in the form of a research grant to the author and Research Studentships to Ph.D. students, and by the Wellcome Trust for a Research Scholarship is also gratefully acknowledged.

REFERENCES Ames, B. N., Goldberger, R. F., Hartman, P. E., Martin, R. G., and Roth, J. R. 1967. The histidine operon. I n “Regulation of Nucleic acid and Protein Synthesis” (V. V. Konigsberger and L. Bosch, eds.) Elsevier, Amsterdam. Ayling, P. D., and Chater, K. F. 1968. The sequence of four structural and two regulatory genes in the Salmonella typhimnrium linkage map. Genet. Res. 12, 341-354.

Balish, E., and Shapiro, S. K. 1967. Methionine biosynthesis in Escherichia coZi: Induction and repression of methylmethionine (or adenosyl methionine) homocysteine methyltransferase. Arch. Biochem. Biophys. 119, 62-68. Bauerle, R. H., and Margolin, P. 1966. The functional organization of the tryptophan gene cluster in Salmonella typhimurium. Proc. Nat. Acad. Sci. US. 56, 111-118. Bccker, M. A,, Kredich, N. M., and Tomkins, G. M. 1969. The purification and characterization of 0-acetylserine sulphydrylase-A from Salmonella typhimurium. J . Biol. Chem. 244, 2418-2427. Bruton, C. J., and Hartley, B. S. 1968. Subunit structure and specificity of methionyl-transfer-ribonucleic acid synthetase from Escherichia coli. Biochem. J . 108, 281-288.

Cantoni, G. L. 1965 S-Adenosylmethionine revisited. In “Transmethylation and Methionine Biosynthesis” (S. K. Shapiro and F. Schlenk, eds.), Univ. of Chicago Press, Chicago, Illinois. Cauthen, Sally E., Foster, M. A., and Woods, D. D. 1966. Methionine synthesis by extracts of Salmonella typhimzirium. Biochem. J . 98, 63C635.

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Chater, K. F. 1969. Linkage and dominance studies of genes controlling methionine synthesis in Salmonella typhimurium. Ph.D. Thesis, University of Birmingham. Chater, K. F. 1970. Dominance of the wild-type alleles of methionine regulatory genes in Salmonella typhimurium. J. Gen. Microbiol. 63, 95-109. Chater, K. F.,and Rowbury, R. J. 1970. A genetical study of the feed-back sensitive enzyme of methionine synthesis in Salmonella typhimurium. J. Gen. Microbiol. 63, 111-120. Chater, K. F., Lawrence, D. A., Rowbury, R. J., and Gross, T. S. 1970. Suppression of methionyl-transfer RNA synthetase mutants of Salmonella typhimurium by methionine regulatory mutation. J. Gen. Microbiol. 63, 121-131. Childs, J. D., and Smith, D. A. 1969. New methionine structural gene in Salmonella typhimurium. J. Bacteriol. 100, 377-381. Clowes, R. C. 1958. Investigation of the genetics of cysteineless mutants of Salmonella typhimurium. J. Gen. Microbiol. 18, 154-172. Cohen, G. N. 1968. “The Regulation of Cell Metabolism” New York. Holt, New York. Dalal, F. R., and Gots, J. S. 1965. Glycine auxotrophs of Salmonella typhimurium. Bacteriol. Proc. p. 89. Davis, B. D., and Mingioli, E. S. 1950. Mutants of Escherichia coli requiring methionine or vitamin BIZ.J. Bacteriol. 80, 17-28. Delavier-Klutchko, C., and Flavin, M. 1965a. Role of a bacterial cystathionine p-cleavage enzyme in disulphide decomposition. Biochim. Biophys. Acta 99, 375-377. Delavier-Kutchko, C., and Flavin, M. 1965b. Role of a cystathionine @-cleavage enzyme in potassium transport. Biochim. Biophys. Acta 99, 377-380. Demerec, M., Gillespie, D. H., and Mizobuchi, K. 1963. Genetic structure of the cysC region of the Salmonella genome. Genetics 48, 997-1009. de Robichon-Szulmajster, H. 1967. RBgulation du fonctionnement de deux chaines de biosynthsse chez Saccharomyces cerevkiae. : threonine-methionine e t isoleucine-valine. Bull. SOC.Chim. Biol. 49, 1431-1462. Dreyfuss, J. 1964. Characterization of a sulphate and thiosulphate transporting system in Salmonella typhimurium. J. Biol. Chem. 239, 2292-2297. Dreyfuss, J., and Monty, K. J. 1963. The biochemical characterisation of cysteinerequiring mutants of SalmoneEla typhimurium. J. Biol. Chem. 238, 1019-1024. Ellis, R. J. 1966. Sulphur metabolism : the usefulness of N-ethyl-maleimide. Nature (London) 211, 1266-1268. Ellis, R. J., Humphries, S. K., and Pasternak, C. A. 1964. Repressors of sulphate activation in Escherichia coli. Biochem. J. 92, 167-172. Foster, M. A., Rowbury, R. J., and Woods, D. D. 1963. Control of the methylation of homocysteine in Escherichia coZi. J. Gen. Miciobiol. 31, xix. Galivan, J., and Huennekens, F. M. 1970. Resolution of the methionine synthetase system from Eschen’chia coli K12. Biochem. Biophys. Res. Commun. 38, 46-51. Greene, R. C., Su, Ching-Hsiang, and Holloway, C. T. 1970. S-Adenosyl methionine synthetase deficient mutants of Escherichia coli K12 with impaired control of methionine biosynthesis. Biochem. Biophys. Res. Commun. 38, 1120-1126. Gross, T. S., and Rowbury, R. J. 1969. Methionyl-transfer RNA synthetase mutants of Salmonella typhimurium which have normal control of the methionine biosynthetic enzymes. Biochim. Biophys. Acta 184, 233-236. Gross, T. S., and Rowbury, R. J. 1970. Biochemical and physiological properties of methionyl sRNA synthetase mutants of Salmonella typhimurium J . Gen. Microbiol. (in press).

E. coli

AND

S. typhimurium S-AMINO ACID

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163

Guest, J. R., Friedman, S., Foster, M. A., Tejerina, G., and Woods, D. D. 1964. Transfer of the methyl group from N5-methyltetrahydrofolates to homocysteine in E. coli. Biochem. J. 92, 497-504. Howarth, S. 1958. Suppressor mutations in some cysteine-requiring mutants of Salmonella typhimurium. Genetics 43, 404-418. Itikawa, H., and Demercc, M. 1967. Ditto deletions in the cysC region of the Salmonella chromosome. Genetics 55, 63-68. Jacoby, G. A., and Gorini, L. 1969. A unified account of the repression mechanism of arginine biosynthesis in Escherichia coli. I. The genetic evidence. J. Mol. Biol. 39, 73-87. Janin, J., Truffa-Bachi, D., and Cohen, G. N. 1967. Subunits of the complex protein carrying the threonine-sensitive aspartokinase activity in a mutant of Escherichia coli K12. Biochem. Biophys. Res. Commun. 26, 429-434. Johnson, D. B., Howells, D. J., and Goodwin, T. W. 1966. Observations on the biosynthesis of thiamine in yeast. Biochem. J. 98, 30-37. Jones-Mortimer, M. C. 1968a. Positive control of sulphate reduction in Escherichia coli. Isolation, characterization and mapping of cysteineless mutants of E. cox K12. Biochem. J . 110, 595-602. Jones-Mortimer, M. C. 196813. Positive control of sulphate reduction in Escherichia coli. The nature of the pleiotropic cysteineless mutants of E. coli K12. Biochem. J . 110, 597402. Jones-Mortimer, M. C. 1968c. Sulphur metabolism in micro-organisms. D. Phil. Thesis, University of Oxford. Jones-Mortimer, M. C., Wheldrake, J. F., and Pasternak, C. A. 1968. The control of sulphate reduction in Escherichia coli by 0-acetyl-L-serine. Biochem. J . 107, 51-53. Kamin, H., Masters, B. S. S., Siegel, L. M., Vorhaben, J. E., and Gibson, Q. H. 1967. Stopped flow studies of NADPH-flavoprotein interaction. Abstr. 7th Znt. Congr. Biochem, Tokyo pp. 187-188. Kaplan, M. M., and Flavin, M. 1966a. Cystathionne-ysynthetase of Salmonella. Catalytic properties of a new enzyme in bacterial methionine synthesis. J. Biol. Chem. 241, 4463-4471. Kaplan, M. M., and Flavin, M. 1966b. Cystathionine-y-synthetase of Salmonella. Structural properties of a new enzyme in bacterial methionine biosynthesis. J . Biol. Chem. 241, 5781-5789. Karlstrom, O., and Gorini, L. 1969. A unified account of the repression mechanism of arginine biosynthesis in Escherichia coli. 11. Application to the physiological evidence. J. Mol. Biol. 39, 89-94. Katzen, H. M., and Buchanan, J. M. 1965. Enzymatic synthesis of the methyl group of methionine. J . Biol. Chem. 240, 825435. Kredich, N. M., and Tomkins, G. M. 1966. The enzyme synthesis of L-cysteine in Escherichia coli and Salmonella typhimurium. J . Biol. Chem. 41, 49554965. Kredich, N. M., Becker, M. A,, and Tomkins, G. M. 1969. Purification and characterization of cysteine synthetase, a bifunctional protein complex, from SaZmonella typhimurium. J . Biol. Chem. 244,2428-2439. Lawrence, D. A,, Smith, D. A,, and Rowbury, R. J. 1968. Regulation of methionine synthesis in Salmonella typhimurium: mutants resistant to inhibition by analogues of methionine. Genetics 58, 473-492. Lazzarini, R. A., and Atkinson, D. E. 1961. A triphosphopyridine nucleotide specific nitrite reductase from Escherichia coli. J. Bwl. Chem. 236, 3330-3335.

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Lee, L. W., Ravel, J. M., and Shive, W. 1966. Multimetabolite control of a biosynthetic pathway by sequential metabolites. J. Biol. Chem. 241, 5479-5480. Lemoine, F., Waller, J. P., and van Rapenbusch, R. 1968. Studies on methionyl-transfer RNA synthetase from Escherichia coli K12. Eur. J . Biochem. 4,213-221. Mager, J. 1960. A TPNH-linked sulfite reductase and its relation to hydroxylamine in enterobacteriaceae. Biochim. Biophys. Acta 41, 553-555. Milner, L., Whitfield, C., and Weissbach, H. 1969. Effect of L-methionine and vitamin BS on methionine synthesis in Escherichia coli. Arch. Biochem. Biophys. 133, 413419. Mizobuchi, K., Demerec, M., and Gillespie, D. H. 1962. Cysteine mutants of Salmonella typhimurium. Genetics 47, 1617-1627. Mudd, S. H. 1965. The mechanism of the enzymatic synthesis of S-adenosylmethionine. I n “Transmethylation and Methionine Biosynthesis” (S. K. Shapiro, and F. Schlenk, eds.), Univ. of Chicago Press, Chicago, Illinois. Miiller-Hill, B., Crapo, L., and Gilbert, W. 1968. Mutants that make more lac repressor. Proc. Nut. Acad. Sci. U.S. 59, 1259-1264. Ozeki, H. 1956. Abortive transduction in purine-requiring mutants of &Salmonella typhimurium. Carnegie Inst. Wash. Publ. 612, 97-106. Pardee, A. B. 1966. Purification and properties of a sulphate-binding protein from Salmonella typhimurium. J . Biol. Chem. 2-41, 5886-5892. Pasternak, C. A. 1962. Sulphate activation and its control in Escherichia coli and Bacillus subtilis. Biochem. J . 85, 44-49. Pasternak, C. A., Ellis, R. J., Jones-Mortimer, M. C., and Crichton, C. E. 1965. The control of sulphate reduction in bacteria. Biochem. J. 96, 270-275. Patte, J. C., Le Bras, G., Loviny, T., and Cohen, G. N. 1963. Retroinhibition et repression de I’homoserine deshydrogenase d’Escherichia coli. Biochim. Biophys. Acta 67, 16-30. Pat%, J. C., Le Bras, G., and Cohen, G. N. 1967. Regulation by methionine of the synthesis of a third aspartokinase and a second homoserine dehydrogenase in Escherichia coli K12. Biochim. Biophys. Acta 136, 245-257. Piperno, J., and Oxender, D. L. 1968. Amino acid transport systems in Escherichia coli K12. J . Biol. Chem. 243, 5914-5920. Richaud, F., and Cohen, G. 1968. Selection of Escheiichia coli mutants devoid of one or both of the activities carried by a multifunctional protein. Biochem. Biophys. Res. Commun. 30, 45-49. Rowbury, R. J. 1964. The accumulation of 0-succinylhomoserine by Escherichia colz and Salmonella typhimurium. J . Gen. Microbiol. 37, 171-180. Rowbury, R. J. 1968. The inhibitory action of a-methylmethionine on Escherichia coli. J. Gen. Microbiol. 52, 223-230. Rowbury, R. J., and Woods, D. D. 1961. Further studies in the repression of methionine synthesis in Escherichia eoli. J . Gen. Microbiol. 24, 129-144. Rowbury, R. J., and Woods, D. D. 1964a. O-succinylhomoserine as an intermediate in the synthesis of cystathionine by Escherichia coli. J . Gen. Microbiol. 36, 341-358. Rowbury, R. J., and Woods, D. D. 1964b. Repression by methionine of cystathionine formation in Escherichia coli. J . Gen. Microbiol. 35, 145-158. Rowbury, R. J., and Woods, D. D. 1966. The regulation of cystathionine formation in Escherichia coli. J . Gen. Microbiol. 42, 155-163. Rowbury, R. J., Lawrence, D. A,, and Smith, D. A. 1968. Regulation of the methionine-specific aspartokinase and homoserine dehydrogenase of Salmonella typhimurium. J . Gen. Microbiol. 54, 337-342.

E. coli

AND

X. typhimuriuin S-AMINO ACID

SYNTHESIS

165

Sanderson, K. E 1970. Current linkage map of SaZmonelkz typhimurium. Bacteriol. Rev. 34, 176-193. Schlesinger, S. 1967. Inhibition of growth of Eschetichzn coli and of homoserine-0transsuccinylasc by a-methylmethionine. J . Bacteiiol. 94, 327-332. Sheppard, D. E. 1964. Mutants of Salmonella typhimzirium resistant to feed-back inhibition by L-histidine. Genetics 50, 611-623. Smith, D. A. 1961. Some aspects of the genetics of inethionineless mutants of Salmonella typhimiii iima. J . Gen. Microbiol. 24, 335-353. Smith, D. A., and Childs, J. D. 1966. Mcthionine genes and enzymes of Salmonella typhimuiizim. Heiedity 21, 265-286. Smith, R. C. 1968. Growth and nucleic acid synthesis of Salmonella typhimuiium inhibited by a-methylmethionine. Con. J . Microbiol. 14, 331-335. Spencer, H. T., Collins, J., and Monty, K. J. 1967. Sequential regulation of cysteine Exp. Biol. biosynthesis in Salmoiiella 1yphininrium. Fed. Pioc. Fed. Amer. SOC. 26, 677. Spizek, J., and Janecck, J. 1969. The effect of cthioninc on thc synthesis of p-galactosidase : formation of an immunologically cross-reacting protein. Biochem. Biophys. Res. Commun. 34, 17-21. Stadtman, E. R. 1963. Enzymc multiplicity and functions in the regulation of divergent metabolic pathways. BacterioZ. Rev. 27, 170-181. Sn, Ching-Hsiang, Grccne, R. C.. and Holloway, C. T. 1970. Regulation of S-adenosylmethionine synthctasc in Exherichin coli K12. Bacteriol. Proc. p. 136. Taylor, A. L., 1970. Current linkage map of Escherichia coli. BacterioZ. Rev. 34, 155-175. Taylor, R. T., and Weissbach, H. 1967a. Isolation of methyl-B,? from Escherichia coli B. N5-methyl-H4-folate homocyateine vitamin BI2 transmethylase. Biochem. Biophys. Res. Commun. 27, 398-404. Taylor, R. T., and Weissbach, H. 1967b. N’-Methyl tetrahydrofolate-homocysteine transmethylase. Partial purification and properties. J . BioZ. Chem. 242, 1502-1508. Trudinger, P. A. 1969. Assimilatory and disimilatory mctabolism of inorganic sulphur compounds by microorganisms. Advan. Microbial Physiol. 3, 111-158. Umbarger, H. E. 1969. Regulation of amino acid metabolism. Aiinu. Rev. Biochem. 38, 323-370. Weissbach, H., and Taylor, R. 1966. Role of vitamin BIZ in methionine synthesis. Fed. Proc. Fed. Amer. Soe. Exp. Bzol. 25, 1649-1656. Wheldrake, J. F. 1967. Intracellular concentration of cysteine in Escherichia C O Z ~ and its relation to repression of sulphate-activating enzymes. Biochem. J . 105, 697-699. Wheldrake, J. F., and Pasternak, C. A. 1965. The control of sulphate activation in bacteria. Biochem. J. 96, 276-280. Whitfield, C. D., and Weissbach, H. 1968. Binding of substrate to N‘-methyl-tctrahydropteroyl-triglutamate-homocysteinetransmethylasc. Biochem. Biophys. Res. Commun. 33, 996-1003. Whitfield, C. D., Steers, E. J., Jr., and Weissbach, H. 1970. Purification and properties of 5’-methyltetrahydropteroyl-triglutamate-homocysteine transmethylase. J . Biol. Chem. 245, 39M01. Wijesundera, S.,and Woods, D. D. 1962. The catabolism of cystathionine by Escherichia coli. J . Gen. Microbiol. 29, 353-366. Woods, D. D., Foster, M. A., and Guest, J. R. 1964. In “Transmethylation and Methionine Biosynthesis” (5.K. Shapiro, and S. Llenk, eds.), Univ. of Chicago Press, Chicago, Illinois.

MUTAGENIC AND LETHAL EFFECTS OF VISIBLE AND NEARULTRAVIOLET LIGHT ON BACTERIAL CELLS* A. Eisenstarkt I. Introduction . . . . . . . . . . . . . . . . 11. Comparison of Biological Effects Produced b y UV, NV-V, and PH-A 111. Mutation by NV-V. . . . . . . . . . . . . . . IV. Membrane Involvement . . . . . . . . . . . . . V. Photodynamic Action (Ph-A) . . . . . . . . . . VI. Sensitization with SBromouracil . . . . . . . . . . . VII. Internal Sensitizers . . . . . . . . . . . . . . . VIII. Possible Target Molecules (Chromophores) for NV-V . . . . IX. Photoreactivation . . . . . . . . . . . . . . . X. Relation of Light-Sensitive Chromophore to Recombination . . . XI. Groa-th Inhibition . . . . . . . . . . . . . . . XII. NV-V Effects on Transforming DNA and on Viruses . . . . . XIII. Action Spectra . . . . . . . . . . . . . . . . XIV. Significance . . . . . . . . . . . . . . . . . XV. Summary. . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . .

. . . . . . .

.

. . . . . . .

.

167 169 174 176 177 179 180 181 183 184 18.5 187 188 188 189 191

I. Introduction

Twenty-eight years ago, Hollaender (1943) focused attention on biological effects of light with wavelengths longer than those emitted by “germicidal” lamps. Although a number of observations had already been reported (Blum, 1941), Hollaender clearly demonstrated, in quantitative terms, that such wavelengths were lethal t o bacteria. The intervening years have offered little in clarifying the molecular mechanisms

* Abbreviations used : W-ultraviolet light (below 300 nm) . NV-V-near-ultraviolet and visible light (above 300 nm). ( I t is recognized that different NV-V wavelengths may produce a multitude of effects. All of these are lumped together in this review; hopefully, future research will sort these out and specific effects will be assigned to specific wavelengths.) PH-A-photodynamic action ; BU-5-bromouracil. PRE-photoreactivating enzyme. t Permanent address : Division of Biological Sciences, University of Missouri, Columbia, Missouri. 167

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A. EISENSTARK

involved in the killing and mutagenesis of bacterial cells by these longer wavelengths. Until a short time ago, the same lack of understanding might have been said of UV” research; but in contrast to NV-V studies, the lethal and mutagenic effects of UV had been described from many aspects in thousands of research papers. Recent discoveries with regard to both W and NV-V effects bring new attention to these older observations. Now that we are aware of pyrimidine dimer formation by UV, and the defined steps in DNA repair following UV, information may now be viewed in a more critical fashion. Perhaps the paucity of NV-V experiments, as compared to those with other radiations, reflects certain skepticisms of scientists, i.e., (a) whether wavelengths above 300 nm are really lethal, or whether the lamps that are used emit a stray, lower UV that escapes detection; (b) if the mode of death and/or mutation by NV-V were the same as by UV, whether there is a serious need to use the former in experiments; (c) whether the longer exposures necessary for experiments with NV-V may present difficulties for quantitative experiments; (d) since there is a huge literature on the effect of light in terms of energy transfer in biological systems (e.g., photosynthesis), and since this still is somewhat of a “blackbox” in terms of biochemical mechanics, whether one should be optimistic that studies of lethal effects of NV-V would lead to new molecular concepts; (e) in view of an extensive literature on photodynamic action of NV-V, whether a cellular component may be acting as an internal sensitizer in NV-V lethality; thus, it may be argued that there is nothing unique about NV-V effects that is not already known in more quantitative terms by the use of external sensitizers. The validity of some of these skepticisms will be examined in this review. This is a particularly appropriate time to focus attention on the effects of NV-V. Not only have important steps been taken recently in our understanding of W genetic damage and repair, but a number of new studies concerning NV-V effects have appeared. The observation in our own laboratory (Eisenstark, 1970) that recombinationless (rec) mutants are highly sensitive to NV-V, raises questions as to whether light chromophores may have roles in repair, recombination, and replication of chromosomes. The specific questions in need of attention may be stated as follows: (a) What biological effects produced by NV-V are the same as those produced by UV? What biological effects are distinctly different for the two? (b) What NV-V wavelengths are most effective for each of the biological alterations (i.e., what is the action spectrum for each of the effects) ? ( c ) What are the critical receptor molecules (chromophores) for NV-V in the cell? (d) How is each chromophore altered, and what

GENETIC DAMAGE BY VISIBLE AND

NEAR-UVLIGHT

169

is the energy chain to other molecules? (e) Is death and/or mutation due to a direct alteration of DNA, or does DNA change represent the end result of a chain of reactions? ( f ) If the DNA is altered, what are the photoproducts? Pyrimidine dimers alone? (g) When damage from light is repaired, is the repair mechanism identical to that of UV repair? (h) If NV-V death is actually a photodynamic action with an internal sensitizer, why are rec mutants more sensitive than wild-type? (i) Is recA strictly a fault in one of the enzymes of the repair process, or could recA mutants involve aberrant chromophores? These might either alter the energy chain, act as hypersensitizers, or as inhibitors of normal replication-recombination function [recB and recC are now known to have endonuclease deficiency, as reported by Oishi (1969) and Goldmark and Lynn (1970) ; also, recA could lack an inhibitor of these two]. II. Comparison of Biological Effects Produced by UV, NV-V, and PH-A

One of the first matters t o be settled is whether NV-V produces the same effect as UV. NV-V death might be the result of stray UV emitted from lamps, or perhaps NV-V and UV alter the same receptor molecules. If either is the case, then this review is comparatively trivial. If, on the other hand, NV-V is an effective lethal and mutagenic agent that strikes a critical macromolecule other than those altered by UV, it is important to identify the event and the molecules. This review will attempt to show that NV-V is indeed different from W, and to evaluate the involvement of certain biomolecules. The work of Webb and Lorentz (1970) supports our conclusions that NV-V produces biological alterations that are uniquely different from those produced by UV. Table 1 lists various observations following non-ionizing irradiation of bacterial cells; similarities and differences produced by UV, PH-A, and NV-V may be noted. Sensitization with BU will be listed under PH-A since the BU studies are few in number and superficially resemble those of PH-A, although it is recognized that the mechanism of action is very different. The list of differences between NV-V and UV (Table 1) warrants the impression that NV-V does something unique as compared to UV. It is difficult to evaluate earlier reports as to which of these differences is critically significant without going to the laboratory and repeating experiments under special conditions, especially the elimination of stray wavelengths. Nevertheless, from this inventory of the many ways that biological effects can be observed, one can now choose critical experiments that might lead to distinguishing the various molecular mech-

170

A. EISENSTARK

anisms involved in radiation damages. Not all of the biological effects listed in Table 1 will be discussed, since many of the observations are self-explanatory, although the significance may remain obscure. To settle the matter as to whether NV-V produces an effect different from UV, the most direct approach would be to identify the receptor molecule of NV-V and to determine its biochemical alteration. With UV, thymine dimers in the DNA are formed as a major photoproduct. To show that NV-V is different, the obvious answer would be to photoinactivate cells and to assay for thymine dimer and other photoproducts. When such an experiment was conducted (Ferron, 1971), photoproducts were formed after blacklight exposure with rf values in one dimensional chromotography similar to those of thymine dimers. If thymine dimers are formed by blacklight, per lethal unit, they are far fewer than the number produced by UV. There is convincing evidence (Table 1) that the lesion of NV-V differs from that of UV. Following UV, the DNA of recA mutants of Salmonella typhimurium and Escherichia coli undergoes rapid degradation (Howard-Flanders et al., 1969). This is not true for NV-V; for the same degree of killing, degradation does not exceed that of unirradiated cells (Ferron and Eisenstark, 1971). While we do not know the event that initiates degradation, a possibility is that a lesion in DNA is a starting site for nuclease activity. Since degradation does not occur for NV-V-treated cells, we may assume that the lesion is different; perhaps the nuclease has no recognition site a t which to start its activity. This difference between UV and NV-V confirms other evidence that degradation is a secondary effect, following the initial lethal event. There are other ways of preventing degradation of recA while still killing cells, including the stopping of protein synthesis and the irradiation of cells without replication forks (Horii and Suzuki, 1968; see also Cramp and Watkins, 1970, for DNA degradation in E . coli B/r). Also, double mutants of uvrA recA degrade their DNA far less than the r e d allele following UV (Ferron and Eisenstark, 1971; Horii and Suzuki, 1970). Interestingly recA recB double mutants do not degrade their DNA following UV exposure (Willetts and Clark, 1969; Goldmark and Lynn, 1970), probably because recB mutation makes the cell endonuclease deficient (see also Hertman, 1969). Following UV, single strand breaks are formed in rec cells, as revealed by alkaline sucrose density gradient centrifugation studies. Such breaks are not formed after rec S. typhimuriuin are exposed to NV-V (Ferron, 1971). The stricter oxygen demand for NV-V killing is an important indication that the biochemical events are different from W killing (Eisen-

TABLE 1 Comparison of Effects of Ultraviolet, Photodynamic Action, and Near-Ultraviolet-Visible Light * Observation

UV (218-300 nm)

DNA degradation in recA mutants Single-strand breaks in DNA

Yes (1) Yes (3) (76)

Mutation

Yes (4)

Oxygen demand for killing Growth inhibition (division delay) Liquid-holding recovery Photoreactivation

Low (23) Moderate (25, 26) Yes for rec (28) Yes (30, 31)

DNA synthesis rate Pyrimidine dimer formation Photoproducts other than T T dimers Induction of lysogenic cells Action spectrum

Slower (37) Yes (38) (38a) (74) Yes (69) (72) Yes Max 260 nm

Ability to support phage multiplicaYes, T2, T1 phage (45, 46) tion after host-cell irradiation Comparison of large and small phage Large phage more sensitive (47) Inactivation of transforming DNA Yes-not pH-dependent respond t o photoreactivating enzyme (PRE) (48)

PH-A

+

[BU UV-yes, 500 times greater than UV alone (3)] Yes (5-19) (71) High (6) Yes (27) No (32) [No with BU (33, 34)]

NV-V (300490 nm)

No (29) No (20) Yes (21, 22) High (24) Very high (20) (73) No for rec at room T (29) Yes, frozen cells (35) No, transforming DNA, room T (36)

No (39, 40, 41) Yes (42, 43, 44) depends upon absorption range of each sensitizer [BU-max. 313 nm (38)]

Yes (49, 50, 70)

Division delay, 300-380 nm (26) (75) Growth inhibition, 338 nm (25) (75) No, T2, T1 (45, 46) Small phage more sensitive (47) More sensitive at low p H ; does not respond to P R E (48)

TABLE 1 (Continued) Observation

UV (218-300 nm)

Shape of killing curve

Little shoulder (20)

Relative killing of recA and recB

recA more sensitive than recB

Linkage of DNA to protein Alteration of protein Salt toxicity Leakage of ions Target molecule (chromophore) Carotenoid protection Membrane damage

(29) Yes (52, 53)

No (20) No (55) Pyrimidine in DNA No (also X-ray) (58)

Fe-sensitization Sensitization to nitrous acid Thiol protection Yes (X-ray also) (61) Polyamine protection Lethality by irradiated medium Yes (63, 64) Interference with transcription tRNA loss of acceptor and inactivation More effective inactivation of growing vs. stationary cells Yes, moderate (24)

PH-A Both single and multiple hit curves (51) Yes (52) Yes (6, 54)

NV-V (300-490 nm) Long shoulder (20) Same sensitivity for recA and recB (29) Yes (20) Yes (55)

Guanine (15, 56, 57) Yes (58) Lysozyme permeability (59J 60)

Yes (61) Yes (62) Yes (11, 66)

* E

m M

3

2

E Yes (11) Yes (36) Yea (65)

Yes (67, 68) Yes, higher (24)

*Numbers in parentheses refer to the following references: (1) Buttin and Wright, 1968; (2) Ferron and Eisenstark, 1971; (3) Hutchinson and Hales, 1970; (4) Witkin, 1969; (5) Calberg-Bacq e t al., 1968; (6) Spikes and Straight, 1967; (7) Bohme and Wacker, 1963; (8) Zampieri and Greenberg, 1965; (9) Kubitschek, 1967; (10) Webb and Kubitschek, 1963; (11) Singer and FrankelConrat 1966; (12) DeMars, 1953; (13) Webb and Tai, 1969; (14) Mathews, 1963; (15) von Lochmann and Stein, 1964; (16) Brenner et al. 1958; (17) Ball and Raper, 1966; (18) Leff and Krinsky, 1967; (19) Richie, 1965; (20) Hollaender, 1943; (21) Webb and Malina, 1967; (22) Kubitschek, 1967; (23) Donini and Epstein, 1965; (24) Eisenstark, 1970; (25) Jagger et al., 1964; (26) Phillips et al., 1967; (27) Teresa et al., 1965; (28) Ganesan and Smith, 1969; (29) Eisenstark, unpublished; (30) Setlow, 1966; (31) Rupert and Harm, 1966; (32) Uretz, 1964; (33) Greer, 1960; (34) Stahl et al., 1961; (35) Brown and Webb, 1970; (36) Cabrera-Juarez, 1964; (37) Rupp and Howard-Flanders, 1968; (38) Grossman and Brown, 1969; (38a) Boyce and Howard-Flanders, 1964; (39) Newmark, 1965; (40) Van Vunakk et al., 1966; (41) Sastry et al., 1966; (42) Geissler, 1963; (43) Smarda, 1964; (44) Freifelder and Freifelder, 1966; (45) Dulbecco and Weigle, 1952; (46) Hill, 1956; (47) Wahl and Laterjet, 1948; (48) Cabrera-Juarez and Herriott, 1963; (49) Bellin and Grossman, 1965; (50) Bellin and Oster, 1960; (51) Harrison and Raabe, 1967; (52) Smith, 1962; (53) Smith, 1966; (54) Spikes, 1968; (55) Bruce, 1958; (56) Simon and Van Vunakis, 1962; (57) Wacker, 1963; (58) Mathews and finsky, 1965; (59) Allison e t al., 1966; (60) Slater and Riley, 1966; (61) Geissler, 1963; (62) Brendel and Winkler, 1966; (63) Wyss et al., 1948; (64) Chopra, 1969; (65) Lorentz and Webb, unpublished; (66) Chandrs and Wacker, 1966; (67) Tsugita et at., 1965; (68) Amagasa and Ito, 1970; (69) Setlow, 1968; (70) Ponce-De Leon and Cabrera-Juarez, 1970; (71) GabreraJuarez and Espinoza, 1970; (72) Donnellan and Setlow, 1966; (73) Jagger, 1970; (74) Setlow and Carrier, 1966; (75) Takebe and Jagger, 1969; (76) Davison and Freifelder, 1966.

u

P

F0 M

*td

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A. EISENSTARK

stark, 1970). This oxygen demand also provides an insight into the biochemistry of NV-V, as noted elsewhere in this review. Another indication that NV-V and UV lesions are different is the fact that transforming DNA of Haemophilus influenme can be reactivated with photoreactivation enzyme after UV, but not after NV-V inactivation (Cabrera-Juarez, 1964). Also, UV-treated host cells can still support growth of phages T2 and T1, whereas this is not so after treating cells with NV-V (Dulbecco and Weigle, 1952; Hill, 1956). This is a good example of the difference between the two types of radiation, but it also points out our lack of knowledge of some of the critical biochemical events following phage infection. A number of additional findings that distinguish UV from NV-V will be emphasized elsewhere in this review. However, the differences listed in Table 1, especially those of DNA degradation following irradiation, oxygen demand, and intracellular phage growth, permits the confidence that the two irradiations strike different chromophores. Ill. Mutation by NV-V

While Hollaender (1943) did not observe mutation by NV-V, several later studies describe increased mutation frequencies following exposures (Table 1 ) . We now need information from a molecular standpoint to gain some insight of the mechanism of NV-V mutagenesis. For example, if one of the events following NV-V is the exposure of single-strand gaps, there is now convincing evidence that one may anticipate an increase in frameshift mutations a t sites within this gap. This is examplified by increased proflavin mutagenesis of T4 phage with a ligase mutation in which joining of DNA pieces is impaired (Sarabhai and Lamfrom, 1969). An obvious test for single-strand gaps would be to NV-V irradiate a light-sensitive mutant containing a frameshift mutation. If singlestrand gaps are formed after NV-V, one would expect occasional corrections of the frameshift, and thus an observable increase in reversion rate. This test may not work for one particular (i.e., the recA lightsensitive) strain, since Witkin (1969) has evidence that recA strains cannot correct frameshifts. If this is correct, a more suitable test strain would be a recB or an hcr with a frameshift mutation. Although hcr mutants are not nearly as NV-V sensitive as rec, they are somewhat more sensitive than wild-type. On the other hand, excision of thymine dimers does not occur in hcr, thus there may be few single-strand gaps, and little correction of frameshifts. Still another view is that if NV-V does not produce frameshifts, this would suggest that it may not produce single-strand gaps in DNA.

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While awaiting insights as to the molecular mechanisms of NV-V mutagenesis, i t is interesting t o note experiments that lead to the conclusion that NV-V wavelengths are indeed mutagenic. These include the work of Kubitschek (1967) , who found that T5-resistant mutants were produced more frequently by NV under aerobic conditions and showed that the mechanism of mutation is different from that of UV. Webb and Malina (1967) presented similar results in which their exposures were restricted to visible light with wavelengths above 400 nm. An interesting aspect of NV-V mutagenesis is that mutation is much higher if E. coli B cells are irradiated while they are dehydrated (Webb and Tail 1969). These investigators (Webb and Tai, 1969; Webb and Bhorjee, 1967) state that light may prevent association between DNA and certain amino acids in protein involved +ith DNA replication and other DNA functions. As expressed elsewhere in this review, one protein candidate might be ligase, which requires NADH (absorption = 340 nm) for activity, and this NADH could be altered by NV-V. Cells are particularly susceptible to NV-V when they are frozen at -2OoC, (Ashwood-Smith, 1965; Zetterberg, 1964) , but no explanation is offered. It should be pointed out that in the case of frozen and dehydrated samples, the action of 254 nm germicidal UV differs from that of NV-V, further evidence that the two act via different mechanisms. Mutagenic action of blacklight is most effective a t relative humidity of 55-65% where the water content is between 15-20%. I n these experiments (Webb and Tail 1969), the number of auxotrophic mutants was scored after irradiation and penicillin screening. The papers by Webb and associates discuss many interesting aspects of the lesions produced by UV and NV-V under conditions of limiting water. It should be emphasized that light-sensitive mutants are defective in some aspects of chromosome function. It is reasonable to anticipate, therefore, that they might have higher, or lower, mutation rates than wild-type, depending upon the nature of the impairment. The above cited case of reduced frameshifts in recA mutants (Witkin, 1969) is a good example. Other cases include radiation-sensitive E . coli ras mutants (Walker, 1969) with greatly increased UV mutability (frameshift?) , a uvr Proteus mirabilis (Bohme, 1967) , a uvr Neisseria meningit i d h (Jyssum, 1968) , and an X-ray sensitive yeast (von Borstel et al., 1968) with increased mutability. We have screened about 60 Salmonella radiation-sensitive mutants (Eisenstark et al., 1969) for streptomycinresistance frequencies. We found no general pattern, but two hcr mutants, out of 30 examined, were high in mutability, as well as two uvr Salmonella anatum cultures (Eisenstark, unpublished). Interestingly, 2 of 15 rec+ revertants of recA S. typhimurium were highly mutable. The reason for

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testing revertants was that phenotypic revertants might represent an overcompensation of the mutant recA defect, and thus lead to excess mutability. It would be interesting to test whether NV-V can induce deletions. In E. coli, deletions can be tested by scoring for phage T1 and T 7 resistance (these become pro) and colicine B and V resistance. Such phenotypes contain deletions, and occur spontaneously (Anderson, 1970). Surprisingly, these deletions are found in equal frequency in wild-type, recA, recB, and recC mutants. Since deletions are thought to occur after a recombinational event, these results are difficult to explain. Induction of lysogeny, also considered a recombinational event and formally a type of deletion, does not occur in recA mutants (Eisenstark et al., 1969). IV. Membrane Involvement

Many cellular chromophores are known to reside in membranes. This has been intensively studied in mitochondria1 membranes where enzymes involved in energy transfer form integral parts of the structure. Also, since bacterial DNA is bound to cell membrane fractions (Ganesan and Lederberg, 1965), it is conceivable that a chromophore may have a role somewhere in the course of chromosome function (i.e., replication, transcription, repair, or recombination). As may be seen in the discussion of repair synthesis, Olivera et al., (1968) have shown D P N involvement in ligase activity. D P N may be located in cell membranes. How would one assay for a role of a chromophore in membrane-DNA interaction? If the chromophore role involves the binding of DNA and membrane, an obvious experiment would be to expose cells to NV-V and to look for quantitative alteration of this DNA-membrane binding. It is conceivable that this could go in either direction: If excess light cripples the chromophore, the membrane could lose its binding force, or, conversely, the membrane might fail to release a particular chromosomal attachment point after completing its function. We have tested this in our laboratory but our preliminary experiments have shown no effect of NV-V on either light-sensitive or wild-type strains (Ferron, unpublished). Another approach would be to test mutants that have an aberrant chromophore, assuming that these may reside in the membrane. There are some E. coli candidates for these, including, ubiA, ubiB, and ubiD (unable to form ubiquinone) (Cox et al., 1968; Cox et al., 1969), to& and chr2 (iron-transport mutants) (Wang and Newton, 1969a,b), hemA, B (hemin) (Sasarman et al., 1968, 1970; Wulff, 1967; Sanderson and

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Hamplova, 1970), and nad, A , B , C (nicotinic acid requirement) (Gholson et al., 1969; Olivera et al., 1968). As yet, there are no reports of altered membrane functions in these mutants. Niacin mutants of E. coli and S. typhimuriunz have been tested for light and UV sensitivity and were found to be negative (Ferron and Eisenstark, unpublished data). The membrane fraction contains DNA polymerase activity (Ganesan and Lederberg, 1965) which could have a chromophore involvement. (It is interesting to note the skepticism in this paper as to whether this polymerase is really involved in replication. See deLucia and Cairns, 1969, for further doubt.) The fact that NV-V is most effective during chromosome replication (Eisenstark, 1970) gives some support to chromophore-polymerase-membrane interaction. The complexity may be even greater when one considers the possibility of a larger unit (analogous to a ribosome) that might exist a t the replication fork. Such an organized unit might consist of a number of cofactors and enzymes, i.e., polymerase, nucleases, a 32-like protein (Alberts and Frey, 1970), and ligase. If one is looking for membrane involvement in NV-V effects, the membrane-bound cytochromes might be examined. O’Hara (1969) has found mutants of E . coli that produce an excess of certain cytochromes, up to tenfold, but it is not known whether these are light sensitive. Another approach would be to look specifically for membrane mutants. Inouye and Guthrie (1969) reported a temperature-sensitive mutant of E. coli that has altered membrane activity attributable to the absence of a membrane protein when cells are grown at the non-permissible temperature. However, this mutant is not radiation sensitive. Another mutant (Azoulay et al., 1967) results in alteration of respiratory particles in E. coli; these particles were shown to reside in membranes. It is not known whether these mutants are radiation sensitive. Mutants that are resistant and tolerant to colicines (tol) appear to have impaired membranes (Bhattacharyya et al., 1970); again it is not reported whether any of these are radiation sensitive. I n PH-A, destruction of membrane-associated enzyme has been noted (Mathews and Sistrom, 1959). Also, PH-A alters membrane permeability in Euglena (Bellin and Ronayne, 1968). V. Photodynamic Action (Ph-A)

Research dealing with light sensitivity of cells, following the addition of dyes, receives periodic comprehensive reviews (Spikes and Straight,

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1967; Spikes and Livingston, 1969; Harrison, 1967) ; therefore, this subject will not be discussed in detail here. PH-A is mutagenic as well as lethal, as cited by these reviewers and by Calberg-Bacq e t al. (1968). Inasmuch as natural internal sensitizers, in lieu of external dyes, may permit the same photodynamic action in growing bacterial cells, certain aspects of this phenomenon will be examined. Upon viewing the list of observations of photodynamic action (Table l ) , it is interesting to note any parallels with NV-V effects without external sensitizer. This gives some indication that an internal, natural sensitizer may account for Nv-V effects. To support this attitude, it should be noted that photodynamic action is observed when sensitizers are added to viruses, subcellular structures, and biomolecules. While NV-V effects on these subcellular components, without sensitizer, may not be absolute zero (see section dealing with viruses and transforming principle, pp. 177-178), the effect of NV-V is greatly reduced when transforming DNA and viruses are treated in vitro as compared to the effect of intracellular irradiation. On the surface, there appears to be a striking parallel between the oxygen demand in PH-A and in NV-V. This impression is emphasized by agreement among photobiologists that the term (‘photodynamic action” is to be restricted to oxygen-involved sensitizations. There are photosensitizers, however, such as psoralens, that do not require molecular oxygen and are actually inhibited by oxygen (Spikes, 1968). The concept that cells may have natural photosensitizers dates back to the first experiments in photodynamic action when Raab et al. (1901) first sensitized paramecia with acridine. Early investigators recognized the similarity of added dyes to natural pigments involved in vision, phototropism, photosynthesis, and photomorphogenesis. The photochemistry of PH-A has been reviewed in detail (Spikes and Livingston, 1969; Spikes and Straight, 1967; Spikes and Ghiron, 1964). Among the interesting observations cited are the effects on a large number of proteins and enzymes, including antisera, some antigens, angiotensinamide hormones, insulin, ricin, toxins, collagen, and fibrinogen. Upon examination, most of these inactivations are attributed to alteration of histidines in the proteins. Purines, pyrimidines, nucleosides, and nucleotides may also be sensitized, and these are in need of special attention because of their possible genetic involvement. In addition to dyes, natural products such as riboflavin and hematoporphyrin, are effective sensitizers. The guanine seems to be particularly reactive (Simon and Van Vunakis, 1962), as found after exposure of bacterial and T 4 DNA. The same is true of tobacco mosaic virus RNA (TMV-RNA) (Singer and Fraenkel-Conrat, 1966). Enzymes do not degrade DNA as effectively after PH-A (Dellweg

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and Opree, 1966), and poly UG is not degraded as well by ribonuclease T1 (Simon et al., 1965). A number of physical-chemical alterations of DNA and RNA have been noted after PH-A, including loss of viscosity (Bellin and Yankus, 1966), depolymerization with breaks in both strands (Freifelder et al., 1961), decrease in sedimentation coefficient of phage T4 DNA (Simon and Van Vunakis, 1962), but not poly UG (Simon et al., 1965). Other effects include 9576 loss of infectivity of TMV-RNA without any change in sedimentation rate (Sastry et al., 19661, suggesting that loss of biological activity does not require scission of the ribosephosphate backbone of RNA. This is supported by the same observation, i.e., loss of biological activity without physical change, in the DNA of transforming principle (Bellin and Grossman, 1965). PH-A decreases heat stability of T4 DNA (Simon and Van Vunakis, 1962). Smith (1962) feels that PH-A causes crosslinks of DNA with protein, as evidenced by decrease in extractability of DNA from cells. Chandra and Wacker (1966) noted that PH-A of poly UG destroys its messenger activity in in vitro synthesis. Among tRNA types, PH-A is most effective in the inactivation of phenylalanine acceptor activity (Tsugita et al., 1965). Although there are additional similar studies, it appears from all of these reports that the main action of PH-A is on the guanine of DNA or RNA. It should be pointed out that there are striking differences between various sensitizing dyes (Simon e t al., 1965). Acridine orange and methylene blue appear to be very different in their effects on DNA and on bacterial membranes. Acetophenone and acetone absorbs in NV and transfers excitation energy to the DNA. This permits Meistrich et al. (1970) to produce thymine dimers selectively without the other UV photoproducts. VI. Sensitization with 5-Bromouracil

While BU differs from other sensitizers in the important way that it becomes an integral part of the DNA, rather than as an adjuvant, it is convenient to consider its photochemistry along with photodynamic action. When 5-bromouracil is substituted for thymine in DNA, the cell becomes sensitive to NV-V, as well as becoming hypersensitive to UV. The maximum peak for the NV-V effect is 313 nm, but visible wavelengths are also lethal. The photochemistry involved is still not understood. An obvious approach would be to identify the photochemical products, as yet undefined. Except for the observation that uracil may be found after illumination (Wacker et al., 1962), no thorough study

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has been reported, as is not the case with W. A further complication is the report that the complementary strand (the one that does not contain BU) may also be damaged after illumination (Eisenberg and Pardee, 1969). A particularly interesting observation is that BU may block transcription rather than to interfere with replication (Menningman, 1967). Why this should lead to drastic lethality, rather than being limited to growth inhibition, is obscure. However, rifamycin, an antibiotic that blocks the initiation of transcription, is also a lethal agent, rather than merely a growth inhibitor. An interesting observation with regard to BU sensitization was made by Webb and Tai (1969). Apparently a certain amount of intracellular water is necessary, otherwise sensitization does not occur. They express the view that water molecules bound to the DNA take part in a photostimulating hydrolysis of BU. Inositol inhibits completely the sensitization by BU, apparently by taking over the role of water in the overall structure of DNA in the dehydrated cell. VII. Internal Sensitizers

It is possible that light-sensitive mutants may contain an excess of a substance that acts as an internal sensitizer in much the same way that acetophenone or a dye transfers light energy to damage DNA (Roth, 1967a,b). There are a number of candidates for natural photosensitizers in bacteria, including carotenoids, porphyrins, cytochromes, cytochrome oxidase, nicotinamide-adenine dinucleotide (NAD) , reduced form of NAD (NADH) , flavins, heme proteins, and other pigmented components. Also, a sensitizer could be a substance that is present in trace quantities so small as to be overlooked in normal analyses, but which still could be critical in energy transmission after illumination. Many species of bacteria are fluorescent, probably due to porphyrins, and in one study (DhkrB et al., 1933), it was suggested that fluorescent bacteria are unusually sensitive to light. Chlorophyll is a derivative of porphyrin and a component of photosynthetic bacteria. Colorless mutants of Rhodopseudomonas spheroides, which lack carotenoids (but not chlorophyll) are highly sensitive to light under aerobic conditions (Sistrom et al., 1956). It is suggested that, in the wild-type, carotenoids prevent the photodynamic sensitization of cells by chlorophyll. Since the mutants lack carotenoids, sensitization occurs a t a high level. Dworkin (1960) suggested that the cell membrane is the site of the

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lethal action. There is another example in which carotenoids may protect against photosensitization. Colorless Sarcina Zutea are killed by light when cells are aerated (Mathews and Sistrom, 1959; Mathews, 1964). In still another case involving a Mycobacterium (Wright and Rilling, 1963), it is uncertain whether the protective pigment is a flavin or a porphyrin. There are a number of interesting details with regard to the lethal effects of light on a respiratory-deficient mutant of the yeast, Saccharomyces cerevisiae (Elkind and Sutton, 1957). They attributed death to a step in the fermentation process, and noted that the mutant formed a new pigmented compound, a possible photosensitizer. Further support for the idea of a photosensitization by a carotenoid comes from the experiments of Burchard and Dworkin (1966), who found light sensitivity of Myxococcus xanthus only under those growth conditions that permitted carotenoid formation. It is interesting to note their comment that the protoporphyrin X I resides in the cell wall-membrane and may be the site of damage. If an internal sensitizer accounts for increased photosensitivity in rec mutants, one should look for an increase of a normal sensitizer, or the synthesis of a new one. I n the case of cytochromes, such E . coli mutants have been found (O’Hara, 1969). Certain cytochromes are formed in as much as tenfold excess, but it is unknown whether there is any change in light sensitivity. It should be noted that NADH absorbed 330-380 nm wavelengths. Its intimate association with the chromosome in replication (Olivera et al., 1968), would make it a candidate as an internal sensitizer in photodynamic action. VIII. Possible Target Molecules (Chrornophores) for NV-V

In choosing a chromophore candidate that might be involved in light sensitivity, perhaps a key paper is that by Epel and Butler (1969) who showed that cytochrome a3 ( a component of cytochrome oxidase) is destroyed by light. In the colorless alga Prototheca zopfii, this destruction of cytochrome a3 by blue light results in inhibition of respiration, cell division, protein synthesis, and nucleic acid synthesis (Epel and Krauss, 1966). They favor the idea that respiration inhibition is the primary effect and the others are secondary consequences. This is supported by the critical oxygen dependence of the phenomenon, since M cyanide protects cells. anaerobic conditions of 2.5 X Is i t safe to dismiss DNA as a chromophore for NV-V? The only support comes from NV-V inactivation of transforming principle

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(Cabrera-Juarez, 1964) and phage (Eisenstark and Ruff, 1970) (see Section XII). It is difficult to rule out the presence of small qualities of contaminating NV-V absorbers, as well as stray, shorter wavelength emissions from fluorescent lamps. In fact, we have found blacklight lamps to have a number of contaminating lines (Ferron and Eisenstark, 1971). Also, there is considerable indication that DNA is not a chromophore. First of all, its absorption spectrum is reduced to almost (but not quite) zero a t 320 nm (Ferron and Eisenstark, 1971). There is no photoprotection of H . influenme transforming principle and phage T 2 DNA (Jagger et al., 1964), although it has been noted that T2-E. coli complexes are sensitive to NV-V (Dulbecco and Weigle, 1952). Although photochemistry has not fully answered the question as to whether DNA can be the direct target of NV-V, the experiments of Ferron (1971) make this doubtful. He finds no single strand DNA breaks, nor protection by DNA solutions, in NV-V inactivation experiments of rec S. typhimurium cells. McLaren and Shugar (1964) point out that, a t 280 nm a t room temperature, nucleic acids in alkaline solution develop a yellow or yellow brown color. This may be the result of fragmentation of the sugars in DNA, giving about 1% absorption above 300 nm. T2 phage at 5.5 X 101O/mlexhibits considerable scattering between 320 and 450 nm, and one wonders if some absorption, albeit very small, might occur. Of course, it must be kept in mind that NAD has known photochemical behavior, with an absorption band of the NADH form a t 340 nm. While this is not part of DNA, the similarity of a portion of its chemical structure and its intimate association with DNA make it an interesting molecule. Results from our own laboratory (Eisenstark and Yoakum, unpublished) prejudice us to favor NADH as a key target molecule in W - V inactivation. As already noted, its maximum absorption peak is 340 nm. When it is added to growing cells, blacklight exposure results in greatly increased lethality, whereas its oxidized form, NAD, protects cells from NV-V. An excess of NADH could explain the hypersensitivity of recA mutants, but we are still uncertain as to how much NAD and NADH enter the cell. McLaren and Shugar (1964), in their detailed discussion of photochemistry, indicate that proteins, particularly, could be involved in NV-V effects. The phenol ring of tyrosine, the endol of tryptophan, and the phenyl of phenylalanine, as well as cysteine in alkaline pH, absorb at 300 nm or above. Leucyltyrosine has good absorption a t 320 nm. When proteins are irradiated at 340 nm, excitation of fluorescence occurs, suggesting the existence of low-intensity absorption bands.

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Recent experiments in our own laboratory (Yoakum and Eisenstark, unpublished) have focused on photoproducts of tryptophan following NV-V exposure. This study was initiated after Lorentz and Webb (in preparation) discovered that nutrient agar plates stored under room lights lose the competence t o yield colonies upon plating of rec cells, although there is no effect when wild-type cells are plated. Our tracking of the plating ingredients implicates a tryptophan photoproduct as the toxic agent for rec cells. Other reports of photoproducts of amino acids and proteins should also be noted (Weil et al., 1951; Bellin and Entner, 1966; Zieve and Solomon, 1966; Paiva and Paiva, 1961). A recent paper by Swenson and Setlow (1970) describes the NV-V inhibition of induction of tryptophanase in E. coli. They conclude that the reaction is photochemical rather than enzymatic since it is greater at 5°C. The peak of activity is a t 334 nm, which is the same as for growth delay and other NV-V effects. An interesting aspect of this study is the extreme sensitivity of this inhibition; more sensitive, in fact, than growth, respiration, and p-galactosidase inhibition. Also, the investigators show that this is not a general inhibition of metabolism. They suggest that pyridoxal phosphate may be the target for the NV since it absorbs a t 334 nm and is a cofactor for tryptophanase. IX. Photoreactivation

The one thing that the phenomenon of photorecovery following UV clearly emphasizes is that the bacterial cell contains molecules that, at least in some forms, interact with NV-V. This subject has been reviewed extensively (Rupert and Harm, 1966; Setlow, 1966), but the following is pertinent to the present discussion. There is more than one post-UV photorecovery phenomenon, and, therefore, more than one chromophore may respond to light. One type of recovery, designated as photoreactivation, involves an enzyme that utilizes thymine dimers as substrate (Setlow and Setlow, 1963). The absorption range for the photoreactivation enzyme (PR) is 300-500 nm. Since this is not the range for a purified protein, one suspects that the chromophore may really be an impurity of the P R enzyme. A peak at 385 nm seems to be characteristic of bacterial P R enzyme (Jagger et al., 1969), which suggests the possibility of a flavin involvement, since flavins have similar absorption peaks. A second type of photoreactivation (Jagger et al., 1969) has an action spectrum peak a t 340 nm, but does not involve the splitting of thymine dimers. It may operate by delaying growth and thus allowing repair

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in a manner similar to liquid-holding recovery (Ganesan and Smith, 1969). This is a revealing point. It indicates that a chromophore with 340 nm absorption is intimately involved with growth, and possibly with DNA replication. A clarifying aspect of the experiments by Jagger et al. (1969) is their use of a mutant ( p h r ) that is missing the PR enzyme, thus separating the two effects. There may be still other types of photorecovery mechanisms as indicated from literature descriptions of phenomena and action peaks (e.g., 313 nm) that do not fit the above two observations. It is unknown whether another target molecule might be involved (Ikenaga et al., 1970; Jagger et al., 1970; Patrick, 1970). Damage by 365 nm irradiation may be photoreactivated, as observed in cells irradiated at O°C (Brown and Webb, 1970). This implicates some dimer formation; however, it does not eliminate the presence of other photoproducts toxic to cells, but where the damaged cell may still be photoreactivated. X. Relation of light-Sensitive Chromophore to Recombination

While a chromophore of W - V lethality has yet to be clearly identified, it is a reasonable assumption that components involved in energy transfer (respiration or phosphorylation) are likely candidates (Wang, 1970). We come to the interesting question of why rec mutants are particularly light sensitive. The answer for recA is not clear, but it would appear that initial experiments should try to discriminate between the following: (1) The possibility that recA mutants possess an aberrant chromophore so that a vital (energy-transfer?) function of DNA replication and recombination does not occur. This aberrant chromophore may be easily crippled by NV-V, to cause death to the cell. (2) A second possibility is that the chromophores are perfectly normal, but that a DNA enzyme, i.e., polymerase, nuclease, or ligase, is abnormal ; when the chromophore is altered by light, the DNA or the enzymes are unable to participate in the normal interaction with the chromophore. (3) That the chromophore merely acts as a transmitter of energy, as in photodynamic action with a dye, to damage the DNA. In this case, the chromophore need not have a direct role in DNA replication or recombination. The damaged DNA of the cell cannot be repaired in recA cells, whereas this is possible in wild-type. I n either of the cases, it would be interesting to know the possible interactions of chromophores, DNA, enzymes, and any other molecule involved in chromosome replication, repair, replication, and transcription. To discuss this, perhaps we might list the enzymes known to be involved

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in these chromosome functions: (1) an endonuclease (nickase) that recognizes an aberration in the linear DNA (thymine dimers or perhaps only a physical or spatial irregularity) and proceeds to break a bond in one of the two chains, (2) an exonuclease that chews away a segment of one DNA strand, (3) a polymerase that restores a strand complementary to the remaining DNA template, and (4) a ligase that joins or seals the new and old DNA by catalyzing the synthesis of a new phosphodiester band between the 5’-phosphoryl and 3’-OH termini of the two DNA chains (Olivera and Lehman, 1967a,b). In any consideration of recombination mechanism, Oishi’s (1969) finding that ATP-dependent nuclease activity is lacking in E . coli recB and recC mutants must be recognized. Also, the purified enzyme and preparation from wild-type cells has DNA-dependent ATPase activity, indicating a multistructured unit. It would appear that nuclease activity is accompanied by an energy-requiring configurational change of the substrate DNA, and it is here that a chromophoric molecule could be involved (Goldmark and Lynn, 1970). These authors also suggest that recA+ codes for an inhibitor of a nuclease (coded by recB+ and recC+), but this inhibitor has not yet been identified. XI. Growth Inhibition

While NV-V effects on bacterial growth are described in numerous papers (e.g., Epel and Krauss, 1966; Jagger, 1967, 1970; Dolphin, 1970), the term “growth inhibition” is poorly defined. In most experiments, the assay method is to score the delay in time for new colony-forming units t o appear following NV-V exposure; i.e., a cell has failed to divide at the time expected had it been growing in the dark, but it finally does divide properly at a later time. This failure, however, could be a t one of several levels. Did the new chromosomal strands fail to elongate? Was there a respiration failure? Did a division-plate in the cell-wall fail to form? All of these could occur, of course, upon NV-V exposure, since these are events in cell growth that are intimately geared to each other, but what was the NV-V-interaction to trigger the growth delay? One must also distinguish between “division delay” and the mere appearance of delay that might be the result of death of a small population of cells (too small to show on colony counts, and not a t all on turbidity measurements or coulter counts, or microscope counts). I n this case, a growth curve following NV-V of cells might give the appearance of a delay that could be confused with division delay. It is obvious that respiration is braked by NV-V, and could be the

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major cause of division delay. The interesting experiments by Kashket and Brodie (1962) indicated that the site of NV action (365 nm) is electron transport to oxygen (oxidative respiration) , and that this is tightly coupled to phosphorylation (Wang, 1970; Brodie and Ballantine, 1960)- In this coupling of respiration to oxidative phosphorylation, especially with regard to NV-V, an important item to note is that either the respiration system or the phosphorylation system is difficult to study in isolation because of the coupling of the two. I n fact, if an itgent slows one, the other system speeds up to equilibrize the two. However, NV-V may damage the coupling mechanism of the two, and thus has a drastic effect (see Jagger, 1970; Lakchaura, 1970; Murtie and Brodie, 1969). While the observed growth inhibition may be triggered by NV-V striking a molecule in the respiration-phosphorylation system, it is important to know whether the next step in division delay is a stoppage of chromosome growth or a failure to start the septum that will eventually separate the two daughter cells. When the effect of NV-V on chromosome growth (Ferron, is measured by noting the rate of uptake of t h ~ m i d i n e - ~ H 1971), DNA chain growth stops at doses well below any lethality. Phillips et al. (1967) linked NV-V growth delay with the photoprotection from UV death that they observed. Either before or after W, NV (300-380 nm) was found to increase survivors. This was attributed to the delay in cell growth; during this delay, repair of UV damage could take place (Rupert and Harm, 1966; J. K. Setlow, 1966; R. B. Setlow, 1968). This information fits a number of other observations, including liquid-holding recovery (Ganesan and Smith, 1968, 1969), in which growth delay rescues the cell. But this growth delay, in terms of our present understanding, really means braking of chromosome growth. Death may occur when the synthesis of new DNA hits a DNA lesion; if this synthesis can be slowed down, there would be an opportunity for repair before the growing fork of DNA reaches the UV lesion. This photoprotection by NV-V is an indication that inhibited chromosome growth is an important aspect of the protection. A convincing aspect of the association of photoprotection with liquidholding recovery is the fact that NV-V does not photoprotect E . coli B,-,, B UV-sensitive strain that probably does not have the property of liquid-holding recovery (Jagger et al., 1964). Also, T2 phage DNA following UV is not photoprotected when injected into E. coli B. Phillips et al. (1967) showed that delay in growth of the size of the cell following UV was coupled with division delay; were they not coupled, cells could grow in size although division had ceased. This was not the case as shown by careful monitering of both cell size and cell

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number with a Coulter counter. A similar experiment should be done with NV-V. Recombinationless (rec) mutants of bacteria are sensitive to both nitrosoguanidine and visible light (Eisenstark, 1970; Eisenstark et al., 1969); note that both appear to strike a t the chromosomal growing point (Cerda-Olmeda and Hanawalt, 1968; Fujisawa, 1970; Webb, 1970). An extensive literature has developed with regard to filament formation following irradiation. It has been observed many times with UV, but results with NV-V are not consistent (Jagger et al., 1964; Phillips et al., 1967). It is interesting to note that, not only does NV-V inhibit the growth of bacterial cells, but it also inhibits HeLa cells a t 360 nm (Klein and Edsall, 1967) and parthenocissus crown-gall tissue cultures (Klein, 1964) . MI. NV-V Effects on Transforming DNA and on Viruses

NV-V inactivation of transforming DNA from H . influenme (Cabrera-Juarez, 1964) and phage (Eisenstark and Ruff, 1970) brings up a critical question as to whether wavelengths above 300 nm have any effect on nucleic acid, phage protein, and any other component that might be present. It is possible that a small amount of stray, under300-nm radiation is responsible for the lethal action, especially since blacklight lamps emit a line at 268 nm (Ferron and Eisenstark, 1971). Cabrera-Juarez (1964), in his NV inactivation studies, introduced a naphthalene solution which is assumed to absorb all wavelengths below 310 nm, including the 268 nm line. Interestingly, inactivation of transforming DNA was not reduced. This was followed by a neat experiment in which NADH was used as a filter, which reduces transmitancy 50% between 300 and 380 nm. As a result of the NADH, the effective NV dosage was cut in half. Assuming the accuracy of the filtering effect of these solutions, these experiments provide evidence that, if inactivation is the result of stray, low wavelengths from these lamps, they are not below 300 nm, and probably not below 310 nm. Further indication that inactivation is not due to wavelengths below 310 nm is the fact that photoreactivating enzyme is not effective as a recovery device after blacklight illumination, whereas this enzyme is effective when UV (254 nm) is used (Cabrera-Juarex and Herriott, 1963). On the surface, this does not fit the observation of Brown and Webb (1970) that blacklightexposed E . coli cells may be photoreactivated. However, there are two important differences : whole cells, rather than transforming DNA, were used, and photoreactivation was carried out a t OOC.

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An interesting case of photo-reactivation in uitro of UV-inactivated transforming DNA should be noted (Rupert et al., 1958). It should also be noted that viruses may carry their own repair genes (Harm, 1964). XIII. Action Spectra

Table 1 (p. 171) lists action spectra for some radiation effects. I n the case of UV, action spectra information is reassuring since the best wavelength for lethality is 260 nm, the adsorption peak for the bases of DNA, thus identifying the effective receptor substance. Unfortunately, this is not so clear for NV-V, where lethal effects have been recorded over a broad range of wavelengths. Hopefully, with the use of sensitive mutants, future action spectra studies may provide a clue as to the receptor. Also, there may be a number of UV-V receptors and each of these might have an optimum absorption a t a wavelength quite different from the others. This merely emphasizes the need for careful identification of the various chromophores in bacteria, and for action spectra studies with monochromatic light. Perhaps a serious oversight in UV-V studies is the failure to understand which wavelengths are emitted by various lamps. For example, when a careful examination is made of the wavelengths emitted by blacklight lamp GE-F15T-BLB (Ferron and Eisenstark, 1971), there are lines other than 365 nm. This finding is emphasized because many investigators attribute their biological observations to this 365 nm emission. There is considerable additional transmission a t 354 nm, and a sharp rise a t 268 nm; the latter may be eliminated with the use of a 0.1 M solution (in benzene) of naphthalene (Ferron and Eisenstark, 1971). Perhaps many of the critical observations listed in Table 1 should be reexamined with the use of monochromatic light. XIV. Significance

Radiobiology, of course, has a long history and occupies the attention of a large fraction of biological research. The value of radiotherapy (Kaplan, 1970) must now receive new consideration in light of recent knowledge of repair mechanisms in radiation damage. As seen from this review, NV-V damages are also repaired, probably by the same mechanisms that repair X-ray and UV damage. Whether NV-V studies will assist in our understanding of these repair mechanisms will depend upon results of current experiments in several laboratories. Certainly, if

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chromophores are identified as participants in DNA replication and repair, insights may be gained. I n at least one current clinical practice, therapy for hyperbilirubinemia, newborn infants are exposed to high-intensity blue light (Behrman and Hsia, 1969). We certainly need to know whether there are other cellular effects that are produced, and whether any of these are damaging. The knowledge of possible mutational involvement is also an obvious necessity. Such matters as optimum wavelengths and dosages and antidotic techniques are in need of further study. Modern technology, of course, increases exposure to NV-V wavelengths. Among other items, attention should be focused on the increasing use of household and industrial fluorescent lamps, as well as blacklight lamps in the entertainment business (e.g., illumination of psychedelic posters). Also, blacklamps are used for suntanning and other home exposures. The press has given much attention to radiations emitted by TV sets, and perhaps the NV-V from these should not be ignored. Potential eye damage is beyond the scope of this review, but it cannot be completely overlooked in any discussion of NV-V effects. It is obvious that there is need for greater knowledge of the molecular biochemical effects of NV-V-emitting devices, if only to set safety standards. Appropriate monitoring and testing schemes are needed, and it is possible that the use of NV-V-sensitive bacterial and human cell-line mutants, such as those described in this review, may serve as biological dosimeters. I n addition to NV-V emitted by the industrial environment and the consumer products of this industrial environment, natural sunlight may have biological effects that are not yet fully understood (Resnick, 1970). Obviously, NV-V has played an important role in evolutionary processes, especially in photosynthesis, but perhaps also in more subtle ways in the long exposures to these wavelengths of cellular components. Genetic effects in germ cells are less likely since epidermal cells would receive the greatest exposure. There are human diseases, such as xeroderma pigmentosum (Cleaver and Trosko, 1970) that may be caused by the inability to repair sunlight damage, which gives further justification for basic studies of NV-V effects, and repair of damage caused by these wavelengths. XV. Summary

This review attempts to separate the known from the unknown with regard to the effect of visible and near-visible light on bacterial cells. It seems clear that wavelengths above 320 nm do something quite different from germicidal UV. The biological distinctions have been described

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in numerous papers; in addition, biochemical lesions and products following NV-V have been shown to be different from those following UV. Still unclear is whether there are different actions for different wavelengths above 320 nm. To determine this, specific chromophores need to be identified, together with identification of the resulting alterations upon exposure to specific monochromatic wavelengths. Upon recognition of a chromophore, it is important to know whether or not this cellular component is acting as an internal sensitizer (as in photodynamic action) to produce the lethal and mutagenic effects. Mutation by NV-V has been described by numerous investigators. However, neither the mode of action nor the types of mutations (i.e., deletions, frameshifts, transitions, transversions) have been determined. Several reports indicate that mutants of bacteria that are sensitive to light may be more mutable than wild type, but this too may need careful reexamination. An interesting aspect of these NV-V studies is the observation that many of the suspected chromophores are integral parts of the bacterial membrane. It is conceivable that chromosome replication (and repair) is engineered via a complex organelle involving membrane components, nucleases, polymerases, and ligases. Among these, an NV-V-absorbing molecule may play a functional role. Supporting this possibility are the statements in several reports that NV-V halts DNA synthesis. Whether this is a primary effect or a secondary reaction is still another matter that needs to be established. There are a number of candidates of 'molecules in bacterial cells that could be NV-V absorbers. Proteins and nucleic acids appear unlikely since they absorb primarily a t wavelengths below 320 nm. On the other hand, the small spillover of absorption into the longer wavelengths perhaps should not be ignored, and the validity of transforming DNA and virus inactivation by NV-V should be settled. NAD and NADH are known light absorbers and should be examined for roles in DNA replication. Photoeffects on bacterial cells have been known for some time, especially photoreactivation following W, but the receptor molecule for even this phenomenon has yet to be identified. Renewed interest in NV-V effects has come from focus on repair mechanisms following irradiation damage. Also, in our own laboratory, the finding that recombinationless strains of S. typhimuriurn are sensitive to light may offer a clue as to the defect in these strains and contribute to our understanding of chromosomal crossing-over. Renewed interest is also based on the fact that, in at least one current clinical practice, as therapy for hyperbilirubinemia, newborn infants are exposed to highintensity blue light. Will this cause cellular damages or mutations? Also, high-intensity NV-V wavelengths are emitted by many sources in modern

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technology, including fluorescent lighting in increasing quantity. In addition to the industrial environment, sunlight may be involved in human diseases, such as xeroderma pigmentosum, and NV-V studies may be of value in understanding of these diseases and in therapy. ACKNOWLEDGMENT Research by author and colleagues supported by National Science Foundation Grant GB-8493. The technical assistance of Elsie Johnson is gratefully acknowledged.

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Oishi, M., 1969. An ATP-dependent deoxyribonuclease from E . coli with a possible role in genetic recombination. Proc. Nat. Acad. Sci. U.S. 64, 1292-1299. Olivera, B. N., and Lehman, I. R. 1967a. Linkage of polynucleotides through phosphodiester bonds by an enzyme from Escherichia coli. Proc. Nat. Acad. Sci. US.57, 1426. Olivera, B. M., and Lehman, I. R. 1967b.Diphosphopyridine nucleotide: A cofactor for the polynucleotide-joining enzyme from Escherichia coli. Proc. Nat. Acad. Sci. U.S. 57, 1700. Olivera, B. M.,Hall, Z. W., Anraku, Y . , Chien, J. R., and Lehman, I. R. 1968. On the mechanism of the polynucleotide joining reaction. Cold Spring Harbor Symp. Quant. Biol. 33, 27-34. Paiva, A. C. M., and Paiva, T. B. 1961. Photooxidative inactivation of angiotensinamide. Biochim. Biophys. Acta. 48, 412414. Patrick, M. H. 1970. Near-UV photolysis of pyrimidine heteroadducts in E . coli DNA. Photochem. Photobiol. 11,477485. Phillips, S. L.,Person, S., and Jagger, J. 1967. Division delay induced in E . coli by near-ultraviolet radiation. J. Bacteriol. 94, 165-170. Ponce-De Leon, M., and Cabrera-Juarez, E. 1970. Photodynamic action on native and denatured transforming DNA from H . influenzae. J. Bacteriol. 101, 681-684. Raab, O.,Danielsohn, P., and Jacobson, R. 1901. Uber die Wirkung fluorescierender Stoffe. Muenchen. Med. Wochenschr.45, 1810-1811. Resnick, M. A. 1970. Sunlight-induced killing in Saccharomyces cerevisiae. Nature 226, 377478. Ritchie, D. A. 1965. The photodynamic action of proflavin on phage T4. Biochem. Biophys. Res. Commun. 20, 720-726. Roth, M. M. 1967a. The photosensitizing ability of prodigiosin. Photochem. Photobiol. 6, 923-926. Roth, M. M. 1967b. Carotenoid pigments and photokilling by acridine orange. J. Bacteriol. 93, 506-507. Rupert, C. S.,and Harm, W. 1966. Reactivation of photobiological damage. Advan. Radiat. Biol. 2, 1-81. Rupert, C. S., Goodgal, S. H., and Herriott, R. M. 1958. Photoreactivation in vitro of W-inactivated H . influenzae transforming factor. J . Gen. Physiol. 41, 451471. Rupp, W.D., and Howard-Flanders, P. 1968. Discontinuities in the DNA synthesized in an excisiondefective strain of Escherichia coli following ultraviolet irradiatian. J. Mol. Biol. 31(2) : 291-304. Sanderson, K.E., and Hamplova, D. 1970. Genetic studies in S. typhimurium. 10th Znt. Congr. Microbiol. (abstr.) p. 52. Sarabhai, A., 2nd Lamfrom, H. 1969. Mechanism of proflavin mutagenesis. Proc. Nat. Acad. Sci. U.S. 63, 1196-1197. Sasarman, A., Surdeanu, M., Szegli, G., Horodniceanu, T., Greceanu, V., and Dumitrescu A. 1968. Hemin-deficient mutants of E . coli K-12. J . Bacteriol. 96, 570-572. Sasarman, A., Sanderson, K.E., Surdeanu, M.,and Sonea, S. 1970. Hemin-deficient mutants of Salmonella typhimurium. J. Bacteriol. 102, 531-536. Sastry, K. S.,Gordon, M. P., and Waskell, L. A. 1966. Photodynamic inactivation of tobacco mosaic virus and its ribonucleic acid by acridine orange. Biochim. Biophys. Acta 129, 4248. Setlow, J. K. 1966. The molecular basis of biological effects of U. V. and photoreactivation. Curr. Topics Radiat. Res. 2, 197-248.

GENETIC DAMAGE BY VISIBLE AND

NEAR-UVLIGHT

197

Setlow, R. B. 1968. Photoproducts in DNA irradiated in vivo. Photochem. Photobiol. 7, 643-649. Setlow, R. B., and Carrier, W. L. 1966. Pyrimidine dimers in ultraviolet-irradiated DNA’s. J. Mol. Biol. 17, 237-254. Setlow, J. K., and Setlow, R. B. 1963. The nature of the photoreactivable lesion in DNA. Nature 197, 560-562. Simon, M. I., and Van Vunakis, H. 1962. The photodynamic reaction of methylene blue with deoxyribonucleic acid. J. Mol. Biol. 4, 488-499. Simon, M. I., Grossman, L., and Van Vunakis, H. 1965. Photosensitized reaction of polyribonucleotides. I. Effects on their susceptibility to enzyme digestion and their ability to act as synthetic messengers. J. Mol. Biol. 12(1) : 50-59. Singer, B., and Fraenkel-Conrat, H. 1966. Dye-catalyzed photoinactivation of tobacco mosaic ribonucleic acid. Biochemistry 5, 2446-2450. Sistrom, W. R., Griffiths, M., and Stanier, R. Y. 1956. Carotenoid-less mutant of Rhodopseudomonas spheroides. J. Cell. Comp. Physiol. 48, 473-515. Slater, T. F., and Riley, P. A. 1966. [Porphyrinl Photosensitization and lysosomal damage [ r a t ] . Nature 209, 151-154. Smarda, J. 1964. Lysogeny and bacteriocinogeny. Folia Microbiol. 8, 254-263. Smith, K. C. 1966. Physical and chemical changes induced in nucleic acids by ultraviolet light. Radiation Res., Suppl. 6, 54-79. Smith, K. C. 1962. Dosedependent decrease in extractability of deoxyribonucleic acid (DNA) from bacteria following irradiation with ultraviolet light or with visible light plus dye. Biochem. Biophys. Res. Commun. 8, 157-163. Spikes, J. D. 1968. Phdodynamic action. I n “Photophysiology,” (A. C . Giese, ed.), pp. 33-64. Academic Press, New York. Spikes, J. D., and Ghiron, C. A. 1964. Photodynamic effects in biological systems. In “Physical Processes in Radiation Biology” (L. G. Augenstein, R. Mason, and B. Rosenberg, eds.), pp. 309-336. Academic Press, New York. Spikes, J. D., and Livingston, R. 1969. The molecular biology of photodynamic action sensitized photoantioxidations in biological systems. Advan. Radiat. Biol. 3, 29-121. Spikes, J. D., and Straight, R. 1967. Sensitized photochemical processes in biological systems. Ann. R e v . Phys. Chem. 18, 409-436. Stahl, F. W., Crasemann, J. M., Okun, L., Fox, E., and Laird, C. 1961. Radiation sensitivity of bacteriophage containing 5-bromouridine. Virology 13, 98-104. Swenson, P. A,, and Setlow, R. B. 1970. Inhibition of the induced formation of tryptophanase in E. coli by near-ultraviolet radiation. J. Bacteriol. 102, 815-819. Takebe, H., and Jagger, J. 1969. Action spectrum for growth delay induced in E . coli B/r by far UV radiation. J. Bacteriol. 98, 677-682. Teresa, G. W., Teresa, N. L., and Milius, P. 1965. Influence of light on the action of acridine orange and proflavin on respiration in E. coli. Can. J . Microbiol. 11, 1028-1029. Tsugita, A., Okada, Y., and Udhara, K. 1965. Photosensitized inactivation of ribonucleic acids in the presence of riboflavin. Biochim. Biophys. Acta 103, 360-363. Uretz, R. B. 1964. Sensitivity to acridine sensitized photoinactivation in Escherichia coli B, B/r, and Bs-l. Radiat. Res. 22, 245. Van Vunakis, H., Seaman, E., Kahan, L., Kappler, J. W., and Levine, J. 1966. Formation of an adduct with tris(hydroxymethy1)aminomethane during the photooxidation of DNA and guanine derivatives. Biochemistry 5, 39863991. von Borstel, R. C., Graham, D. E., La Brot, K. J., and Resnick, M. A. 1968. Mutator activity of a X-radiation-sensitive yeast. Genetics 80, 233.

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von Lochmann, E. R., and Stein, W. 1964. Zur Inaktivierung durch Thiopyronin mit und ohne Licht. Naturwissenschaften 51, 59. Wacker, A. 1963. Molecular mechanisms of radiation effects. Progr. Nucl. Acid Res. Mol. Biol. 1, 369499. Wacker, A., Dellweg, H., and Weinblum, D. 1962. On the radiation-sensitizing action of 5-bromouracil. J. Mol. Biol. 3, 787-789. Wahl, R., and Latarjet, R. 1948. Inactivation de Bacteriophages par des radiations de grande longueurs d’0rde (3,400-6,OOOA). Ann. Inst. Pasteur Park 73, 759771. Walker, J. C. 1969. E. coli ras locus: 1t.q involvement in radiation repair. J . Bacteriol. 99, 713-719. Wang, C. C., and Newton, A. 1969a. Iron transport in E. coli: Relationship between chromium sensitivity and high iron requirement in mutants of E. coli. J. Bucteriol. 98, 1135-1141. Wang, C. C., and Newton, A. 1969b. Iron transport in E. coli: rolls of energydependent uptake and 2,3-dixydroxy-benzoylserine. J. Bacteriol. 98, 1142-1150. Wang, J. H. 1970. Oxidative and photosynthetic phosphorylation mechanisms. Science 167, 25-30. Webb, R. B., and Kubitschek, H. E. 1963. Mutagenic and antimutagenic effects of acridine orange in E . coli. Biochem. Biophys. Res. Commun. 13, 90-94. Webb, R. B., and Lorentz, J. R. 1970. Lethal effects of long wavelength ultraviolet and visible light on cells. Phofochem. Photobiol. 12, 283-289. Webb, R. B., and Malina, M. M. 1967. Mutagenesis in Escherichia coli by visible light. Science 156, 1104-1105. Webb, S. J. 1970. Sequential mutations induced in E. coli by black light. 10th Int. Congr. Microbiol. (abstr.) p. 53. Webb, S. J., and Singh Bhorjee, J. 1967. The effect of 3000-4000 A light on the synthesis of p-galactosidase and bacteriophages by Escherichia coli B. Can. J . Microbiol. 13, 69-79. Webb, S. J., and Tai, C. C. 1969. Physiological and genetic implications of selective mutation by light at 320-400 nm. Nature (London) 224, 1123-1125. Weil, L., Gordon, W. G., and Buchert, A. R. 1951. Photooxidation of amino acids in the presence of methylene blue. Arch. Biochem. Biophys. 33, 90-109. Willetts, N. S., and Clark, A. J. 1969. Characteristics of some multiple recombination-deficient strains of Escherichia coli. J. Bucteriol. €00,231-239. Witkin, E. M. 1969. Ultra-violet induced mutation and DNA repair. Ann. Rev. Genet. 3, 525-552. Wright, L. G., and Rilling, H. C. 1963. The function of carotenoids in a photochromogenic bacterium. Photochem. Photobiol. 2, 339-342. Wulff, D. L. 1967. 8-Aminolevulinic acid-requiring mutant from Escherichia coli. J. Bacteriol. 93, 1473-1474. Wyss, O., Clark, J. B., Haas, F., and Stone, W. S. 1948. The role of peroxide in the biological effects of irradiated broth. J. Bacteriol. 56, 51-57. Zampieri, A,, and Greenberg, J. 1965. Effect of heat and plating medium on survival of Escherichia coli after treatment with radiometic chemicals. J. Bacteriol. 89, 931-936. Zetterberg, G. 1964. Mutagenic effects of ultraviolet and visible light. I n “Photophysiology.” (A. C. Giese, ed.), Vol. 2, pp. 247-281. Academic Press, New York. Zieve, P. D., and Solomon, H. M. 1966. Effect of hematoporphyrin and light on human fibrinogen. Amer. J. Physiol. 210, 1391-1395.

GENETICS OF P2 AND RELATED PHAGES

.

1 Elizabeth Bertani and Giureppe Bertani Microbiol Genetics laboratory. Korolinrko Inrtitutet. Stockholm. Sweden

I . Introduction . . . . . . . . . . . . . . . . . . 200 I1. Natural History . . . . . . . . . . . . . . . . . 201 A . Origin of P2 . . . . . . . . . . . . . . . . . 201 B. Host Bacteria for P2 . . . . . . . . . . . . . . 201 C . P2-Related Phages . . . . . . . . . . . . . . . 201 I11. The Free Phage and Its DNA . . . . . . . . . . . . . 202 A . The Phage Particle . . . . . . . . . . . . . . . 202 B . The Phage DNA . . . . . . . . . . . . . . . . 202 C The Physical Map of P2 DNA . . . . . . . . . . . . 204 D . Comparative . . . . . . . . . . . . . . . . . 205 I V . Mutational Types . . . . . . . . . . . . . . . . 207 A. Properties of the Virus Particle . . . . . . . . . . . 207 B. Essential Functions . . . . . . . . . . . . . . . 207 C . Lysogeny . . . . . . . . . . . . . . . . . . 209 D . Comparative . . . . . . . . . . . . . . . . . 211 V . Recombination . . . . . . . . . . . . . . . . . 212 A. Frequency . . . . . . . . . . . . . . . . . 212 B. Different Recombination Mechanisms . . . . . . . . . 212 C . Negative Interference . . . . . . . . . . . . . . 214 D . Recombination Involving the Prophage . . . . . . . . . 214 E . Arrangement of Genes on the P2 Chromosome . . . . . . . 215 F. Comparative . . . . . . . . . . . . . . . . . 217 VI . Replication . . . . . . . . . . . . . . . . . . 217 A . Intracellular Forms of P2 DNA . . . . . . . . . . . 217 B. Point of Origin and Direction of Replication . . . . . . . 218 C . Phage and Bacterial Functions Needed by P2 for Replication . . 218 D . Involvement of Gene A in Replication . . . . . . . . . 219 E . Comparative . . . . . . . . . . . . . . . . . 220 VII . Regulation . . . . . . . . . . . . . . . . . . . 220 A. Functions Involved in Multiplication . . . . . . . . . . 220 B . Factors Affecting Lysogenization . . . . . . . . . . . 221 C. Split-Operon Control of the inl Gene . . . . . . . . . . 222 D . Induction . . . . . . . . . . . . . . . . . . 223 E . Comparative . . . . . . . . . . . . . . . . . 224 VIII . The Lysogenic State . . . . . . . . . . . . . . . . 225 A. Chromosomal Sites . . . . . . . . . . . . . . . 225 B. Chromosite Preference . . . . . . . . . . . . . . 225

.

199

200

L. ELIZABETH BERTANI AND QIUSEPPE BERTANI

C. Eduction of Host Cell Genes D. Immunity to Superinfection E. Comparative. . . . . References . . . . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

230 230 231 232

1. Introduction

Temperate bacteriophages-those that are able to establish a symbiotic relationship (lysogeny) with the host bacteria-are a popular subject of research in molecular genetics. I n such systems, the genetic material of the virus appears in a number of forms: compact and inert in the free phage particle or extended and variously active in the host cell as (a) vegetative phage following infection of a sensitive bacterium; (b) superinfection pre-prophage, following infection of an immune bacterium; (c) prophage, integrated into the host cell chromosome in an established lysogen; and (d) plasmid, in carrier cells. These forms of the genetic material of the virus may be studied with various appropriate physical methods, while genetic theory supplies the thread that connects them. Besides the macromolecular mechanisms found also in virulent bacteriophages, like adsorption to the cell, injection of the nucleic acid, assembly of the progeny virus particles, etc., the lysogenic systems offer the possibility of studying other interesting macromolecular mechanisms, like the insertion of viral DNA into the host cell chromosome, the repression of phage functions in the lysogenic condition, the stabilization of the carrier state, etc. The temperate bacteriophage most widely studied to date is phage A. The temperate phage P2, whose biology and genetics are reviewed here, promises to be an equally rewarding subject for intensive study. I n the first place, P2 appears to be rather different from A in a number of biologically interesting properties : it is not inducible, it interacts with a variety of host chromosomal sites, it recombines very little, etc. Furthermore, A and P2 may be taken to be the prototypes of two main groups of temperate Enterobacteria phages in nature. We isolated from nature (the Los Angeles County Hospital) 42 temperate phages, able to attack Eschem'chiu coli but all dissimilar in some property from each other. Of these, 16 were serologically related to P2, and 12 to A. I n this review attention will be focused on phage P2, but-whenever possible-comparative information on a number of less-well-studied, P2related bacteriophages will be supplied. On some points, more thorough coverage and older references may be found in other reviews (Bertani, 1958; Campbell, 1969; and Calendar, 1970).

GENETICS O F

P2

AND RELATED PHAGES

20 1

II. Natural History

A. ORIGINOF P2 Wild-type P2 is a line of phage isolated from the Lisbonne and Carrere strain of E. coli (G. Bertani, 1951). This bacterial strain, isolated in 1923, is the oldest known lysogen. It carries at least two other bacteriophages, besides P2, which have been called P1 and P3. Phage from this strain was used in very early work. I n more recent times, independent phage isolates from the Lisbonne and Carrere strain have been studied by the Beumers (phage H+, most probably identical to P2, and phage H-, most probably identical to P l ) , who have been interested primarily in the adsorption properties of these phages (see for example, Beumer, 1961) and by Frbdbricq (phage a, most probably identical to P2), who made a first attempt to localize this prophage on the bacterial chromosome (Frbdbricq, 1953). B. HOSTBACTERIA FOR P2 Strains of Shigella were traditionally used to detect the phages of the Lisbonne and Carrere strain: one, called Sh, has been used by us. P2 grows just as well on E. coli C, which is a generally good phage indicator, with no known restriction mechanisms, and is now the standard indicator for this phage. The efficiency of plating of P2 on E. coli K-12 is 5 to 10 times lower: the reason for this has not been studied. On E. coli B, P2 grows well, once it has adapted to the restriction and modification system of this strain (Bertani and Weigle, 1953; see review by Arber and Linn, 1969) . Circumvention of restriction systems and alteration of phage-adsorbing capacity make Salmonella typhimurium LT2 fully sensitive to P1 (B. A. D. Stocker, personal communication) and likewise to P2 (G. Bertani, unpublished). For other aspects of restriction and modification in phage P2 see also Christensen (1964) , Hattman (1964), and Uetake et al. (1964). P2 grows also on some Serratia strains (Bertani et al., 1967), which is interesting because the base ratio of the DNA of P2 differs substantially from that of Xerratia. Details of techniques for growing P2 are found in Bertani and Bertani (1970). The bacterial strains mentioned here will be referred to in the text by their abbreviations, Sh, C, K, B, and Sa (for Serratia).

c. P2-RELATED

PHAGES

Some information is available concerning several temperate phages, independently isolated from nature and related serologically and in other

202

L. ELIZABETH BERTANI AND GIUSEPPE BERTANI

ways to P2. These phages are listed in Table 1. The first three were selected from a larger sample for their noninducibility (Jacob and Wollman, 1956). The last seven were selected from our collection mentioned above (Section I ) , because they formed good plaques and were serologically related to P2. The phages listed are all serologically related to P2, and are not inducible by ultraviolet light. Different immunity specificities are represented in this group of phages but their number appears to be limited. These phages are all-unlike the A-related phages testedhelpers for the “satellite” phage P4, discovered by Six (1963). P 4 is a defective phage that can produce mature phage particles only if a helper phage is present in the same cell. The P 4 particles produced are serologically identical to whichever phage was used as helper. It is interesting t o note that phage P1, which differs from P2 in numerous prcjperties (it is larger, has more DNA, is slightly inducible, can transduce, recombines efficiently, does not usually attach to the host chromosome, does not help P4, can grow lytically on rep bacteria, etc.), nevertheless is fairly closely related to P2 in neutralization tests (R = 0.2, see Table 1). The two phages have in fact very similar tail base plates (D. H. Walker, personal communication). Both were isolated from the Lisbonne and Carrere strain, and it might be that a t some point in evolution they came to share some tail genes. I n what follows, however, P1 will not be considered among the P2-related phages.

I l l . The Free Phage and Its DNA

A. THE PHAGE PARTICLE Phage P2 has an isometric icosahedral head (of approximately 60 nm diameter) and a tail (approximately 135 nm long) with a contractile sheath, a base plate, and tail fibers (Anderson, 1960; D. H. Walker, personal communication). The particles are composed of protein (62% by weight) and of DNA (38%) and have a buoyant density of 1.43 to 1.44 in CsCl gradients. Most preparations of P2 contain a small percentage of noninfectious, tailless, or otherwise abnormal particles, separable by density from the main type (D. H. Walker, personal communication). B. THEPHAGE DNA Each particle of P2 contains a piece of double-stranded, nonpermuted DNA about 10-13 p long (depending on how the sample is prepared

GENETICS O F P 2 AND RELATED PHAGES

203

TABLE 1 Temperate Bacteriophages Related to P2

Phagea

Serological relatedness Immunity to P2b specificityC

Notesd

18 186

0.1-0.3 0.01-0.05

W

299

0.1-0.3

P2

EM

W+

1

W

u,

PK P2 H y dis P3

0.1-0.3 1 0.1-0.3

P2 dis P3

h

P4 +D5 +Dl24 +Dl45 +Dl60 +D218 +D252 +D266

0.1-0.3 0.1-0.3 0.1-0.3 0.1-0.3 0.2-0.5 0.1-0.3 0.1-0.3

EM

f,

P4 P3 dis 145 P3

EM

EM EM i,i,

EM

Referencese Jacob and Wollman (1956) Jacob and Wollman (1956) ; Baldwin et al. (1966) Jacob and Wollman (1956); Geisselsoder and Mandel (1970) Glover and Kerseman (1967); Pizer et al. (1968) Jesaitis and Hutton (1963) Cohen (1959) Bertani (1951); D . H. Walker (personal communication) Six (1963); Inman et al. (1971)

k, I

m

P3 dis P2

n

All, except 186, grow on C. All help P 4 (see text), although 186 may do so only to a very limited extent (E. W. Six, personal communication). All are noninducible by UV light (inducibility less than 3 % at a dose that induces more than 90% of bacteria lysogenic for A). With the exception of P4 (Lindqvist and Six, 1971), all are unable to multiply lytically in a rep host (Calendar et al., 1970). Original observations with a P2 specific antiserum a t a K p 2 neutralization constant of 0.3-3.0 per minute. The table lists the values of R = ratio of K for the given phage to K p 2 . Original observations based on tests of all possible combinations: lysogen us. superinfecting phage. Host: C, except for 186. E M signifies that the virus has been studied in the electron microscope. I n all cases the virus particles were practically indistinguishable from those of P2, the only exception being P4. Additional references are given in the text. Immunity specificity not fully tested: 186 is however heteroimmune in respect to P2, P3, P 2 H y dis, and 18 (E. W. Six, personal communication). Phage 186 was grown on K. 0 W+ is probably not the same phage as W of Jacob and Wollman (1956). P 2 H y dis is not a fully independent natural isolate: it arose as a recombinant between P2 and a defective prophage, or other genetic structure, present in B. P4 is a defective phage (see text). i The tail of P4 is identical to that of its helper; the head is smaller. Phage 18 does not form plaques on C(+D124). Phage +Dl24 grows poorly a t temperatures above 37"C, and helps P4 only at 30°C. ln Phage +Dl45 helps P 4 only at 30°C. " Phage P3 does not form plaques on C(+D252). J

204

L. ELIZABETH BERTANI AND GIUSEPPE BERTANI

for electron microscopy), molecular weight 22 million daltons, based on a molecular weight for A DNA of 31 million (Mandel, 1967; Inman and Bertani, 1969; A. K. Kleinschmidt, personal communication). This corresponds to approximately 33,000 base pairs or a maximal coding capacity for 30 to 70 different proteins (with an average of 350 to 150 amino acids per protein). Like the DNA of the E . coli host bacterium, P2 DNA contains the four normal bases in approximately equimolar proportions (Bertani and Bertani, 1970). When extracted from the free phage, the DNA is linear, that is, has free ends. These, however, like the ends of phage A DNA, can join reversibly to form rings or dimers, presumably due to the presence of short, complementary, single-stranded terminal segments (cohesive ends) (Mandel, 1967; Inman and Bertani, 1969). The cohesive ends of P2 DNA are much more stable than those of h DNA: in 2 M ammonium acetate, they separate a t 76OC, whereas those of A DNA do so a t 63*C (Itandel and Berg, 1968b; Inman and Bertani, 1969). This reaction may be followed not only by electron microscopy, but also by measurement of DNA infectivity. The “helper” assay, developed by Kaiser and Hogness (1960) for A DNA infectivity has been modified for P2 DNA by Mandel (1967). I n the presence of calcium ions, the DNA itself (without helper) is infective (Mandel and Higa, 1970).

C. THE PHYSICAL MAPOF P2 DNA Heterogeneity of DNA base composition may be demonstrated by a number of methods. It may turn out to be of fairly general occurrence, a t least in viruses and bacteria (see Skalka et al., 1968; Yamagishi, 1970). It possibly reflects evolutionary phenomena where the evolutionary units are functionally organized segments of DNA, rather than independent genes. These questions have been discussed by Hershey (1969). Electron microscopical techniques have been developed for the determination of the position along a piece of double-stranded DNA of those segments presumably richer in adenine and thymine which denature preferentially upon raising the temperature (Inman, 1966), or increasing the alkalinity of the medium (Inman and Schnos, 1970). If the DNA is pure and homogenous, more specifically, if it is not circularly permuted, so that all the molecules have the same base sequence, it is possible to construct from such data denaturation maps like those represented in Fig. 1. One can see that the results with the two methods of denaturation are concordant, that the maps for the two unrelated phages (P2 and A) are quite different, and that the heterogeneity in DNA base pair composition along the phage chromosomes is pronounced.

GENETICS OF

P2

205

AND RELATED PHAGES

C

:;I_ 4

0.6

0.4

I

,_ , _ 2

4

~

8

6

10

12

14

16

18

1 .o-

-

0.8-

0

0.40.4

0.2

0.2-

1

2.5

I

5.0

1 I

7.5

10.0

12.5

15.0

17.5

P h y s i c a l map d i s t a n c e ( m i c r o n s )

F I ~ 1. . Denaturation maps of DNA of phages P2 and A. A. P2, denaturation at 534°C (from Inman and Bertani, 1969). B. P2, denaturation a t high p H (from Schniis and Inman, 1971). C. A, denaturation at 54.0"C (from Inman, 1967). D. h, denaturation at high pH (from Inman and Schnos, 1970).

Thermal denaturation of P2 DNA, followed by means of optical density measurements, gives highly differentiated curves and permits the recognition of a t least three DNA fractions: I, corresponding t o 20% of the DNA mass, with 34% GC content; 11, 16%, with 45% GC content; and 111, 64%, with 58% GC content (Inman and Bertani, 1969).

D. COMPARATIVE The P2-related phages that have been studied with the electron microscope (see Table 1 for references) are morphologically indistinguishable from P2, with the exception of the defective phage P4, which has a smaller head. The straight tail with contractile sheath clearly distinguishes this group of phages from A. Particles of 299 and W+ have

206

L. ELIZABETH BERTANI AND GIUSEPPE BERTANI

buoyant densities very close to that observed for P2 (Geisselsoder and Mandel, 1970; Glover and Kersaman, 1967). Particles of P2 Hy dis have a buoyant density slightly higher than that of P2: if this is attributed exclusively to a higher DNA content, the DNA of this phage ought to be 2% in excess of that of P2 (Cohen, 1960). Particles of P2 Hy dis are also more sensitive to heat than P2 (Cohen, 1959), which is consistent with the finding that in several phages (references in Ritchie and Malcom, 1970) and in P2 itself (G. Bertani and D. H. Walker, unpublished) losses of DNA (deletions) increase thermal stability of the particles. The molecular weights of the DNA obtainable from phages 186 and 299 have been estimated at 20 million (Wang, 1967) and 21 million daltons (Geisselsoder and Mandel, 1970), respectively, hence very near the 22 million obtained for P2. Phage P4, on the other hand, contains only about 30% of the amount of DNA typical of a P2 particle (Inman et al., 1971). The DNA molecules of phages 299, 186, and P4 also have cohesive ends (Baldwin et al., 1966; Wang, 1967; Mandel and Berg, 1968a; Inman et al., 1971) and the formation of mixed dimers of P 2 DNA with the DNA of either 299 or 186 has been reported (Mandel and Berg, 1968a,b). Neither of these phages can form dimers with A DNA (Baldwin et al., 1966). Like P2, the cohesive ends of 186, 299, and P2 H y dis DNA begin to separate at temperatures much higher than those required for A DNA (Wang, 1967; Mandel and Berg, 196813; Inman and Bertani, 1969). It is thus likely that these four phages have similar base sequences in their cohesive ends. The cohesive ends of 186 DNA are approximately 17 nucleotides long, hence 5 nucleotides longer than those of A DNA, and partial sequences have been determined (Wu, 1970). Phages 299, 186, and P2 Hy dis may be used equally well as helpers in the infectivity assay for P2 DNA. Since A cannot be used, it has been suggested that the “helping” process involves an interaction between the cohesive ends of the DNA being taken up and of that injected by the helper phage (Mandel and Berg, 1968a). The denaturation map of P2 H y dis (Inman and Bertani, 1969) shows a minor difference in respect to that of P2 near one end. This is consistent with the observation of a small, but definite difference in the melting curves for the two DNAs. The heat denaturation of 299 DNA has been analyzed photometrically (Geisselsoder and Mandel, 1970) : the pattern obtained resembles very much that of P2 DNA. One of the two subfractions of P2 DNA fraction I, however, appears to be missing in 299, and this is consistent with the slightly higher average GC content found for 299 DNA.

GENETICS OF P 2 AND RELATED PHAGES

207

IV. Mutational Types

A. PROPERTIES OF THE VIRUS PARTICLE

P2 requires calcium ions for optimal adsorption. Mutants with altered calcium requirement are known (cui, I , and rd in Table 2 ) . Host range mutants (able to adsorb to resistant mutants of the host bacterium) of P2 have been isolated (G. Bertani, unpublished), but they are rather unstable upon storage. Only one such mutant has been used to any extent (Chase, 1964). Defective virus particles are formed by certain conditional mutants, under nonpermissive conditions (see Section IV, B, 2 ) .

B. ESSENTIAL FUNCTIONS A number of conditional mutants of P2, of both the t s and am (or sus, suppressor sensitive) type, have been isolated and arranged by means of complementation tests into some 18 cistrons (Lindahl, 1969a, 1971). All these genes presumably specify proteins which have to do with some aspect of the lytic multiplication cycle of the phage, including structural proteins for the phage particle. 1. Early Functions

Two “early” essential genes have been identified by Lindahl (1969a, 1970, 1971) : cistrons A and B. Infection with phage carrying a mutation in either of these genes under nonpermissive conditions (high temperature or absence of suppressor) does not result in killing of the bacterial host, which may instead become lysogenic. Furthermore, little if any incorporation of radioactive label into phage DNA can be detected in such cases (E. Ljungquist, personal communication ; Lindqvist, 1971). 2. Late Functions

Phages carrying mutations in other essential genes (cistrons D through H, and J through T: Lindahl, 1969a, 1971) lyse the bacterial host cell

under nonpermissive conditions. In these cases the phage DNA multiplies (E. Ljungquist, personal communication; Lindqvist, 1971) and the formation of incomplete or defective phage progeny particles may be demonstrated by means of in vitro reconstitution (Edgar and Wood, 1966) of active phage particles. I n this test, lysates obtained under nonpermissive conditions of mutants in genes L through Q can give rise to active phage particles when mixed with similar lysates of mutants in genes D through H , J, R , and T (G. Lindahl, personal communication). Since

208

L. ELIZABETH BERTANI AND QIUSEPPE BERTANI

TABLE 2 P2 Mutational Types and Their Abbreviations

am cai C

cc co2 di8

fun

int

1

4J m

old

rd

8af IS

Amber or suppressor sensitive mutants: form plaques only on host bacteria carrying a suppressor gene. A variety of mutations belonging to different cistrons (see text).' Calcium independent in adsorption, but also unstable in the presence of ca1cium.c Mutant cl (or simply c) forms clearer plaques (especially on Sh) than wildtype phage.b I t lysogenizes a t reduced frequency,d but its lysogens are stable,)*eJ even though they have reduced immunity to superinfection.iv' In cistron C.amh Mutants CS and c4 are like c l , from which they differ only quantitatively in lysogenizing capacity and immunity 1evel.j Mutants c6 through c9 form clearer plaques at high temperature (37 to 42°C) than a t 30'C.' Also in cistron C.' Forms slightly clearer plaques than wild-type on citrate agar." Adsorption and stability are unaffected. Lysogenizes normally, but the lysogens produce little or no phage. Affects the activity, the specificity, or the synthesis of the int gene product.= Not a mutation: symbolizes the immunity specificity of the defective prophage present in strain B, which is different from that of P2." P2 Hy dis is the hybrid carrying that specificity. Unable to convert the host bacterium to high sensitivity to 5-fluorouracil.0 Recessive. Unable to lysogenize by itself, although it permits survival of some of the infected cells. Turbid plaques. Recessive: the presence of wild-type phage permits int to lysogenize. Lysogens for intl,~intl4, intd0, intdl, and i n t 3 l ~ produce spontaneously less than 0.1% of the normal amount of phage; lysogens for inti3 and int.90~produce 1 to 30% of the normal amount. Makes plaques larger when combined with rdl .b It is very strongly dependent on calcium for adsorption: it gives no plaques on citrate agar." Forms plaques slightly larger than wild type and has larger burst size.c Adsorption and stability are unaffected. Forms minute p1aques.h Able to lysogenize a recombination defective mutant, lyd, of E. coli Cr; the wild-type phage cannot. Also unable, as prophage, to interfere with the multiplication of phage X.* Mutant rdf (or simply rd) forms smoothly round plaques on Sh.' It is insensitive to high concentrations of citratee; rdda resembles rdl in plaque appearance, but is not necessarily allelic to it. Not a mutation. Symbolizes the genetic change resulting from an interaction (presumably recombination) between P2 and chromosite 11, and affecting the site affinity of the phage.6 Previously symbolized as site specificity SII. Temperature sensitive, generally unable to form plaques a t temperatures from 37 to 42'C. A variety of mutations belonging to different cistrons (see text).h

GENETICS OF

P2

AND RELATED PHAGES

209

TABLE 2 (Continued) vir

Xt

Symbolizes a variety of mutants, all unable to establish lysogeny. Mutant virl is a “weak”a or immunity-sensitive virulent: it gives no plaques on lysogenic indicat0rs.f.k It is recessive: it may become and remain prophage if a second, wild-type prophage is also present.%In cistron C.Osh Mutant virl4 is another weak virulent, isolated however in P2 Hy dis, and therefore sensitive to dis rather than P2 specific immunity.” Mutants virl9 and vir20 (isolate 88 of@)are conditional virulents: lysogenize C to some extent, but not Sh.0 Assigned to cistron Zh.Mutants virSv and vir.@‘ are “strong,”a or immunityinsensitive virulents. They are presumably operator mutations for the early functions A and B.’ Mutant vir82, another strong virulent, is a deletion (G. Bertani and D. H. Walker, unpublished). Mutant vir6 is an “intermediate” virulent: gives plaques on lysogens with variable efficiency dependent on the immunity level of the 1ysogen.f Produces curing of lysogens in high frequency.m Double mutant virl virlSw also behaves as an intermediate virulent. Extratemperate: gives very turbid plaques and lysogenizes much more efficiently than wild

G. Bertani (1953a). * G. Bertani (1954). ~ B e r t a n et i al. (1969). d L . E. Bertani (1959).#Six (1959). f Bertani and Six (1958). L. E. Bertani (1960).h Lindahl (1969a). Lindahl (1971). jL. E. Bertani (1961). kL. E. Bertani (1965).’L. E. Bertani (1968). G. Bertani (1953b). Cohen (1959). 0 Bertani and Levy (1964). P Lindahl (1969b). q Choe (1969). Sironi (1969). a Lindahl et al. (1970). ‘Six (1971). u Six (1966). vL. E. Bertani (1957). G. Bertani (1962). zG. Lindahl and M. G. Sunshine (personal communication). ~

a

@

the mutation 1, which affects adsorption, is located very close to some mutations in cistron G , it is assumed (Lindahl, 1969a) that the genes of the D-J, R, T group specify tail structures, i.e., mutants in such genes can still supply normal phage heads in reconstitution experiments. Conversely, genes of the L-& group would be responsible for the formation of the phage head. Gene K appears to specify a protein required for lysis (G. Lindahl, personal communication). C. LYSOGENY 1 . Prophage Attachment and Detachment

As for other phages, the attachment or “integration” of phage P2 into the bacterial chromosome at specific sites (“chromosites”) requires at least one phage gene product, as shown by the occurrence of int mutants (see Table 2 ) . The fact that lysogens carrying an int prophage (obtained by complementation) are defective in phage production in-

210

L. ELIZABETH BERTANI AND GIUSEPPE BERTANI

dicates that this gene is needed also for the excision of the prophage from the host chromosome. Another gene, cox, affecting excision, but not integration of P2, and different in map location from int, has been identified recently (G. Lindahl and M. G. Sunshine, personal communication). The specificity of prophage attachment (i.e., the probability with which a P2 phage is integrated a t one or the other of the available chromosites) is affected by the genetic constitution of the phage. An important role must be played by the DNA base sequence in the “episite,” the segment of phage DNA where the reciprocal exchange with the bacterial chromosome postulated by the Campbell model takes place. The saf variants (see Table 2) very probably represent changes in the episite. 2. Maintenance

Like other temperate phages, P2 produces a specific immunity repressor whose function is to block the transition of the prophage or of a superinfecting phage into vegetative phage. Gene C specifies the repressor and gives a t least three classes of mutants: (a) mutants with reduced lysogenizing ability and imparting, as prophages, low levels of immunity t o superinfection (example: c l ) ; (b) temperature-sensitive mutants, whose lysogens are normal a t 3OoC, but are abortively induced a t high temperatures (example c5) ; and (c) weak virulent mutants, having lost all ability to establish lysogeny, in a.bsence of complementation (example: v i r l ) . Another class of mutants, (example: virl9, vir20) assigned to a gene called 2, are host dependent: they are unable to lysogenize Sh, but do lysogeniee E . coli C. The function of gene 2 in Sh is not clear. 2 mutants complement both C (L. E. Bertani, 1960) and int mutants well (Choe, 1969). Also, they do not affect immunity specificity, since P2 H y dis virZ recombinants may be obtained easily (Cousin, 1963). Among P2 mutants having lost the ability to lysogeniee, one not uncommonly finds the immunity-insensitive type (like vir3). These are probably operator mutants in the early function operon (Lindahl, 1971), and are deletions in many cases (G. Bertani, unpublished). One immunity-insensitive mutant is known which reverts to the immunity-sensitive state (L. E. Bertani and L. Falt, unpublished). 5.

Lysogenic Conversion

Formally this term covers any effect of the prophage on the lypogenic cells, especially if unessential to the maintenance of the lysogenic state. In general, bacteria lysogenic for P2 are more sensitive to 5-fluorouracil

GENETICS OF

P2

AND RELATED PHAGES

211

and its derivatives than the corresponding nonlysogenic strain (L. E. Bertani, 1964). P2 mutants (fun, for fluorouracil nonconverting) which have lost this property, but are otherwise normal, have been isolated. Another property of bacteria lysogenic for P2 is their inability to support the growth of phage A, although adsorption is unaffected. The old mutants of P2 have lost this property. The connection between this effect, and the property by which old mutants have been defined in the first place (their ability to overcome the effects of the Zyd mutation in the host bacterium) is not yet clear. The old’ allele is incompatible with lysogeny in a lyd host even when an already established prophage is introduced by means of bacterial crosses into a Zyd cell from a lyd+ donor lysogen (Sironi, 1969), and this suggests that the old gene is functional also in the lysogenic condition. P2 lysogens also restrict the growth of phage T2 and related phages (Bertani, 1953a; Lederberg, 1957; Smith et al., 1969). This restriction, however, is not affected by old mutations.

D. COMPARATIVE There is very little information on mutation types in P2-related phages. P2 H y dis differs from P2 in that part of the genome which is concerned with immunity and specificity of immunity, as shown by the fact that P2 mutants in cistron C or strong virulent mutants lose such properties in acquiring the dis immunity specificity from the defective prophage of strain B (Cohen, 1959). Nevertheless one can obtain from P 2 H y dis mutant types exactly corresponding to the c, weak and strong virulent mutants of P2 (Cohen, 1959). Heat-inducible mutants have been isolated from phage 186 (Baldwin et al., 1966), and abortively inducible ones from phages 299 (Golub and Zwenigorodsky, 1969) and 18 (Golub and Reshetnikova, 1970). Immunity-insensitive mutants are known for P4 (Lindqvist and Six, 1971). I n general, several of the P2-related phages yield immunity-insensitive mutants easily: this is very common for example with +D145, where such mutants may be found as “spontaneous” plaques in a lawn of the corresponding lysogen. Like P2, prophage W+ also excludes A, and mutants of W+, analogous to the old mutants, have been isolated (Kerszman et al., 1967). The restriction of A in the two systems is different, however. Whereas it is possible to isolate mutants of A that are able to grow on either P2 (Lindahl etal., 1970) or W+ (Glover and Aronovitch, 1967) lysogens, those selected to grow on W+ lysogens are still unable to overcome P2-restriction (Kerszman et aZ., 1967). Like P2, W+ prophage also restricts the

212

L. ELIZABETH BERTANI AND GIUSEPPE BERTANI

multiplication of phage T2, but again the two systems are not quite identical in this respect (Pizer et al., 1968). V. Recombination

A. FREQUENCY Genetic recombination of P2 in the course of vegetative multiplication has been studied extensively by Lindahl (1969a,b). P2 has the remarkable property of recombining very little in such experiments, as compared with phages having roughly the same DNA size and burst size. For example, genes at opposite ends of the genetic map of A (which has 30% more DNA than P2) may show recombination frequencies of up to 13%, whereas the maximum observed for P2 is 0.3% under normal conditions. P2 is the exception: the temperate phage P22 of Salmonella, which has as much DNA as A (Thomas, 1966), gives recombination frequencies up to 20-25% (Gough and Levine, 1968) ; phage P1, with twice as much DNA (Ikeda and Tomizawa, 1965) gives up to 6-776 (Scott, 1968). The only other DNA phage known to have very low recombination frequencies (maximum 0.2%) (Tessman, 1965) is 513 and its relative 4x174: these phages have much less DNA than any of the phages mentioned above.

B. DIFFERENT RECOMBINATION MECHANISMS This peculiarity of P2 becomes even more striking when the contributions of the various mechanisms of recombination are analyzed. Genetic recombination in molecular terms is without doubt a complicated reaction sequence. Indeed, in bacteria several mutations are known each of which may reduce drastically the frequency of recombination: these mutations are thought to affect proteins which either are required for the normal recombination process, or may interfere with it as a result of mutation. Some phages are known to carry genes whose products are required for or may be involved in recombination: these products might be homologous to corresponding bacterial gene products, or be highly specific for the phage. Moreover, several temperate phages possess genes whose products are required for the unique reciprocal recombination event between the phage DNA and the host chromosome, that leads t o prophage integration. Recombination of this type (int) can take place also between two phage chromosomes, but is always localized to the episite. Phage is known to use all three recombination pathways: the host

GENETICS OF P 2 AND RELATED PHAGES

213

system (called rec), its own system of general (i.e., not site-specific recombination (called red), and the int system. (The use of the word system should not be taken to imply a completely independent reaction pathway in each case.) Using appropriate combinations of mutant phages, it is possible to study the contributions of each of these pathways to the total recombination frequency. Mutations of the red type are not known for P2, but the use of int mutants permits the measurement of the residual recombination, when the int pathway is inactive. One finds (Lindahl, 196913) that the recombination frequency between markers located one on each side of the episite is then reduced as much as one hundredfold, and that the bulk of the recombination observable in P2 between markers a t the extremes of the vegetative map is really due to recombination in the episite. The amount of genetic recombination across the episite which is due to the int pathway is greater for A (2% recombination frequency, Signer et al., 1969) than for P2 (0.3%,Lindahl, 1969b) . Since however this type of recombination is strongly dependent on the amount of the int gene product, and on other conditions, the difference between the two phages in this respect is noticeable, but not tremendously striking. One may also recall that the two phages are both able to lysogenize with good efficiency. Where the difference is really striking instead is in the amount of recombination remaining in the absence of the int pathway as can be already guessed from the figures given above: for P2 the maximum non-int recombination frequency is about 0.03%, for A it may be as high as 10%. When A int red double mutants are used, or when A red is used and the recombination frequency is measured over a segment outside of the episite, the amount of recombination observed is much less than in the red+ control, and is attributed to the operation of the recombination system of the host. Indeed, if in addition a recA host mutant is used, this residual recombination all but disappears. The two contributions, from the host and from the red pathway, although not additive, appear to be of similar magnitude (see Echols and Gingery, 1968). It is remarkable that in P2 the total amount of all non-int recombination is already lower than the residual recombination in due to the host pathway. This phenomenon remains unexplained. One can think of a compartmentalization within the cell during the multiplication process of P2 DNA, so that it would be unusually difficult for P2 DNA molecules of different parentage to meet. Alternatively, the low recombination frequency of P2 could be the result of the greater stability of the cohesive ends of the P2 DNA molecule when paired: it has been suggested (Baldwin et al., 1966) that the formation of circular dimers may be a prerequisite for recombination to take place. P2 DNA might form circular

214

L. ELIZABETH BERTANI AND GIUSEPPE BERTANI

monomers immediately following infection, and remain in this form during most of the multiplication cycle, making the formation of dimers relatively rare (Mandel and Berg, 1968a). Alternatively, one could imagine that dimers that are formed fail to monomeriae again later. More generally, a structural constraint might impose a selection against the majority of recombinant classes, which thus would not be recovered as viable phage particles in a standard cross. A limitation of the data discussed in this section is that they were obtained with host C for P2 and host K for A: the possibility of host effects on recombination has not been fully investigated.

C. NEGATIVE INTERFERENCE Negative interference-a common phenomenon in bacteriophage crosses (see Visconti and Delbriick, 1953)-is very strong in P2 (Lindahl, 1969b): the frequency of recombination over a map segment among phage particles having undergone recombination over another map segment may be increased several thousandfold. This suggests that the “resistance” to recombination is not due to some general property of P2 DNA, but rather to an obstacle-the difficulty of meeting for two molecules, or of forming or splitting a dimer-such that, once this is removed, recombination can occur normally over the whole molecule. When one of the two segments used to calculate negative interference includes the episite, the interference observed is not as high (data in Lindahl, 1969a), and-given the limited information available-could even be all attributed to the non-int component of recombination included in measurements of recombination frequency across the episite. Ultraviolet irradiation of the phage used in crosses increases very strongly the frequency of non-int recombination, whereas the factor of increase observed in recombination across the episite is smaller, and may all be due to the effect of UV light on the contribution of the non-int pathway (see Lindahl, 1969a, Fig. 2). Very high negative interference has been observed also in phage S13 (Baker and Tessman, 1967).

D. RECOMBINATION INVOLVING THE PROPHAGE With temperate phages it is possible to perform other types of recombination experiments than the standard cross-mixed infection of a sensitive bacterium-discussed up to this point. One can study the following: (a) recombination between prophages a t allelic chromosites in bacterial

GENETICS O F P 2 AND RELATED PHAGES

215

conjugation or transduction experiments ; (b) recombination between a superinfecting phage and the prophage, using as superinfecting phage either an immunity-sensitive or an immunity-insensitive phage ; (c) recombination between two immunity-sensitive phages superinfecting an immune cell; (d) recombination between two prophages, carried by the same host cell, either a t different chromosites, or attached in tandem. Some of these approaches have revealed important information on the prophage state, and will be mentioned again later on. Others have not yet been sufficiently exploited to deserve discussion. Some observations however are relevant to the questions raised in the previous sections. I n bacterial crosses, with P2 in the prophage state in both parent bacteria, the amount of recombination within the prophage appears to be normal, i.e., approximately as expected in proportion t o the DNA length represented by the prophage in the bacterial chromosome (Wiman et al., 1970). Recombination between the prophage and a superinfecting, immunityinsensitive mutant phage has been studied by Chase (1964) and Eastburn (1969), even though their primary concern was the repair of damage produced by UV irradiation in the superinfecting phage due to interactions with the prophage. A great deal of reactivation, as shown by smaller slopes for the survival curves as a function of the irradiation dose, can take place in such a system, if the prophage is closely related genetically to the superinfecting phage. Which chromosite is occupied by the prophage seems to be irrelevant. Relatively high frequencies of recombination accompany the reactivation. The mutation lyd in the host (Sironi, 1969), which reduces very much the amount of recombination in the host cell, does not affect prophage-dependent reactivation (Eastburn, 1969) or even vegetative phage recombination (Lindahl, 1969b).

E. ARRANGEMENT OF GENES ON

THE

P2 CHROMOSOME

A detailed genetic map of P2 has been constructed by Lindahl (1969a,b, 1971). Because of the disproportionately greater amount of recombination a t the episite due to the int recombination pathway, the map, as obtained, shows a long segment, corresponding to 80 to 90% of the total, completely devoid of genes. This is, of course, an artifact, and to obtain a picture which might more adequately represent the real distribution of genes along the DNA, the map of Fig. 2 has been corrected for int recombination, i.e., it represents the map one would obtain if all phage used in the crosses were unable to use the int pathway. The map resembles in some features that known for phage A. I n both phages the genes involved in the synthesis of the structural proteins

216

c-

L. ELIZABETH BERTANI AND GIUSEPPE BERTANI

--w

___)

[-

---* episite

W

= 0001 %

recombination frequency

FIQ.2. The genetic map of P2 according to Lindahl (1969a,b, 1970, 1971). The distances represented are based on two-factor crosses. Because of inconsistencies between different sets of crosses (different hosts), the distance T to D may be in error by a factor of 2. The gap in the map represents the episite: and is roughly proportional to the recombination frequency obtainable in a cross between int phages. In crosses between int+ phages this gap would be almost ten times longer than the whole map as given here. The segments with arrows under the map indicate the known transcription units.

are clustered in two groups in the left part of the map (head components to the left, tail components to the right), whereas a few genes that are necessary for phage DNA synthesis ( A and B in P2, 0 and P in A), lie towards the right end of the map, to the right of gene C which specifies the immunity repressor. In both systems, the int gene is immediately to the right of the episite, which, for P2, is between genes D and C (Calendar and Lindahl, 1969). In several other respects the maps of P2 and h appear to differ. T o date no genes comparable to N or red of A have been noted in P2. Attempts to detect a phage-specific, A-like exonuclease in the Pa-related phages 186 and 299 (Shuster et al., 1967) have proved negative. Like N and red, the gene coding for the exonuclease in h. is located in a segment between int and C: it is thus possible that phages of the P2 family do not have the corresponding genes (see also Section VII). Second, the gene thought to be responsible for phage endolysin production in P2, gene K , lies in the left half of the chromosome, whereas the gene specifying endolysin is at the far right in the A map. Third, analogs of the cII and cIII genes, which participate in the establishment of lysogeny in A, have not been reported as yet in P2: the Z gene of P2 clearly does not correspond in its map location to either cII or cIII. Although some of these differences may be the result of insufficient information, it is not unlikely that the gene composition of P2 may be simpler than that of A since the length of DNA available is 30% shorter. A surprising feature of the P2 map is the location of two genes, old and fun, which, in addition to the repressor gene C, appear to be active in the lysogenic state. Both are far from the C gene, and are themselves

GENETICS O F

P2

AND RELATED PHAGES

217

separated by a run of genes for structural proteins, which ought to be repressed in the lysogenic state. This suggests that there are a t least three operons in P2 which are regularly transcribed in lysogenic cells, whereas only one, containing the C gene, is known for A. Gene Z might belong to the same operon as fun. The orientation of the denaturation map of P2 (Fig. 1) in respect to the genetic map (Fig. 2) is not yet known with certainty. There is some evidence however that the one assumed in the figures is the correct orientation, as discussed by Inman and Bertani (1969) on the basis of differences in denaturation maps and curves between P2 DNA and P 2 H y dis DNA.

F. COMPARATIVE There are no published data on the recombination properties of P2-related phages, with the exception of what has been mentioned already concerning P2 Hy dis. A few am mutants have been isolated from either of phages 4D218 and +D266, and crossed (G. Bertani, unpublished) in strain C. I n both cases the recombination frequencies obtained were quite low. This would suggest that the low recombination frequencies observed in P2 are not a peculiarity of this phage, but may represent a general property of this family of phages. In prophage-dependent reactivation experiments, P2 H y dis prophage may rescue efficiently irradiated superinfecting P2 (Chase, 1964) , and this holds for the inverse phage combination (Eastburn, 1969). I n the same type of experiment,, phages P2, P3, PK, and W+ have been tested in all possible heterologous combinations, without obtaining any detectable reactivation (Eastburn, 1969). When hybrids between two of the phages were tested against one of the parents, however, some reactivation did occur. These results would indicate that this type of reactivation requires a high level of genetic homology between the superinfecting phage and the prophage, and a t the same time strengthens the assumption that P2 H y dis is identical to P2 over a large part of its genome. No prophage dependent reactivation could be observed between P2 and P 4 (Eastburn, 1969). VI. Replication

A. INTRACELLULAR FORMS OF P2 DNA Covalently closed, circular forms of P2 DNA have been found in vivo. Upon infection of a sensitive host, most of the parental DNA

218

L. ELIZABETH BERTANI AND GIUSEPPE BERTANI

is converted to a form that sediments more rapidly in sucrose gradients than does DNA extracted from phage particles and is resistant to denaturation in alkali (Calendar et al., 1970; Lindqvist, 1971). This material, which is similar in its properties to the “supercoiled” DNA found following infection with A, also appears following superinfection of an immune host (Lindqvist, 1971), i.e., in the absence of replication. Although the conversion of linear to circular phage DNA may take place prior to replication, the circular structures are also actively involved in DNA synthesis. Radioactive label, added during the multiplication cycle, is incorporated into the rapidly sedimenting form (Calendar et al., 1970; Lindqvist, 1971). Furthermore, in pulse-chase experiments, the labeled circular DNA appears to be later converted to the linear form (E. Ljungquist, personal communication ; Lindqvist, 1971). B. POINTOF ORIGINAND DIRECTION OF REPLICATION Circular forms of vegetative P2 DNA have been observed with the electron microscope by Schnos and Inman (1971), who have applied the technique of denaturation mapping to the study of replication in both P2 and A (Schnos and Inman, 1970). Both the region of the chromosome in which replication begins and the direction i t proceeds can be identified by this technique. Circular, partially replicated phage DNA is first prepared from infected bacteria and then denatured to a limited extent. For each molecule, the pattern of denatured regions compared with the established denaturation map permits the identification of the point that corresponds to the ends of the linear molecule, and of the relative positions of the branched points delimiting the replicated segment. In A, replication begins in the right half of the chromosome, in the region of the P gene, and, surprisingly, appears to proceed in both directions. I n P2, replication begins near one end of the molecule and proceeds in only one direction. If the orientation of the physical with the genetic map of P2 is as suggested in Inman and Bertani (1969), the replication would begin in the region of the A and B genes and proceed to the right.

C. PHAGEAND BACTERIAL FUNCTIONS NEEDED BY P2

FOR

REPLICATION

The observations quoted in Section IV, B, 1 suggest that the products of genes A and B are necessary for normal phage DNA replication. I n addition, P2 is absolutely dependent on at least one host function

GENETICS O F

P2

AND RELATED PHAGES

219

that is not required by A. Denhardt et al. (1967) isolated bacterial mutants ( r e p ) unable to support the multiplication of phage +X174. In these strains, the single-stranded 4x174 DNA is converted to a double-stranded form by synthesis of a complementary strand, but further DNA synthesis is blocked. The same strains are unable to support multiplication of phage P2. Although the fast-sedimenting form of P2 DNA can be found following infection of a rep host, there is no uptake of radioactive label into the phage DNA (Calendar e t al., 1970). Thus, it would appear that P2 and the double-stranded form of +X174 share some step in replication that is not shared with A.

D. INVOLVEMENT OF GENEA

IN

REPLICATION

Lindahl (1970) has reported that mutants in gene A do not complement any other mutants except B mutants and then only when the concentration of salt in the medium is adequate. Lindahl tends to believe that even in this case the complementation found is due to “leakiness.” More surprisingly, gene A mutants cannot be complemented by any other mutants or even by wild-type phage. Following mixed infection under nonpermissive conditions, but adequate salt concentration with an A mutant and a B mutant (or wild-type P2), the yield contains almost exclusively B mutant type (or wild-type P2). The A gene thus appears to code for a protein that cannot be shared between chromosomes, i.e., acts only in cis configuration. Possible explanations for the inability of the product of gene A to act in trans have been discussed by Lindahl (1970) : they include replication of P2 in a “compartment” that is impermeable to the A gene product; rapid inactivation of the A gene protein; synthesis of A gene protein in close proximity to the A gene, followed by rapid binding of the product to the chromosome; or a configurational change in the chromosome of A gene mutants that blocks their replication. Whatever the explanation, the unusual properties of the A gene product could account for the inability of immunity-sensitive phages to replicate in the presence of immunity repressor. In general, when a lysogen is reinfected or superinfected with phage that is homoimmune to the prophage carried by the lysogen, no lysis is observed and no DNA synthesis of the superinfecting phage can be detected (Bertani, 1954; Wolf and Meselson, 1963). Thomas and Bertani (1964) showed that a n immunitysensitive phage did not replicate in a homoimmune lysogen even when an immunity-insensitive derivative of the same phage was actively multiplying in the same cell and all factors necessary for multiplication

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were present. This specific block of replication by the immunity repressor has been demonstrated for both P2 and A, but has never been entirely explained. Assuming that gene A is under direct control of the immunity repressor, an immunity-sensitive phage would not be able to replicate in a homoimmune lysogen because it could neither make its own A gene protein nor utilize that produced by a co-superinfecting, immunityinsensitive phage, since the A gene product acts only in cis. Although this explanation is adequate for the case of P2, an analogous gene having the same behavior as gene A has not yet been demonstrated for A.

E. COMPARATIVE Although the defective phage P4 needs a helper phage to complete its multiplication cycle, it is able to replicate its D N A also in the absence of helper (Lindqvist and Six, 1971). Covalently closed, circular forms, containing either parental or newly synthesized P4 D N A have been detected by sedimentation analysis (Lindqvist and Six, 1971) and observed with the electron microscope (R. B. Inman, personal communication). All the P2-related phages tested so far are, like P2, unable to multiply in rep hosts (see Table 1) with the exception of P4. The latter multiplies normally in such hosts when a helper phage is present, and replicates its D N A even in the absence of helper (Six and Lindqvist, 1971).

VII. Regulation

A. FUNCTIONS INVOLVED IN MULTIPLICATION The early gene B appears to be directly under the control of phageimmunity repressor. It has been shown by complementation experiments (Bertani, 1968) that gene B is expressed following exposure of a P2 c6 lysogen to high temperature, although there was little or no increase in the activity of six other genes (D, F, H, L, M , or 0) concerned with the synthesis of phage structural proteins. Two independent, spontaneous, immunity-insensitive mutations (vir3 and vir24) have been localized by crosses a t a site between the genes C and B (Lindahl, 1971). This site is probably the receptor for the immunity repressor and the operator for the A and B genes. The presumed BA operon would then be transcribed from left to right. I n P2 there is no evidence for a second early function operon controlled by the immunity repressor as observed in A.

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Since phages that have mutations in genes A or B do not lyse or kill the bacteria, it is likely that the expression of late functions in P2 depends in some way on the activity of these genes. The activation of late genes in P2 has not been studied in detail, but from what information is available (Bertani, 1968; L. E. Bertani, unpublished; G. Lindahl, personal communication), there is little or no activation of prophage genes in immune lysogens by superinfecting immunity-insensitive P2 phage. The late genes of P2 prophage can be efficiently activated, however, by phage P4 (Six, 1963; Six and Connelly, 1966; Six and Lindqvist, 1971), although again the mechanism is not yet established. The direction of transcription of genes in the left half of the P2 chromosome has been studied by Lindahl (1971) using polar am mutants. According to this test, if an am mutation in a given gene results in decreased expression of adjacent genes, that gene is assumed to be transcribed first. Of five groups of late genes, there were four that appeared to be transcribed in the rightward direction and one that might be transcribed in the opposite direction. The direction of transcription of the old and fun genes, which are active in the prophage state, has not been determined. B. FACTORS AFFECTING LYSOGENIZATION

It is characteristic of temperate phages that infection of a sensitive cell may have a t least two very different outcomes: lysis of the cell with phage production, or establishment of lysogeny. Although the outcome is probably determined by fluctuations in competing reactions, the mechanisms which lock the infected cell in one or the other pathway have not been completely elucidated. Phage P2 can establish lysogeny in all the bacterial strains that support its growth. Lysogenization occurs very efficiently in C and Sh (5-15% of the infected cells) (Bertani, 1957, 1959, 1962), but rarely in Sa (Bertani et al., 1967). At least in C and in Sh, the probability of establishment of lysogeny is a constant-under a set of cultural conditions-for an infected cell, independently of the multiplicity of infection. This is in contrast to other phages which show the Boyd effect, i.e., lysogenize more efficiently when the bacterium is multiply infected, and suggests that P2 DNA multiplies severalfold in most cells before becoming a stable prophage. When the phage has been irradiated with Uv light, a Boyd effect is noticeable also for P2 (L. E. Bertani, unpublished). It would be desirable to study the effect of multiplicity of infection on lysogenization with the mutants in cistrons A and B , which show

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no or very little DNA replication, but do lysogenize in a non-permissive host. As with other phages, the frequency of lysogenization depends very much on cultural conditions. A transient inhibition of protein synthesis or exposure to acridines in the course of infection increases very strongly the frequency of lysogenization in P2 (Bertani, 1957). A similar effect is obtained if the infected cells are exposed to a small dose of UV (Bertani, 1959). These phenomena are unexplained, but are ripe for a reexamination with more-advanced techniques.

CONTROL OF C. SPLIT-OPERON

THE

int GENE

Although the int gene of P2 occupies approximately the same position on the chromosome as that of A, its expression seems to be controlled in an altogether different way. I n A the int gene is under the control of the immunity repressor. Two experiments suggest, however, that the corresponding gene in P2 is expressed independently of the presence or absence of immunity repressor. First of all, P2 phage, superinfecting an immune lysogen, attaches readily a t the preferred P2 attachment site, provided the superinfecting phage is int+ (Six, 1966; Bertani, 1970). This can be interpreted to mean that the int gene of a superinfecting phage is expressed even when repressor is present. On the other hand, when a P2 c5 lysogen is derepressed by exposure to high temperature, the prophage does not detach efficiently (Bertani, 1968) unless the derepressed lysogen is simultaneously superinfected with wild-type P2. Again, to obtain detachment of the prophage, the superinfecting helper must be id+, suggesting that the prophage is unable to express its int gene even when derepressed. Thus, the expression of the int gene in P2 appears to depend more on the state of integration of the phage, i.e., whether it is superinfecting phage or integrated prophage, rather than on whether repressor is present or not. I n order to explain these observations, it has been proposed (Bertani, 1970) that the int gene in P2 belongs to an operon that extends to either side of the episite and that it is split off from its primary promoter site when the phage integrates into the chromosome. This type of regulatory mechanism is probably of secondary importance in the case of large phages like P2, most of whose genes are controlled by repressor. It might be of greater importance, however, for smaller viruses, such as polyoma or SV40, which have only enough DNA to code for five or six genes. Certainly it would seem more economical than using one gene to make a special regulatory substance to control the expression of the other four or five.

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D. INDUCTION One of the properties that P2 and the other related phages have in common is non-inducibility by UV light. P2 has also been tested for induction by other treatments, such as fluorodeoxyuridine (Bertani, 1964), mitomycin C (Levine, 1961), and thymine starvation (L. E. Bertani, unpublished) with negative results. Ultraviolet light induction of phage A has been explained by assuming that some product (“inducing substance”), either a DNA precursor or degradation product, formed as a result of the irradiation, complexes with and inactivates the phage repressor (Goldthwait and Jacob, 1964; Ben-Gurion, 1967; Hertman and Luria, 1967 ; Geissler, 1970). The same sequence of events may also precede the rare, spontaneous production of phage by A lysogens, as shown by the fact that rec- A lysogens are not only no longer induced by UV light, but also have greatly reduced rates of spontaneous phage production (Brooks and Clark, 1967). In the case of P2, however, there is no detectable decrease in the immunity of a lysogen following irradiation with UV light (L. E. Bertani, unpublished). Furthermore rec- P2 lysogens produce as much phage spontaneously as rec+ strains (Calendar, personal communication ; Laffler and Luria, personal communication). Thus, the events that lead to inactivation of the P2 immunity repressor must be quite different and the P2 repressor may be insensitive to the hypothetical “inducing substance.” The latter is consistent with the observation that double lysogens for P2 and A are inducible by UV light (G. Sironi and M. A. Pedrini, personal communication; L. E. Bertani, unpublished). The experiment is complicated by the fact that A can not multiply in the presence of a P2 prophage, but this difficulty was overcome either by using a P2 old mutant instead of wild-type P2 or by using a mutant of A that can circumvent the interference by P2 prophage. This result rules out the possibilities that, for example, the inducing substance is not formed in the presence of a P2 prophage or that P 2 produces so much repressor that there is not adequate “inducing substance” to inactivate all the repressor molecules present, because in these cases the P2 prophage should also protect A from induction. Even if the P2 repressor reacted with the hypothetical “inducing substance,” irradiated P2 lysogens most likely would not produce phage. It is possible to isolate mutants of P2 that make temperature-sensitive repressors (Bertani, 1968). When lysogens carrying such mutants are placed at 42OC, they do not produce phage unless they are superinfected

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with more homologous phage. Similar observations have been made concerning zygotic induction in P2. I n conjugation experiments, the transfer of a P2 c prophage into a nonlysogenic recipient may result in loss of recombinants without any detectable phage production (Kelly, 1963). Thus, inactivation or removal of P2 repressor does not necessarily initiate phage multiplication. As already discussed, this is most likely because the int gene is not efficiently expressed by a P2 prophage. A temperature-inducible derivative of P2 that gives good yields of phage when the lysogen is placed a t 42OC has been isolated (Calendar, personal communication) from the abortively inducible mutant P2 c6; it carries a second mutation that permits phage multiplication following derepression of the lysogen. Although inactivation of the P2 immunity repressor is not sufficient to trigger the conversion of prophage into multiplying phage, P2 lysogens do produce phage spontaneously with a certain low probability. Furthermore, it is known that spontaneous phage production depends on the int gene product, since lysogens carrying int mutant's have reduced phage production. Thus, in P2, spontaneous phage production could result from an accidental lowering of the immunity level, coincidental with some other prophage-localized event, such as exceptional transcription of the int gene. A formally equivalent hypothesis has been proposed by Six (1959) on the basis of the effect of prophage dosage on the rate of spontaneous lysis.

E. COMPARATIVE The defective phage P4 is able to establish itself as prophage even in the absence of helper phage, suggesting that it has all the genes necessary for integration (Lindqvist and Six, 1971). Moreover, stable lysogens or carrier cells may also be obtained following infection of a nonlysogen with P4 virl, an immunity-insensitive mutant of P4 (Lindqvist and Six, 1971). Strains doubly lysogenic for A and W+ phage are inducible: as in the case of P2 described above, the presence of a W+ prophage does not interfere with the induction of A by UV light (Kerszman et al., 1967). Abortive induction of a lysogen carrying a temperature-sensitive clearplaque mutant of phage 299 has been described (Golub and Zwenigorodsky, 1969), and this suggests that split-operon control of integration and excision may also be present in this phage. I n addition, Golub et al. (1970) have isolated a temperature-sensitive mutation in an essential gene, which prevents the loss of immunity a t high temperature in an abortively inducible prophage.

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225

A mutant of phage 186, 186 p , that is temperature inducible and gives good yields of phage, has been reported (Baldwin et al., 1966). VIII. The Lysogenic State

A. CHROMOSOMAL SITES On the basis of linkage between prophage and bacterial markers in crosses, it has been possible to demonstrate that in hosts C and K prophage P2 is attached to the bacterial chromosome (Bertani and Six, 1958; Kelly, 1963). Less-direct evidence indicates that this is true also for Sh (Bertani, 1954), and probably also for Sa (Bertani et al., 1967). P2 can attach as prophage at any of a number of different chromosomal sites, some of which have been precisely localized. The taxonomy of P2 chromosites is summarized in Table 3. It has been found that (1) in C, there is a very strong preference for one chromosomal site, called I, with several “second choice,” but still highly specific (because of demonstrated repeat occupancies) sites; (2) in K , there is no such definite preference-instead, two sites, H and 11, are occupied with about equal probability; (3) site I1 occurs both in C and in K, but H and I are not allelic (the genetic maps of C and K are largely homologous; see Wiman et al., 1970). For Sh there is no evidence from crosses, but results of superinfection experiments (see later) indicate a strong preference for one site, as in C (Bertani, 1954; Bertani and Six, 1958). For all cases where it has been studied (sites I and I1 in C ; sites H and I1 in K ) , prophage P2 appears to insert itself according t o the Campbell model, with the same episite being used in all cases, independently of the chromosite (Calendar and Lindahl, 1969). These experiments localized the episite between genes D and C, i.e., the same segment in the P2 map where int recombination is effective. The orientation of the prophages in respect to the host chromosome is different in the two locations I and 11. B. CHROMOSITE PREFERENCE The mechanism behind the chromosite preference pattern is incompletely understood. Most likely it reflects recognition of base sequences on the chromosome, which must be partly different from one another, by a base sequence on the phage DNA, the specific integration enzymes, or both. If chromosite I in C is replaced with the homologous chromosomal segment of K by transduction (Sunshine and Kelly, 19671, the

10 10 Dl

TABLE 3 Known P2 Chromosites in Eschetichia coli

Chromosite

1.

11.

He

111. IVh V through IX' Ek

Map location Between the histidine operon and methionine gene metG6.d a t about 4 x 0 0 of the E. coli map clockwise, from the conventional origin, tht. Cotransducible with metG, to a small extent also with his.c.bJProphage gene C is near the metG endb Between metE (a methionine gene) and thu (rhamnose)ba t 8s00 to 8 x 0 0 of the map, clockwise, from the origin. Cotransducible with both markers.c*bProphage gene C is near the metE end6 Between shiA (shikimate) and his,c.g,b a t about *$ioo of the map, clockwise, from the origin. Cotransducible in good frequency with his.c-b Prophage gene C is near the his end Between man (mannose) and his, a t about 3%00 of the map, clockwise, from the origind Weakly linked to tr?, (tryptophan) and metEc Not precisely localized Not precisely localized

Independent occurrences studied

Notes

10,a 8," 3,d 6,f Found in C, where it is the preferred site for P2. It is possible that a homologous, but much less 11: 7,jand efficient chromosite exists in K (see Section many others VIII, C.) l , 1,s ~ 4,L 10,~ Found in K and C. This might possibly be the same site studied by Fr6dhricq (1953)for his 14' isolate a

2," 3"

Found in K, where it is occupied about as often &s 1I.c This site could not be distinguished from I in the crosses of Table 4, referencen

1,s lf?,1'

Found in C. Possibly also in Kf

lA 1 each'

Found in C Found in C Found in C. Obtained with P2 suf

lk

The data in the third column must not be taken as a random sample of chromosite occupancies. Moreover, the techniques used in chromosite recognition varied a great deal, and 80 did the reliability of the identification. For chromosite I, data from superinfection experiments are excluded. Bertani and Six, 1958. * Calendar and Lindahl (1969). Kelly (1963).d Wiman, et al. (1970). e Bertani (1962). f Sunshine and Kelly (1967).g M. G.Sunshine (personal communication). A Six (1960).i Six (1966).i Choe (1969). Six (1971).

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resulting strain, although mostly C, becomes like K in respect to preference, i.e., the phage will lysogeiiize at one of several sites with roughly equal probabilities. The reciprocal situation, where the preferred chromosite I of C is introduced into K, has not yet been studied. There is also evidence for genetic changes in the phage which modify the chromosite preference. Six (1963, 1966, 1971) has shown that some phage which is produced from a prophage established in site I1 differs from the “wild-type” phage, in having an aItered site preference. This phage can be recognized in superinfection experiments in that it establishes double lysogeny more often than wild-type phage. Six was able to show that this difference is not the result of a preexisting phage mutation, but rather the consequence of an interaction between the prophage and chromosite 11. The new property (called saf) is inherited like any other genetic property in the course of vegetative multiplication. One would expect that saf phage has a sequence of bases in its episite partly different from the corresponding sequence in the wild-type phage, and probably more similar, or identical, to the sequence in chromosite 11. The lysogens in site I1 studied by Six did not produce a homogeneous population of phage in respect to the saf property, but rather a mixture of saf and s a p phage. If however one uses saf phage to establish lysogeny in site 11,the lysogens obtained produce pure saf phage. Similarly, heterogeneity of phage produced by a lysogen has been observed with a saf phage lysogenized in site I. Figure 3 presents two models which could explain the observations made to date. Phage produced by K lysogens in site H behaves like phage from site I in respect to site specificity when tested in C ; saf phage may be obtained from K site I1 as well as from C site 11. It should be stressed that these phenomena of site specificity are completely independent of immunity specificity: Six (1971) has repeated many of his experiments using P2 H y dis (which differs from P2 primarily in its immunity specificity) and found superimposable results. In C, as far as experience goes, no singly lysogenic clone has been obtained following infection of sensitive cells that did not have a prophage in site I. Sometimes, however, two prophage copies are established simultaneously, in which case two diff ercnt prophage sites are recognizable in the doubly lysogenic clone obtained, one being I, and the other being a “second choice” site, like I1 (Bertani, 1962). This situation is very different from what one observes in A, where as a rule doubly lysogenic strains carry the two prophages next to each other, in “tandem,” and are rather stable (Calef et al., 1965). Stable tandem dilysogcns are obtained in P2 only when the phage carries an int mutation (Choe, 1969; Bertani, 1971). I n a P2 tandem dilysogen

228

L. ELIZABETH BERTANI AND GIUSEPPE BERTANI

A. e-

-

/--

B.

FIG. 3. Possible schemes to explain chromosite preference and formation of suf phage. In both schemes one assumes a simple Campbell model, where the higher the similarity in base sequence between episite and chromosite, the more frequent the integration at that chromosite, and where episite in the wild-type phage and chromosite I are identical in base sequence by definition. According to scheme A (Six, 1966) the integration crossover can take place anywhere within the paired sites. Chromosite I1 differs from chromosite I over a certain stretch; as a consequence chromosite I1 consista of three rmbsegments: a and c, which are identical to the corresponding parts of chromosite I, and b, which is different. When a s a p phage is integrated in chromosite I (or, mutatis nautandis, a suf phage in chromosite 11) it makes no difference where in the site the reciprocal crossover occurs: the phage produced will be always the same.

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one of the two operons containing the int gene is reconstituted through joining of the two prophages, and will thus be active even in the lysogenic state (see Section VII, C ) . If the active operon contains a n int+ allele, the lysogen will be unstable because of int recombination between the prophages (Bertani, 1971). These facts suggest the following possibilities: ( 1 ) that P2 prophage tandems occur quite often in the course of lysogenization, but are rapidly reduced to monolysogens or (more rarely) to two-site dilysogens ; (2) that potential second choice chromosites may exist also for A prophage, but they are never effectively occupied because the second prophage establishes preferentially a tandem which is in this case a stable one (suggested by E. W. Six, personal communication). Similar relationships hold for the results of experiments where a lysogen in site I is superinfected with a homoimmune phage. Here, if the superinfection preprophage establishes itself, it either replaces the existing prophage (the more common event) or i t attaches a t a second-choice chromosite (Bertani, 1954; Bertani and Six, 1958; Six, 1960, 1961). Substitution of the existing prophage (as indicated by the replacement of prophage genetic markers) probably can take place as a result of ordinary recombination between the superinfecting phage and the resident prophage, but the more common event is one requiring int recombination (Bertani, 1970). This suggests that also in homoimmune superinfection a tandem structure is first formed in site I and then segregates to form stable single lysogens, some of which happen to carry the superinfecting ~~

When a snft phage integrates in chromosite I1 (or a s a j phage in site I) the needed reciprocal crossover may occur eithcr in a or in c : when phage is produced by such lysogens, presumably following excision of the prophage by a similar reciprocal crossover, the type of phage produced will depend on where the crossovers occurred. Integration in a and excision in n, or integration in b and excision in b, will give only the original phage type, whereas integration in u and excision in b, or integration in b and excision in a, will produce snf phage. Such lysogens therefore will produce always a mixture of snf and snf' phage. According to scheme B, integration occurs in three steps: (1) a specific enzyme makes single stranded cuts at the ends of episite and of chromosite, (2) exchange of partners takes place between the complementary single stranded ends thus produced (analogous to the cohesive ends of the mature phage DNA), (3) a ligase repairs the cuts, thus inserting covalently the prophage into the host chromosome. Lack of identity in the sequences a t the interacting sites will cause imperfect pairing, hence a hcteroduplex segment, which might then be recognized by degradative and repair enzymes with the final result that one of the two single stranded ends in each pair will be remodelled becoming identical in sequence to its partner. The lysogens obtained in such cases may be of all possible types, i.e., produce only saf phage, only sap phage, or a mixture of the two, and the frequencies of the various types will depend on the particular specificities of the repair enzymes involved.

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L. ELIZABETH BERTANI AND GIUSEPPE BERTANI

phage type, rather than the preexisting type. This possibility is supported by the finding (Six, 1971) that in superinfection of a normal lysogen with saf phage it is possible to obtain dilysogens with the two prophages very near each other, presumably in tandem, but not when both prophage and superinfecting phage are sap, as though tandems of two s a f f prophages were extremely unstable. As in other temperate systems, superinfection of a P2 lysogen with a related heteroimmune phage often leads to curing, i.e., loss of both phages and survival of the bacterium (Cohen, 1959; Six, 1960). It is not a t all clear why curing should be so common in heteroimmune superfection, and very infrequent in homoimmune superinfection.

C. EDUCTION OF HOSTCELLGENES

An interesting property of P2 has been observed in strain K : this is the removal, called eduction, of a piece of host chromosome adjacent to chromosite H (Kelly and Sunshine, 1967). The bacteria which have lost this segment of genome are easily recognizable, because they are histidine-dependent: the histidine operon is in fact very near site H, clockwise from it, on the bacterial chromosome. I n addition to the genes of the histidine operon, in all cases examined, the eductants have also lost two other genes, gnd and rfb, on the other side of the histidine operon (M. G. Sunshine and B. L. Kelly, personal communication). Eduction takes place spontaneously with low probability in bacteria lysogenic for P2 prophage in site H, simultaneous to the loss of the prophage. If the phage is an int mutant, eduction does not occur; if the lysogenic cell is superinfected with homoimmune int+phage, eduction takes place a t a very high frequency (M. G. Sunshine, unpublished). Since the end of the segment lost seems to be constant or nearly constant in all eductants, and it roughly corresponds to where site I should be if K were completely homologous to C, all these data would be easily explained if a site partially homologous to I existed also in K ; eduction could then result from a reciprocal exchange following pairing between one end of the prophage in H and this hypothetical site, with the help of the int recombination pathway (M. G. Sunshine and B. L. Kelly, personal communication).

D. IMMUNITY TO SUPERINFECTION A temperate phage does not give plaques on bacteria lysogenic for the same type of phage: the superinfecting phage is said to be immunity-sensitive. Its DNA becomes a superinfection preprophage, which

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does not multiply and is diluted out, apparently without being rapidly degraded, among the progeny of the superinfected bacterium (Bertani, 1954). The superinfection preprophage P2 is not completely inactive: it synthesizes immunity repressor (gene C) (Bertani, 1965), it converts to fluorouracil-sensitivity (gene fun) (Bertani and Levy, 1964), and it expresses the int gene (Bertani, 1970). I n addition, a small fraction of superinfection preprophages may participate in other reactions: prophage substitution, double lysogenization, and also vegetative multiplication. This last occurs with a small but definite probability for any superinfecting phage particle, which is unable itself, because of mutation, to make immunity repressor during its lifetime as preprophage. Although this probability appears to depend on the amount of immunity repressor present in the cell, it does not seem to be the result of a saturation of the repressor previous to vegetative multiplication of the superinfection preprophage (Bertani, 1965). Within this theoretical framework, the frequency of lytic reactions following superinfection with a weak virulent mutant may be used as a measure of the level of immunity in a lysogenic strain. Other more empirical methods consist in measuring the killing of lysogenic cells following exposure to increasing amounts of superinfecting phage (Bertani, 1961) or in studying the efficiency of plating of the lysogen for a set of P2 mutants having various degrees of immunity insensitivity-"intermediate" virulent mutants (Bertani and Six, 1958; Six, 1963). I n general one finds the following: (1) strains lysogenic for a c mutant have a lower immunity level than lysogens carrying the wild-type prophage; (2) a doubly lysogenic bacterium is more immune than the corresponding singly lysogenic bacterium ; and (3) a monolysogen in site I1 is more immune than the corresponding monolysogen in site I (Six, 1966; B. Ronn, personal communication). Point (1) is obvious and is consistent with gene C being the structural gene for the immunity repressor, point (2) refleck the presence of a double set of prophage genes, and point (3) can also be explained as a gene dosage effect if one considers that the origin of replication of the E. coli chromosome is said to be not far, counterclockwise, from chromosite 11, replication proceeding clockwise. This predicts that, on the average, in an actively multiplying cell population, there will be more than one copy (at most 2) of chromosite I1 for each copy of chromosite I.

E. COMPARATIVE Little is known about lysogeny in P2-related phages; P2 Hy dis and derivatives have been studied most, and, except for immunity specificity, behave very much like P2 (Bertani and Six, 1958; Six, 1960; Six, 1971).

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L. ELIZABETH BERTANI AND GIUSEPPE BERTANI

They cure P2 lysogens efficiently, when superinfecting, and this is reciprocal (Cohen, 1959; Six, 1960). The defective prophage existing in strain B, from which P2 H y dis may be obtained by recombination with P2, has been localized at a site near the histidine operon, clockwise from it (Cousin, 1964). This site, for all we know, could be homologous to site I. Apparently the inducible phage 424 has an attachment site very close if not identical to the site of this defective prophage (Cousin, 1964). Jacob and Wollman (1956) localized a site for each of the prophages 18, 186, and 299 on the bacterial chromosome. PK and 299 resemble P2 in their site specificity in C (Six, 1971) and in their ability to educe the histidine genes (M. G. Sunshine, personal communication). P3 (Bertani and Six, 1958; Kelly, 1963), W+ (Six, 1971), and P4 (M. G. Sunshine, D. Usher, and E. W. Six, personal communication) are known to attach to the host chromosome a t sites different from I ; in the P3 and P4 cases, these sites are also different from 11, 111, and H. Phage 186 does not seem to interact with P2 chromosites: doubly lysogenic bacteria for the two phages may be obtained, and no extensive curing is noted (G. Bertani, unpublished). ACKNOWLEDGMENTS In addition to several colleagues, mentioned in the text, who have allowed us

to refer to some of their still-unpublished results, we wish to thank particularly Dr. Erich W. Six for many discussions during the preparation of this review, Dr. Gunnar Lindahl for supplying extensive mapping data prior to publication, Dr. R. B. Inman for permission to reproduce his in part still-unpublished DNA denaturation maps, and Dr. Richard Calendar, for constructive comments on the manuscript. Our work has been supported by a joint grant from the Swedish Medical and Natural Sciences Research Councils, and the Swedish Cancer Society.

REFERENCES Anderson, T. F. 1960. On the fine structures of the temperate bacteriophages P1, P2 and P22. Proc. Eur. Reg. Conf. Electron Micros., Delft, 1960 2, 1008-1011. Arber, W.,and Linn, S. 1969. DNA modification and restriction. Ann. R e v . Biochem. 38, 467-500. Baker, R., and Tessman, I. 1967. The circular genetic map of phage S13. Proc. Nut. Acad. Sci. U.S. 58, 1438-1445. Baldwin, R. L., Barrand, P., Fritsch, A., Goldthwait, D. A., and Jacob, F. 1966. Cohesive sites on the deoxyribonucleic acids from several temperate coliphages. J . Mol. Biol. 17, 343-357. Ben-Gurion, R. 1967. On the induction of a recombination-deficient mutant of Escherichia coli K-12. Genet. Res. 9,309-330. Bertani, G. 1951. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacteriol. 62, 293-300.

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Bertani, G. 1953a. Infections bactkriophagiques secondaires des bacGries lysog6nes. Ann. Inst. Pasteur, Paria 84, 273-280. Bertani, G. 1953b. Lysogenic versus lytic cycle of phage multiplication. Cold Spring Harbor Symp. Quant. Biol. 18, 65-70. Bertani, G. 1954. Studies on lysogenesis. 111. Superinfection of lysogenic Shigellu dysentetiue with temperate mutants of the carried phage. J. Bucteriol. 67, 696-707. Bertani, G. 1958. Lysogeny. Advan. Virus Res. 5, 151-193. Bertani, G. 1962. Multiple lysogeny from single infection. Virology 18, 131-139. Bertani, L. E. 1957. The effect of the inhibition of protein synthesis on the establishment of lysogeny. Virology 4, 53-71. Bertani, L. E. 1959. The effect of ultraviolet light on the establishment of lysogeny. Virology 7, 92-111. Bertani, L. E. 1960. Host-dependent induction of phage mutants and lysogenization. Virology 12, 553-569. Bertani, L. E. 1961. Levels of immunity to superinfection in lysogenic bacteria as affected by prophage genotype. Virology 13, 378-379. Bertani, L. E. 1964. Lysogenic conversion by bacteriophage P2 resulting in an increased sensitivity of Escherichia coli to 5-fluorodeoxyuridine. Biochim. Biophys. Acta 87, 631-640. Bertani, L. E. 1965. Limited multiplication of phages superinfecting lysogenic bacteria and its implications for the immunity. Virology 27, 496-511. Bertani, L. E. 1968. Abortive induction of bacteriophage P2. Virology 36, 87-103. Bertani, L. E. 1970. Split-operon control of a prophage gene. Proc. Nut. Acad. Sci. U.S. 65, 331-336. Bertani, L. E. 1971. Stabilization of P2 tandem double lysogens by int mutations in the prophage. Virology (in press). Bertani, L. E., and Bertani, G. 1970. Preparation and characterization of temperate, non-inducible bacteriophage P2 (host: Escherichia coli). J. Gen. Virol. 6, 201-212. Bertani, L. E., and Levy, J. A. 1964. Conversion of lysogenic Escherichia colt by non-multiplying, superinfecting bacteriophage P2. Virology 22, 634-640. Bertani, G., and Six, E. W. 1958. Inheritance of prophage P2 in bacterial crosses. Virology 6, 357-381. Bertani, G., and Weigle, J. J. 1953. Host-controlled variation in bacterial viruses. J. Bacteriol. 65, 113-121. Bertani, G., Torheim, B., and Laurent, T. 1967. Multiplication in Serrutia of a bacteriophage originating from Escherichiu coli: Lysogenization and host-controlled variation. Virology 32, 619-632. Bertani, G., Choe, B. K., and Lindahl, G. 1969. Calcium-sensitive and other mutants of bacteriophage P2. J. Gen. Virol. 5, 97-104. Beumer, J. 1961. Isolement et ktude chez les Shigella des rdcepteurs aux bactkriophages du bacille de Lisbonne. Me'm. Acad. Roy. Med. Belg. 4 (31, 1 4 7 . Brooks, K., and Clark, A. J. 1967. Behavior of X bacteriophage in a recombinationdeficient strain of Escherichia coli. J. Virol. 1, 283-293. Calef, E., Marchelli, C., and Guerrini, F. 1965. The formation of superinfectiondouble lysogens of phage in Escherichiu coli K12. Virology 27, 1-10. Calendar, R. 1970. The regulation of phage development. Annu. Rev. Microbiol. 24, 241-296. Calendar, R., and Lindahl, G. 1969. Attachment of prophage P2: gene order at different host chromosomal sites. Virology 39, 867-881.

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Calendar, R., Lindqvist, B., Sironi, G., and Clark, A. J. 1970. Characterization of REP- mutants and their interaction with P2 phage. Virology 40, 72-83. Campbell, A. M. 1969. “Episomes,” 193 pp. Harper, New York. Chase, M. 1964. Reactivation of phage P2 damaged by ultraviolet light. Ph.D. Thesis in Microbiology, University of Southern California. Choe, B. K. 1969. Integration defective mutants of bacteriophage P2. MoZ. Gen. Genet. 105, 275-284. Christensen, J. R. 1964. Further studies of host-controlled modification of bacteriophages P2 and T1. Virology 24, 270-277. Cohen, D. 1959. A variant of phage P2 originating in Escherichia coli, strain B. Virology 7, 112-126. Cohen, D. 1960. Analyse de I’hybridation du phage P2 et d’un prophage dkfectif d’Escherichia coli B, par la mkthode de centrifugation en gradient de densitk. C.R. Acad. Sci. 250, 946-948. Cousin, D. 1963. Etude d‘un prophage dkfectif de la souche B d’Escherichiu coli. Thirse, Facultk des Sciences, Universitk de Paris. Cousin, D. 1964. Localisation gknetique d’un prophage dkfectif de la souche B d’Escherichia coli. Ann. Inst. Pasteur, Paris 106, 8474366. Denhardt, D. T., Dressler, D. H., and Hathaway, A. 1967. The abortive replication of +X-174 DNA in a recombination-deficient mutant of E. coli. Proc. Nut. Acad. Sci. US. 57, 813-820. Eastburn, J. 1969. Rescue by a prophage of a superinfecting irradiated phage. MS Thesis in Microbiology, University of Iowa, Iowa City, Iowa. Echols, H., and Gingery, R. 1968. Mutants of bacteriophage X defective in vegetative genetic recombination. J . MoZ. Biol. 34, 239-249. Edgar, R. S., and Wood, W. B. 1966. Morphogenesis of bacteriophage T4 in extracts of mutant-infected cells. Proc. Nut. Acad. Sci. U.S. 55, 498-505. Frkdkricq, P. 1953. Transfert gknbtique des propriktks lysogknes chez E. coli. C. R. SOC.Bwl. 67, 2046-2048. Geisselsoder, J., and Mandel, M. 1970. Physical properties of phage 299. Mol. Gen. Genet. 108, 158-166. Geissler, E. 1970. Zum Mechanismus der (W-)-Induktion von Prophagen. Stud. Biophys. Berlin 19, 185-206. Glover, S. W., and Aronovitch, J. 1967. Mutants of baoteriophage lambda able to grow on the restricting host Escherichia coli strain W. Genet. Res. 9, 129-133. Glover, S. W., and Kerszman, G. 1967. The properties of a temperate bacteriophage W+ isolated from Escherichia coZi strain W. Genet. Res. 9, 135-139. Goldthwait, D., and Jacob, F. 1964. Sur le mecanisme de l’induction du dkveloppement du prophage chez les bactkries lysoghes. C. R. Acad. Sci. 259, 661-664. Golub, E. I., and Reshetnikova, V. N. 1970. [A new case of prophage defective induction.] Mikrobiologiya 39, 1046-1050 (in Russian). Golub, E. I., and Zwenigorodsky, V. I. 1969. Defective thermal induction of a non-inducible bacteriophage. Virology 39, 919-921. Golub, E. I., Orlowa, G. G., and Reshetnikova, V. N. 1970. Repressor function of late phage gene. Virology 42, 538-539. Gough, M., and Levine, M. 1968. The circularity of the phage P22 linkage map, Genetics 58, 161-169. Hattman, S. 1964. The control of host-induced modification by phage Pl. Virology 23, 270-271. Hershey, A. D. 1969. Genetics Research Unit, Report of the Director. Carnegie Inst. Washington Year. 67, 555-568.

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Hertman, I., and Luria, S. E. 1967. Transduction studies on the role of a rec+ gene in the ultraviolet induction of prophage lambda. J . Mol. Biol. 23, 117-133. Ikeda, H., and Tomizawa, J. 1965. Transducing fragments in generalized transduction by phage P1. I. Molecular origin of the fragments. J. Mol. Biol. 14, 85-109. Inman, R. B. 1966. A denaturation map of the A phage DNA molecule determined by electron microscopy. J. Mol. Biol. 18, 464-470. Inman, R. B. (1967). Denaturation maps of the left and right sides of the lambda DNA molecule determined by electron microscopy. J. Mot. Biol. 28, 103-116. Inman, R. B., and Bertani, G. 1969. Heat denaturation of P2 phage DNA: compositional heterogeneity. J . Mol. Biol. 44, 533-550. Inman, R. B., and Schnos, M. 1970. Partial denaturation of thymine- and 5-bromouracil-containing X DNA in alkali. J. Mol. Biol. 49, 93-98. Inman, R. B., Schnos, M., Simon, L. D., Six, E. W., and Walker, D. H., Jr. 1971. Structural properties of P4 bacteriophage and P4 DNA. Virology 44, 67-72. Jacob, F., and Wollman, E. L. 1956. Sur le processus de conjugaison e t de recombinasion chez Escherichia coli. I. L’induction par conjugaison ou induction zygotique. Ann. Inst. Pasteur, Paris 91, 48Wi10. Jesaitis, M. A., and Hutton, J. J. 1963. Properties of a bacteriophage derived from Escherichia coli K235. J . E z p . M e d . 117, 285-302. Kaiser, A. D., and Hogness, D. S. 1960. The transformation of Eschen’chia coli with deoxyribonucleic acid isolated from bacteriophage Xdg. J. Mol. Biol. 2, 392-415. Kelly, B. 1963. Localization of P2 prophage in two strains of Escherichia co2i. Virology 19, 32-39. Kelly, B. L., and Sunshine, M. G. 1967. Association of temperate phage P2 with the production of histidine-negative segregants by Escherichia coli. Biochem. Biophys. Res. Commun. 28, 237-243. Kerszman, G., Glover, S. W., and Aronovitch, J. 1967. The restriction of bacteriophage A in Escherichia coli strain W. J . Gen. Virol. 1, 333-347. Lederberg, S. 1957. Suppression of the multiplication of heterologous bacteriophages in lysogenic bacteria. Virology 3, 496-513. Levine, M. 1961. Effect of mitomycin C on interactions bteween temperate phages and bacteria. Virology 13, 493-499. Lindahl, G. 1969a. Genetic map of bacteriophage P2. Virology 39, 839-860. Lindahl, G. 1969b. Multiple recombination mechanisms in bacteriophage P2. Virology 39, 861666. Lindahl, G. 1970. Bacteriophage P2: replication of the chromosome requires a protein which acts only on the genome that codod for it. ViroEogy 42, 522533. Lindahl, G. 1971. On the control of transcription in bacteriophage P2. Unpublished data. Lindahl, G., Sironi, G., Bialy, H., and Calendar, R. 1970. Bacteriophage lambda; abortive infection of bacteria lysogenic for phage P2. Proc. Nut. Acad. Sci. U.S.66, 587-594. Lindqvist, B. 1971. Vegetative DNA of temperate coliphage P2. Mol. Gen. Genet, 110, 178-196. Lindqvist, B. H., and Six, E. W. 1971. Replication of bacteriophage P4 DNA in a nonlysogenic host. Virology 43, 1-7. Mandel, M. 1967. Infectivity of phage P2 DNA in presence of helper phage. Mol. Gen. Genet. 99, 88-96. Mandel, M., and Berg, A. 1968a. Cohesive sites and helper phage function of P2, lambda, and 186 DNA’s. Proc. Nut. Acud. Sci. U.S. 60, 265-268.

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Mandel, M., and Berg, A. 196813. Melting temperatures of the cohesive ends of some non-inducible coliphage DNA's. J. Mol. Biol. 38, 137-139. Mandel, M., and Higa, A. 1970. Calcium-dependent bacteriophage DNA infection. J. Mol. Biol. 51, 501-521. Pizer, L. I., Smith, H., Miovic, M., and Pylkas, L. 1968. Effect of prophage W on the propagation of bacteriophages T2 and T4. J. Virol. 2, 1339-1349. Ritchie, D. A., and Malcom, F. E. 1970. Heat-stable and density mutants of phages T1, T3 and T7. J. Gen. Virol. 9, 35-43. Schnos, M., and Inman, R. B. 1970. Position of branch points in replicating A DNA. J. Mol. Biol. 51, 61-73. Schnos, M., and Inman, R. B. 1971. The starting point and direction of replication in P2 DNA, J. Mol. Biol. 55, 31-38. Shuster, R. C., Breitman, T. R., and Weissbach, A. 1967. The failure of three temperate coliphages to direct synthesis of a new A-type deoxyribonuclease. J. Biol. Chem. 242, 3723-3725. Scott, J. R. 1968. Genetic studies on bacteriophage P1. Virology 36, 564-574. Signer, E. R., Weil, J., and Kimball, P. C. 1969. Recombination in bacteriophage A. 111. Studies on the nature of the prophage attachment region. J. MoE. Biol. 46, 543-563. Sironi, G . 1969. Mutants of Escherichiu coli unable to be lysogenized by the temperate bacteriophage P2. Virology 37, 163-176. Six, E. W. 1959. The rate of spontaneous lysis of lysogenic bacteria. Virology 7, 328-346. Six, E. W. 1960. Prophage substitution and curing in lysogenic cells superinfected with heteroimmune phage. J. Bacteriol. 80, 728-729. Six, E. W. 1961. Inheritance of prophage P2 in superinfection experiments. Virology 14, 220-233. Six, E. W. 1963. A defective phage depending on phage P2. Bacteriol. Proc. p. 138. Six, E. W. 1966. Specificity of P2 for prophage site I on the chromosome of Escherichia coli strain C . Virology 29, 106-125. Six, E. W. 1971. Prophage integration specificity for P2-like phages. Unpublished data. Six, E. W., and Connelly, C. 1966. Helper-dependent multiplication of defective phage P4. Bacteriol. Proc., p. 112. Sip. E. W., and Lindqvist, B. H. 1971. Multiplication of bacteriophage P4 in the absence of replication of the DNA of its helper. Virology 43, 8-15. Skalka, A., Burgi, E., and Hershey, A. D. 1968. Segmental distribution of nucleotides in the DNA of bacteriophage lambda. J. Mol. Biol. 34, 1-16. Smith, H. S., Pizer, L. I., Pylkas, L., and Lederberg, S. 1969. Abortive infeotion of Shigella dysenteriae P2 by T2 bacteriophage. J. Virol. 4, 162-168. Sunshine, M. G., and Kelly, B. 1967. Studies on P2 prophage-host relationships. I. Alteration of P2 prophage localization patterns in Escherichiu coli by interstrain transduction. Virology 32, 644-653. Tessman, E. S. 1965. Complementation groups in phage 513. Virology 25, 303-321. Thomas, C. A., Jr. 1966. The arrangement of information in DNA molecules. J . Gem. Physiol. 49, (Part 2), 143-169. Thomas, R., and Bertani, L. E. 1964. On the control of replication of temperate bacteriophages superinfecting immune hosts. Virology 24, 241-253. Uetake, H., Toyama, S., and Hagiwara, S. 1964. On the mechanism of host-induced modification. Multiplicity activation and thermolabile factor responsible for phage growth restriction. Virology 22, 202-213.

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Visconti, N., and Delbriick, M. 1953. The mechanism of genetic recombination in phage. Genetics 38, 5-33. Wang, J. C. 1967. Cyclization of coliphage 186 DNA. J. MoZ. Biol. 28, 403-411. Wiman, M., Bertani, G., Kelly, B., and Sasaki, I. 1970. Genetic map of Eschen'chia coZi strain C. MoZ. Gen. Genet. 107, 1-31. Wolf, B., and Meselson, M. 1963. Repression of the replication of superinfecting bacteriophage DNA in immune cells. J . Mol. Biol. 7, 636-644. Wu, R. 1970. Nucleotide sequence analysis of DNA. I. Partial sequence of the cohesive ends of bacteriophage A and 186 DNA. J. Mol. Biol. 51, 501-521. Yamagishi, H. 1970. Nucleotide distribution in the DNA of Escherichiu coli. J . MoZ. BioZ. 49, 603-608.

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Howard B Newcornbe Biology and Health Physics Division. Atomic Energy of Canada limited. Chalk River. Ontario

I . Introduction . . . . . . . . . . . . . . . . . . I1. Resistance, Sensitivity. and Repair . . . . . . . . . . . A . Cell Stage and Sensitivity . . . . . . . . . . . . . B . Genetic Control of Sensitivity . . . . . . . . . . . . C . Repair Mechanisms and Sensitivity . . . . . . . . . . D . External Modification of Sensitivity . . . . . . . . . . E . Fractionals and Repair Mechanisms . . . . . . . . . . F. Molecular Basis of Resistance, Sensitivity. and Repair . . . . . G . Biological Significance of Repair Mechanisms . . . . . . . I11. Nature of the Genetic Changes . . . . . . . . . . . . A . Gene Mutations. . . . . . . . . . . . . . . . B . Chromosomal Aberrations . . . . . . . . . . . . . C . Lethal Sectoring . . . . . . . . . . . . . . . . D . Induced Recombination . . . . . . . . . . . . . E . Gene Conversion and Paramutation . . . . . . . . . . F . Miscellaneous Genetic Effects . . . . . . . . . . . . IV . Scientific and Practical Uses of Induced Genetic Changes . . . . . A . Identification of the Chromosomes That Carry Particular Genes . . B . Measurement of Gene Length . . . . . . . . . . . . C . Studies of the Effects of Different Dosages of Particular Genes and Chromosomes . . . . . . . . . . . . . . . . 1). Studies of Meristems . . . . . . . . . . . . . . E . Practical Uses in Insect Control and Plant Breeding . . . . . 1 ' . Quantitative Studies Involving Differences in the Exposures . . . . A . Dose-Response Relationships . . . . . . . . . . . . B. Dose-Rate and Fractionation Effects . . . . . . . . . . C . Ion-Density Effects . . . . . . . . . . . . . . . D . Transmutation Effects and Incorporated Radioisotopes . . . . 1'1. Importance of the Cellular Consequences of Genetic Changes . . . . A . Cell Death . . . . . . . . . . . . . . . . . B . Carcinogenesis . . . . . . . . . . . . . . . . VII . Importance of the Hereditary Consequences in Individuals and Populations . . . . . . . . . . . . . . . . . . A . Effects on Quantiative Characters . . . . . . . . . . . B . Genetic Deaths and the Sex Ratio . . . . . . . . . . C . Effects on the Fitness of Populations . . . . . . . . . . I). Human Implications . . . . . . . . . . . . . . VIII . Conclusions . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . 239

240 241 241 243 245 247 250 252 255 256 256 258 260 261 263 264 266 266 266 267 267 268 269 270 272 274 275 276 276 277 279 280 281 283 286 288 290

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I. Introduction

A review may serve to acquaint a reader with recent developments, and i t may seek to evaluate current success in achieving the aims that prompted the work. The present account will attempt to perform both functions, in terms of the genetic consequences of exposing cells, organisms, and populations to ionizing radiations. Discussion is restricted to studies reported over the 3-year period 1967-1969. The review is written less for the specialist interested in particular genetic effects, than for the investigator concerned with the field as a whole. When X-rays were first shown to cause hereditary changes it was hoped that the processes of organic evolution might be better understood through studies of the induced mutations, and also that the practical uses of the radiations would be facilitated by detailed knowledge of the lethal and hereditary effects of the induced chromosomal rearrangements and gene mutations. Work currently in progress reflects these early aspirations. Radiations are being used, as in the past, to investigate the origins and nature of genetic diversity and the role of selective forces in perpetuating or suppressing newly arisen changes. They are being applied to studies of the structure of genes and chromosomes, and the changes which these structures are capable of undergoing. With more practical ends in view, the genomes of living cells are being altered by irradiation to improve agricultural crop plants, and to cause death or sterility for the purpose of destroying cancer cells or controlling insect pests. By far the strongest stimulus for research in radiation genetics, in terms of financial support, has occurred because of concern over the consequences for man of the increasingly widespread use of major sources of radiation and radioactive materials as a result of the development of nuclear energy. I n view of the magnitude and purpose of this investment of effort in science, by society, it is particularly fitting to consider in what measure basic studies have succeeded in contributing to knowledge of the importance to man of the changes that are induced in the genetic materials of his own germ plasm and body cells. A number of recent reviews and general articles, dealing mainly with work published prior to the period covered by the present account, should be mentioned in passing. These relate to the following topics: radiation genetics (Wolff, 1967), radiosensitivity and repair (IAEA, 1966a, 1968a ; Sobels 1969a), mutation in microorganisms (Auerbach, 1967), mammalian radiation genetics (Green, 1968a ; Searle, 1967), human radiation cyto-

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genetics (Evans et al., 1967), transmutations of radioisotopes (IAEA, 196813), and uses of induced mutations (Stubbe, 1967). In Wolff’s review (1967) it was stated that recent work has given rise to “no conceptual changes that seem likely to affect the field.” If this comment still holds true, it constitutes a special reason to reexamine current strategies in radiation genetics.

H. Resistance, Sensitivity, and Repair

Differences in sensitivities to radiation-induced genetic change have been of theoretical interest from the beginning, and the extents of such differences are of current practical importance in relation to the protection of man from radiation injury. Relevant research has paid particular attention to such differences in the various stages of the cell cycle, in cells of different genetic make-ups, and under various external circumstances. Current interpretations have been concerned especially with repair systems operating at the molecular and chromosomal levels.

A. CELL STAGEAND SENSITIVITY The yields of chromosomal aberrations and gene mutations from a given radiation exposure have long been known to vary with the stage in the cell division cycle. The recent evidence serves mainly to confirm that, for widely different organisms and cell types, a period of increasing sensitivity occurs in late prophase after DNA synthesis has been completed. Only limited evidence has been presented concerning the possible role of repair mechanisms in contributing to these changes. However, it does appear that repair of the initial lesions and fixation of genetic damage tend to occur preferentially or most rapidly during the synthesis of new DNA or shortly thereafter. Among female germ cells, for example, immature oocytes of mice in the prolonged (dictyate) prophase stage show low sensitivity to the induction of mutations at specific loci by chronic exposure to neutrons or y-rays (Searle and Phillips, 1968). By way of contrast, mature late prophase oocytes of the insects Drosophila and Dahlborninus have been shown to be markedly sensitive to the induction of chromosomal translocations and visible mutations, respectively (Traut, 196713; Baldwin, 1968, 1969a,b). For both of these insects, the sensitivity of the oocytes continues to increase as they approach full maturity (Zimmering and

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Scott, 1968; Baldwin, 1968, 1969b). An apparent exception to this general rule, i.e., a seemingly low yield of induced translocations from the otherwise sensitive late oocytes of Drosophila, has been accounted for by Traut as due to an association a t high doses between translocation induction and induced lethal changes; at low doses the yield of translocations reaches the expected high level. Among male germ cells, the spermatogonia (premeiotic stem cells) of mice have been shown to be exceedingly insensitive to the induction of changes leading to X-chromosome loss (LBonard and SchrSder, 1968), although they are capable of yielding some transmissible chromosome aberrations (Griffin and Bunker, 1967). The spermatids (postmeiotic cells) of mice are the most sensitive stage for the induction of translocations (LBonard and Deknudt, 1968b), but losses of ring-X chromosomes from male germ cells of Drosophila represent an exception in that the greatest sensitivity to radiation-induced loss occurs in the earlier spermatocyte stage (Leigh, 1969). To account for this, it has been proposed that the major mechanism involves induced crossing-over rather than chromosome breakage or nondisjunction. After the sperm nucleus enters the egg it passes through a period of high sensitivity to induced genetic damage during the process of changing into the paternal pronucleus. This sensitivity has been observed in Drosophila for the induction of recessive lethals (Graf et aE., 1969) and in mice for the induction of translocations (LBonard and Deknudt, 1967a). The possibility that the high sensitivity could be connected with the replacement of arginine-rich histone by lysine-rich histone, suggested by Graf and his co-workers, is of interest but would be difficult to test experimentally. Among somatic cells, enhanced sensitivity to radiation-induced chromosome breakage during late prophase (i.e., in Gz as compared with G, and S) has been confirmed in such diverse sorts as human lymphocytes (Prembree and Merz, 1969a), cultured lines of marsupial cells (Bick and Brown, 1969), and those of Vicia root tips (Scott and Evans, 1967). An exception occurs, however, in Paramecium. Two kinds of X-ray-induced mutations are recognized in this organism; one type remains largely repairable until the occurrence of DNA synthesis, while the second, which probably includes 2-hit chromosomal aberrations, tends to be rapidly fixed in its final mutant form. For the induction of both types, the G, period is much more sensitive than the G, (Kimball and Perdue, 1967). Sensitivity to radiation-induced genetic change is not always associated with less-rapid repair. In human lymphocytes, for example, repair in the form of restitution of chromosome breaks takes place more rapidly,

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rather than less so, during the G, period in which the chromosomes are particularly sensitive to X-ray-induced breakage (Prembree and Merz, 196913). Also, the rate of fixation of radiation-induced genetic damage in Paramecium, which might be expected to be associated with rate of repair and degree of resistance, occurs mainly during the S period and to only a limited extent in the sensitive GI and the resistant G, stages (Kimball and Perdue, 1967). I n this organism, some of the damage induced in G, is converted to an irrepairable form prior to the next S stage, and i t appears that the rates of fixation and repair, and the sensitivity to genetic damagc, all vary over the mitotic cycle but not necessarily in similar ways. Radiation-induced lesions, of course, differ in the above respects from those induced by such chemicals as the alkylating agents. For the latter, fixation of the damage in Paramecium occurs only in the S stage. Also there is no detectable interaction of X-ray-induced with chemically induced chromosomal breaks in the cells of Vicia root tips (Scott, 1968).

B. GENETICCONTROLOF SENSITIVITY Resistance and sensitivity to the lethal effects of radiation are in some instances substantially influenced by identifiable single gene differences, this being true for both X-rays and ultraviolet radiation. Although this review is concerned primarily with the effects of ionizing radiations, some discussion of sensitivity to ultraviolet light is unavoidable, since the same mutations may affect the responses to both agents. Recent work has added to the numbers of mutant genes known t o affect the responsc to radiation, and for certain of the genes the locations in linkage maps have been determined. I n addition, polygenically determined differences in sensitivity have been described, and correlations of resistance and sensitivity with polyploidy and with variation in the amounts of nuclear material have been investigated further. Only in the microorganisms has i t been possible to map by linkasge methods the gene loci a t which mutations to resistance and sensitivity occur. In the case of Escherichia coli, the position of the locus responsible for the unique resistance of the B/r mutant to both X-rays and UV was determined only after transduction of the gene to a crossable strain of E . coli. It was then shown to lie between genes for the fermentation of lactose and arabinose (Donch e t al., 1969); other loci for UV resistance and sensitivity have also been mapped (Donch and Greenberg, 1968; Chung and Grecnberg, 1968). I n addition, tests of allelism in yeast have shown that some 22 loci are associated either with combined X-ray and UV sensitivity or with UV sensitivity alone (Cox and Parry,

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1968) and 3 other loci are associated with X-ray sensitivity alone (Resnick, 1969a). Less is known about the genetic basis of radiation sensitivity in higher organisms, but a natural population of Drosophila has been shown to be polymorphic for a number of loci on the second and third chromosomes that influence in an additive fashion the degree of sensitivity (Parsons e t al., 1969) ; the genes of the X chromosome appear to have little effect. Such differences in sensitivity are presumably widespread, and inbred strains of silkworm have been shown to differ as much as eightfold with respect to sensitivity of embryos to killing by radiation (Murakami, 1969). Less readily interpretable, although presumably under genetic control, is an observed increase in sensitivity to both X-rays and UV in a cell line from an irradiated culture of Chinese hamster cells. The karyotypes in this line have been analyzed (Todd and Hellewell, 1969) but it is the stability of the phenotype that most strongly suggests that a deletion of genetic material may be responsible. The genetic redundancy associated with polyploidy has long been known to contribute to radiation resistance in certain instances. Further evidence of this kind has been obtained recently from studies of diploid, tetraploid, and hexaploid species of Solanuin (Yamagata e t al., 1969). However, the expected differences were observed only with acute irradiation and not with chronic. Also, substantial interspecific differences were observed within the same levels of polyploidy. Such evidence points to genetic make-up as more important than either genetic redundancy or interphase chromosome volume in determining degree of radiation resistance and sensitivity. The idea that the total DNA content, or the volume, of the nucleus or of the chromosomes may influence sensitivity to both the mutagenic and the lethal effects of ionizing radiation has been extended by the recent studies of Sparrow and his co-workers (see Baetchke e t al., 1967; Sparrow et al., 1968; Unterbrink e t al., 1968). Over a wide range of plant species the mutation rates per roentgen as measured by petal spotting show a close correlation with the amount of DNA per chromosome, and the sensitivity to killing (LD,,,) appears to be linearly related to the susceptibility to induced mutation. Among the plants used for this particular series of studies, the sensitivities to induced genetic change covered a 75-fold range and the DNA contents per chromosome a 50-fold range. Other studies have explored sensitivity to radiation-induced killing among 120 diverse sorts of organisms ranging from viruses to higher plants and animals. These were found to fall into eight discrete groups when the doses required to produce equal killing were plotted against

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chromosome volume. The significance of the groupings is not yet clear and they bear no simple relationship to the phylogenetic classifications.

C. REPAIRMECHANISMS AND SENSITIVITY Where variations in radiation sensitivity are determined by single gene differences, it is customary to look for correlations that will reveal something of the nature of the repair processes. The characteristics that have been found to be altered in X-ray sensitive mutant strains of microorganisms include (a) UV sensitivity, (b) susceptibility to induced and spontaneous mutation, (c) absence of photoreactivation and multiplicity reactivation, (d) absence of ligase activity, (e) loss of ability to excise DNA, and ( f ) recombination deficiency and other anomalies of meiosis. Increased sensitivity may, of course, sometimes mean simply that more initial damage is inflicted. However, the conclusions that are usually drawn are that repair processes are involved and, depending on the numbers and natures of the correlated characteristics, that such processes are multiple and presumably necessary for normal functioning of the cell even in the absence of radiation. Of the many known radiation sensitive mutants of yeast, by far the majority are sensitive either to ultraviolet and X-rays or to ultraviolet alone (Cox and Parry, 1968; Laskowski et al., 1968; Nakai and Matsumoto, 1967; Snow, 1968). Only recently have a few loci been shown to yield mutants that are sensitive to X-rays but not to UV (Resnick, 1969b), This latter phenomenon has not so far been reported for bacteria, with the partial exception of one mutant of E . coli which is unable to repair X-ray-damaged phage DNA although it is quite capable of repairing UV-damaged phage and X-ray damage to its own DNA (Kato and Kondo, 1967). Examples of mutations to X-ray and to UV sensitivity alone are normally interpreted as indicating a t least two separate pathways in the repair mechanisms. The mutations to combined X-ray and UV sensitivity are regarded as evidence for a common step in these pathways and multilocus control as indicating a multiplicity of steps in each. An association between X-ray sensitivity in yeast and an increase in the spontaneous mutation rate (von Borstel e t al., 1968) has been interpreted by Resnick (196913) as indicative of an association between repair mechanisms and a wide variety of normal phenomena. Somewhat curiously, an analogous observation with respect to susceptibility t o induced mutation runs in the opposite direction. The rate a t which mutations to prototrophy occur in E . coli under the influence of 7-rays is reduced in the susceptible strains, rather than increased, a t least when

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the comparison is made between samples in which equal amounts of killing have been induced (Stern, 1969). Since the difference in mutation rate when expressed in this manner is greater in the presence of oxygen than in its absence, it is theorized that oxygen acts to change potential mutations into lethal damage so that the two processes are in competition with each other. A number of X-ray-sensitive mutants of microorganisms are even more obviously deficient for various components of the repair systems. These include a strain of Neurospora that lacks the ability to respond to photoreactivating light (Tuveson and Mangan, 1968), a strain of bacteriophage T4 that exhibits reduced multiplicity reactivation (Boyle and Symonds, 1969), and a strain of E . coli in which the enzyme ligase fails to function at certain restrictive temperatures so that DNA produced during either normal replication or repair synthesis fails to be integrated into an intact strand (Pauling and Hamm, 1969). A variety of possible associations between the yield of radiation-induced mutations, on the one hand, and the repair and/or recombination processes, on the other, are suggested by studies with E . coli. However, the various inferences sometimes appear to be mutually exclusive. Where loss of the ability to carry out excision repair is associated with no change in the yield of X-ray-induced mutations (Kondo, 1969), it has been inferred that the potentially mutagenic damage resulting from the exposure is not excisable in either the normal or the deficient strain. Where the opposite association is found and loss of ability to carry out excision repair is associated with a gross reduction in the yield of 7-ray-induced mutations, i.e., to 5% of that in the normal strain (Bridges et al., 19681, it has been inferred that the induced mutations in the normal strain are caused by errors in the excision repair. Similarly, from one recombination deficient strain of E . coli that yields no X-rayinduced mutations (Kondo, 1969), it has been inferred that the induced mutations in the normal strain are due to errors in recombinational repair. (See also Witkin, 1969, for further discussion.) Such ready explanations of opposing effects, on the basis of known systems, seem almost too facile, and one is inclined to suspect that an unknown system may actually be involved. That there may be repair mechanisms that make use of some sort of genetic recombination to get rid of radiation-induced damage has been suggested by Howard-Flanders and his co-workers (1968). They observed recombination deficiency in some bacterial mutants that are X-ray- and W-sensitive. Recent data from Salmonella support this evidence (Eisenstark et al., 1969), and some sensitive mutants of yeast have also been found to be recombination deficient (Resnick, 1969a).

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Conversely, i t has been claimed that some mutants of yeast selected for recombination deficiency are also X-ray sensitive or have mutant genes that are allelic with those for X-ray sensitivity (Rodarte e t al., 1968) and that a similar association has been observed also in Ustilago (Holliday, 1967). Curiously, none of the UV-sensitive mutants of yeast identified by Snow (1968) was recombination deficient. However, there are examples from a variety of organisms of associations of UV sensitivity with effects on meiosis, e.g., in yeast (Cox and Parry, 1968), Aspergillus (Lanier e t al., 1968) and Ustilago (Holliday, 1967), and on sporulation in yeast (Puglisi, 1967).

D. EXTERNAL h~ODIFICATION O F SENSITIVITY The yields of radiation-induced changes in the genetic material of the cell, e.g., gene mutations and chromosomal breaks and rearrangements, may be modified by a wide variety of supplementary treatments, either during the radiation exposure or administered shortly after its completion. Current research continues to contribute to the already substantial body of knowledge on this subject. Much of the appeal of such work has to do with the hope of throwing light on the nature of the processes leading to gene mutation and chromosome rearrangement, and the method by which the initial lesions are repaired before they become “fixed” in some final form. The effects of oxygen still receive considerable attention, and a variety of other agents such as nucleic acid base analogs, antibiotics, energy sources, and even simple storage, have been tested and shown to be effective in altering the yield of radiation-induced genetic changes. Post-irradiation exposure of Drosophila to oxygen, as compared with an oxygen-free atmosphere, decreases the frequencies of such induced changes as mutations and chromosome translocations in early spermatids (Watson, 1967), detachments of attached X chromosomes in oocytes (Seeley and Abrahamson, 1969), mortality in early and late oocytes (Sankaranarayanan 1969a,b), and conversion of potential recessive lethal mutations into dominant lethals (Rinehart, 1967). Effects of this kind are not confined to Drosophila; in barley, restitution of chromosome breaks is favored by the presence of oxygen after irradiation especially in combination with an elevated temperature (Kumar and Natarajan, 1967). Such post-irradiation effects of oxygen are interpreted in terms of a possible stimulation of metabolic activity and, more particularly, of repair or restitution of premutational damage and chromosome breaks. The apparent absence of post-irradiation oxygen effects in mature

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spermatozoa of Drosophila (Sankaranarayanan, 1967b), and especially when these are irradiated in the female (Sobels, 1969b), might be taken as supporting this interpretation if one regards the presence of cytoplasm as important for nuclear repair. The presence of oxygen during irradiation has long been known to increase the yields of genetic changes. An exception, however, has recently been reconfirmed. I n the case of radiation-induced losses of X chromosomes in Drosophila, the oxygen effect is not observed where the X chromosome contains an inverted region or is a ring chromosome. The latter observation has recently been repeated with a different strain of flies carrying ring Xs (Leigh, 1968). The structural feature shared by such oxygen-refractory X chromosomes is thought to be associated with the distribution of heterochromatin about the centromere, but whether the losses are due to chromosome breakage as is commonly assumed is still uncertain. Other supplementary treatments should be mentioned. a. Triphosphates. Triphosphates of adenosine and cytosine have been shown to be active in reducing the amount of radiation-induced genetic damage in two quite different sorts of organisms. In Drosophila, ATP injected near the testes prior to irradiation reduces the yield of dominant lethals from all postmeiotic stages except spermatozoa, although no effect was observed on the induction of recessive lethals or of translocations and deletions involving the X chromosome (Mittler and Walsh, 1967). I n Tradescantia, the triphosphates of both adenosine and cytosine are effective in reducing the yields of radiation-induced chromosomal aberrations (Beatty and Beatty, 1967). It is believed that the triphosphates act by furnishing energy for the repair processes. b. Caffeine.Administered prior to or after irradiation, caffeine increases the yields of induced recessive lethals from all stages of spermiogenesis in Drosophila, except for mature spermatozoa in which the yield is reduced (Mittler and Callaghan, 1969). In barley, post-irradiation treatment with caffeine enhances the yield of chromosomal aberrations by as much as two- to threefold (Yamamoto and Yamaguchi, 196913). It has been variously suggested that caffeine may act as a metabolic inhibitor or anoxia inducer (in view of the effect on mature spermatozoa) and, from the results of chemical studies with bacteria, that it may specifically inhibit the excision enzyme of dark repair and possibly repair replication. c. Halogenated Analogs. Halogenated analogs of the nucleic acid bases have been shown not only to sensitize when incorporated into DNA prior to irradiation, but also to be active when applied after exposure to ionizing radiation. Bromouracil as a post-irradiation treatment was

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found to increase the yield of egg-color mutations in silkworms by twoto threefold (Murakami and Ito, 1969), and an analogous effect of 5-fluorodeoxyuridine has been observed with respect to induced chromosomal aberrations in barley (Yamamoto and Yamaguchi, 1969a). In the silkworm, the enhancement, which is known as “co-mutagenesis,” is believed to occur because the bromouracil becomes incorporated into the DNA in the place of thymine during the repair synthesis that follows the excision of DNA in which there are radiation-induced lesions. The effect in this organism is greatest when the insects are fed on bromouracil 8 hours after the irradiation. The use of fluorodeoxyuridine in barley is thought to cause the chromosome breaks to remain open (i.e., unrestituted) longer so that the ends are available for rejoining with other broken ends. A recent example of sensitization, as distinct from postirradiation modification, is an enhancement by 5-iodo-2’deoxyuridine of the yield of radiation-induced chromosome bridges in cultured calf thyroid cells that had been grown in the presence of the agent (Weijer and Giblak, 1968). The mechanisms involved in this sort of sensitization have been widely discussed in the earlier literature. d. Sulfaguanidine. When present during irradiation, sulfaguanidine is reported to reduce substantially the normally high proportion of incomplete, as compared with complete, chromatid exchanges induced in root tip cells of Vicia during G, phase (Lazhnyi, 1968). No corresponding effect on the ratio of fragments to reunions was observed among the chromosome aberrations induced in G,. The interpretation of the phenomenon is unclear. e. Actinomycin D . Actinornycin D has been shown, by means of fractionated exposures to X-rays, to inhibit restitution of chromosome breaks induced during S phase in human lymphocytes (Prembree and Mere, 1969a). This antibiotic is believed to inhibit DNA-dependent RNA synthesis and subsequent protein synthesis. It also blocks DNA synthesis by complexing with guanine. f. Dimethyl Sulfoxide. When present during irradiation, dimethyl sulfoxide reduces the yield of aberrations induced in the chromosomes of Vicia root tips to about half (Kaul, 1969). The mechanism of action is unknown, but “radical scavenging” has been suggested as a possibility. This agent has been found previously to protect animals against the lethal effects of irradiation. g. Storage. In addition to the above array of chemically active agents, simple storage of cells in a nondividing state has been shown to reduce the amount of genetic damage induced by radiation, in organisms as remote from one another as cereals and mammals. Induced mutation frequencies in rice and induced chromosomal aberrations in barley were

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both lowered when hydration of neutron irradiated seeds was delayed for 10 days after the exposures (Rao et al., 1968). Only among the chlorophyll mutations of rice were there inconsistencies, some rates being higher and some lower as a result of the delay. I n mice, oocytes had been shown earlier to be capable of repairing neutron-induced damage that would otherwise lead to specific locus mutations, provided that sufficient time is allowed before fertilization. A similar effect has now been confirmed for X-ray-induced damage (Russell and Kelly, 1968) ; and in yeast, prolongation of the interval between X-irradiation and cell division resulting from treatment with p-mercaptoethanol has been found to decrease lethal sectoring and increase survival (James and Werner, 1969b). Such studies of the external modification of radiation-induced genetic injury are of special interest where they point fairly directly to interpretations in terms, for example, of molecular processes of repair or of restitutions of chromosome breaks. However, one is impressed by the almost infinite array of potentially possible experiments, involving different supplementary treatments, organisms, cellular states, and genetic end points. I n view of this, it seems almost inevitable that unexplained phenomena and plausible speculations should continue to greatly outnumber genuine new insights.

E. FRACTIONALS AND REPAIR MECHANISMS The occurrence of mutations affecting some but not all of the lines of descent from an irradiated genome attracted attention in the past as indicating either that the genes and chromosomes were not single structures a t the time of exposure, or that the processes leading to genetic change had remained in some way incomplete until replication had taken place. More recently, induced mosaic or fractional mutants have been studied in the hope that they might serve to indicate a failure on the part of the repair processes to restore one or other of the strands to reflect in a complementary fashion the precise information contained in the mate. On this view, the non-mosaic or complete mutants are regarded as indicative of “repair” of nonmutated strands so that they become strictly complementary to their mutated counterparts (see Auerbach 1967; Dubinin and Soyfer, 1969). Because other possible explanations of the occurrence of complete mutants appear improbable in the light of experimental evidence, much of the current data relating to fractional mutations has been interpreted in these terms. Although many organisms are suitable for studies of the phenomenon, most of the recent data have come from yeast, fruitflies, and silkworms.

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Not all mosaics are associated with point mutations. I n silkworms, the frequency of mosaics for mutations at loci controlling the color of the serosa cells of the eggs rises exponentially with increasing dose to mature spermatozoa, indicating a 2-hit origin (Tazima and Onimaru, 1969). If one interprets the mosaics that are represented only by small mutant areas of the egg as due to chromosome breakage and loss of a fragment, roughly half may be assigned to this cause. I n Drosophila, the dumpy locus mosaics are known to be unassociated with chromosome breakage. However, the yield of mosaics in this organism reaches a plateau at very low doses, indicating that the processes may differ substantially from those associated with ordinary mutations. No single current hypothesis adequately accounts for the various data from different organisms. Induced mosaicism is not restricted to gene changes, associated and unassociated with chromosomal rearrangement, but includes as well chromosome rearrangements unassociated with gene mutations, as has been demonstrated for Drosophila (Abrahamson et al., 1968). The proportions of fractional to complete mutations have been known for some time to vary with the radiation dose in the case of yeast, i.e., being greatest when the radiation dose is low. Only recently, however, has the analogous observation been made for Drosophila (Matsudaira et al., 1967). With exposures of less than 1500 R to males, fractional mutations at specific loci outnumbered the corresponding whole-body mutations by more than 2: 1, whereas a t higher doses the proportions were reversed. With different chemical and physical mutagens the ratio varies even more widely, as has been shown in fission yeast (Nasim and Auerbach, 1967; Auerbach, 1967; Nasim, unpublished). In this organism, X-rays produce the lowest observed proportion of fractionals (around 20%), followed by UV and various chemical mutagens, with hydroxylamine at an extreme position in the spectrum producing mutants of which 90% occurred as sectored colonies. Evidence that repair mechanisms may be involved in the production of whole-colony mutants is based on studies of UV-induced mutations in radiation-resistant and radiation-sensitive lines of yeast (Nasim, 1968a; Haefner, 1967a; Resnick, 1969b). I n Nasim’s experiments, for example, two slightly sensitive lines of fission yeast showed high proportions of sectoring, i.e., in the direction expected if repair activities were low. However, a line which is highly sensitive to UV did not conform to the theory in that it exhibited an unexpectedly low induced-mutation frequency coupled with a not unexpected high frequency of inducedlethal sectoring. From this it has been inferred that there are other

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sorts of repair which are not involved in maintaining identical information in the two DNA strands. There are other examples of the failure of mosaic frequency to be correlated in a predictable way with differences in sensitivity. I n silkworms, differences in capacity for repair of premutational damage have been regarded as accounting for the changing proportions of mosaics induced by irradiating different stages of spermatogenesis (Tazima and Onimaru, 1969). However, the most sensitive stage in the male germ cells, late meiotic prophase, yields a substantial excess of complete mutations, whereas the most resistant stage, mature spermatozoa, yields mainly mosaics. Furthermore, in comparisons of a sensitive with a resistant strain of silkworms, irradiation of spermatids results in an excess of complete mutants from the former and of mosaics from the latter, i.e., again in the opposite direction from that predicted by simple theory. Although repair processes may well influence the proportions of complete and mosaic mutants, the predictive value of the simple theory is low as applied to a range of organisms. This does not necessarily imply that the simple theory is incorrect, but merely that it is inadequate to account for the biological complications in the various organisms. Interpretation of the phenomenon of mosaic mutation is rendered still more difficult by the observation that some lines of descent from irradiated cells continue to produce mosaic sub-lines over many cell generations. Two recent accounts indicate that such replicating instabilities may be induced in fission yeast, not only by chemical mutagens and W light, but by X-rays as well (Nasim, 1967, 1968b, 1969). Cells from red-and-white mosaic colonies, resulting from mutagenic treatment of a parental red (adenine-deficient) strain, give rise to further red-andwhite sectored colonies known as secondary mosaics. It is mainly the cells from nonmutant red sectors that yield the replicating instabilities, but in a few cases both the mutant and the nonmutant portions of the same mosaic colony yield cells that give rise to further mosaic colonies. I n the case of some X-ray-induced replicating instabilities the frequencies of secondary mosaics ranged up to nearly 1%.The molecular basis of the phenomenon is still a matter for speculation.

F. MOLECULAR BASISOF RESISTANCE, SENSITIVITY, AND REPAIR In the past, interest in the chemical bases of resistance and sensitivity has centered on the induction by ionizing radiation of fsee radicals and other chemically active products from irradiated water and organic molecules. I n studies of the short-lived free radicals, chemical explanations have been sought for the enhancement or protection resulting from the

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presence of such agents as oxygen and sulfhydryl compounds during the exposure. Investigations of the long-lived free radicals produced by irradiation of dry materials such as plant seeds have been concerned with measurements of the amounts formed and their persistence as detected by electron spin resonance techniques, in relation to the extents of the chromosomal damage and the lethal effects. I n recent years, however, the emphasis in chemical studies of induced changes in the genetic materials has shifted to the molecular mechanisms involved in the repair of damaged DNA. Because the radiation-induced genetic injury in dry seeds is closely correlated with the total free radicals, and because these may persist in the seeds over prolonged periods, it has sometimes been suggested that the genetic damage which accumulates in unirradiated seeds on prolonged storage may have a similar origin. However, tests using dry seeds that have been stored for up to 48 years have shown that neither the signal amplitudes nor the shapes of the electron spin resonance patterns change significantly with increasing duration of storage (Conger and Randolph, 1968). There have been relatively few recent chemical investigations of the nature of the changes induced in DNA by ionizing radiations, such as chain breaks and base damage, in vitro or in vivo. Similarly, molecular studies of the steps in dark repair-i.e., recognition of the damage, incision, excision, replacement, and rejoining of the strands-have been carried out largely following exposure to ultraviolet (see Moseley, 1968, for review). The same may be said for studies of the various types of radiation sensitivity in microorganisms, e.g., involving reduced ability to divide, loss of host cell reactivation of bacteriophage, and recombination deficiency, with the exception of those referred to earlier in this review (see Strauss, 1968). It is widely held that radiation-induced lesions of DNA and their repair play an important role in determining whether chromosomes will break and whether cells will live or die. Relevant to this belief are studies of the rates at which radiation-induced breaks in the DNA are repaired a t different stages in the cell division cycle. Such studies have been carried out with human kidney cells using alkaline sucrose gradient centrifugation to detect single-strand breaks (Lohman, 1968) , following on earlier work with leukemia cells (Lett et al., 1967). Rejoining of the DNA breaks was found to be most effective in early S phase, and least so in G,. This suggests a possible parallel with the special high sensitivity of chromosomes during metaphase to radiation-induced breakage that becomes visible at the next cell division. There are a number of similar parallels. The DNA breaks observed

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in the above study resemble chromosome breaks in that they increase linearly with radiation dose, and the DNA is protected by cysteamine against the induction of breaks in the same way that chromosomes are protected. Also, other studies of radiation-induced breaks of DNA in mammalian (Chinese hamster) cells have demonstrated that the processes by which rejoining takes place occur a t all stages of the cell cycle, including mitosis (Humphrey et al., 1968). This chemical finding is reminiscent of genetic evidence that radiation-induced premutational damage in Paramecium can be repaired, or fixed as final mutations, at all stages of the cell cycle (Kimball and Perdue, 1967). In bacteria, the effect of ionizing radiation on the synthesis of DNA has been shown to differ from that of UV in a number of ways. Less inhibition of DNA synthesis results from exposure to X-rays (Achey, 1968), and ionizing radiation actually induces the formation of new starting points for DNA replication, although UV has no such effect (Billen, 1969). The additional replication is not associated with repair synthesis and is not an artifact of the methodology, but is a result of semiconservative synthesis starting in regions of the chromosome other than that occupied by the growth point at the time of irradiation (Hewitt et al., 1969; Billen et al., 1967; Billen and Hewitt, 1967). A chemical approach to the study of excision and repair of X-rayinduced damage to DNA, using metabolic inhibitors, is currently in its infancy. In the uniquely resistant Micrococcus radiodurans, Driedger has demonstrated that when repair of radiation-induced damage takes place in the presence of phenethyl alcohol a secondary form of degradation of DNA occurs after the repair has been completed (Grayston and Driedger, 1969; Driedger, unpublished). From what is known of the mode of action of phenethyl alcohol this implies a lingering form of radiation damage to the membranes which cannot be repaired in the presence of the drug. Nearly half a million rads are required to produce detectable killing of M . radiodurans, and high sublethal exposures result in the excision of 30% of the DNA. It is inferred that overlapping excisions of the two strands of DNA are somehow avoided, although many would be expected on a random basis. So far, such manipulations of cell metabolism have been carried out primarily to explore the anatomy of the repair processes, and they have yielded information only on such short-term consequences of the supplementary treatments as can be detected by chemical means. To assess the biological implications it will be necessary to compare the later consequences of modifying the repair in various ways, as indicated by the amounts of lethal and genetic damage. More especially, the chemical

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and the biological end points will need to be studied in parallel in the same organism. Little of the research reviewed has involved observations of both the early chemical end points and their later biological consequences. One exception is a recent study which has sought to relate an environmentally determined modification of the sensitivity of the DNA to induced breakage to a difference in cell survival. When oxygen is present during the irradiation of E . coli, the amount of breakage of the DNA has been shown by means of sucrose gradient centrifugation to be increased (Achey and Whitfield, 1968). The difference is almost certainly a direct reflection of an increase in the initial physical damage to the DNA, since the cells were maintained in a chilled state during the irradiation and until analyzed. Because this oxygen effect is so closely correlated with O2 enhancement of X-ray-induced cell killing and inhibition of DNA replication, a simple cause-and-effect relationship must be inferred.

G. BIOLOGICAL SIGNIFICANCE OF REPAIR MECHANISMS Although much of the evidence relating to repair mechanisms points to differences between species and even between strains of the same species, there is ample indication that such mechanisms must be widely distributed over living forms and that they are of major significance for survival. I t seems unlikely, moreover, that they exist solely to protect cells and organisms against damage from ionizing radiation or even from UV. As has been suggested earlier, the associations with such normal cell functions as genetic recombination and spontaneous mutation indicate a much more general role for the repair mechanisms. A further example will serve t o strengthen this inference. Two recent studies have established that an hereditary disease of humans, known as xeroderma pigmentosum, is associated with an inability of the cells of affected individuals to excise UV-induced thymine dimers and repair the altered DNA strands, and that this deficiency is in turn due to a defect in the endonuclease-mediated chain breakage which is thought to be an initial step in dimer excision (Cleaver, 1968; Setlow et al., 1969). Because the persons with this disease develop fatal skin cancers when exposed to sunlight, the excision process in normal individuals plays an obviously important role in protecting them from an otherwise lethal effect of ultraviolet, What is less apparent is that it may protect them also against other hazards of a more endogenous sort. Quite apart from the skin cancers resulting from exposure to sunlight, a proportion of those affected by xeroderma pigmentosum may exhibit poor physical

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development or stunted growth, and sometimes severe neurological abnormalities and mental retardation. From this example in man and from the associations observed in microorganisms it is evident that repair mechanisms are widespread in nature, and that they serve a multiplicity of purposes in addition to protecting against radiation injury. Ill. Nature of the Genetic Changes

The changes that occur in a gene when it mutates under the influence of ionizing radiation may be studied by a number of methods, including, in particular, fine mapping of the altered region by means of breeding tests for linkages with other changes in the same region, and tests of the ability of the altered gene to complement the functions of other mutant alleles of the same locus. For studies of the nature of induced chromosomal aberrations, radioactive pulse-labeling and radioautography sometimes complement the more conventional cytological techniques. And techniques have been devised for the study of less familiar sorts of induced genetic change, such as the method of pedigree analysis for the detection of lethal sectoring in the lines of descent from an irradiated cell. A variety of other induced genetic changes are discussed in the recent literature. These include gene conversion, paramutation, recombination and chiasma formation, euchromatization, plastid mutation, nondisjunction, gynogenesis and androgenesis and, in a somewhat different category, mutations induced indirectly in unirradiated genetic materials by irradiated chemicals. Of these various genetic effects of irradiation, lethal sectoring is of special interest as being an essentially new phenomenon discovered within the past 5 years, and as representing a genetically mediated form of induced lethality that is widespread and yet has escaped detection for so long. A. GENEMUTATIONS Radiation-induced mutations involving losses of normal function at known gene loci have long been regarded as including a proportion that are due to actual removal of genetic material. Recent evidence from Neurospora, Drosophila, and E . coli provides detailed support for this view. Although simple deletions of chromosome material are usually thought to form a size continuum from very small to very large, evidence from

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fine mapping by genetic recombination in Neurospora indicates that the distribution is far from uniform. Among X-ray-induced multilocus deletions in the region of the ad-3 gene there is a deficiency of long deletions and an excess of short ones, presumably due to a decreasing probability that two independent breakage ends will be able to fuse when they are distant from one another (de Serres and Webber, 1967). Furthermore, as might be expected for 2-hit alterations, the yield of recessive lethals associated with multilocus deletions in this region is less a t low than at intermediate dose rates (i.e., a t 10 R/minutes vs. 100 R/minutes) . Such a dose-rate effect would not be expected for the induction of “point” mutations, and is not observed; the total yield of these from the ad-3 region is independent of dose rate. Nevertheless, there is evidence that the dose rate does make a difference a t the molecular level to the spectrum of induced mutations (Malling and de Serres, 1967, 1968; de Serres, 1968). Different types of point mutations tend to be induced by very high as compared with very low dose rates (1000 R/minUtes vs. 10 R/minutes) . The different recessive lethal mutations for which this effect is observed are distinguished from one another by their varying abilities to complement the activities of other such alleles of the same locus as indicated by restoration of viability when both are present in the same cell. The direction of this effect is not what might be expected. Capacity for “complementation” is presumably greatest where the least genetic material has been lost or inactivated, and very high dose rates have been shown to favor the production of the least-restrictive (i.e., nonpolarizing) category which is known from studies of chemically induced reverse mutations to be associated largely with single base-pair substitutions. Low dose rates on the other hand favor the induction of noncomplementing mutants which are associated with a variety of genetic changes, including deletions or insertions of genetic material. An intermediate class of mutants (polarized complementing) , which also includes deletions or insertions of genetic material but in smaller proportions, shows no change with dose rate. The direction of this effect may not be readily interpretable, but that the different complementation patterns are correlated with the natures of the radiation-induced mutations a t the molecular level has been well established. Independent evidence from the fine mapping of X-ray-induced recessive lethals in a segment of a Drosophila chromosome confirms that the great majority involve two or more functional (i.e., allelic) units of the chromosome, whereas most chemically induced mutations affect only one such unit (Lifschytz and Falk, 1968). It is inferred not only

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that X-ray-induced recessive lethals are due to chromosomal aberrations, but that even the visible mutations induced by X-rays are due to deficiencies at nonessential loci, a point of view held by Stadler over 20 years ago on the basis of data pertaining to mutations in maize microspores. That a proportion of X-ray-induced mutations map as deletions has been demonstrated also for E . coli, but in these studies W and nitrous acid were much more effective in producing the kinds of deletions that the experiment was designed to detect, namely those leading to restoration of function in a particular mutant gene (Schwartz and Beckwith, 1969). However, this could be because the deletions induced by the X-rays tended to remove too much genetic material and to be lethal as a result. The efficiency with which X-rays delete portions of genetic material would readily account for its especially high mutagenicity in the case of induced reversions of the white-ivory mutation of Drosophila (Bowman, 1969). This mutation is believed to be associated with addition of genetic material to the white locus. Spontaneous reversion and presumed loss of the additional material occurs readily during meiosis provided there is synapsis, and much less readily during mitosis. As might be predicted, radiation greatly enhances somatic reversion but has a lesser effect on meiotic reversion.

B. CHROMOSOMAL ABERRATIONS Most recent studies of the mechanisms of chromosome breakage and reunion have either been prompted by, or have made reference to, a novel interpretation of chromatid discontinuities proposed by Revell in 1966. The interpretation of radiation-induced breaks and rearrangements involving whole chromosomes as seen at mitotic metaphase has not been questioned, but Revell did argue that apparent discontinuities of single chromatids were not simple breaks as had commonly been supposed. From studies with root tip cells of the broadbean Vicia faba he concluded that the great majority (95-990/0) were “achromatic lesions” which failed to give rise to acentric fragments a t anaphase. For the small fraction that could be confirmed as due to breakage by the observation of chromatid fragments released a t anaphase, he argued that the doseresponse relationships indicated a 2-hit origin, i.e., that they were the products of exchanges of breakage ends involving two points of breakage. Evidence that most metaphase chromatid lesions represent true breaks has been presented by Conger (1967). He found in Tradescantia micro-

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spores that less than a third of the chromatid gaps induced during the S and G, phases failed to be confirmed at anaphase as productive of real chromatid deletions (compared to 60 to 300 times that proportion for V i h a ) . No reason could be advanced for this large discrepancy, which is apparently associated with an intrinsic species or cell-type difference. An independent study of the chromosomal aberrations induced by X-rays in Trillium pollen and root tip cells has shown that the numbers of incomplete chromatid rearrangements increase as the irradiation time approaches mitosis, a finding that could in itself be accounted for equally well by either the exchange hypothesis or the classical hypothesis (Grant, 1967). An observed excess of sister chromatid unions induced during or shortly after DNA replication would not have been predicted on either hypothesis alone. Thus, the results of this study do little to settle the question. Two recent reports argue more strongly that terminal chromatid deletions arise as a result of 2-hit exchanges. Comparisons of the observed and expected proportions of the possible sorts of chromatid aberrations, using data from Tradescantia, have been interpreted as supporting the exchange hypothesis (Savage et al., 1968). A detailed account of the manner in which the predictions were made will not be attempted in the present review. However, a similar conclusion has been drawn from dose-response studies using cultured cells of the wallaby (2n = 11) by Brewen and Brock (1968). They obtained 2-hit dose-effect curves, not only for the obvious two-lesion aberrations such as rings and dicentrics, but for terminal deletions as well, a result strongly supporting the exchange hypothesis. At present, because of the diversity among the relevant data, there is little unanimity of opinion concerning the importance of exchanges in the production of terminal chromatid deletions. Relevant to the exchange hypothesis, but less directly concerned with resolving the current disagreement, is a study of the “strandedness” of the chromatids of higher organisms. It is not known whether a chromatid consists of more than just a single DNA strand, and whether radiation-induced sister strand exchanges do, or do not, involve more than just the subunits of one DNA double helix. Heddle (1968) has considered this question in the light of earlier radioautographic data on the relative frequencies of so-called single and twin exchanges. These are scored a t the second metaphase following the interphase at which the newly synthesized strands of DNA are labeled, and the names refer to the fact that one, or both, of the chromosomes in which the labeled strands are represented as chromatids may exhibit an exchange of label along the lengths of its component chromatids. Tritiated thymidine has

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been used both to label the strands and to provide the radiation that induces the sister exchanges. Heddle not only interprets the data as compatible with the idea that the chromatids may be multistranded, but points to exceptions to the semiconservative pattern of the replication that would be difficult to explain if the chromatids are not multistranded. A similar conclusion has been reached more recently from a demonstration that chromatid aberrations of V . faba occurring a t the second postirradiation mitosis which were once regarded as due to delayed chromatid breakage (Montesi et al., 1967), could not arise from errors in the replication of previous chromatid aberrations (Heddle, 1969a).

C. LETHALSECTORING Of the genetic consequences of exposure to ionizing radiation currently being studied, most have been known for decades. “Lethal sectoring” is a notable exception having been described first in 1965, independently and a t about the same time, by James and Haefner. The phenomenon is important, and it occurs in widely different organisms. With doses of radiation that produce little or no killing in sells of yeast, as indicated by their colony-forming ability, more than half may give rise to clones of descendants that contain clusters of dead cells. Thus, lethal sectoring can be regarded as representing a major destructive effect of irradiation that had somehow previously escaped detection. The phenomenon has now been reported in bacteria, yeast, algae, and mammalian cell cultures (see James, 1967a,b, 1968; James and Saunders, 1968; James and Werner, 1967; James et al., 1968; Haefner, 1967b; Haefner and Striebeck, 1967; Miltenberger et al., 1966) and is presumably found throughout the natural world. Detection and detailed study of lethal sectoring is normally carried out by the use of microdissection methods to establish pedigrees for the vegetative descendants from irradiated cells. In the case of yeast, for example, the products of successive cell divisions on nutrient agar are simply dissected away from each other and replanted in identifiable locations on an agar surface, the process being repeated for each dividing pair of cells over a number of generations. The damage may be regarded as dominant since it is expressed in irradiated diploid and polyploid yeast. It is heritable over many cell generations but the unstable cells that initiate lethal sectoring characteristically revert to normal and produce mostly stable lines (James and Saunders, 1968) , Sublines occur which sector persistently (James, 1967a,b). These have been tested in crosses for the presence of nuclear genes that might cause the effect but, perhaps because of technical diffi-

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culties, the results have so far been inconclusive. However, lines which have recovered reveal a high incidence of lethal mutations. Recovery of unstable cells has been found to be a multi-step process. Closely related cells tend to resemble each other in the likelihood of full recovery, and partially recovered cell lines exhibit both a greater propensity to recover fully and a diminished rate of production of lethal sectors. None of the interpretations so far advanced adequately accounts for all aspects of the phenomenon. It seems unlikely that delayed expression of nuclear damage such as chromosome breakage, delayed mutation due to an induced mutagen, or the delayed production of homozygotes for induced recessive lethals as a result of mitotic recombination, could be the cause. So far, the possibility of misassortment of an extrachromosoma1 constituent has received the most attention ; lethal sectoring is preceded by a reduced rate of cell division in the earliest generations after irradiation, suggestive of some sort of metabolic deficiency. It has been variously suggested that lethal sectoring may be associated in some manner with repair activity, or the lack of it. Thus, spontaneous lethal sectoring occurs at an unusually high rate in UV-sensitive lines of bacteria (Haefner and Striebeck, 1967) and fission yeast (Nasim and Saunders, 1968), implying an association with the absence of a repair mechanism. Conversely, Beam (1967) has suggested that lethal sectoring might represent a form of repair in which damaged genetic material is eliminated through segregation. However, if this were the case the phenomenon might be expected to occur only rarely in haploids; in fact, in haploids it is rare after X-irradiation (Haefner, 1967a; Glickman, 1969) but common after exposure to UV. That the radiation injury leading to lethal sectoring is capable of being repaired has been demonstrated by James and Werner (1969a,b). When the interval from irradiation to the first post-irradiation cell division is prolonged by exposure to P-mercaptoethanol, the amount of induced lethal sectoring is reduced. Similar prolongation of the interval between the first and second post-irradiation divisions is also effective, indicating that the damage is not necessarily “fixed” a t the time of DNA synthesis.

D. INDUCED RECOMBINATION Ionizing radiations have long been known to increase the frequency of genetic recombination during both mitotic and meiotic cell division. Recent reports describe this phenomenon, or its cytological counterpart, increased chiasma frequency, in fruitflies, fish, algal cells, phage, and

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grasshoppers. I n addition, investigations of induced somatic cell changes in the petals of flowers suggest that they may have a similar origin. In female Drosophila, ionizing radiation acts to increase crossing-over near the centromere and to decrease it in the regions that are far from the centromere. Recent studies of this phenomenon have been concerned with comparisons of the effects of radiation with those of toher agents such as heat (Chandley, 1968) and antibiotics (Scholefield and Suzuki, 1967; Hayashi and Suzuki, 1968) which also affect crossing-over. With both heat and X-rays there is a period of special sensitivity when the oocytes are believed to be synthesizing DNA, and both agents are thought to act by interfering with meiotic synapsis in oocytes and mitotic pairing in oogonia. Both effects would decrease crossing over in the distal region thus permitting more to take place near the centromere. The effects of the antibiotic mitomycin C even more closely resemble those of ionizing radiation, in that the increase due to temperature occurs only in a region of the chromosome that is adjacent to heterochromatin, whereas the increases due to radiation and to the antibiotic involve a much longer region (Hayashi and Suzuki, 1968). The similarity is not unexpected since radiation and mitomycin C both cause extensive DNA breakage and degradation. An apparent lack of association of the radiation effect with proximity to heterochromatin has been claimed for stocks of Drosophila having structurally different types of chromosomes (Puro, 1969). However, Roberts (1969) observed that where 90% of the heterochromatin of the X chromosome had been transferred by an inversion from the region of the centromere to the distal end, the regions of X-ray-induced increase and decrease in crossing-over were also reversed. Crossing-over does not normally occur in male Drosophila but may be induced by irradiation, and has been shown to occur not only in spermatocytes but in spermatogonia and stem cells as well (Bateman, 1968; Kale, 1967). The most sensitive stage is the spermatocyte. The response varies linearly with dose to the spermatocytes, but as the 1.5 power of dose to spermatogonia. A radiation-induced increase in crossing over has been observed also in the guppy (Schroder, 1969b), and there is evidence that it occurs in the spermatogonial cells a number of division cycles prior to meiosis. I n the alga Chlainydornonas two short stages in meitoic prophase have been shown to be sensitive to the influence of X-rays on genetic recombination (Lawrence, 1967). These are believed to correspond to leptotene and pachytene. On the basis of this and similar observations in plant cells, Lawrence suggests that crossing-over takes place in a t least two steps. The first is thought to involve structural change a t

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a number of loci in the pre-leptotene stage and, the second, interactions a t pairs of such sites at pachytene to bring about crossing-over. From the effects of inhibitors of DNA synthesis on crossover frequency, it is inferred that both steps are associated with DNA synthesis. An X-ray-induced increase in crossing-over has been studied in phage T4 (Campbell, 1968). This is believed not to be due to “forced” crossing-over to avoid lethal damage (i.e., to the inviability of noncrossover genomes) since there is little multiplicity reactivation. The phenomenon has also been studied cytologically in grasshoppers by scoring chiasma frequencies in the long chromosomes a t meiotic metaphase following irradiation. The sensitive periods for the induction of additional chiasmata by X-rays in the desert locust Schistocerca gregaria have been shown to occur during the leptotene-zygotene stages of meiosis and during premeiotic mitoses (Westerman, 1967). Similar observations in another grasshopper, Melanoplus, have confirmed the radiosensitivity of the leptotene-zygotene stage and have demonstrated in addition a heat-sensitive period during the late zygotene-pacyhtene stage (Church and Wimber, 1969). Although these findings differ from those of Lawrence in Chlam.ydomonas, they nevertheless tend to support his two-step theory of induced crossing-over. I n the past there have been numerous reports of radiation-induced crossing over in mitotic as well as meiotic cell divisions. Recent evidence suggests that mitotic recombination may occur in the cells of flower petals and stamen hairs (Mericle and Mericle, 1967). I n a study of radiation-induced mutations for flower color in Tradescantia, which appear as red spots or sectors in otherwise blue flowers and stamen hairs, it was noted that these changes were associated with little or no chromosome fragmentation, thus making it unlikely that they are due to terminal deletions. Of the remaining possible mechanisms, namely gene mutation, chromosome nondisjunction, and mitotic crossing-over, the latter seemed particularly likely because of the common occurrence of multiple sectors in the stamen hairs. The presence, for example, of two red cells separated by a nonniutant cell in a developing hair seems most readily interpretable in terms of mitotic crossing-over. E. GENECONVERSION AND PARAMUTATION When a gene locus assumes the characteristics of a homologous locus in a heterozygous diploid cell, without the usual indications that crossing-over has taken place, it is said to have been “converted” by its homolog. Similarly, where a gene undergoes heritable change as a result of passage through a heterozygote containing another allele of

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the same locus, without actually being converted to the other allele, it is said to have undergone “paramutation.” Exposure to ionizing radiation is known to alter the frequencies of both gene conversion and paramutation. In yeast, gene conversion has now been reported to occur during mitotic cell division, at the hi-1 locus when this is present in the heteroallelic state. Such conversions resemble those that occur during meiosis, and their frequency is increased by exposure to X-rays. The yield increases linearly with the dose a t low doses and less than linearly a t higher doses. The conversion is believed to take place either just prior to or just after DNA replication (Hurst et al., 1967). Paramutation has been studied mainly in the R locus of corn, which controls pigmentation of the aleurone layer of the seeds. Certain alleles of this locus are changed heritably when transmitted through plants heterozygous for certain other alleles of the same locus. The process has been shown to be modified in a t least three different ways by exposures to X-rays (Shih and Brink, 1969; Shih, 1969). First, in alleles that have lost some of their pigmenting potential through paramutation this function may be partially restored by treatment with X-rays, diethyl sulfate, or ethyl methanesulfonate ; where the pollen is so treated X-rays are the more efficient agent, and where seeds are treated the chemicals are more effective. Second, irradiation of the so-called paramutable alleles, while in the pollen, results in either an increase or a decrease in the susceptibility to subsequent paramutation, depending on the allele irradiated. Third, irradiation of the so-called paramutagenic alleles which cause other alleles to undergo paramutation tends to reduce their potency. A relatively simple mechanism is thought to account for most of the induced changes relating to paramutation. The R locus is regarded as having two parts, one specifying an enzyme and the other, consisting of a heterochromatic region made up of a variable number of common units called metameres, repressing to various degrees the function of the first. Changes are believed to occur through misreplication of the metameres to alter their number. X-rays would be expected in most but not all instances to produce their effect by causing losses of metameres through deletion.

F.

~ ~ I S C E L L A N E O UGENETIC S EFFECTS

Not all radiation-induced hereditary changes fit neatly into the above categories. Some involve mutations of extrachromosomal genes, some have to do with consequences affecting whole genomes, some are mediated

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in unusual ways, and some represent mutations of nuclear genes but are worth mentioning separately because of their distinctive or unusual expressions. A few recent reports relate to changes of these less usual kinds. Mutations affecting the chlorophyll content and morphology of chloroplasts are in part nuclear in origin and in part extranuclear. Such hereditary alterations have been known for a number of decades to occur in fern prothalli grown from irradiated haploid spores. Recently it has been shown that only 6% of the chloroplast variants induced in spores of the fern Osmunda regalis were due to mutations of nuclear genes (Howard and Haig, 1968). Both the radiation-induced mutation rate and the killing were found to be influenced by fractionation of the exposure, presence or absence of oxygen, the quality of the radiation, and the physiological state of the spores, in essentially the same ways. Similar plastid mutations have been studied also in another fern, Pteris (Mehra and Palta, 1969). An unusual genetic effect of ionizing radiation has been reported in which the normal heterochromatization of certain chromosomes is reversed (Nru, 1969). I n male mealy bugs the paternal set of chromosomes becomes heterochromatic early in development whereas in the females both sets remain euchromatic. When males are irradiated after hatching, sectors of the testes are found to contain cells in which both sets of chromsomes are euchromatic. These abnormal cells fail to undergo the second meiotic division and, as a result, produce diploid sperm. The radiation-induced change is believed to involve damage to a gene locus, with a rate of occurrence corresponding to about 0.3 mutations/million cells/rad. Heavy irradiation of eggs or sperm prior to fertilization has long been known to result in the production of embryos from the unirradiated nucleus alone. This so-called Hertwig effect, which was discovered first in amphibia, has recently been successfully used in fish together with temperature shocks after fertilization to produce diploid embryos by chromosome doubling which are homozygous for the genes a t all loci (Purdom, 1969). The object is to obtain the equivalent of inbred lines for possible commerical breeding, without having to carry out many generations of inbreeding. Gynogenetic embryos have been produced from irradiated sperm and unirradiated eggs of trout, plaice, and flounder, and the proportions of diploids among these are increased by the cold shocks to 60% of all embryos, from 1%. Androgenetic embryos from irradiated flounder eggs and unirradiated sperm were all haploid, whether or not temperature shocks were used. There have been numerous reports that chromosome breakage may

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be caused indirectly by exposure of cells to irradiated sucrose or glucose. Further confirmation of this effect has come from studies of root tip cells of Vicia and Tradescantia treated with glucose which had been exposed earlier to doses of y-radiation in the vicinity of a million rads (Ma, 1968). Most prevalent were breaks involving the centromeres of metacentric chromosomes ; in addition, chromatid breaks occurred. The former are attributed to long-lived products of radiolysis, and the latter to short-lived products. I n Vicia, early interphase is the stage of greatest sensitivity to this indirect effect.

IV. Scientific and Practical Uses of Induced Genetic Changes

Radiation-induced gene mutations and chromosomal rearrangements have been employed in studies of problems that have little to do with radiation as such. Examples of both the scientific and the practical uses, appearing in the recent literature, have covered a wide range of topics, including the identification of cytologically distinguishable chromosomes with particular linkage groups, the measurement of gene length, the effects of the presence of different numbers of particular genes or chromosomes, the growth of meristems, the control of insect pests, and the improvement of crop plants. OF THE CHROMOSOMES THATCARRY A. IDENTIFICATION PARTICULAR GENES

Some 150 X-ray-induced translocations in the housefly have been used by Wagoner (1967 and 1969) to relate the five genetic linkage groups with appropriate members of the five pairs of autosomes. The X and Y chromosomes carry no known genes and are ent,irely heterochromatic. Translocations involving an autosome with the Y chromosome were of special value in verifying the relationships because the genes of the translocated autosome are in this circumstance inherited only by males.

B. MEASUREMENT OF GENE LENGTH At the other end of the scale of sizes, X-ray-induced mitotic recombination has been used, along with other tests, to estimate the length of the adenine-8 locus of yeast (Esposito, 1968). Interest in the question arose because this locus has a much lower spontaneous mutation rate than the adenine-6 locus and previous studies had suggested that muta-

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tion rate and gene length might be correlated. The test showed, however, that the low spontaneous mutability could not in this instance be accounted for in terms of the size of the gene.

C. STUDIES OF

THE

EFFECTS OF DIFFERENT DOSAGES OF

PARTICULAR GENESAND CHROMOSOMES

Gamma-rays have been used to induce translocations in the chromosomes of the plantain, Plantago insularis, for the purpose of studying the effects of the duplications and deficiencies of chromosome material that occur through segregation in the translocation heterozygotes (Stebbins et al., 1967). This plant is particularly suitable for such work because of its small chromosome number ( n = 4) and rapid life cycle. I n a similar manner, radiation has been used to induce tandem duplications of the lozenge region of the X chromosome of Drosophila, for the purpose of studying the effects of different dosages of the mutant and normal alleles at the lozenge locus (Bender, 1967). Also, radiationinduced non-disjunction has been employed to obtain a strain of Drosophila afinis characterized by XO males, to demonstrate that artificially produced Y-less males are fertile like those that occur naturally in other strains of the same species (Voelker, 1967).

D. STUDIES OF MERISTEMS Radiation-induced mutations in cells of plant meristems, when they produce visible changes in color in the resulting sectors of the plants, permit knowledge to be gained of the rates a t which cells divide and the numbers of primordial cells from which various organs are derived. Using induced mutations of Tradescantia from blue t o pink color, which occur a t a rate of 4.8 x 10-'/meristematic hair cell/R, it has been possible to show that the stamen hairs grow mainly from the terminal as distinct from the interstitial cells (Ishikawa and Sparrow, 1968). B y irradiating oats heterozygous for a gene for albinism it has been shown that the highest leaf on the plant has the highest number of induced white stripes and is therefore derived from more primordial cells than are the other leaves (Ichikawa and Ikushima, 1967). Similar observations have been made, with similar findings, for two other cereals, i.e., for the top ear in corn (Sarvella and Grogan, 1967), and for the primary spike in wheat (Donini et al., 1968). By way of contrast, the same approach when applied to Gladiolus indicated that the top flowers came from the least number of cells in the irradiated corms, and some-

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times more than one of the top flowers came from just a single cell, whereas a number of irradiated stem cells might contribute to the lower flowers (Buiatti et al., 1969).

E. PRACTICAL USES IN INSECT CONTROL AND PLANT BREEDING As the uses of radiation-sterilized males for the control of insect pests have been reviewed recently (IAEA, 1969), a single example will serve to illustrate this application. Studies in Greece on control of the olive fruitfly Dacus oleae, showed that the presence of radiation-sterilized flies among unirradiated flies in a ratio of 8:l reduced the percentage of damaged olive fruit to about one-twentieth that in comparable controls (Manikas and Baldwin, 1969). A number of papers have appeared on the uses of radiation-induced genetic changes in agricultural plant breeding since the last major review of such work (IAEA, 1966b). These have dealt chiefly with induced quantitative variation but have been concerned also with specific gene mutations, aneuploidy, and a method for selfing lines that are normally self -incompatible. Increases in the variance of agriculturally important characteristics and increased responses to selection as a result of radiation-induced mutations have been reported for hexaploid wheat (Good, 1967), barley (Aastveit and Gaul, 1967) , autotetraploid Brassica (Gupta and Swaminathan, 1967) and peanuts (Gregory, 1968). Particularly detailed studies have been carried out in the latter organism showing (a) that induced mutations with small effects may be exposed for investigation when large detrimental changes have been removed from the genome, (b) that the frequency of such mutations increases as an exponential function of their diminishing effect, and (c) that the induced quantitative variation is largely additive. Furthermore, hybrids between superior selections from a single population of irradiated peanuts tend to exhibit hybrid vigor. In oats, mutations increasing the variance for plant height, heading date, and seed weight, have been found to be induced more efficiently by alternate treatment with a physical and a chemical mutagent (thermal neutrons and ethyl methanesulfonate, i.e., EMS) than by either agent alone (Joshi and Frey, 1967). This is interpreted to mean that the gene loci or the directions in which they mutate are to some extent mutagen-specific, and that for the purposes of plant breeding it is worthwhile to look for effective combinations of mutagens. Radiation has also been used to produce mutations at specific loci as in the case of DDT resistance in barley (Wallace et al., 1968). Most

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barley is susceptible to DDT, and resistance is recessive and dependent on one or two pairs of genes. Among a large number of radiation-induced mutants tested, varying degrees of resistance were represented, ranging from full resistance to virtually normal susceptibility. Radiation-induced reciprocal translocations of watermelon chromosomes have been used to produce seedless fruit (Oka et al., 1967). In the past, seedless watermelons have been obtained by crossing diploid male parents with autotetraploid female parents to produce triploids. The present alternative approach is being explored because of the low yields of triploid seed, the difficulties of germinating them, and the late maturity of the triploid fruit. So far, only partial reduction of seed formation has been achieved in hybrids with reciprocal translocations, but further reductions should be possible with more complex chromosome configurations. Radiation-induced aneuploidy has been studied also in peanuts (Patil, 1968) , but with a view to their use in plant improvement. A related use of radiation for plant breeding, but not directly to alter the genetic materials, has been described in Lilium longiflorum (Hopper and Peloquin, 1968). Irradiation of the styles of this plant with doses of the order of 2G70 k R permits the growth of pollen tubes from selfincompatible pollen. The method can facilitate the development of inbred lines as well as the exploration of the biochemical nature of this pollenstyle interaction. V. Quantitative Studies Involving Differences in the Exposures

The responses of the genetic materials to ionizing radiations may vary with the total dose administered, the time course of the exposure, and the distribution of the ion pairs in space. Studies of such response differences serve to indicate something of the nature of the processes by which the induced changes occur, and, in particular (a) the numbers of damaging events or ion clusters required to cause a given change, (b) the occurrence and rate of repair or restitution of a lesion in the interval between the arrival of successive ion clusters which interact with each other, and (c) the amount of localized damage, in terms of the density of the ion clusters, needed to produce most efficiently a primary lesion of the genetic material. Current experiments involving differences in the amount, distribution and quality of the ionizing radiation necessarily bear a close resemblance to those carried out over the past four or five decades. Nevertheless, many of the questions that have prompted such studies have still not been adequately answered.

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A variation on this general theme arises where the sources of the ionizing radiations are radioactive atoms that have been incorporated into the genetic materials of the cells. In this situation, not only the effects of the ion pairs are of interest, but more especially the effects of the transmutations of the atomic nuclei of one element into those of another when these are a part of the structure of the genetic material itself. A. DOSE-RESPONSERELATIONSHIPS Interest 'in the dose-response relationships for induced genetic changes has been associated with (a) evidence of a 2-hit component in the response curves for induced changes where this has been found, (b) a diminishing yield per unit dose a t the high-dose end of the curve, and (c) linearity and deviations from linearity in the response over the lowdose range. This last sort of information has special implications for radiation protection wherever large numbers of people are exposed to low doses of radiation. Evidence of a 2-hit origin for most 2-break chromosomal rearrangements induced in human peripheral leukocytes has been obtained by Bender and Bruen (1969). This result differs from that obtained earlier by other workers. At high doses, the yield of translocations and dominant lethals in mice falls off in the same manner as that for Russell's specific locus mutations (Lyon and Morris, 1969; see also Oftedal, 1968). As with the specific locus mutations, the yield is restored to the expected value if the radiation dose is fractionated; this is attributed to the synchronizing effect of the early fraction on the cell cycle so that a more homogeneous cell population is exposed to the later fraction. For a different reason, a part of the genetic damage from high doses of radiation to mouse liver cells is prevented from being expressed as abnormal anaphases in regenerating livers (Conger and Curtis, 1968). I n this case, the more abnormal cells tend not to divide as readily or as soon as the less abnormal and the normal cells. A difference in the degree of saturation observed in neutron and X-ray dose-response curves for chromosome exchanges, which was earlier attributed to a difference in the number of exchange sites for the two sorts of radiation, is now interpreted as due to the different effects of distortion on a basically linear and a basically quadratic dose-eff ect curve (Savage, 1967,1969). An unexpected linear relationship has been found between X-ray dose and the induction of chromosomal translocations in spermatogonial stem

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cells of mice (Ldonard and Deknudt, 1967a,b,c, 1968a, 1969). These rearrangements have been observed cytologically and the curve has been explored down to 25 R, indicating that they are produced by only one electron track and not by the interaction of two. Similarly, i t has bcen demonstrated that the incidence of dominant lethals induced in mouse spermatozoa increases linearly with radiation dose over the range from 100 R to 1500 R; this has since been shown to be true for doses from 10 R to 100 R (Ldonard and Deknudt, 1967a). Curiously, the slope of the dose-response curve appeaw to be about 40% greater in the lower dose range than was found in the earlier study. Whether this seeming discrepancy implies a hypersensitivity a t low doses is not clear. A now well-established hypersensitive response in the low-dose range, however, has been observed a t the variegated ( V ) locus of the tobacco plant, for chronic 7-irradiation. This hypersensitivity has now been confirmed for acute r-irradiation, using two different gene loci (V and R ) controlling, respectively, variegation and red anthocyanin pigmentation (Sand and Smith, 1968). The effect is substantial, and the yield of mutations per unit dose increases at the lower doses by factors of 5 and 9 for the V and R loci, respectively, over the range from 180 rad down to 15 rad. These studies were based on somatic mutations producing colored spots in the petals of the tobacco flowers. It seems quite unlikely that differential killing of the more mutable cells could account for the effect, in view of (a) the low doses, (b) an observed shifting ratio of the two sorts of mutations over the different doses, and (c) the absence of any detectable distortion of the flower shape even a t the high-dose end of the range. Since chromosome losses, and chromosome gains through nondisj unction, are important causes of birth defects in human infants, special interest exists in the response relationships for these other sorts of genetic change. Unfortunately, little is known about the mechanisms by which they are produced, on which predictions of the likely response curves might be based. However, recent work by Traut and his co-workers has demonstrated for losses of X chromosomes from mature oocytes of Drosophila a curvilinear relationship above 200 rad and a close approximation to linearily for the points at 100 and 200 rad (Traut, 1967a, 1968; Traut and Scheid, 1969; see also Kiriazis and Abrahamson, 1968). The conclusions reached are that a t relatively low doses the main mechanism of chromosome loss requires only one break, and that radiation-induced losses may therefore be expected to occur at rates that are proportional to dose in the low-dose range. An approach by which the yields of chromosomal changes leading

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to congenital malformations in vertebrate embryos might be studied at even lower doses has been described in some detail by Newcombe and McGregor (1967). B. DOSE-RATEAND FRACTIONATION EFFECTS A reduction in the effectiveness of a given total dose of radiation when this is fractionated or administered a t a lower rate is generally regarded as indicating that more than one ion track is required to produce the effect, and that repair or restitution of the damage caused by an earlier track may sometimes forestall interaction with the damage caused by a later one. Where such an effect is not expected but is nevertheless observed, or when an effect in the reverse direction is detected, it is usually inferred that differential cell killing is responsible, unless this can be ruled out. Three examples of so-called reverse dose-rate effects have been reported recently, all relating to visible mutations. (a) In mouse spermatogonia, fast neutrons have been shown to be less effective in inducing specific locus mutations and dominant visibles when delivered a t highdose rates (Batchelor et aZ., 1967) ; the result confirms earlier findings. (b) In tobacco plants, mutations of the unstable variegated ( V ) locus, scored as colored sectors in the petals, are induced in greater numbers by exposure to low as compared with high-intensity gammas (Sand and Smith, 1967). This effect is unlikely to be due to differential cell killing, because not only was the total dose low (24 R ) , but for the stable R locus scored in the same tissue the induced mutation rate was apparently unaffected by the difference in intensity; it is concluded that the mutable V gene is genuinely hypersensitive a t low intensities by as much as fourfold. (c) In the parasitic wasp Dahlbominus, a small reverse dose-rate effect has been reported for induced mutations in mature oocytes, affecting eye color and body malformations (Baldwin, 1968, 1969b). Again, it seems quite unlikely that differential cell killing can be responsible for the effect since more of the host cocoons were parasitized and more progeny were produced following acute as compared with chronic exposures. An unexpected fractionation effect in the conventional direction (i.e., less effect when the dose is distributed in time) has been reported for recessive lethal mutations induced in DrosophiZa spermatocytes, and has been interpreted as due to cell selection (Tates, 1968). A similar effect has been reported also for somatic mutations in the flowers of Cosmos, the mutation rate being very low after chronic y-irradiation as compared with acute X-irradiation (Gupta and Samata, 1967).

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For dominant lethal mutations induced in mouse spermatogonia, fractionation of the exposure neither increased nor decreased the yields detectably (Sheridan, 1968a) although an earlier report had shown such an effect and had attributed it to selective killing in a heterogeneous cell population. Translocations induced in mouse spermatogonia have shown a striking dose-rate effect with both y-rays and X-rays, over the range of intensities from 0.02 to 100 R/minute (Searle e t al., 1968). Also, X-rays of 250 kVp were more effective than gammas, and about twice as effective at the lower dose rates. This study was based on direct cytological observation at first meiotic metaphase, and the findings are not unexpected in view of similar results from many of the earlier cytological studies using more favorable materials. The importance of the present experiment lies in the demonstration of such an effect by direct microscopic observation of the altered chromosomes before cell selection could intervene. The methods of dose fractionation and dose protraction have been applied also to investigations of X-chromosome losses that occur in Drosophila following irradiation of mature and immature oocytes (Traut, 1968). The responses differ for the two sorts of cell. For mature oocytes there is no dose-rate effect and the response varies linearly with total dose, as would be expected if the losses were due to chromosome breakages resulting from single hits. Immature oocytes, however, showed both a reduction in yield of induced losses with prolongati,on of the dose, and also a multi-hit dose-response curve. It is inferred that the high restitution rate for chromosome breaks a t this early stage makes i t unlikely that the damage will be permanent unless two or more breaks are produced near together in time and space so that sister union competes favorably with restitution. The resulting anaphase bridge formation is thought to yield XO males where the X chromosome is involved, and dominant lethals where an autosome is involved. In human lymphocytes from peripheral blood, differences in the dose rates for exposures to fast neutrons had little effect on the frequencies of induced chromosomal aberrations (Scott et al., 1969). Differences of 1000-fold in the neutron dose rate (from 0.057 to 50 rad/minute at doses up to 150 rad) did not alter the frequency in cells irradiated in vitro after stimulation with phytohaemagglutinin. The small dose-rate effect observed for cells irradiated in the nonstimulated condition was thought to be due to differential onset of cell division in the less heavily damaged cells. An observed linear response for the induction of dicentrics by X-rays confirms Evans’ earlier finding and might be expected in view of the absence of a dose-rate effect.

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C. ION-DENSITY EFFECTS The relative biological efficiencies (RBEs) of radiations of different qualities producing different linear ion transfers (LETS) along the tracks, have been of interest for some time as yielding information about the nature of the biological targets. In addition they have practical importance in the setting of maximum acceptable levels for human exposures. Current findings may be regarded as contributing a few additional but important items to an existing large but not wholly satisfactory catalog of RBEs. Two studies of fast neutron exposures of Drosophila have yielded RBEs of 2.1 and 2.2 for the induction of recessive lethals in premeiotic and postmeiotic male germ cells (Lamb et al., 1967), and RBEs of < 2 and 3, respectively, for the induction of sex-linked lethals in female germ cells, and for mutations affecting egg-to-adult viability (Dickerman, 1967). In mice, fast neutrons are reported to be as much as 20 to 25 times as effective as X-rays in inducing translocations in spermatogonia, of kinds that can be scored cytologically in the descendant spermatogonia (Searle et al., 1969). These high values relate to chronic exposures, whereas for acute exposures the RBE values are in the vicinity of four. A similarly high RBE, i.e., 23, had been observed earlier for the induction of specific locus mutations in mouse spermatogonia by exposures to low-intensity neutrons (Searle and Phillips, 1967). In human peripheral lymphocytes irradiated in vitro, the RBE for induction of dicentrics is 3.5 for fast neutrons as compared with X-rays (Scott et al., 1969). This value is largely independent of the total dose, as would be expected in view of the linear dose-response relationships for the induction of dicentrics in these cells by both X-rays and neutrons. Under other circumstances the RBE for neutrons may vary widely with total dose. This has been shown for chromosome exchanges in Tradescantia microspores (Savage, 1967), and the value is not always greater than unity. The reason for this is that the response to increasing doses of neutrons falls off at high doses whereas that for X-rays varies linearly with dose. The result is that the RBE for neutrons declines to less than unity at doses above a few hundred rad. Other examples of studies of the efficiencies with which neutrons induce genetic changes in plants relate to (a) chromosomal aberrations in seeds of Nigella (Ahnstrom et al., 1969) (b) chimeras for specific locus mutations in bread wheat (Rana and Swaminathan, 1967), and (c) chlorophyll mutations in barley seed (Leroy, 1968). An RBE of 20 has been reported in bread wheat; neutrons of high energy have been found to be more efficient for the induction of exchange aberrations, and less

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efficient for the induction of simple chromosome breaks than are lowenergy neutrons. An interesting effect of a-particles occurs when they are produced in plants containing unusual amounts of boron by exposure to neutrons. I n the plant Lycopersicon, induced chlorophyll mutations show twice as large a boron effect as do mutations at specific loci (Ecochard and de Nettancourt, 1969). The difference is believed to be due to a tendency for the boron to be located preferentially at some distance from the nucleus, and not to any differential response to high-LET radiation.

D. TRANSMUTATION EFFECTS AND INCORPORATED RADIOISOTOPES Of the genetic effects of radioactive nuclides incorporated into living organisms, those due t o the action of radioactive 32Pare of special interest. When atoms of this isotope occupy a place in the DNA, their transmutations to 32S alter the architecture of the genetic material in ways that have little to do with the damage caused by the ionizations which occur at the same time. Recent work on the effects of the incorporation of a2Pinto yeast, Drosophila, maize, and lilies will be described. I n addition there is a single report on the effects of the incorporation of tritium (3H) into Drosophiln. I n yeast, high proportions of two unrelated kinds of variants, i.e., radiosensitive mutants and abnormally segregating strains, have been described as arising from haploid cells heavily labeled with 32P (Moustaechi et nl., 1967). In the latter strains, various heterozygous marker genes are caused to segregate abnormally a t meiosis to produce other than the usual 2:2 ratios in the ascospores. Mutations of a gene, or genes, controlling duplication of genetic material early in meiosis, are thought to account for the effect. That the transmutation effect of the 32P disintegrations is important has been demonstrated for the production of mosaics for sex-linked lethals in Drosophila (Lee e f al., 1967). The experiment involved minimizing the conscquenccs of the p-particles that accompany the transmutations, by storing the 39P labeled spermatozoa in unlabeled females so that the particle tracks tend to be dissipated outside the sperm cells. Of a number of radioactive isotopes tested for mutagenic activity in maize and Lilium, 32P and 35S have been found to cause losses of genetic markers, whereas W a and R9Srwere much less effective, presumably because lower amounts were incorporated into the cells under study (Steffensen, 1968). That a radioactive isotope does not need to be incorporated into the

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DNA in order to cause genetic damage has been clearly shown for the production of recessive lethals by tritiated thymidine in Drosophila (Oftedal and Kaplan, 1969). As might be expected in view of the lengths of the tritium p-tracks (i.e., 1-p average and 8-p maximum), mutations were induced in the germ cells after incorporation of the thymidine into the cytoplasm but before it could be synthesized into new DNA. (For a review of the biological effects of incorporated radioisotopes the reader is referred to IAEA, 1968.) VI. Importance of the Cellular Consequences of Genetic Changes

To an irradiated organism, the most important consequences of the resulting alterations in the hereditary characteristics of its body cells occur when these lead either to cell death or to cancerous changes. For neither sort of consequence are the relationships with the better-known kinds of induced changes in the genetic materials well understood. Induced gene mutations occur too infrequently to contribute substantially to cell death, especially in diploid cells where their effects would tend to be recessive. Gross chromosomal aberrations on the other hand have long been known to be responsible for some of the deaths that are observed after cells irradiated in interphase or prophase have had an opportunity to undergo mitosis. The importance of this mechanism of cell killing continues to be debated. For a review of the subject the reader is referred to Davies and Evans (1966). A related problem concerns the radiation-induced changes that are not lethal to the cell but which alter its hereditary characteristics in such a way as to permit it to give rise to a malignant growth. A. CELL DEATH Doubt has been cast on the importance of the contribution from genetic changes to the total amount of radiation-induced cell death. In HeLa cells, for example, a lack of concordance has been demonstrated between the variations in sensitivity which occur over the GI phase, as relating respectively to cell lethality and chromosome breakage (Terasima and Ohara, 1968). Sensitivity to radiation-induced cell death increases by about fourfold over this period, whereas sensitivity to the induction of chromosomal aberrations remains unchanged and the induction of dicentrics (believed to be dominant lethal) actually declines. Reports of studies of sublethal changes add little to the above picture. Survivors of irradiated lines of hamster cells have been described which

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form small colonies, are radiation sensitive, and occur with a regularly increasing frequency following progressively higher doses (Todd, 1968). These defective lines show no cytologically detectable alterations in karyotype. Some of the most convincing evidence that chromosome breakage and the resulting aneuploidy following the next anaphase are of major importance in causing radiation-induced cell death comes from organisms in which the chromosomes have diffuse centromeres. There are several of these among the algae, grasses, and certain insects. Because the functions of the centromeres are distributed over the full lengths of the chromosomes, breakages and rearrangements of the chromosome material do not lead to losses of fragments or to anaphase bridges. Fragments behave as independent chromosomes a t anaphase, and there is no such thing as a dicentric chromosome or an acentric fragment. From time to time there have been reports that cells of organisms which have diffuse centromeres are highly radioresistant. The most recent example of this sort of study relates to an insect, a Lepidopteran species known as the cabbage looper (North and Holt, 1967). Unfortunately, none of the work to date with organisms having chromosomes with diffuse centromeres represents a serious attempt to relate quantitatively the degree of radioresistance, under various circumstances, to the fact that the chromosomes are substantially immune to radiationinduced losses o i large amounts of material. Such studies could, however, do much to clarify the distinction between the lethal effects that are a result of chromosomal imbalance and those that are not. B. CARCINOGENESIS When a multicellular organism is irradiated, the induced breakages and rearrangements of chromosomal material that do not interfere with mitosis may persist and give rise to aneuploid lines of cells. Such aneuploid clones have been found to linger over many years in the lymphocytes of heavily irradiated humans (Buckton et al., 1967; Ishihara and Kumatori, 1967; Sasaki and Norman, 1967; Schmid and Bauchinger, 1969). There is still no general agreement as t o whether cells in which chromosomal changes have occurred have an increased likelihood of giving rise to cancers, but the view that they do has received considerable support in recent years. A number of lines of evidence suggest that chromosomal breakage and rearrangement may be a cause of cancer, rather than just a consequence of it. With a few notable exceptions (Conen, 1967; Nowell et al., 1967) almost all mammalian tumors exhibit chromosomal anomalies,

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and these often occur in obvious clones of cells (Conen, 1967; Lejeune, 1967). The kinds of chromosomal alterations observed in one tumor are moreover frequently distinguishable from those of another, even where by clinical and histological standards the tumors appear to be identical. Not only are the physical and chemical carcinogens capable of causing chromosomal breakage and rearrangement, but the same may be said of the various DNA and RNA viruses which are known to produce tumors (Nichols et al., 1967). In the special case of chronic granulocytic leukemia, which is associated in almost all instances with a characteristically shortened version of a particular chromosome, even when the disease occurs following irradiation (Kamada and Uchino, 1967), it seems most likely that the chromosome change is in fact the cause of the cancer, when it occurs in stem cells of the bone marrow. When this so-called Philadelphia chromosome is seen in skin cells and lymphocytes of irradiated people there is no apparent association with an increased risk of leukemia (Ishihara and Kumatori, 1967), although an association with polycythemia Vera has been reported (Levin et al., 1967). For tumors in general, other possible mechanisms may be more difficult to rule out. These include (a) activation of an oncogenic virus, (b) depression of the immune response, (c) alteration of the hormone level, and (d) nonspecific cell killing with a consequent stimulation of regeneration (Fialkow, 1967; Upton et al., 1967). It remains a distinct possibility that some fraction of radiation-induced chromosomal aneuploidies may serve to “liberate” the descendent clones from the usual constraints on cell proliferation so that they are permitted to take a first step toward cancerous growth. The possibility of a causal relationship between chromosomal anomalies and cancer induction provides special reason for an interest in the yields of chromosomal rearrangements from low doses of radiation. A linear response has been reported over the range from 5.6 R to 48 R for the induction of chromosome rings and dicentrics in human leukocytes grown in culture, indicating that such exchanges may be caused by single tracks (Schmickel, 1967). The precision with which such changes may be scored even when induced in vivo has led Heddle (1969b) to propose their use for biological dosimetry after an exposure has occurred. Although the rearrangements may be scored with precision, there is unfortunately no information relating quantitatively the numbers of induced chromosomal changes and the risks of subsequent malignancies. This account would not be complete without a reminder that ionizing radiations are capable of inducing tumors not only in animals but also

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in plants. There arc many examples of this, and two recent reports describe the induction of tumors in tobacco plants (Conklin and Smith, 1968) and in Arabidopsis (Hirono et al., 1968). As yet, however, evidence from the plant tumors has contributed little or nothing to a n understanding of the possible primary or secondary role of chromosomal anomalies in tumor induction. VII. Importance of the Hereditary Consequences in Individuals and Populations

Much of the social incentive and financial backing for the truly large volume of work carried out over the past two-and-a-half decades on the genetic effects of ionizing radiations was forthcoming because of the importance of the subject for the well-being of man himself. Various official and unofficial estimates have been made of the genetic injury that might result from exposing human populations to given amounts of radiation. Nevertheless, the uncertainties in such estimates still range over at least two orders of magnitude, and there are major questions a t issue that are unlikely to be answered by current approaches. It is in this area particularly that reassessment of the aims of research on the genetic consequences of irradiation may be most needed. One unsettled question relates to the possible harm arising out of the effects of an increase in mutation rate on the quantitative or polygenic traits. A second concerns the reality and social significance for man of the so-called genetic deaths that are believed to occur eventually whenever a mutant gene causes even a slight average reduction in the level of adaptation of its carriers to their environments. Another unanswered question has to do with the extent of the contribution from the recurrent mutations of natural origin to the present “load” of hereditary diseases and disabilities of known kinds occurring in human populations. Since i t is this mutation-maintained fraction of our genetic troubles that would increase as a result of an artificially-induced elevation of the mutation rate, the question is of practical as well as scientific importance. Evidence from relevant studies of laboratory organisms is currently confused. Not only do the questions posed for the laboratory populations differ from those of special concern for human populations, but conflicting experimental results are often obtained from the same organism owing to differences in the circumstances of the tests. Thus research has provided only limited guidance concerning the human implications of genetic

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change, and the generality of population phenomena described in the laboratory is frequently in doubt. A. EFFECTS ON QUANTITATIVE CHARACTERS Few of the observed effects of induced increases in mutation rate on quantitative traits of experimental animals and plants are either clearly deleterious or beneficial. Furthermore, the evidence from different experiments often points in opposite directions. Studies of the effects of parental or ancestral irradiation on the characteristics of mammalian litters, for example, have yielded quite conflicting results. Increases in the weights of litters of mice from irradiated sires have been reported by Singer and Touchberry (1967) and a similar result has been obtained with rats (Taylor and Chapman, 1969a,b), whereas other experiments with mice (Green, 1968b) and with Duroc pigs (Cox, 1967, 1968) have indicated a decrease in body weight, coupled in the case of the pigs with a decrease in the thickness of the body fat. I n the experiment with pigs, no corresponding effect on either body weight or body fat was observed in another strain, known as Hampshire, under otherwise similar conditions (Cox, 1968). I n view of the levels of statistical significance associated with most of the above results, the seeming discrepancies cannot be regarded as due to chance. In one respect the results of such studies appear to be in agreement. After large accumulated exposures to successive generations of mice, Green (196813) found no effect on embryonic mortality or fetal abnormality. Similarly, King (1968) found no lingering effect of ancestral irradiation on litter size in mice, apart from the first-generation effect due to dominant lethals. Similar results have been obtained from studies of guppies (Schroder 1969a,c). For a variety of other organisms and quantitative traits, there are reports of increases in the variance of the characteristics as a result of radiation-induced genetic changes. This is true for body weight in the insect Habrobracon (Dalebroux and Kojima, 1967), bristle number in Drosophila (Jones, 1967), plant size and flowering time in Arabidopsis (Chatergee and Gardner, 1967; Brock, 1967), seed set and straw length in rye (Aastveit, 1967), and corolla length in tobacco plants (Ando and Vencovsky, 1967). Such increases in variance were sometimes associated with changes in the mean values as in the case of a lowered seed set and reduced straw length in rye (Aastveit, 1967). The above evidence implies that the genetic effects of radiation on quantitative traits may be important, but it does not indicate clearly

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whether these effects would be to the detriment or the benefit of an exposed population.

B. GENETICDEATHS AND

THE

SEX RATIO

If simple theory is correct, a population irradiated over many generations should build up in its gene pool an accumulation of genetic damage which, after the irradiation had stopped, would decline only gradually as carriers of the harmful genes died prematurely or failed to reproduce. However, few experiments have yielded results that can be interpreted quantitatively in this simple fashion. Because of the lack of such data, it is not uncommon to equate the numbers of genetic deaths in unspecified future generations directly with the numbers of induced mutations. For this reason, there has been considerable interest in the rates of induced mutation in a variety of organisms. These rates are sometimes measured for particular gene loci, hopefully of representative kinds, and sometimes for whole genomes or for particular chromosomes such as the X chromosomes of mammals and flies. In special circumstances, attempts have been made to assess the numbers of recessive or dominant lethals induced in the X chromosomes of men and mammals from observations of the shifts in the sex ratio among offspring from irradiated fathers or mothers. A similar approach has also been used in an attempt to detect an accumulation of mutations in the X chromosomes with advancing age of parents or grandparents. It is possible, of course, to estimate for human populations the number of genetic deaths that would result from a given radiation exposure, using the induced mutation rates observed in lower organisms. Such estimates have limited value, but not primarily because of uncertainties in the extrapolation from, for example, mice to men. Instead, the chief reason is that we do not know to what extent the eliminations of deleterious genes occur through deaths and disabilities at times in the human life cycle that make them socially important. If, for example, they occurred mainly between fertilization and implantation, their social importance might be small. The absence of such information greatly reduces the value of the available quantitative data on the susceptibility of genes, and genomes, to induced mutation, at least as applied to assessment of the importance in man of a radiation-induced increase in mutation rate. For the above reason no attempt will be made here to summarize the actual data from recent studies of induced-mutation rates. A list of such studies will be sufficient for present purposes. These relate to recessive visible and recessive lethal mutations in rats (Taylor, 1968 ;

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Haverstein and Chapman, 1968), recessive lethal mutations in mice accumulated over 14 generations of irradiation (Sheridan and Wirdell, 1968), recessive visibles and specific locus mutations in guppies (Schroder, 1969d), eye color mutations and lethal mutations associated with body malformations in the wasp Dahlbominus (Baldwin, 1968, 1969b), and color mutations in the stamen hairs of Tradescantia (Nayar and Sparrow, 1967; Ichikawa e t al., 1969). Other relevant studies have been mentioned elsewhere in this paper. Of interest also is a study of spontaneous mutation rates a t specific loci in mice (Schlager and Dickie, 1967), a study indicating that induced mutation rates in Drosophila may be grossly underestimated because of associations with chromosomal alterations affecting fertility (Lefevre, 1967), and a demonstration that dominant lethal mutations induced in maize pollen do not, as was once thought, affect their own transmission through interfering with pollen germination and fertilization (Pfahler, 1967). There is in addition a description of a recessive lethal mutation in mice which has adverse dominant effects on implantation rates, intrauterine death rates, litter size, and the mating powers of males (Sheridan, 1968b). Attempts to study mutation rates through observations of changes in the sex ratio are of interest because of the promise which the method once held out. On simple genetic theory one would expect that recessive lethals accumulated in an irradiated female parent would tend to kill male offspring preferentially because these inherit the maternal X chromosome in a hemizygous state (i.e., in mammals and Drosophila). Similarly, when a male parent has been irradiated, dominant lethals induced in his X chromosome would be expected to kill female offspring preferentially because these inherit the paternal X chromosome whereas sons do not. Tests of the correctness of this simple theory continue to yield equivocal results. In rats, the expected effect on the sex ratio is observed among offspring from irradiated males but not among those from irradiated females (Haverstein et al., 1968). Similar studies with swine, however, show neither of the expected effects (Mullaney and Cox, 1969). An attempt to apply the method to detect the effects of a supposed accumulation of spontaneous mutations in aging human parents and grandparents has failed to reveal any significant association (Cann and Cavalli-Sforza, 1968). This result is in contrast with earlier findings but these could be accounted for in terms of socioeconomic stratification. A mathematical approach has been applied recently to indicate the minimum size of a sex-ratio study capable of detecting an effect of a given mutation rate (Traut, 1969).

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I n view of the amount of interest that centers around the induced and spontaneous mutation rates in man and in lower organisms, it is disappointing that current and past studies of this kind have yielded so little insight into the importance of the mutations once they have occurred.

C. EFFECTS ON

THE

FITNESS OF POPULATIONS

Numerous experiments have been carried out to test the “fitness” of irradiated populations, in terms of their abilities to survive, propagate, and compete. Such studies are commonly motivated by an interest in the role of mutation in the evolutionary process, and as a consequence of this the findings tend to have limited value in helping to answer the kinds of question that are raised about the genetic effects of irradiating human populations. In particular, although induced deaths and disabilities might be regarded as tolerable for a Drosophila population provided they were balanced by induced increases in fertility, the same cannot be said for a human population, which takes a special interest in the welfare of its individual members. Undoubtedly, population experiments could be designed to answer such questions as: (a) How many deaths will result from exposing a population of a given size to a particular dose of ionizing radiation? (b) How will these deaths be distributed over the generations following the radiation exposure? and ( c ) At what stages in the life cycle will they occur? It is even possible that data from experiments which have already been carried out could be used to answer such questions, but that the methods of analysis have tended to obscure the answers when the questions are posed in this form. The difficulty will become more apparent as we proceed. A surprising proportion of studies with Drosophila populations have yielded what might be regarded as evidence of a beneficial effect of a radiation-induced increase in mutation rate. The results should not be interpreted, however, as indicating that a human population in which such effects occurred would necessarily regard the benefit as adequately compensating for the detriment to certain of its individual members. The seeming benefit has, moreover, often been conditional upon rather special circumstances. Thus, irradiation has been reported to improve viability in genetically homogeneous cultures of Drosophila but to reduce it in mixed cultures (Weisbrot, 1967). Similarly, some of the induced recessive lethal and sublethal changes have been shown t o be retained in extremely crowded cultures because of the advantage they bestow in the heterozygous state and in spite of the associated reductions in

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egg-to-adult viability in the homozygotes (Salceda, 1967). The possible role of metabolic products excreted into the medium in influencing advantage and disadvantage under conditions of competition has been discussed by Weisbrot (1969). There is also evidence that small amounts of radiation-induced heterozygosity may be beneficial where large amounts would be harmful. I n inbred populations of Drosophila the naturally occurring genetic variability, as indicated by the spread in the numbers of sternopleural bristles, is correlated linearly with fitness in the form of productivity. Where radiation is used to increase the amount of heterozygosity, a similar relationship is observed (van Delden and Beardmore, 1968). However, the radiation-induced heterozygosity is only beneficial when the amount is small. As might be expected, more benefit was associated with natural heterozygosity than with that induced by radiation, in the case of the populations that showed the least reproductive fitness ; however, for the initially better populations the reverse was, somewhat surprisingly, true. Small amounts of radiation-induced heterozygosity have likewise been shown to increase the ability of inbred populations of Drosophila simulans to compete with unirradiated populations of Drosophila melanogaster, whereas larger amounts have the opposite effect (Blaylock, 1969). There seems, moreover, to be no general rule regarding such effects. That a large radiation-induced increase in heterozygosity would increase viability in a population that is already highly heterozygous would seem doubly unlikely. Nevertheless, such an effect has been reported following relatively high total doses (6000-12000 R ) , the advantage among individuals heterozygous for chromosomes irradiated in the spermatogonial stage being more than sufficient to compensate for the obviously deleterious effects of the irradiated chromosomes when homozygous (Falk, 1967a). The result has not been satisfactorily explained on the basis of current theory. There are also other special circumstances that may determine whether the induced changes are advantageous or disadvantageous. That irradiated chromosomes may produce beneficial, i.e., heterotic, effects when inherited via the male parent, and detrimental effects when inherited via the female parent, has been demonstrated by Falk (1967~).He interprets the difference as due to selective transmission of the more beneficial changes via the male parents. Also, the presence of sigma virus has been shown to enhance the deleterious effects of X-ray-induced mutants in the heterozygous condition (Baumiller, 1967). There are other experiments with Drosophila that provide no evidence of genetic benefit from irradiation. I n populations maintained in an uncrowded condition to minimize competition, chronic irradiation has

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been shown to produce a substantial decline in egg-to-adult viability (Sankaranarayanan, 1967a). The rates of recovery to near-normal viability after irradiation was stopped varied in different lines. Many took only a few generations to recover, and the few lines which under uncrowded conditions maintained low levels of egg-to-adult viability quickly recovered when subjected to crowding and intense selection. In another type of experiment involving competition between two genetically similar but phenotypically distinguishable populations of D. melanogaster both advantageous and disadvantageous mutations were found, but on the average the induced changes were strongly disadvantageous when in a heterozygous genetic background (Dyer, 1969a,b,c). Further experiments with Drosophila which have yielded measurements of the degree of detriment associated with induced deleterious and lethal changes, when in the heterozygous state, include those of Tobari and Murata (1969) and of Falk (196713). An apparently beneficial effect of the introduction of radiation-induced heterozygosity into an inbred line has been clearly demonstrated in another kind of insect, Tribolium (Wisnieski and Crenshaw, 1969). Irradiated (R) and unirradiated (C) inbred lines were crossed in the four possible combinations and the productivities of the female offspring from these different crosses were taken as an indicator of genetic fitness. When allowance is made for a lingering maternal effect (female offspring from C X R, unirradiated male by irradiated female, crosses being much less productive than those from R x C) the advantage arising out of the radiation-induced heterozygosity becomes apparent. Females from R x C crosses are superior in productivity to those from C X C, and females from R x R crosses are superior to those from C X R. Studies with mammals have yielded even less-readily interpretable information than have those with insects. In mice, comparisons of lethal-free animals with the carriers of recessive lethals indicated no overall deleterious effect on viability in the heterozygote (Luning, 1969). However, one family showed increased intrauterine death in female carriers of a recessive lethal, and the heterozygous males had a reduced mating ability. Other studies of irradiated mouse populations have sought to detect a disadvantageous effect as indicated by the ability to withstand a test exposure to radiation (Sheridan 1967, Sheridan and Ronnbach, 1967). No differences were found in the LD,,s or in the number of litters or the litter sizes after the test exposures, when irradiated lines were compared with unirradiated. Also in a very thorough study of populations of mice irradiated over as many as 35 generations, no change in body weights or mortality could be detected (Spalding et al., 1969). Although these results might seem to imply that induced

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mutations are only occasionally harmful, they unfortunately fall short of establishing such a far-reaching conclusion.

D. HUMAN IMPLICATIONS Although geneticists have consistently pointed to the importance of avoiding harmful changes in the human germ plasm, relatively few investigators have thought of their radiation experiments as providing data that would have a quantitative bearing on the problem of limiting the harm to an acceptable level. This is particularly true of studies of the fitness of irradiated populations of laboratory organisms. The kind of answer required for such a purpose is usually thought of in terms of the numbers of descendants who will die or be otherwise harmed by the genetic changes induced in a population of a given size by a given radiation dose. Of the induced deaths, only those that occur after the early stages of development have been completed would be regarded as socially important, and there is considerable interest also in the manner in which these would be distributed over the generations. It seems unfortunate that even the results of those experiments with fruitfly populations which might perhaps have been interpreted in this simple fashion to provide numerical estimates of individual injury for at least one species of organism have been used almost exclusively for other purposes. Even where the uncertainties are great, numerical estimates of the amount of genetic harm to man that might arise from a given radiation exposure provide at least some indication of whether current practices in the use of radiation and radioactive materials are sensible or not. Such estimates have usually been based either on (a) the spontaneous mutation frequencies for known genetic diseases of man, or their prevalence in the population, coupled with a probable “doubling dose” for mutations in general, or (b) the induced mutation rates over a whole genome, or per locus, in conjunction with estimates of the numbers of loci in the human genome. A number of recent papers are relevant to these two approaches. From studies of single-gene traits in man, Stevenson and Kerr (1967) have drawn the conclusion that the spontaneous rates for the “visible” mutations have been overestimated in the past by about one order of magnitude. The reason given is that the conditions which have been used for this purpose have been especially selected for ease of study because of their high mutation rates. His evidence is based on 49 traits which have various frequencies in the population, and it indicates that the less mutable substantially outnumber the more mutable. As a result

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it would appear that published estimates of 50 X mutations per gamete for autosomal dominants and 10 X 10-G for X-linked recessives may be too high by tenfold. If this is so, the estimation of genetic risks to man may be affected in two ways. Those estimates which are based on a representative spontaneous mutation rate for human gene loci, in conjunction with the probable number of loci responsible for the bulk of the simply inherited diseases and a rate-doubling dose derived from animal experiments, would have to be reduced by the above factor of ten. Estimates which are based on the assumption that a dose of radiation which doubles the mutation rate a t a number of representative gene loci will double the prevalence of the more common single-gene diseases, may also be too high for a different reason. There are examples which suggest that the higher the spontaneous rate of mutation of a locus the less will be the factor by which the rate is increased by a given radiation exposure. If this is correct it would imply that more radiation is required to double the mutation rates for common diseases than those for the rarer diseases. The lines of reasoning taken in three recent mathematical papers (Sved e t al., 1967; King, 1967; Milkman, 1967) also have a bearing on attempts to estimate the numbers of radiation-induced genetic casualties in man, based on the current Toad” of hereditary diseases and handicaps, or rather on that part of it that is not maintained as balanced polymorphisms by opposing or fluctuating selective forces. Since such selective balances involve lack of “fitness” on the part of homozygotes as compared with heterozygotes, it was thought that there must be some limit to the number of balanced polymorphisms (and to the multiplicity of less-fit homozygotes) that a population can tolerate without an excessive amount of lethality and reproductive failure. This reasoning would tend to emphasize the role of mutation in maintaining the genetic troubles of the human race, and the harm that would arise from a radiationinduced increase in the mutation rate. However, a consensus is reached in the above three papers, that the presence of a large number of balanced polymorphisms in a population is not incompatible with a high level of fitness. If so, a somewhat larger part of our genetic troubles may be maintained by selection (i.e., favoring genes that have net beneficial effects in the population as a whole, but which may be harmful in particular settings) than has heretofore been supposed. Conversely, the part maintained by recurrent mutations, i.e., the part which is SUSceptible to a radiation-induced increase, may be smaller than has been thought. Direct tests to determine whether the genetic diseases of man are

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maintained by the pressure of selection or by recurrent gene mutations have in the past been laborious in the extreme, involving as they do the collection of extensive reproductive histories of the families of affected individuals. Considerable advances have been made, however, in the use of the vital registration syst,em to provide reproductive histories, and of routine records containing diagnostic information to provide histories of ill-health (Newcombe, 1967, 1969a,b,c). Since records of these kinds are to an ever-increasing extent being processed by computers, it is rapidly becoming possible to interrelate those pertaining to individuals and families throughout large populations. Practical tests involving a file of some 800,000 vital and health records for the Canadian province of British Columbia have demonstrated the feasibility of extracting fertility data for families of individuals affected by various hereditary and nonhereditary diseases. Thus, for the future a clearer distinction should become possible between the hereditary conditions that are maintained by selection and those that remain prevalent due to recurrent mutations of natural origin. Until this distinction can be made with greater certainty it remains difficult to predict what the effects of an artificially induced increase in mutation rate are likely to be, in terms of particular human diseases. VIII. Conclusions

One of the objectives in reviewing progress in such a broad area over such a short period of time has been to look for possible deficiencies in the current effort as relating to important gaps in present knowledge. The reviewer recognizes that no two geneticists would prepare identical lists of such deficiencies; nevertheless, several suggestions are presented for the readers’ consideration. a. Chromosome Breakage and Cell Death. Although radiation-induced chromosomal aberrations have been known for about 40 years to be capable of causing cell death, and the phenomenon of breakage and rearrangement has been studied intensively for its own sake during the late 1930s and early 1940s, we still do not know to what extent it is the major cause of death among irradiated cells, except in special circumstances, Suitable biological materials exist in which deaths due to chromosomal and nonchromosomal causes could be distinguished (e.g., pollen mother cells of diploid and tetraploid Tradescantia and the subsequent pollen tubes, and organisms with diffuse centromeres) but these have not been fully exploited for this particular purpose. b. D N A Breakage and Repair. Although chemical studies of radia-

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tion-induced breakage of DNA in the cell, and of the subsequent excision and repair synthesis are currently in vogue, it seems possible that such phenomena may likewise be in danger of being investigated largely for their own sakes without establishing quantitatively their biological significance in terms of the death or survival of irradiated cells and the production of chromosomal anomalies and gene mutations. c. Quantitative Inheritance. Although studies of the effects of radiation-induced increases in genetic variability have been carried out in both plants and animals, the orientations of the work with the two sorts of organism have tended to differ to an extent that limits sharply the generality of the findings as applied to problems of crop improvement, evolutionary mechanisms, and radiation protection in man. Despite the amount of related work there is, for example, no clear indication of the extent to which the continuously variable or “quantitative” characters of man, such as intelligence, would be affected by a radiation-induced increase in the mutation rate. d. Aneuploidy and Cancer. Although much effort has been devoted to observations of chromosomal anomalies in cells that have become cancerous, it is still not known whether particular sorts of chromosomal imbalance, or aneuploidy, play a causal role or are merely a result. I n principle a reverse kind of screening, in which various aneuploid lines of cells that have not been derived from tumors are tested for tumor-forming ability, might yield more conclusive answers if enough lines could be screened. e. Genetic Deaths and Their Importance in Man. The Muller-Haldane principle that on the average each mutation leads eventually to a genetic death (i.e.l to the elimination of a mutant gene through death a t some stage in the life cycle, or through a failure to produce) has not been seriously challenged, However, we still have no idea whether such genetic deaths in man occur mainly in the early prenatal stages, and would therefore be unnoticed, or whether they are the result of harm a t later stages in the life cycle when it would have much greater social importance. j . Effects of the Mutation Rate in Maintaining Specific Diseases in the Human Population. Although about six out of every hundred liveborn individuals have trouble at some time in their lives that is due largely or in part to genetic causes, there is still little direct evidence concerning the fraction of this total that is maintained by recurrent mutation. Until the role of the naturally occurring mutations in determining human health and well-being is better understood it will be difficult to predict the consequences of a radiation-induced increase ,in the mutation rate in a human population.

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Wolff’s statement in 1966 that recent work in radiation genetics had given rise to “no conceptual changes that seem likely to affect the field” would appear still to be true. However, it is also clear that some of the important gaps in present genetic knowledge arise because existing concepts have not yet been followed to their logical conclusions. Sometimes these conclusions need to be stated in quantitative terms, and a detailed accounting may be required as an integral part of the relevant research. A concept, like a biological “conceptus,” will tend to become valuable only as it acquires “flesh.” Instead of direct attempts to formulate concepts, some effort a t reassessing the aims of radiation genetics may be more urgently needed, and new theories should then be likely to follow. The early aspirations of radiation geneticists served in their time to stimulate research from which has come the present wealth of data and the current repertoire of concepts. Important deficiencies in this accumulated body of knowledge and theory become strikingly apparent, however, whenever attempts are made to apply it to problems that are of social as well as scientific importance. Geneticists warned long ago, for example, that the quality of the human germ plasm could be eroded by exposure to man-made radiation. As a result of this warning, the need is now recognized to assess accurately the impact of radiation from nuclear technology and other sources on the genetic health of our society. Unfortunately, the extent of our current ignorance limits the attempted assessments to the status of educated guesswork. It seems unlikely that current studies of traditional kinds will provide more than a small part of the knowledge and insight required for this purpose. However, the development of approaches of greater relevance to the problem should result in the collection of new kinds of data which will in turn require the formulation of new concepts.

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GENETIC FACTORS IN AGING* H. J. Curtis Biology Deportment, Brookhoven National Laboratory, Upton, New York

I. Introduction. . . . . . . . . . 11. Factors Affecting Aging . . . . . . 111. Theories of Aging . . . . . . . . A. Rate-of-Living Theory . . . . . . B. Collagen Theory . . . . . . . C. Autoimmune Theory . . . . . . D. Somatic Mutation Theory . . . . . IV. The Evidence Favoring a Mutation Theory A. Indirect Evidence . . . . . . . B. Direct Evidence. . . . . . . . V. A Composite Theory of Aging . . . . The Nature of the Induction Steps . . . VI. The Nature of the Mutations . . . . . VII. The Life Span of Dividing Cells . . . . VIII. Summary . . . . . . . . . . References . . . . . . . . . .

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I. Introduction

All forms of life, from the simplest bacteria to man, go through a definite life cycle. This involves many parts, and many different functions contribute to the sequence of events. One part of the cycle, especially in higher plants and animals, is referred to as aging. This leads to the death of the individual so this has often been used as a definition of aging. However, aging in most biological systems and especially in man, is recognized as being much more than that. I n man, the hair gets gray, the muscles weak, the eyesight dim, the hearing poor, and he develops all the other infirmities which are associated with old age. But these manifestations, annoying and limiting as they are, are not the factors which now lead man and domestic animals in Western societies to the death of the individual. They may contribute some small

* Research carried out a t Brookhaven National Laboratory under the auspices of the U.S. Atomic Energy Commission. 305

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part to the cause of death, but the real cause is the degenerative diseases. These diseases include cancer, cardiovascular disease, and the autoimmune diseases. Thus, aging in man is a very complex process, and must be analyzed as such. It is foolish to think of a single cause of aging, just as it is common knowledge that automobiles wear out for a wide variety of different reasons, but some makes of cars are more likely to fail in one part, and others in other parts. I n the same way, the genetic constitution of an individual predisposes him to succumb to a particular disease, but there is still a chance he may contract any of the other degenerative diseases. It is interesting to speculate as to the causes of aging in different forms. A mouse lives about two years and man about 70 years. We say they are genetically programmed to live this long. We even know that 'some strains of mice are genetically programmed to live much longer than other strains, and the sons and daughters of long-lived parents also have a long life expectancy. There is thus no question but that the factors determining the life expectancy of the individual reside in the germ plasm, but the question is how this information manifests itself. If one compares the chromosomes of mice and men, by any of the methods of modern biology, no significant difference is found, and yet there is a 30-fold difference in life expectancy. Mice develop about the same infirmities as men, and die of about the same degenerative diseases, but they do this about 30 times faster. I n 1908 Rubner pointed out that there is a rough correlation for all mammals between cellular metabolism and longevity. In other words, the total number of calories produced by a gram of tissue in a mouse is about the same as for a gram of tissue in a man. The difference is that it must be produced faster in a mouse in order to maintain the body temperature since the surface-tovolume ratio is so much larger. This correlates with the observation (Johnson et al., 1961) that rats forced to live in a cold environment increase their metabolism and decrease their life span. In general these animals die from the same causes as do animals kept in a warm environment. However, this effect is rather small, and many other lines of evidence lead to the conclusion that the metabolic rate plays only a small part in aging. But we are far from understanding the reason for this small part. It then seems clear that genetic factors play a dominant role in the phenomenon of aging, but environmental factors can certainly alter the course of the process, sometimes rather drastically. It appears that aging is a process which causes the gradual deterioration of the individual,

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and makes him prone to develop the infirmities of old age as well as the degenerative diseases. II. Factors Affecting Aging

The idea that aging is something other than the sum of the aging symptoms comes from a consideration of the various factors which can affect it. The first of these is radiation. If one irradiates an animal when young with a moderate dose of X-rays, for example, it is found that virtually no effects of the radiation may be noticed until the animal starts to grow old, at which time it is observed that it appears to grow old faster than its unirradiated control, and will die sooner. It contracts the same degenerative diseases as the control, but it merely contracts them sooner. Thus it appears that the entire aging process is accelerated, and the phenomenon has come to be known as radiation-induced aging. Likewise, mice can be treated in such a way as t o become very obese, and they too suffer accelerated aging. If these same mice are restricted in their food intake, they cannot become obese and they live a8 long as their normal controls. It is clear that it is simply a question of eating too much. On the other hand, if normal mice have their diets carefully restricted, the aging process is retarded. If rats are kept in a cold room so their metabolism must be increased to maintain their body temperature, they will age faster. There is some evidence that exercise will retard the aging process, but the evidence is somewhat equivocal. I n the human, a t least, psychological factors appear to be of considerable importance. For example, if the spouse of an elderly person dies, then the probability of his dying during the ensuing year is greatly increased. I n all these things it is not a question of an acceleration of one or another symptom of aging by the procedure, but an acceleration of all the symptoms by each of the procedures. Obviously there will be more acceleration of some symptoms than others, and this will vary for the different procedures followed. However, the general picture is so striking as to give one confidence that there is an underlying aging process which proceeds steadily, creating increasingly fertile ground for the development of the symptoms of aging. 111. Theories of Aging

A. RATE-OF-LIVING THEORY There have been many theories to account for the gradual decline and demise of the individual. First is the rate-of-living theory, which

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postulates that there is only a limited amount of vitality and that the individual can choose to use it up quickly and live a short life, or slowly and live a long one. This is very much like the wear-and-tear or stress theory of aging, which postulates that the various stresses to which all individuals are subject finally add up, rendering the individal nonfunctional in some vital part. Most of the research designed to prove these theories have shown either that stress and exercise have virtually no effect on longevity, or that they tend to prolong life. In an extensive series of experiments on mice (Curtis and Gebhard, 1959; Curtis, 1966a) it was shown that a chemical stress repeated as often a8 three times a week throughout the life of the animal would keep the animals sick most of their lives but would not affect their longevity. Also, a more severe stress, which gradually killed off the treated mice, when discontinued left the remaining mice with as great a life expectancy as their untreated controls. Studies on the human (Fox and Skinner, 1964), conducted on comparable groups of laboring vs. sedentary persons, have tended to show that the ones who perform reasonably hard work during most of their lives have a greater life expectancy than their sedentary controls. Thus there seems no real experimental basis for the stress theory, and indeed gerontologists are now advising everyone to take as much exercise, both mental and physical, as possible for as long as they are able.

B. COLLAGEN THEORY The collagen theory postulates that with time the collagen of the body, which makes up a large fraction of the body protein, becomes more and more rigid and interferes with normal organ function. It is quite possible that this does occur and it may interfere with the exchange of nutrients in the capillary circulation of all organs. This would lead to inefficient gas exchange in the lungs, poor oxygenation in the muscles and brain, etc. This then plays a part in the “nuisance” aspects of aging, but probably has little if anything to do with the etiology of the degenerative diseases.

THEORY C. AUTOIMMUNE The autoimmune theory postulates that a change (perhaps a mutation) takes place in some of the cells of the immune recognition system, such that they do not recognize certain somatic cells as belonging to the individual. An immune reaction then takes place which leads to the various autoimmune diseases. These are important diseases but they

GENETIC FACTORS I N AGING

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are only one class of the degenerative diseases, so the autoimmune theory must be taken as applying to only one part of the aging problem. Since it seems highly likely that these diseases are initiated by a mutation in one or more somatic cells, this theory can be considered as one part of the somatic mutation theory.

D. SOMATIC MUTATION THEORY The somatic mutation theory postulates that spontaneous mutations occur in the somatic cells, causing them and their progeny to perform a different function from that performed in the original tissue. It is usually understood, and will be followed here, that any heritable change in a cell is considered a mutation. This could include point mutations, chromosome aberrations, or even dedifferentiation. Changes in the genome which might take place could lead to a cell with uncontrolled growth characteristics (cancer), to one with an altered cholesterol metabolism (arteriosclerosis), or to a clone of altered immune cells which do not recognize some other body cells as belonging to that individual (autoimmune diseases). This theory is very attractive conceptually since it can explain, in general terms, most if not all the phenomena of aging. But the question is, is it true? IV. The Evidence Favoring a Mutation Theory

A. INDIRECT EVIDENCE One of the most prominent and serious symptoms of aging is cancer, and the mutation theory of carcinogenesis is very old. One could almost state axiomatically that a cancer represents a mutated clone of cells. As previously indicated, it is usually considered that the autoimmune diseases arise as a result of mutation in a somatic cell. This view is greatly strengthened by an observation by Holmes (1965). She used the NZB strain of mice, which is associated with the development of hemolytic anemia, an autoimmune disease, in a high percentage of the animals. If spleen cells from an old mouse with anemia are injected into young mice of that strain, they immediately develop anemia. It is difficult to think of any mechanism other than mutation in spleen cells which could cause this effect. However, the mice must be genetically predisposed, so it is quite likely that mutations both in the immune system and in the target organ would be necessary to initiate the disease.

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Werthessen (1962) found that atherosclerotic plaques exhibit an altered cholesterol metabolism, and that this alteration is maintained by the progeny of these cells when grown in tissue culture. This strongly implies that atherosclerosis starts with a mutation in an endothelial cell in an artery. Some of the most prominent features of aging involve muscular weakness, diminished brain function, etc. Shock (1961) has attributed these to cell loss, and certainly lethal mutation in a somatic cell is about the simplest mechanism one can imagine to explain this random cell death. There are undoubtedly other factors involved, but it is apparent that in a general way mutations in somatic cells can reasonably explain the symptoms of aging. But this does not constitute proof.

B. DIRECT EVIDENCE The work in connection with the development of atomic energy during World War I1 proved the thesis, which had long been suspected, that various forms of ionizing radiation, when administered in sublethal doses to young animals, caused them to die of “old age” a t an earlier age than their controls. The irradiated animals not only looked older than their controls, but they died of about the same diseases, except that they contracted them sooner. This has been confirmed many times. Since radiation is known to be a very potent mutagenic agent, it was immediately postulated that the accelerated aging, and by inference also natural aging, was due to mutations in somatic cells. However, in spite of the attractiveness of this concept, it remained merely an attractive theory for many years. Eventually Failla (1957) and Szilard (1959) presented theoretical treatments of the idea. The first experimental attempt to verify the concept produced negative results (Curtis and Gebhard, 1958): a chemical mutagen, nitrogen mustard, even when injected into mice repeatedly, failed to shorten the life span. However, it was later shown that this mutagen affects only a very small percentage of the somatic cells, so one would expect only a very small or undetectable effect. The next experimental effort involved scoring chromosome aberrations in somatic cells of mice. The best cells to use are liver cells, since they seldom undergo division but can be forced into mitoses by partial hepatectomy. Cells are scored for aberrations in late anaphase and early telophase, and most aberrations consist of bridges or micronuclei (Stevenson and Curtis, 1961). I n this way large numbers of cells can be scored without undue labor. It has been shown in plants that the

31 1

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aberrations scored in chromosomes of somatic cells are proportional to the gene mutations scored in the next generation (Caldecott, 1961). With the use of this method in liver cells, many correlations have been established between factors which affect aging and the development of chromosome aberrations. First, the number of chromosome aberrations increases steadily with age in normal animals (Fig. l ) , and may reach values as high as 80% in some very old animals. This is a very constant finding in all animals so far studied. Next, it is found that all types of ionizing radiation increase the aberration frequency dramatically, and it returns to control levels only after a period of many months in mice (Fig. 1 ) . Next, y-rays were administered to mice at a low dose rate and the effect on the chromosomes compared with that of X-rays administered a t a very high dose rate. It is well known that radiation administered a t a high dose rate is about four times as effective in shortening the life span as at low dose rate. The results of this experiment are shown in Fig. 2, and it will be observed that the high dose rate is also roughly four times as effective in producing chromosome aberrations. On the other hand, when this experiment was repeated using neutrons instead of X- or 7-rays, it was found that there was no difference in chromosome aberration frequency between high and low dose rates for this radiation (Fig. 3 ) . This correlates well with the fact that the life-shortening effect of neutrons is independent of dose rate. From these and a number of other experiments employing radiations, it is apparent that there is a quantitative as well as qualitative relation between the degree of life shortening produced by a given radiation or radiation regime and the chromosome aberrations which it induces. It would be very surprising if there were not a causal relation between them. Two different inbred strains of mice may have quite different life spans, even though both exhibit a wide spectrum of senile pathology. If normal aging is due to the development of mutations, the short-lived mice should develop chromosome aberrations at a higher rate than do long-lived ones, and indeed such is the case (Fig. 4). Whereas there are exceptions to this relation even between inbred strains of mice (see below), as a general rule it seems valid. An experiment has been performed to compare mice and dogs in this regard (Curtis et al., 1966a). It was found that dogs develop chromosome aberrations in liver much more slowly than do mice, and of course they also age much more slowly. These experiments lend potent support to the concept that mutations in somatic cells are involved with aging. However, there are exceptions to this general rule which cannot be

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ignored. It has been found (Curtis et al., 1966b) that mice of the C58 strain, which develop leukemia at an early age and have a median life span of 10 months, develop aberrations as slowly as long-lived strains. One must assume that these mice have a genetic defect which does not affect chromosome stability, and is probably in the nature of a point 100

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mutation. In another experiment, F, hybrid mice were compared with their parent strains with respect to chromosome aberration frequency (Curtis et al., 1966b). It was found that they develop aberrations a t a rate intermediate between those of the parent strains, but they lived longer than either of the parent strains. One must conclude that hybrid vigor is caused by something other than the stabilizing of the chromosome structure. All these experiments show that mutations in somatic cells play an important role in aging. However, they also clearly demonstrate that chromosome aberrations, a t least as scored by the methods employed here, do not tell the whole story. This emphasizes the fact that aging in a mammal is a very complex syndrome, and it is futile to try to explain it by one simple theory. V. A Composite Theory of Aging

The experiments just discussed show that mutations in somatic cells are very important in the development of the aging syndrome, but they also indicate that there must be other factors involved. There are also other facts which preclude the acceptance of a simple mutation theory. There is every reason to believe that the spontaneous mutation rate in somatic cells remains relatively constant throughout life. If aging were due to a mutation, for example to form a cancer, then one would expect the death rate from that cancer to be relatively constant with age. This is, of course, a t complete variance with the facts. The form of the mortality curve, however, suggests a multi-hit phenomenon, so one can assume that each of the symptoms of aging result not from a single change in a somatic cell, but from a series of such changes (Curtis, 1966b). The process is represented schematically in Fig. 5. According to this scheme, a series of improbable events is required to transform a cell into one which may lead to one of symptoms of aging, such as cancer. The entire aging Syndrome is then represented by a matrix of several thousand or even hundreds of thousands of possible reactions. These reactions are proceeding simultaneously, and eventually one will reach its goal (e.g., a cancer). Which one arrives there first depends upon the genetic constitution of the individual, which determines the rate constants, upon probability, and upon environmental factors which can change some rate constants. From the scheme of Fig. 5, one can compute the rate of formation of type 2 cells (e.g., Az) as dAz = r ~ ~ A l d tB ~ A z d t

GENETIC FACTORS IN AGINU

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FIG.5. A schematic representation of the composite theory of aging. A series of events take place in s great many of the somatic cells of the body, and each event has its own probability constant. The end result of each process is a cell which contributes to the aging process. Since Az << Al and assuming a Then, since A1 is very large

+ p, the

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Az = aaAit Similarly, assuming A3 << A2 Aa = BaAzt = CtapaAIt' One can then generalize with the equation (Curtis, 1967) "dt

= Kt(n-1)

(1)

where N is the number of persons developing a disease, n the number of events required for induction, and K a constant which depends on the rate constant for each step. Applying this equation to mortality data, the predicted form of the curve is remarkably close to the observed one (Fig. 6). Further, if one plots mortality data for individual degenerative diseases, the form of the curve predicted by Eq. (1) is also remarkably close to the observed one. The data fit this equation far better than the simple exponential form as implied by the Gompertz plot. Indeed, after one has applied this equation to many degenerative diseases and found an excellent agreement with the epidemiological data, one gains considerable confidence that the underlying assumptions must generally be correct. This general concept is not new, since the multiple factor theory of carcinogenesis was formulated many years ago. Also, Armitage and Doll (1954) proposed this concept, which has been extended by Burch

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FIQ.6. Log age-specific death rate vs. age for males in the United States. Points derived from Eq. (1) are a h shown, assuming an average of six steps in the induction process. (From Curtis, 1967.) (1963, 1968) and Burnet (1965). There are a number of diseases for which the simplifying assumptions involved in the formulation of Eq. (1) are not valid. Burch has formulated a more precise set of equations for dealing with special diseases, and has obtained excellent agreement between observed and predicted curves. The curve of Fig. 6 indicates that an average of six events is required to produce death from one of the degenerative diseases. Burch (19681, in his extensive analysis of a great many degenerative diseases, finds a great difference in the numbers of events required to initiate various degenerative diseases. For example, early onset childhood leukemia may be initiated by two events, whereas cancer of the prostate requires 18. However, most of them require about six.

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There are many other arguments favoring this general approach, although it must be admitted that it may not be the whole story. It suffers from being very general and complex, but this is the nature of the aging process.

THENATUREOF

THE

INDUCTION STEPS

There seems little doubt now that one of the first steps in the process must be a mutation in a somatic cell. The principal arguments favoring this view come not only from the chromosome studies but from the fact that mutagenic agents, especially radiations, invariably cause life shortening by accelerating the appearance of the degenerative diseases. The reason for believing a mutation must be one of the first steps is that radiation produces a decreasing effect on life shortening as the radiation is applied later in life. When radiation is given to very old animals, it has virtually no life shortening effect (Lindop, 1965). A number of other experiments support these views (Curtis, 1969). A subsequent step in the induction process must be a stimulus for cell division if such is needed. Obviously a single cell by itself is not going to cause much trouble t o a whole organism, but a whole clone of aberrant cells, such as a cancer, can be fatal. Something must start the process of clonal replication, otherwise a single aberrant cell may remain unexpressed in a tissue for many years, as is known to be the case for radiation carcinogenesis. It was shown by Cole and Nowell (1964) that if radiation preceeds a stimulus for regeneration in the liver, hepatomas appear in abundance, whereas this is not true if the order is reversed. Berenblum and Trainin (1963) have emphasized the importance of the order of application of chemical carcinogens. Finally, Maini and Stich (1961) showed that only those azo dyes which will both break chromosomes (cause mutation) and initiate mitosis cause many liver tumors; either factor alone is rather ineffective. We can only speculate concerning the nature of the other steps, but there is abundant evidence that there are other steps. For example, many benign tumors remain quiescent for years before suddenly becoming malignant. Again, benign experimental tumors may be transferred to other animals many times before suddenly becoming invasive. One might postulate that several of the first steps are mutations. However, present indications (Lindop and Rotblat, 1961) are that the lifeshortening effect is a linear function of radiation dose. If two or more mutations were required, one would not expect a linear response. There

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was then the possibility that a mutation followed by a mitotic stimulus and followed by another mutation might be required. However, an experiment specifically designed to test this, using liver tumor induction in mice as the test (Curtis, 1969), indicated that this was not the case. It is by no means necessary to think of the total sequence depicted in Fig. 5 as occurring in the target tissue. The immune recognition system, which eliminates cells which do not belong to that individual from the body, must be altered to permit the uncontrolled growth of a tumor, for example. I n the case of the autoimmune diseases, it may be that the primary step is a mutation in a cell in the immune system which initiates a clone which does not recognize particular parenchymal cells as belonging to that individual. The resulting immune reaction can be very unfortunate. Further, it is by now well demonstrated that virus plays a very important part in the etiology of some, perhaps all, cancers. Thus the activation of a virus may be one of the steps of Fig. 5. It is interesting to compute the probability (mutation rate?) for the steps in these reactions. It was assumed, for simplicity, that all the steps have an equal probability. It has been estimated (Burch, 1963) that for the reactions considered here there are about 10" cells a t risk for the human. It can then be computed that the probability for each step is of the order of per cell per year. This can be compared to the mutation rate computed from the spontaneous appearance of genetic anomalies in the human population. For individual diseases this rate is of the order of per gamete, considering them to be due to a single mutation in dominant genes. Then over a generation time of 30 years this gives an approximate value of 3 X per gamete per year. This is more than a thousand times smaller than the value for somatic cells derived from Eq. (1). It is difficult to know how to reconcile these values, and perhaps one should not try. A mutation which leads to ti cancer in a somatic cell may well occur a t a completely different spontaneous rate than a mutation which may cause phenylketonuria. It is obviously not possible to estimate mutation rates from measurements of chromosomal aberrations. However, chromosome aberration studies indicate that in the liver of old mice more than half the cells have visible chromosomal defects. For each visible defect it is reasonable to postulate several point mutations, so on this basis every liver cell would be expected to contain several mutations, indicating a high mutation rate. Finally, one may postulate that genetic anomolies are also due to multi-hit events, and in this case values very much higher than 3 X lo-' per gene per year would be required. I n view of these uncertainties, a mutation rate of 10-~per gene per year for human somatic cells may be quite reasonable.

GENETIC FACTORS IN AGING

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VI. The Nature of the Mutations

It seems highly likely that the chromosome aberrations scored in this work are not the actual lesions responsible for the degenerative diseases. One can only hope that they give some index of the true mutation rate. This view was greatly strengthened by a recent experiment using liver tumor induction as an indication of mutation rate (Curtis et aZ., 1968). As noted previously, if a dose of radiation is given in order to produce mutations in the liver, and this is followed by a dose of CCI, to induce liver regeneration, then liver tumors are produced in a high percentage of the animals. Even if the dose of CCI, is delayed for 9 months there is still a high yield, indicating that the mutations originally produced had remained dormant until unmasked by the regeneration. However, if these animals are scored for chromosome aberrations, it is observed that they gradually return to control levels over the 9month period. This clearly indicates that chromosome aberrations in liver cells can be repaired over a period of some months, whereas the mutations (point?) responsible for the initiation of this tumor are never repaired. It should be emphasized that this applies only to this particular tumor; but certainly indicates that chromosome aberrations may be a poor indicator of genetic damage. However, it is almost the only one available. It is interesting to note the very high aberration frequency which may be present in cells which appear to be functioning normally. For example, if one gives a rather large but not fatal dose of neutron radiation to a group of mice, one finds in liver cells a chromosome aberration rate which approaches 100% and this persists for the life of the mouse. Apparently liver function is normal. This must mean that all the cells are full of mutations, but very few of them are in genes controlling liver function. A very high percentage of genes in a liver cell, for example, are not necessary for normal cell function. However, if the cell is forced into division, the damaged chromosomes may so interfere with the process that the cell dies. This is why the radiation sensitivity of cells in wiwo varies so widely; those cells which must divide often, such as those of the bone marrow, are very sensitive, while those which seldom if ever divide, such as brain cells, are very insensitive. It is interesting to note that the chromosome aberration frequency in liver cells of mice increases steadily with age, and that these cells divide seldom if at all. This indicates that spontaneous mutations in these cells are not caused by an error of chromosomal replication, but must be due to some mechanism independent of cell division. This agrees

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with the conclusion of Novick and Szilard (1950) based on work with bacteria. The sequence in Fig. 5 which leads to cell death deserves special mention. A change in a cell which transforms it from a normal somatic cell to a malignant one certainly requires a very special and subtle change. It is a very rare event. However, a lethal mutation must be very common, since a single mutation in a gene affecting any of a great many vital cellular functions would cause cell death. Thus this process might be expected to require only one step. In most cell systems in mammals, the death of a single cell presents no problem. I n the skin, for example, a dead cell would soon be replaced. However, in organs with fixed postmitotic cells, such as the brain, this would represent a permanent loss. It is then significant that in assessing decrement in organ function, Shock (1961) concluded that for organs with postmitotic cells, such as muscle or kidney, functional loss was a linear function of age. This then implies a “one-hit” curve, which means that a great many of the “nuisance” aspects of aging result from single mutations in somatic cells. Shock found a decrement in organ function between young adulthood and old age of the order of 30% for most organs, and if, as Shock believes, this is due to cell loss, this means the loss of millions of cells from every organ. It has been estimated that the normal person loses about 10,000 brain cells per day. Probably all these cells do not die a genetic death, but many of them must. If they all died a genetic death, it would indicate a lethal mutation rate of about per cell per year, agreeing well with the other values derived from Eq. (1). VII. The Life Span of Dividing Cells

It was assumed for many years that aging took place in tissues composed of fixed postmitotic cells, and that tissues in which the cells were undergoing continual division did not age. A dividing cell system can eliminate aberrant cells by cell selection a t the time of mitosis, just as a suspension of bacteria can continue indefinitely as long as it is supplied with nutrient. This concept has recently been challenged on two counts. First, if one examines the total aging syndrome it will be evident that dividing cell systems are as much involved in the etiology of the symptoms of aging as any others. Skin cancer and leukemia are common examples of this. The second challenge originated from the work of Hayflick (1965) based on tissue culture studies of human fibroblasts. He found that

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embryonic fibroblasts in culture are able to undergo about 50 divisions before they develop chromosome aberrations and the culture dies out. If cells are taken from older persons and cultured, there will be fewer divisions before the culture dies out, indicating that some of the 50 possible divisions had already taken place. These and other studies led to the idea that a differentiated mammalian cell is capable of only a limited number of divisions, and this in some way is an important factor in the aging process. The experiments on which this conclusion are based have been confirmed a number of times, but the interpretation is .far from clear. This work was done on in vitro systems, and it is doubtful that cells in vivo would respond in this way. There have been many attempts to perform serial transplants of various cells from old animals to young ones, and when these animals get old to transfer this same tissue to young animals again. The tissue eventually dies, but as the skill of the investigator improves the grafts live longer. One is tempted to conclude that if the conditions were perfect perhaps the cells could live indefinitely. I n any event, all mouse cells tested so far have been found to live longer than the whole mouse, so if this phenomenon exists it probably does not contribute to aging. In a recent series of experiments Curtis and Tilley (1971) failed to find any evidence that bone marrow cells of mice cannot continue division indefinitely. The integrity of the cells was judged chiefly by chromosome aberrations, since chromosomal disintegration was found to accompany the deterioration of the cell cultures. It was found first that there is no increase in chromosome aberration frequency with age in these cells. Also, agents which kill bone marrow cells, like radiation, or cytotoxic agents, like vinblastine, can repeatedly be made to kill off a large fraction of these cells (see Fig. 7 ) . But regeneration takes place immediately and the cells seem unchanged. It was estimated that in some of these experiments as many as 1000 extra doublings were forced on the stem cells but when these mice became old the chromosome structure was indistinguishable from that of young mice. Even in a special strain of mice in which there is a genetic instability in the bone marrow cells, there was no evidence of a decreased vitality with time. From these experiments the conclusion seems clear that if there is some limitation on division capacity, it plays no part in the normal aging process. These results definitely show that chromosome aberrations are occurring continually in bone marrow cells and are being eliminated by cell selection a t the time of cell division. However, point mutations are probably not eliminated in this way, so they must continue to accumulate in these cells and give rise in this tissue to the symptoms of aging,

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HOURS AFTER 62.5rads OF NEUTRONS

FIG. 7. Chromosome aberration frequency in bone marrow cells of mice, strain C57BL/KT, irradiated with 626 rads of neutrons vs. time after irradiation. Control points are at zero time. Young mice were 177 days old and old ones 797 days old. (From Curtis, 1969.) such as leukemia. These experiments should then finally dispose of the idea that aging occurs only in fixed, postmitotic cells. I n addition, these experiments strongly indicate that the spontaneous mutation rate remains constant throughout life for these cells. The results of chromosome studies on liver cells (Curtis, 1966a) are compatible with this observation. This gives important confirmation to the conclusions of Burch (1968) and Curtis (1969) , derived from studies of mortality curves, that the mutation rate in somatic cells remains constant throughout life. VIII. Summary

It is clear that aging in the mammal is largely controlled genetically. Aging has two rather distinct components: the “nuisance” factor, for example, muscular weakness, and the mortality factor resulting from the development of degenerative diseases. Diseases such as arthritis obviously have components of each. Both result a t least in part from mutations in somatic cells. The “nuisance” symptoms of aging can be explained as due to cell loss in postmitotic somatic cells, which is probably caused by lethal mutations in single genes. The degenerative diseases are initiated by very rare cellular events, each disease probably requiring about six such events for initiation, only one or two of which are mutations. The nature of the other steps is largely unknown. Probably some of the steps take place in cells not in the target organ. Cells in the immune recognition system must also be altered to allow the development

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of aberrant cells in the target organ. All somatic cells undergo spontaneous mutation, and once a cell has suffered a mutation which may eventually lead to a degenerative disease it is not eliminated from the cell pool by cell selection. It may take a very long time before the mutation becomes manifest because a number of other quite improbable events must also take place. The reason a man lives so much longer than a mouse, for example, is because his spontaneous mutation rate and the probability of occurrence of the other cellular changes is very much lower.

REFERENCES Armitage, P., and Doll, R. 1954. The age distribution of cancer and a multi-stage theory of carcinogenesis. Brit. J. Cancer 8, 1-8. Berenblum, I., and Trainin, N. 1963. New evidence on the mechanism of radiation leukaemogenesis. I n “Cellular Basis and Aetiology of Late Somatic Effects of Ionizing Radiation” (R. J. C. Harris, ed.), pp. 41-52. Academic Press, New York. Burch, P.R. J. 1963. Carcinogenesis and cancer prevention. Nature 197, 1145-1151. Burch, P. R. J. 1968. “An Inquiry Concerning Growth Disease and Ageing.” Oliver and Boyd, Edinburgh. Burnet, M. 1965.Somatic mutation and chronic diseases. Brit. Med. J. 1,338346. Caldecott, R. S. 1961. Seedling height, oxygen availability, storage and temperature ; their relation to radiation induced genetic injury in barley. I n “Effects of Ionizing Radiations on Seeds,” pp. 3-24. International Atomic Energy Agency, Vienna. Cole, L. J., and Nowell, P. C. 1964. Accelerated induction of hepatomas in fast neutron irradiated mice injected with CCL. Ann. N . Y . Acad. Sci. 114, 259-265. Crowley, C., and Curtis, H. J. 1963. The development of somatic mutations in mice with age. Proc. Nut. Acad. Sci. U.S. 49, 626628. Curtis, H. J. 1966a. “Biological Mechanisms of Aging.” Thomas, Springfield. Curtis, H.J. 1966b. A composite theory of aging. Gerontologist 6, 143-149. Curtis, H. J. 1967. Biological mechanisms of delayed radiation damage in mammals. I n “Current Topics in Radiation Research” (M. Ebert and A. Howard, eds.), Vol. 111, pp. 141-174. North-Holland Publ. Co., Amsterdam. Curtis, H. J. 1969. Somatic mutations in radiation carcinogensis. In “Radiation Induced Cancer,” pp. 45-56. International Atomic Energy Agency, Vienna. Curtis, H. J., and Crowley, C. 1963. Chromosome aberrations in liver cells in relation to the somatic mutation theory of aging. Radiation Res. 19, 337-344. Curtis, H. J., and Gebhard, K. L. 1958. Comparison of life shortening effects of toxic and radiation stresses. Radiation Res. 9,104-109. Curtis, H. J., and Gebhard, K. L. 1959. Radiation induced aging in mice. Prog. Nucl. Energy, Ser. 6 2, 210-216. Curtis, H. J., and Tilley, J. 1971. The life span of dividing mammalian cells in vivo. J. Gerontol. 26, 1-7. Curtis, H. J., Tilley, J., and Crowley, C. 1964. The cellular differences between acute and chronic neutron and gamma ray irradiation in mice. I n “Biological Effects of Neutron and Proton Irradiations,” Vol. 11, pp. 143-155. International Atomic Energy Agency, Vienna.

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Curtis, H. J., Leith, J., and Tilley, J. 1966a. Chromosome aberrations in liver cells of dogs of different ages. J. Gerontol. 21, 268-270. Curtis, H.J., Tilley, J., Crowley, C., and Fuller, M. 1966b. The role of genetic factors in the aging process. J . Gerontol. 21, 366-368. Curtis, H. J., Czernik, C., and Tilley, J. 1968. Tumor induction as a measure of genetic damage and repair in somatic cells of mice. Radiation Res. 34, 315-319. Failla, G. 1957. The aging process and carcinogenesis. Ann. N.Y. Acad. Sci. 71, 1124-1135. Fox, A. M.,and Skinner, J. S. 1964. Physical activity and cardiovascular health. Amer. J. Cardwl. 14, 731-746. Hayfiick, L. 1965. The limited in vitro lifetime of human diploid cell strains. Exp. Cell Res. 37, 614-636. Holmes, M. C. 1965. Coombs test conversion in young NZB mice induced by transfer of lymphoid cells from Coombs positive donors. Aust. J. Ezp. Biol. Med. Sci. 43, 399405. Johnson, H. D.,Kintner, L. D., and Kibler, H. H. 1961. Effects of 48°F and 83°F on longevity and pathology of male rats. J . Gerontol. 18,29-36. Lindop, P. J. 1965. Radiation and lifespan. In “The Scientific Basis of Medicine Annual Reviews,” pp. 91-109. British Federation of Postgraduates, Oxford Univer. Press, London and New York. Lindop, P. J., and Rotblat, J. 1961. Long term effects of a single whole body exposure of mice to ionizing radiation. Proc. Roy. SOC.,London Ser. B 154, 332349. Maini, M. M., and Stich, H. F. 1961. Chromosomes of tumor cells. 11. Effect of various liver carcinogens on mitosis of hepatic cells. J . Nut. Cancer Znst. 26, 1413-1424. Novick, A.,and Szilard, L. 1950. Experiments with the chemostat on spontaneous mutations of bacteria. Proc. Nut. Acad. Sci. U.S. 36, 708. Shock, N. W. 1961. Physiological aspects of aging in man. Ann. Rev. Physiol. 23, 97-122. Stevenson, K. G., and H. J. Curtis. 1961. Chromosomal aberrations in irradiated and nitrogen mustard treated mice. Radiat. Res. 15, 774-784. Szilard, L. 1959. On the nature of the aging procesa. Proc. Nut. Acad. Sci. US. 45, 3042. Werthessen, N. T. 1962. The site of the primary lesion in atherosclerosis. Angwlogy 13, 520-530.

TOWARD A GENERAL THEORY OF GENETIC RECOMBINATION IN DNA* Rollin

D.

Hotchkiss

The Rockefeller University, New York, N e w York

I. The Nature of Recombinational Deviations . . . . . 11. Evolution of Recombination Models . . . . . . . 111. Biochemical Requirements of a Recombination Model . IV. A Molecular Model Providing Asymmetric Recombination V. Molecular Events Leading to Resolution. . . . . . VI. Later Steps in Resolution of Recombination Complexes . VII. Some Comparisons and Generalizations . . . . . . VIII. Conversions and Transformations . . . . . . . References . . . . . . . . . . . . . .

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Genetic recombination in various systems has exhibited the following manifestations among others: (1) creation of new linked marker arrays representing apparently reciprocal rearrangements of the arrays preexisting in the parental genomes: (2) such rearrangement within a single genetic locus, resulting in the production of new allelic phenotypes representing that locus ; (3) the occurrence of fine structural recombinations as above with either expected, or excessively increased or reduced numbers of associated recombinations of adjacent markers (negative or positive interference) ; (4) excessive recombination rates between certain mutant sites (nonadditivity) ; ( 5 ) nonreciprocal exchanges giving in single events exclusively one recombinant product, or producing it more often than its reciprocal ; (6) apparently nonreciprocal recoveries of excessive copies of one parental allele compared to another (gene conver-

* This article is dedicated to the memory of Dr. Milislav Demerec, whose lifelong study of genetic loci in a variety of organisms underlies our concepts of the continuity of genetic maps. In addition much gratitude is felt for his efforts and achievements in providing at the Cold Spring Harbor laboratories an atmosphere in which the sciences of genetics, biochemistry, and physics could productively interact. This study was aided by support from the National Science Foundation through Grant GB-6696. 325

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sion): and (7) polarity in the way in which the inequalities of (4), ( 5 ) , or (6) are affected by linear map position. Genetic recombination models, too numerous for brief review, have been suggested, accounting sometimes with more, sometimes with less, generality for these observations. Some of them are discussed by Fogel and Hurst (1967), Taylor (1967), Stahl (1969), Whitehouse (1969,1970), and Holliday and Whitehouse, (1970). The first three observations listed require mainly single or double exchanges a t greater or smaller intervals, as accounted for in all models. The remaining items present various versions of inequalities in exchanges that violate the easy a priori assumption of essential randomness.* The nature of the violation may be seen below to be somewhat general. The idea of an excess or deficiency in a recombinant class [item (6)] has meaning only in relation to some measure of what might have been. Several measures have been used or implied in various systems to estimate the normal expectation: (1) the number of opportunities for confrontations, (2) the number of actual contacts between homologs, (3) the number of viable progeny, (4) the number of postrecombinant genomes, ( 5 ) the number of reference marker recombinations, or (6) the segregation pattern within the primary products of a tetrad. The variable reliability and form of these different measures has hampered correlation of studies in the different systems. The number of opportunities or confrontations may be difficult to know if the parents (as vegetatively multiplying phages) are not constant or controlled. The

* It should be pointed out from time to time that the assumption of randomness within a class of events is an opportunistic assumption which permits interesting and useful quantitative hypotheses to be made and tested. It derives from ignorance of specific causation, is not in itself a logical or mechanistic model in spite of itR usefulness, and calculations made with its use should be considered as containing an assumption. To begin with, randomness cannot even be proposed until there are empirical findings which show t h d the “predicted” events are possible and occur a t all. As previously pointed out (Hotchkiss, 1958; Hotchkiss and Evans, 19581, such a process as exchange between polynucleotide chains does not occur randomly among all of the links of a chain (carbon-carbon, for example) but presumably is random only within a certain class of linkages having a demonstrated (not predicted) lability. These are tacitly taken to be phosphate diester linkages, and even at these bonds there are detailed choices (3’ or 6’, carbon-oxygen or oxygen-phosphorus) of unequal probability. I t is not known how such probabilities are further modified by the adjoining pyrimidine or purine bases. In this and many other examples the brave assumption of ‘‘random probability” merely means the worldly complaisance, “probably equal within certain classes of approximately equal probability.” The classes for which such assumptions are useful usually become smaller and smaller subclasses as further biochemical specificities are continually revealed to be governing them. Probability calculations sometimes can point toward the need to define these subclasses.

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number of surviving DNA fragments in such asymmetric exchanges as transformation, transduction, or mating may be affected in unknown degree by degradation, elimination, and repair. Reference markers are basically needed to reveal the number and the regularity of the events or populations (e.g., the transformation and transduction events, or yields, the number of normal matings, zygotes, or meiotic tetrads involved) from which the recombinant progeny are being derived. 1. The Nature of Recombinational Deviations

All of the mentioned aberrations of recombination [items (4) to (7)] can be viewed as manifesting a similar aspect: a marker region is found to be excessively represented among one part of the progeny. In nonadditivity or “map distortion” the excess is among recombinant progeny bearing particular other neighboring sites. Nonreciprocality in a small progeny leads to an excess of one recombinant array, or is seen as a gene conversion from which a particular marker segregates 3: 1, instead of a normal 2:2. Ascospore sets may then be further modified: by additional replication, 3 : 1 ( X 2 ) giving 6:2; or, if already replicated, being joined by additional normal pairs, 3(+2) : 1(+2) giving 5:3 sets. We may consider how such gene conversion events would be easily overlooked if occurring in mass populations rather than closed sets such as ascospores: (1) if flanked by two markers, a converted region would produce an excess, perhaps imperceptible, of “double crossovers”; (2) if occurring more frequently with an adjacent crossover, or if flanked by only one marker, it would appear as an excess of recombinants (map expansion) in the next adjacent interval; or (3) if not demarcated by available markers, conversion would pass unrecognized and not be considered a normal property of the system. Thus, it is possible to propose that the general finding underlying all “abnormal” recombinations amounts to an apparently excessive survival or copying of a limited region from one parent, coupled with a deficiency in recovery of that from another parent. We may propose to consider them all formally as intranuclear transformations, or gene conversions, inasmuch as the mechanism is not precisely known for any of these aberrations. This concept obliges us to look for correspondence and lack of correspondence among the phenomena; as mentioned, the different modes of measurement make this rather difficult. We will return to this topic later, If one seeks to ascribe these aberrations to some modified exchange, other than gene transformation or conversion, one is forced to speak of nonreciprocal exchanges-this being merely a description of the findings rather than a mechanism.

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II. Evolution of Recombination Models

I n early chromosome models for symmetrical recombination, breakage points were assumed to coincide exactly on each parental element so that fusion would regenerate intact recombinant products. Perhaps the unpleasant strictness of this requirement, together with the unequivocal demonstration of aberrant tetrads, stimulated the resurrection of the idea of the copying-switch or “copy-choice”-which, occurring in replication, did not require a material loss of one parental strand but only an extra copying of one parental element at the expense of the other. Models of this period were mainly obliged to account for what could formally be deduced from genetic exchanges or vaguely derived from cytological observations. The last decade or so has furnished two important restrictions concerning molecular events in genetic processes-the semi-conservative replication of chromosomes and DNA, and also the discovery that large segments of preformed DNA exchange their neighbors in the various recombination processes. It became clear that copy-choice alone would not account for genetic recombinations, and that recombination models would have to be more detailed and biochemically realistic. Considerations based upon molecular features of DNA structure have tended to introduce new degrees of freedom and increase the number of conceivable models for recombination. Among the new features in recent models are: (1) the single DNA-strand break or reunion (less destructive of structure than a chromosomal or chromatid breakage) ; (2) the postulation of complementary hydrogen bonding regions as aligning mechanisms allowing candidate strands to be brought into near register for rejoining (removing the requirement for ends accidentally to find each other) ; (3) assumption of limited regions of DNA strand biosynthesis to coordinate exact points for breakage and reunion, postulation of either; (4) adventitious alternation of template in this biosynthesis to provide extra duplicates of certain regions; or ( 5 ) selective “repair in” or “repair out” of base inhomologies, to account for increase or decrease of individual allele recoveries; and (6) use of the directionality of chromosome synthesis to give polarity to some of these steps. It is an unfortunate feature of the present situation that with resort to all of these degrees of freedom, most of the currently available models can become overlapping or near equivalents. Indeed, a charitable application of some of these eminently reasonable new features to certain of the old models can convert them as well into the newer type. It does not seem, then, to be an appropriate time to “disprove” any of

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the models, but rather, hopefully, a time for finding generalizing principles. It will be my intention to refine further a molecular model for recombination by seeking-rather than freedoms-additional limitations which seem to derive from DNA structure and from accumulating knowledge about enzymes presumed to be involved in the recombination process. Among the ways in which proposed models differ in detail is in the sequence postulated for the nearly equivalent steps of strand-breakings, homolog-findings, and rej oinings among the four single strands usually considered. One other type of difference is in the component which it is permitted to sacrifice or waste: a parental segment, the use of a template, or a mismatched base residue, etc. Among the different models, two factors contribute to require waste of parental DNA strands: inequality of the parental entities (fragment vs. chromosome, etc.) , and any requirement for DNA synthesis accompanying recombination (balanced by an equivalent sacrifice, unless whole chromosomes are assumed to be replicated). Concerning some of these requirements current biochemical knowledge seems to the author to suggest some choices and to tend to eliminate certain others. Finally, different models tend to appear the more unlike because they are developed to explain different aspects of recombination. It will be useful here to relate or adapt all models to the case of a potentially reciprocal exchange between parental markers that span a region within which excessive recovery (or conversion) of another marker or markers occurs. Additional reciprocal exchanges, sacrifice of one product, and so on, can for the purpose be treated as minor variations suiting the result to particular systems, but imposing in themselves no important difficulties. As is usual, the parents will be considered to be two preexisting, or separately generated, homologous reaches of DNA duplex. The products will be considered to be one or two DNA duplexes with possibly altered segments but with the polarity of DNA strands preserved. Ill. Biochemical Requirements of a Recombination Model

Briefly described, some properties of real DNA systems which seem relevant to recombination are: (1) existing enzymes capable of opening (incision or nicking), shortening (excision), extending (polymerase), and joining (ligase) DNA chains under natural cellular conditions; (2) these DNA-monitoring enzymes collectively act to repair altered (e.g., irradiated) DNA, and at least some of the components of these systems (defective in recombination-deficient, radiation-sensitive mutants) facilitate

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recombinations; (3) the DNA monitoring enzymes in their primary actions open, modify, or join only single DNA chains a t a time; (4) most or all DNA-monitoring enzymes act upon a specific substrate-recognition site: an interrupted DNA double strand (Fig. 1); and ( 5 ) the polarity (3’- to 5’-phosphate diester direction) of DNA strands will almost certainly be preserved in viable-progeny strands. There is therefore a demonstrated connection between the effects of known enzymes or genetic blocks upon repair of damaged DNA and upon genetic recombination. The common component of DNA structure upon which many of the enzymes exclusively act, is a nearly perfect hydrogen-bonded duplex (as indicated in Fig. 1) interrupted or

473 I I I I J I I I I I I I

Common recognition site for DNA enzymes (x-y = H-bonded base poirs) Exomples, D-F (z=dtscontinuity,etc)

D -

t * @ =

t*=l

FIQ.1. Binding or recognition sites for DNA-monitoring systems. Arrow heads represent 3’-hydroxyl ends of single DNA chains. Escherichia coli DNA polymerase binds to the sites encircled (except that on D). At sites A, A’, B (and the same in E, F) it can activate the following processes : adding on or removing mononucleotidyl residues, exchanging of pyrophosphoryl residues a t 3’-ends, or shortening by hydrolysis of a 5‘-phosphate chain end. At single-chain openings (nicks) i t can shorten a 5’-end, and maintain or elongate a 3’-end until a DNA ligase is available to seal up a nick (such as at A, A’ when only one phosphate is missing) and thereby exclude further degradation. If elongation occurs along its own template, the result is ‘‘repair,” if along a heterologous template, copy-switch conversion. Polymerase neither binds to nor attacks double-stranded regions of DNA, and does not attack a single exposed 5‘-phosphate end (as C). Incision occups at discontinuities or abnormalities such as site Z on D, and creates gaps such as A or A‘. Excision systems act like DNA polymerase to widen such gaps. All of the systems require the hydrogenbonded double-strand structure in order to act, but act only upon one of the strands.

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terminated as shown, in either strand. These sites are found in DNA molecules a t the point of single-strand breaks or gaps, and may be generated in recombination processes. One may suspect that some form of such a site can be a signal for initiation of the enzymatic processes setting recombination in motion (Fig. 2 ) . If a genetic model is to become a reasonable molecular model, the sound principle of the economical hypothesis must be adapted from genetics to prescribe minimal use of those biochemical events which are less probable. It has been customary for example in genetic models to assume no recombination event whenever with the markers available no genetic rearrangement could be detected. With simple detectors, simple models were possible. I n a broad way, one can say, however, that as regions are more fully defined with markers, the more one finds complex behavior and modified rates in the exchanges of genetic recombination. It is obvious, for example, that nonreciprocal local exchanges will not be recognized unless they are flanked by markers sufficiently nearby to preserve the bias they produce. If we could form some estimate of the number of actual molecular events occurring in recombination, with or without marker exchange (“molecular recombinations”) we would be able more justly to assign priorities to the more probable and less probable choices among real chemical events. For example, we might find that a molecular exchange which is very probable may seldom coincide with a genetic exchange: nevertheless, to include rather than exclude such a mechanism in our molecular model is necessary if we are to arrive at what is a biochemically realistic as well as a genetically minimal hypothesis. I n particular, I would propose that transfers of single-strand DNA segments are a common accompaniment of close contact between homologous DNA regions. If the tracts transferred are of limited size and somewhat random in occurrence they could often bypass a marker site and lead to a molecular insertion that is not necessarily detected as a genetic one. Those that are detected would appear as double crossovers, and from the conservative point of view, would traditionally be supposed to be as rare as the opportunity of demonstrating them. Recent isotopic studies (Lacks, 1962; Fox and Allen, 1964; Notani and Goodgal, 1966; Bodmer and Laird, 1968) indicate that physically recombined DNA strands occur very frequently in bacteria undergoing transformation, even when only a small percentage of the cells shows detectable specific genetic transformation. We must consider such breakage and rejoining to be a demonstrated potentiality of DNA strands in living cells. Size-limited transformation of local regions at short range, whenever heterologous DNAs are in close contact, was specifically urged

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(Hotchkiss, 1958; Hotchkiss and Evans, 1958) as an explanation for nonadditivity or expansion of genetic maps, high negative interference, aberrant tetrads, and other aspects of nonreciprocality. Before considering further the possible unity of such effects, let us continue to explore how strand exchanges may appear in biochemically and physically realistic terms. IV. A Molecular Model Providing Asymmetric Recombination

For the sake of brevity we may deal with only a single primary case, two DNA duplexes with single-strand breaks preexisting or developing in approximately corresponding positions in each parent, indicated in Fig. 2 and those following. Until occurrence of the first break (at A) there is essentially no site able to bind DNA polymerase, but that break produces a site immediately attractive to known affinities of the enzyme. Several things can happen thereafter, more or less governed by the (somewhat) random hydrogen bond openings and closings between complementary regions, but rendered irreversible through action of the polymerase (or ligase) enzyme. Degradation of 5’ or 3’ chain ends, or rebuilding of the latter, can occur within the same duplex, corresponding to postulated “repair” mechanisms. Events significant for recombination will in general require interaction of the strand with elements of the other parental DNA. We may picture the liberated single strand, carrying a coating of DNA polymerase as something like a poisoned arrow, able to activate certain events when it makes contact with a polynucleotide site of appropriate homology. The process thus far outlined satisfies one criterion which I believe is a fundamental requirementthat the two parents enter unequally into the individual confrontation, in order that they may often be unequally represented in the ultimate products. One parent strand essentially “attacks” the other parent and may succeed in setting up a heterozygous “recognition site” for DNA enzymes (see Fig. 1 above) as portrayed a t 11, Fig. 2. This structure is vulnerable to polymerases, which, if similar to Eschen’chia coli DNA polymerase (Kornberg, 1969) will be able either to build up or degrade the heterozygous overlap. Degradation at A will essentially produce an excision repair of the lower parent. Synthesis at A along the new template, however, will produce a genetically recombinant strand (111) which may now be conserved (Fig. 3) either by continuing and joining the corresponding other homolog (IV,) or returning to its own strand (IVb) as a double crossover. The choice IV, may be determined by the possible existence and widening by

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FIG.2. Schematic diagram of probable behavior of DNA molecules, containing broken strands, under the influence of DNA polymerase. An end (A) if detached from its complement can move about until it finds a structure with a t least 8 to 10 suitable complementary bases for pairing. I n the case shown the lower strand has “attacked” the heterologous complementary strand above, forming hydrogen bonds (vertical dashes) and loosening the binding between that one and its partner. The two 3’-hydroxyl-ended (arrow on right) internal segments may be viewed as in competition for that partner; if the attacking strand returns to its own original partner, the balance is settled in favor of the status quo. However, DNA polymerase, indicated by ovals (II), will be attracted to the unsatisfied ends or chains and force the balance toward another decision. In the case shown, biosynthesis (dashed lines) upon the 3’ end at A and shortening at A’, causes the system more and more to favor the attack by the lower chain upon the upper duplex. In the meantime the lower strand has acquired information copies from the upper duplex.

polymerase of an appropriate nearby strand break in the homolog (A’ in Fig. 2 ) , while the choice of IVb may result from the possible polymerase 5‘-hydrolase action in widening the original gap a t A, or from the failure to find a break at A’. (It will be proposed below that the growth along A tends to produce a break at A’.) One mode of completion of the recombination of IV, is pictured in Fig. 4, following three alternative topological assumptions ; segregation with original partners, segregation with new partners, and destruction of one duplex (V,, VI,, and VII,, respectively). Equivalent treatment of the possibility IV, will be pictured in the “b” series below (Fig. 5 ) . The segregations depicted in Fig. 4 require cumbersome unwinding steps, as do other replication and segregation models. The second junc-

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tion-finding and rejoining of the abandoned segments-requires no special triggering, being the result of an attack, synthesis, and joining implicit in the original break and junction. If it fails, the nonreciprocal recombinant is produced as in VII,. The successful rejoining of the residual fragments in the same manner as the original pair produces reciprocal recombinant half-duplexes in which as indicated there is a possibility of conversion (3 copies of one segment) in the crossover region due to the regions of break and resynthesis not being necessarily in register. V. Molecular Events leading to Resolution

The principal other postulate I will make in this model is that intertwining of helices will naturally lead to certain strand breaks. It is possible to picture known enzymological specificities as the “driving force” leading to the secondary openings and rejoinings of strands which complete the recombination. These would create a reasonable alternative to the elaborate unwinding called for in Figs. 4 and 5. Let us consider in more detail the already mentioned openings and rejoinings. An attacking strand together with polymerase could grow and become helically wound along its new t e m p l a t e t h e complementary half-helix which we will call the “borrowed strand” of the (first) attacked or recipient DNA-and progressively displace corresponding segments of the homolog. As each mononucleotide unit is added, the polymerase, having no affinity for double-helical regions, loses contact with that site, moves along one step, and combines instead at the new end (as indicated in Fig. 1; based upon findings of Kornberg, 1969). At some point, corresponding let us say roughly to A’ in Fig. 2, as the borrowed strand is being abducted by competitive pairing from association with its former partner, the complex will be forced toward a more or less irreversible “decision point.” If a preformed break does not exist, this structure (“opposed helices” on one continuous strand, the two complements overlapping, one or both trying to grow) must eventually achieve some kind of resolution. I n the figures, I have indicated the assumption of a chain break or nick in the homologous strand (at A’) and the sets of decision determined by it. This break makes relatively easy the joining of the homologous ends as in Fig. 3 (IV,). Other possibilities for resolution must be considered: (1) an existing break in the borrowed template strand instead; (2) no preformed breaks, but a developing strain as the two homologs compete for the borrowed strand, the imperfect pairing leading, like irradiation damage, to an incision break in one of the strands near the junction; (3) degradation of one of the

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t

FIG.3. Two alternative outcomes for the situation attained a t 111, in Fig. 2.

In IV. the foreign strand has invaded the upper duplex and grown along the

helical groove (much as assumed for RNA transcripts) until i t reaches or creates a strand break in that homolog. At this point a DNA ligase recognition site is produced and there is either competition for joining, or if polymerase has shortened the homolog, the oppositely derived homologs may readily be joined, completing a onestrand crossover in a relatively stable link. The displaced strands and the displaced polymerase are now able to make approaches or attacks upon other strands. In IVb the growing attacking strand has not succeeded in finding or creating a strand opening in the upper duplex. Here, having completed a “copy-choice,” it withdraws from that template and returns to its original partner; further outcomes are suggested in Fig. 5.

strands from its end down to the junction; or (4) a spontaneous early break, in the borrowed strand, as assumed by Whitehouse (1963), makes the initial attack easier and symmetrical in his model and its successors. The same first two breaks and mutual hybrid formation, also assumed by Boon and Zinder (1969), became in their model the elegant basis of an unsymmetrical replication fork in which the remnant end of the borrowed section also elongates for a time. The extra break (4) then, does not lead to resolution but eventually also requires further resolving steps such as we are now discussing. Reflection will suggest that most of the forms of resolution just outlined lead back to the same few alternatives already proposed. The forced incision [as in (2) ] is presumed to result from unsuccessful pairing where two overlapping homologs compete for a .continuous strand; it would seem most likely that one of the unfulfilled homologs would be broken, rather than the oversatisfied continuous strand. Alternatively, opening the attacking homolog amounts to suppressing or degrading its growing end, but may allow it shortly afterward to attack again. Therefore one of the most common results may be the strand incision forced at A’ in the homologous strand (possibility 2), followed perhaps

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be degradation (3), leading at first to the single crossover strands (IVa, Fig. 3) as treated herewith in detail. If, nevertheless, some fraction of the breaks open up the borrowed strand, we have the situation of case (1)-in which the hybrid pair suffers a rather lethal double termination and might be free to unwind, giving a return of the recombinant strand to itself similar to that indicated in Fig. 3 (IVJ , eventually producing a heteroduplex containing one parental and one recombinant strand, the latter a double crossover made by “copy-choice.” Otherwise, complex additional bond breakages and uncoilings are required for resolution, probably resulting in removal by repair of the whole recombinant region and rebuilding of parentals. This would also be expected if degradation occurs on the borrowed strand after a breakage, or on the free end of the attacking strand. Therefore the outcomes indicated in Fig. 3 may represent all or most of the probable mechanisms which actually lead to recombinants, with the product IVb less frequent and somewhat more liable to destructive removal than product IV,. To return to the second junction, that achieved by the abandoned segments: the breakage of the attacked homolog (at A’) liberates an end which can in turn now become an attacking end. Since it is also a 3‘-end, it can therefore, unlike the donor 5’-end which was abandoned at the original break, be biosynthetically extended along the donor template, and in the manner just outlined eventually be able to break and join its homolog. However, if not very much degraded before it grows, it will have preserved a third segment of the recipient genome. Conversion will therefore have occurred at the left end of the recombinant region just where the original attacking strand had to copy the borrowed template before it could complete its attack upon the recipient. A strand break is not less arbitrary or gratuitous if the rationale for it is essentially: Manifestly strand breakages must somehow occur: we will assume them to occur randomly over nearby regions of each of four involved strands. The number of arbitrary strand breakages we have assumed is fewer than in almost any other model for recombination. [Boon and Zinder (1969) have also pointed out, but not insisted, that some breaks might be the result of others and of pairing.] The principal arbitrary assumptions we have made are : the preexistence of a single break in parental DNA and a tendency of the 3‘-hydroxyl chain end at the break to explore nearby double helices and insinuate itself along a complement in another parental duplex. The remaining steps seem spontaneously probable : sufficient openness of helices to permit local synapse or recognition, a likelihood of the attacking strand thus diverting a borrowed strand competitively from its original part-

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ner-much as RNA transcripts are considered to behave when in the presence of their polymerase-and setting up thereby of a recognition site a t which DNA-polymerase (or similar) enzymes can elongate the attacking strand by local synthesis along the borrowed template. Competition between the growing copy and the resident homolog should then produce, or make use of, further single-chain breakage and in a proportion of cases irreversibly favor the newly formed recombinant. Finally, unwinding, exonucleolytic trimming, and rejoining of the heteroduplexes to make polymerase-resistant double helices are specified with little detail, but much as in DNA repair and replication generally. Summarizing thus far, we have presented arguments suggesting that the first stages of resolution processes concerned with the active (right) end of the hybrid complex lead to either: two recombinant single strands of like polarity not precisely reciprocal (these are shown untangled in Fig. 41, or, probably less often, the double crossover in one strand (a copy-choice: see Fig. 5 ) . Both types are likely to carry molecular conversions at the left ends of the crossover region, i.e., toward the starting or &-ends of the recombinant strands. The conversion will normally consist of regions copied from the attacked or recipient parent (in both types). But if the attacked homolog should be excessively degraded before its rescue, conversions may (in series a ) consist of donor parent segments-presumably never of both, except that further crossovers may lead to further conversions. It will be noted that these restrictions define a basis for polarity effects in conversions, for their nonreciprocality as events and for unequal yields of reciprocal types in populations. Such polarity does not require precise initial breakage of two strands a t operator sites as proposed in the later Whitehouse models (Whitehouse and Hastings, 1965; Whitehouse, 1966, 1967), but only a frequent attack upon one strand starting relatively often at preferential sites which may include an operator or transcription origin. VI. later Steps in Resolution of Recombination Complexes

But we have not yet taken account of the full resolution process. Extensive untangling and unwinding processes such as are assumed for segregation of replicated chromosomes can be postulated, but their mechanism is not very fully known. Complete unwinding a t both right ends-probably unlikely unless swivel points or strand breaks are close-would permit rewinding as suggested in V, (and V b ) , which in effect extends the hybrid regions toward the right. Unwinding of left ends, under similar restrictions, would extend the hybrids to the left

338

ROLLIN D. HOTCHKISS

/

< ma

- _ _

-

__

+-

/

- - --

-- - - - -- -

/a-

_-

-- -- -

FIO.4. Three alternative outcomes for the segregation of the crossed-over duplexes of IV., Fig. 3. By axial (“speedometer cable”) rotation the intact heterozygote unwinds from either the (right) upper former partner (V.) or the (left) lower one (VL). The former partners are either degraded (VIL), or the polymerase in a similar manner helps these strands (V. or VI.) to complete themselves and join by a ligase-susceptible finishing step. Heteroduplexes are formed; the regions of new synthesis are not precisely overlapping, therefore, in parts of these regions unequal replication (molecular conversion) occurs. The eventual segregation patterns for these duplexes would be (indicated top to bottom, a t three successive positions from left to right; U-upper class, 1, lower class parentage) : For V. For VI.

UUl1,

UlUl,

UUUl, UUUI,

lUUl UUll

The central converted region, here yielding 3: 1 parental segregation, if occurring late in a half-tetrad would of course with a normal 2:2 pair make a 5:3 set. Another sequence of synthesis could give Ulll regions. but by this pathway never both UUUl and Ulll regions in the same pair of duplexes. In case the other strands are degraded only a simple exchange (VII. or equivalent) is detected.

and lead to the possibilities VI, and VII,, and would be additionally required for VIb. It seems far more likely that in the complex produced by rejoining the broken strands of IV, (a still-entwined precursor form of V, and VI,) additional incisions will occur and obviate the unwinding. These would be expected at the left end of the hybrid region as the result of distortion in the competitive “opposed helices” a t this end (Fig. 6 ) . In general, there are four unpaired points susceptible to breakage and repair. It may be that the donor strand (C) complementary to the original

339

GENETIC RECOMBINATION IN DNA

- -- - _ m b

- - - - - _ _ _ _ -

-

-

-- -

-

-- - -

-__- +-+4

FIG.5. Three alternative outcomes for the segregation of the self-returned case, Ivb of Fig. 3. Simple repair of the broken strands might be possible (V, and VIb). Segregation however, is complicated by the interim winding of the synthesized heterozygous portion of the attacking strand into the upper template helix. The schematically simple segregation suggested in Vb requires another strand break and unwinding of this central overlap. The exchange of partners as in VIb is even more improbable, but still feasible if there are swivel points for rotation (necessary for recombinations rather generally) which could allow all four ends of the complex to be dangled and rotate from this small purchase. It seems more likely that the very “repair” enzymes involved in producing this structure would act to remove the entangled heterologous strand. Ultimately, then a simple copychoice transformation or conversion would be registered (VIIb). This does not however explain the incorporation of donor nucleotide tracts in actual transformation (see text). The outcome Vb is the same as the copy-choice achieved by the model of Paszewski (1970) which begins as this one (scheme Ivb, Fig. 3) but assumes a break and degradation of the borrowed region, and preservation of the copy. The break a t A’ in the homolog (Fig. 2) is not requisite for any of these variations, but it may assist the degradation for VIIb.

attacking strand, being first to be abandoned and last to become hybrid, will be most subject to incision. It could then counterattack in turn and as described (Fig. 6 legend) lead to a reciprocal crossover with at least one region of conversion. If, however, equilibration of the bonding in the intertwined helices has rendered them dynamically symmetrical as suggested in Fig. 6, there would be essentially equal probability of any one of the four strands

340

ROLLIN D. HOTCHKISS

.

D

IX

A

n’

FIQ.6. Diagrams representing in linear and helical schematic form the essential features of the biochemically based recombination model. In earlier steps (Figs. 2, 3) the attacking strand has been lengthened (A to A’), by copying recipient information, forced or found a break in its homolog, and joined it at A’. This liberated a 3’-ended strand (B) from the (original) recipient which then in a similar way attacked and joined the donor helix farther along at B‘. Gene conversion has occurred in the region from A to B, as the result of a tendency for 3‘ ends to be lengthened (as heterologous copies) while 5’ ends are unchanged, or, if anything, shortened. The intertwined region a t the left will now tend to be broken a t an unpaired site near the original point of separation of complementary strands in the primary attack. After some equilibration and possible lateral migration of the opened-out zone, any one of the strands C, D, E, or F will be more or less equally subject to breakage by repair systems. One half of the possibilities give outside marker recombination; one half do not. Breakage at C, for example, will result in an attack by the 3‘ end of that strand upon D; it will copy template F, then cause opening of D. Strand D will thereafter attack and join strand C. The four sequences with approximately equal probability are : C breaks, then D breaks, then E breaks, then F breaks, then

D; outside marker recombination C; outside marker recombination F ; no outside recombination E; no outside recombination

(conv. (conv. (conv. (conv.

DF) CE) DF) CE)

Secondary conversions will depend upon the relative lengths of regions of synthesis and those of synapse; if because of “strain” both helices are opened a t the same

GENETIC RECOMBINATION IN DNA

341

being opened. A subsidiary break in its homolog would then be precipitated. I n about one half of the cases the originally active pair of homologs would be involved, and thus in about half of the conversions lead t o heteroduplexes containing doubly crossed-over strands (no outside marker recombination). The problems of disentangling would be minimal. It will be recalled that one of the greatest difficulties of recombination models has been to explain how among conversions outside marker recombinations are hardly more frequent than their absence. Thus, reasonable consideration of the biochemical properties of the known systems can lead to single or double crossover recombinants, exchanging or not exchanging outside markers. I n each case the exchanges are reciprocal on the large scale for outside markers but not reciprocal at the finer internal level. Conversions a t this finer level are likely for markers a t the initiating (or left) end of the recombinant region. They should reproduce a region (not a site) primarily derived from only one parent, often the recipient, and even when more complex, would be nonreciprocal events. If initial breakage and attack tends to start from a particular strand in both parents, conversions in mass may be reciprocal but show polarity toward one side. If there are preferential regions having even moderate tendency to initiate more frequent attacks, the mass data will show polarity effects. It is doubtful if the available data are extensive enough to demonstrate only a single zone of polarity per operon as proposed by Whitehouse and Hastings (1965) and Whitehouse (1966). It seems more reasonable at present to suppose that there are several, possibly overlapping, polar zones of different intensity, as is also suggested by Paszewski (1970), who likewise supposes a structural basis for polarity direction. VII. Some Comparisons and Generalizations

Space does not permit, and the time may be premature, to attempt to relate the present model quantitatively to data accumulated on conversions in Ascomycetes. Nor can we stop to compare it in detail with numerous previous models. This presentation is rather an attempt at realistic redefinition of some chemical and physical limitations which may restrict the too-gratuitous postulation of strand breakages, findings, place or at nearly the same time by repair, then the synthesis and secondary conversions will be slight. As indicated in parentheses, secondary conversions may extend or parallel the original conversion (which produced an excess of DF markers), or convert to the other parentral type CE, in another strand at another region.

342

ROLLIN D. HOTCHKISS

pairings, biosyntheses, and rejoinings in liberal combinations that do little more than restate the foreknown genetic results. Holliday (1964) proposed complementary pairing to secure the register of broken co-parallel strands for rejoining; Whitehouse (1963) used it to hold anti-parallel broken strands, and allowed synthesis a t the ends to achieve register and joining (see Fig. 7) ; we have here tried to define

md

+

-

4 I /

d

\

.-

*

--

-

-

FIQ.7. Conceivable alternative patterns for recombination, considered less probable than those outlined in Figs. 2 to 6 (not late stages of those models). Scheme IV, is essentially the single-crossover model of Whitehouse and Hastings (1965) and approximates the later models of Whitehouse (1966, 1967). Oppositely directed DNA strands break at the same level (at the operator in later models) and become templates for localized synthesis, eventually joining their homologs. A crossover of outside markers results, as in IVa, having intermediate regions of unequal overlaps, or conversion, and would be ordinarily far more common than conversion ‘‘without” (i.e., with double) outside exchange. The site and sequence of breakages are devised to suit the purposes of the model but not justified as biochemically probable. Local synthesis begins from the broken ends, which thus develop affinity for the oppositely derived end. A switch of template is assumed which then exactly positions the hybrid pair so that recombined helices are set up. In some fashion the remaining ends, and later also the growing ends, open up two homologous sites in the previously unbroken strands, and join them to make two recombinant intact helices. If the arbitrary first steps are assumed, two of the four required rejoinings could have a mechanism and polarity similar to those outlined under Fig. 3. The other two, joining the other helix, remain in the present form arbitrary.

a probable mode for just such joining and fusion. All of the recent models illustrate the proposal of Pritchard (1960), who much earlier suggested that recombination might involve both incorporation of DNA segments by breakage-fusion and some synthesis with change of template, or copy-choice. To a large extent, the model presented in this paper achieves both of these by processes, believed to progress spontaneously (in the cellular environment) with a minimum number of arbitrarily assumed steps.

GENETIC RECOMBINATION IN DNA

343

Older recombination models account for asymmetric outcomes, such as conversions, by providing individual asymmetric resolution steps from largely symmetrical initial complexes. Recently, Stahl (1969) proposed replicated diploid loops which could recombine asymmetrically, discard the other halves and somehow disentangle. Pamewski (1970) (see legend, Fig. 5 ) , Boon and Zinder (1969) (see Fig. 8 ) , and the present author emphasize the asymmetric availability of a template from one parent t -

“

tt

+Cf c

,

/

.---- - -- - - -- El

/---\

FIG.8. Another possibility for resolving the recombinations, alternative to Figs.

3 to 6. This is a representation of the model of Boon and Zinder (1969). The

exchange of template and attack begin asymmetrically as in Fig. 2, 111, but as in Whitehouse (1963) and Pasaewski (1970),the borrowed strand is also assumed to require a break. This permits one end of it to attack the other donor strand and join it, and its residual end together with the initiating donor strand to set up a “replication fork.” This fork progresses to B further point where three subsequent breaks and joinings produce either a double crossover plus parental (shown in IVI) or a pair of single crossovers. Conversion can occur at sites of unequal overlap. The published model expressly disavows specific prescription of the molecular mechanism. While the early opening of the borrowed strand is considered improbable in the present paper (on the basis of competition for pairing; see text), if it be assumed, i t leads easily to the second attack and joining shown at the bottom of IV,. Replication can be assumed to follow a “fork,” although detailed enzymatic mechanism remains unknown, but there is little explanation of the need for an interruption of the fork and return to fuse with the fragmented parent.

to the primer (3’-hydroxyl strand end) from another parent. In the Whitehouse models such contributions are bilateral and therefore initially symmetrical. We have further suggested here that a single-strand end can attack an unbroken double helix; some arguments and evidence from the literature for special activity of strand ends and this thesis have been summarized elsewhere by Hotchkiss (1968). The heteroduplexes produced as suggested in this article would yield conversions with 5 :3 (postmeiotic) segregation ratios in Ascomycetes,

344

ROLLIN D. HOTCHKISS

and not 3: 1 (or 6:2) ratios unless one of two things were true: unless (1) they were produced as primary products before replication, or (2) they were made homologous by postrecombinational repair. The same considerations apply to most other models, except that of Boon and Zinder (1969) which postulates a simultaneous copying that with linear arrays can give directly 3 : l conversion of both strands with or without outside marker recombination. Stahl (1969) also assumes sufficient duplicate copies that selective discard can give any desired ratio of marker survival. The proposal that repair is stimulated by mismatched base pairs is at present questionable and unsupported (Stahl, 1969; Paszewski, 1970; see however, Holliday and Whitehouse, 1970) and quite different from the more accepted proposition we employ here, that unpaired segments may be opened up by repair systems. The correction of mismatched base pairs is postulated in an extreme sense by Fogel and Mortimer (1969), who propose that conversions occur by repair of hybrid regions without obligatory relation to recombination. Their view is based upon the large number of conversions they obtained without outside marker recombination in a well-marked yeast region. Their proposal differs explicitly from our model (Figs. 3 to 6) and those of other workers presented here (Figs. 7 and 8) in that the biosynthesis and copying they postulate is that used to replace an excised repaired region but not to extend broken ends. There are some implications which may eventually make possible experimental test of some of the principles I have applied to recombination. For our model it is possible to consider one parental strand as the initiating or attacking parent in the first step. At this stage it is also a donor of molecular segments as a sort of investment necessary to achieve hybrid pairing. It would be helpful to form some estimate of the relative chain lengths involved in the initial anchoring and those added by copying of the neighbor, before breakage and fusion of the homolog. The success of that broken homolog in finding and opening the residual donor strand fragment will determine how long the segment of the duplicated recipient will be that remains as a conversion region. If the homolog is degraded back past the ruptured end of that fragment, then it will probably grow back with an additional (converted) copy of the donor parent instead. Presumably, if the intact donor complement is available for pairing there is little likelihood (at a 3’-end) of losing so much as that of the recipient strand. Conversions from this stage should therefore be found at one end only of the junction between parental segments, and should most commonly represent the primary recipient or attacked genome.

GENETIC RECOMBINATION I N DNA

345

If these are the preponderantly characteristic first steps in both double and single crossovers, it should eventually be possible to detect their regularity underlying the still-later steps which complicate the picture. We have argued the greater probability of the third breakage occurring in the opposite donor strand and producing single crossovers; in these there should be an opportunity for conversions of these strands also to recipient type (at a different site, farther to the left). The double crossovers would tend to have conversions a t the left ends also, but in this case perhaps representing either parent (therefore in effect lengthening or shortening the crossed-over region). Some evidences of these events are suggested in the observation of “transition regions” (of isotope density) by Bodmer and Laird (1968) -newly synthesized DNA adjoining markers newly incorporated in bacterial transformation. Unfortunately, as we shall see below, the intrinsic asymmetry of transformation processes tends to specialize their role as indicators of how recombinations in general occur. VIII. Conversions and Transformations

Transformations and gene conversions, observed in widely different systems, have some features in common. It is clear that both involve the short-range transfer of specific and exact units of information from one parent. Another correspondence is revealed in recent ascospore analyses by Fogel and Mortimer (1969), which show the high frequency of double-site conversions in yeast, e.g., 50% of the conversions involving an arginine site also involving conversion of another locus about 500 nucleotide residues distant. The included DNA molecular weight, about 2 X lo5 daltons, is of the same order as that conveyed in transformations of various species. I n the yeast system as in bacterial transformations (Hotchkiss, 1958; Hotchkiss and Evans, 1958; Ephrussi-Taylor and Gray, 1966) “cotransfer” decreases as marker pairs become more separated. The postulate of marker incorporation efficiency as attributable to differential susceptibility of different markers to repair, is a t least in harmony with the mechanism proposed by Fogel and Mortimer (1969) for gene conversions. The overall frequency of 1.7% conversion observed by the latter workers shows that it is a significant feature of DNAto-DNA confrontation when suitable markers are available. The term, gene conversion, has been used for those cases in which a gene is modified in the near presence of an allele to resemble that allele, with both the modifier and the modified gene surviving. The nonreciprocality of conversions derives from considering them as potential

346

ROLLIN D. HOTCHKISS

exchanges, during which, however, one parent gene region disappears and another appears in duplicate. Transformation events also include the characteristic disappearance of one parental-the recipientgene region, but the donor parent is a chromosome fragment, and after donating a segment is regularly sacrificed; thus any equivalent failure of this parent to acquire genes reciprocally from the other is obscured or swallowed up in the greater “nonreciprocality” that all of its not donated genes are lost. Genetic transformation of a bacterial genome by homologous DNA, like transduction and perhaps bacterial conjugation, presents an opportunity to study recombinations in which one parent is of simpler constitution than the other. (Viral recombinations may involve whole genomes, but the number of parental genomes confronting each other are seldom actually known, because of sampling variation and explosive replication.) This may mean that some complications of recombination processes may be simplified or circumvented in transformations. The algebra of DNA strand recovery in transformations appears to be 4

become 2

(rather than 4 return as 4)

I n fact, in Pneunaococcus the effective donor unit seems to be a singlestranded intermediate (Lacks, 1962) and in Bacillus subtilis, some kind of denatured complex (Venema et al., 1965). I n several species it is clear that the molecular units incorporated are exclusively single strands (Fox and Allen, 1964; Notani and Goodgal, 1966; Bodmer and Laird, 1968). The elimination of the other strand appears to precede recombination in some cases (Lacks, 1962) although it has been proposed (Fox, 1966) , or, in other cases demonstrated (Steinhart and Herriott, 1968) , that some of the exchange occurs a t recombination itself. Transformation processes demonstrate that DNA fragments may “attack,” in the sense of this paper, an intact DNA duplex, and it seems clear that the retained donor unit must be the attacking strand. Processes involving its discarded complement must be supposed either to be unavailable or to result in degradation: for example, those which only regenerate the attacking strand, the copy-choice series b, Figs. 3 and 5. Intermediate IV, (Fig. 3) however may be produced; the recipient (upper) homolog is opened and joined, but the thereby liberated (left) end of this strand in beginning a counterattack finds either no donor homolog to hybridize with, or a degraded o n e - o r else the hybrid is especially susceptible. When this counterattacking strand is itself sufficiently shortened to find and counterattack the unpaired donor strand a t the point where that originally attacked the recipient helix, it can rejoin at that point, stabilizing a double-crossover insertion of a donor

GENETIC RECOMBINATION IN DNA

347

segment in one strand of that helix. This is just what is found, both for the donor genes and the donor atoms, in transformation. One of the most characteristic expectations from the hypotheses offered in this paper could be tested specifically and rather critically in transformation systems, and possibly ultimately in a reciprocal recombination. It is that nucleotide 3’-chain ends should relatively often-approaching a maximum of 50% in transformation-be incorporated into the recombinant, sometimes intact and always more so than the 5’-ends. The latter would usually be considerably shortened and probably never be fully incorporated. Both isotopic and genetic marker incorporation could be tested. In bacterial mating it must be assumed that the transferred DNA brings in many adventitious 3’-ends. Meanwhile information favorable to the main hypothesis has already appeared. Chevallier and Kopecka (private communication, 1970) have confirmed and extended the observations of Bernardi and Bach (1968) showing that DNase I1 produces greater reduction of transforming marker uptake per breakage than does DNase I. The former enzyme leaves 3’-phosphate ends and these would require additional dephosphorylation before they are eligible for known building and joining systems. It does not seem advisable a t this point to attempt to explain results from in vitro produced artificially hybrid DNA, since such hybrids probably contain many breaks, gaps, overlaps, and impaired ends, all of which may greatly stimulate additional repairs when they are introduced into a recipient cell. These results are very interesting from the standpoint of repair, however. The results of transformations, then, are in keeping with the biochemical pattern outlined here as a general model for recombinations. Their genetic effects, however, come from the actual introduction of donor DNA gene regions into recipient chromosomes, and they probably occur by steps which can yield single and double crossovers when whole genomes interact. Gene conversions are probably different, resulting, I propose, from asymmetric replication and discard of parental segments a t the crossover borders of biparental zones in almost all recombinations. In many recombinations they are detectable only as frequency disturbances, if at all, and in transformations they appear only as local zones of DNA resynthesis.

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348

ROLLIN D. HOTCHKISS

Boon, T., and Zinder, N. D. 1969. Proc. Nat. Acad. Sci. U.S. 64, 573-577. Ephrussi-Taylor, H., and Gray, T. C. 1966. J. Gen. Physiol. 49, 211-231. Fogel, S., and Hurst, D. D. 1967. Genetics 57, 455-481. Fogel, S., and Mortimer, R. K. 1969. Proc. Nat. Acad. Sci. US.62, 98-103. Fox, M. S. 1966. J. Gen. Physiol. 49, 183-196. Fox, M. S., and Allen, M. K. 1964. Proc. Nut. Acad. Sci. U.S. 52, 412419. Holliday, R. 1964. Genet. Res. 5, 282-304. Holliday, R., and Whitehouse, H. L. K. 1970. Mol. Gen. Genet. 107, 85-93. Hotchkiss, R. D. 1958. Symp. Soc. Exp. Biol., 13, 49-59. Hokhkiss, R. D. 1968. Cold Spring Harbor Symp. Quant. Biol. 33, 857-870. Hotchkiss, R. D., and Evans, A. H. 1958. Cold Spring Harbor Symp. Quant. Biol. 23, 85-97. Kornberg, A. 1969. Science 163, 1410-1418. Lacks, S. 1962. J . MoZ. Biol. 5, 119-131. Notani, N., and Goodgal, S. H. 1966. J. Gen. Physiol. 49, 197-209. Paszewski, A. 1970. Genet. Res. 15, 55-64. Pritchard, R. H. 1960. Symp. SOC.Gen. Microbiol. 10, 155-180. Stahl, F. W. 1969. Genetics 61, (suppl.), 1-13. Steinhart, W. L., and Herriott, R. M. 1968. J. Bacteriol. 96, 1718-1731. Taylor, J. H. 1967. I n “Molecular Genetics” (J. H. Taylor, ed.), Part 11, pp. 95-135, Academic Press, New York. Venema, G., Pritchard, R. H., and Venema-Schroeder, T. 1965. J . Bacteriol. 89, 1250-1255.

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BIBLIOGRAPHY Publications of M. Demerec

1921 Heritable characters of maize. X. Zebra striped leaves. J . Hered. 12, 406407. 1922 With Emerson, R. A., Eyster, W. H., and Anderson, E. G . Studies of somatic mutations in variegated maize pericarp. Anat. Rec. 23,90-91 (abstr.). 1923 Inheritance of white seedlings in maize. Genetics 8,561-593. Heritable characters of maize. XV. Germless seeds. J . Hered. 14,297-300. 1924 A case of pollen dimorphism in maize. Arner. J. Bot. 11, 465-469. Genetics of four groups of seedling characters which affect the development of chlorophyll in maize. Anat. Rec. 29, 133-134 (abstr.). With Emerson, R. A. Inheritance of white seedlings in maize. Anat. Rec. 29, 134 (abstr.). Genetic relations of five factor pairs for virescent seedlings in maize. Cornell Univ. Agr. Exp. Sta. Mena. 84, 1-38. 1925 Reddish-a frequently “mutating” character in Drosophila virilis. Anat. Rec. 31,345 (abstr.) . With Satina, S. Manoilov’s reaction for identification of the sexes. Science 62, 225-226. Inheritance of pale green seedlings in maize. Genetics 10, 318-344. 1926 Reddish-a frequently “mutating” character in Drosophila virilis. Proc. Nut. Acad. Sci. U.S.A. 12, 11-16. 349

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DEMEREC

With Brink, R. A., and MacGillivray, J. H. Effect of the waxy gene in maize pollen-a reply to criticisms. Genetics 11,3840. Notes on linkages in maize. Amer. Natur. 60, 172-176. Heritable characters of maize. XXV. Piebald seedlings. J. Hered. 17, 300-306. The effect of selection on the frequency of mutation of the gene for miniature-alpha in Drosophila virilis. Anat. Rec. 34, 169-170 (abstr.) . Miniature-alpha-a second frequently mutating character in Drosophila virilis. Proc. Nat. Acad. Sci. U.S.A. 12, 687-690. 1927 Magenta-alpha-a third frequently mutating character in Drosophila virilis. Proc. Nat. Acad. Sci. U.S.A. 13,249-253. Heritable characters of maize. XXIX. Midcob color. J. Hered. 18, 420-422. A second case of maternal inheritance of chlorophyll in maize. Bot. Gaz. (Chicago) 84, 139-155. 1928 A possible explanation for Winge’s findings in Lebistes reticulatus. Amer. Natur. 62, 91-94. The behavior of multiple genes. Verh. V Int. Kongr. Versrbungswiss. (Berlin 1927) pp. 183-193. Mutable characters of Drosophila virilis. I. Reddish-alpha body character. Genetics 13, 359-388. 1929 Mutable genes in Drosophila virilis. Proc. Int. Congr. Plant. Sci., 1st Ith. N.Y. 1, 943-946. Cross sterility in maize. 2. Indulct. Abstamm. Vererbungsl. 50, 281-291. Genetic factors stimulating mutability of the miniature-gamma wing character of Drosophila virilis. Proc. Nat. Acad. Sci. U.S.A. 15, 834-838. Changes in the rate of mutability of the mutable miniature gene of Drosophila virilis. Proc. Nat. Acad. Sci. U.S.A. 15, 870-876. 1930

A genetic factor affecting germinal mutability of miniature-alpha wing character of Drosophila virilis. I n “The Laws of Life,” Mem. Vol. 60th Birthday Prof. Dr. V. Ruzicka, pp. 45-56. Aventinum, Prague, Czechoslovakia.

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With Farrow, J. G. Non-disjunction of the X-chromosome in Drosophila virilis. Proc. Nut. Acad. Sci. U.S.A. 16, 707-710. With Farrow, J. G. Relation between the X-ray dosage and the frequency of primary non-disjunctions of X-chromosomes in Drosophila virilis. Proc. Nat. Acad. Sci. U.S.A. 16, 711-714. 1931 The behaviour of two mutable genes of Delphinium ajacis Proc. Int. Bot. Congr. 5th pp. 196-97 (abstr.). Behaviour of two mutable genes of Delphinium ajacis. J. Genet. 24, 179-193. The gene (resum6 of a lecture). The Biological Laboratory 3 , 2 9 3 2 . 1932 Effect of temperature on the rate of change of the unstable miniature-3 gamma gene of Drosophila virilis. Proc. Nut. Acad. Sci. U.S.A. 18, 430-434. Rate of instability of miniature-3 gamma gene of Drosophila virilis in the males in the homozygous and in the heterozygous females. Proc. Nut. Acad. Sci. U.S.A. 18, 656-658. Changes in the instability of miniature-3 gene of Drosophila during ontogeny. Proc. Int. Congr. Genet. 6th 2,43. Exhibits. Proc. Int. Congr. Genet. 6th 1,68-73.

1933 Miniature-5 a new unstable gene in Drosophila virilis. Amer. Natur. 67, 68-69 (abstr.). What is a gene? J. Hered. 24,368-378. The effect of X-ray dosage on sterility and number of lethals in Drosophila melanogaster. Proc. Nut. Acad. Sci. U.S.A. 19, 1015-1020. 1934 Gene deficiencies as cell lethals in Drosophild melanogaster. Amer. Natur. 68, 165. Effect of X-rays on the rate of change in the unstable miniature-3 gene of Drosophila virilis. Proc. Nat. Acad. Sci. U.S.A. 20, 28-31. Biological action of small deficiencies of X-chromosome of Drosophila melanogaster. Proc. Nut. Acad. Sci. U.S.A. 20,354-359. With Lebedeff, G. A. Spindle-fiber attachment end of the X-chromosome of Drosophila virilis. Cytologia 5, 391-394. The gene and its role in ontogeny. Cold Spring Harbor Symp. Quant. Biol. 2, 110-115.

352

DEMEREC

1935 Role of genes in a cell. Amer. Natur. 69,61-62 (abstr.) . Role of genes in evolution. Amer. Natur. 69, 125-138. Relative importance of various genes to the organism. Science 81, 420 (abstr.) . Unstable genes. Botan. Rev. 1,233-248. Woods Hole summer meeting of the Genetics Society of America. Collecting N e t 10,261, 264-265. Behavior of chlorophyll in inheritance. Cold Spring Harbor Symp. Quant. Biol. 3, 80-86. With Lebedeff, G. A. The gene. Carnegie Inst. Washington Yearb. 34, 40-43. 1936 With Hoover, M. E. Deficiencies in the forked region of the X-chromosome of Drosophila melanogaster. Amer. Natur. 70, 47 (abstr.) . With Hoover, M. E. Three related X-chromosome deficiencies in Drosophila. J . Hered. 27, 206-212. Frequency of “cell-lethals” among lethals obtained a t random in the X-chromsome of Drosophila melanogaster. Proc. Nut. Acad. Sci. U.S.A. 22, 350-354. Heredity and radiation. Radiology 27, 217-220. The nature of mutations. I. Collecting N e t 21,9-10. With Hoover, M. E. The gene. Carnegie Inst. Washington Yearb. 35, 40-45. 1937 A mutability stimulating factor in the Florida stock of Drosophila melanogaster. Genetics 22, 190 (abstr.) . Differences in mutability in various wild-type lines of Drosophila melanogaster. Science 85, 442 (abstr.) . With Fricke, H. The influence of wave-length on genetic effects of X-rays. Proc. N a t . Acad. Sci. U.S.A. 23,320-327. Relationship between various chromosomal changes in Drosophila melanogaster. Cytologk, Fujii Jubilee Vol., pp. 1125-1132. Frequency of spontaneous mutations in certain stocks of Drosophila melanogaster. Genetics 22, 469-478. With Kaufmann, B. P. Frequency of induced breaks in chromosomes of Drosophila melanogaster. Proc. Nut. Acad. Sci. U.S.A. 23, 484-488.

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With Slizynska, H . Mottled white 258-18 of Drosophila melanogaster. Genetics 22, 641-649. With Kaufmann, B. P. The gene. Carnegie Inst. Washington Yearb. 36, 44-51. 1938 Hereditary effects of X-ray radiation. Radiology 30,212-220. With Bauer, H., and Kaufmann, B. P. X-ray induced chromosomal alterations in Drosophila melanogaster. Genetics 23,610-630. Eighteen years of research on the gene. Carnegie Inst. Wash. Publ. 501, 295-314. With Kaufmann, B. P., and Hoover, M. E. The gene. Carnegie Inst. Washington Yearb. 37, 40-47.

1939 With Hoover, M. E. Hairy-wing duplication in Drosophila melanogaster. Genetics 24, 68 (abstr.) . L’importanza di alcuni loci per l’organismo. Sci. Genet. (Turin) 1, 123-128. With Hoover, M. E. Hairy wing-a duplication in Drosophila melanogaster. Genetics 24, 271-277. The nature of X-ray-induced lethal changes in the notch region of the X-chromosome of Drosophila melanogaster. Science 89, 401 (abstr.) . Chromosome structure as viewed by a geneticist. Amer. Natur. 73, 331-338. With Kaufmann, B. P., and Sutton, E. The gene. Carnegie Inst. Washington Yearb. 38, 185-191. 1940 With Kaufmann, B. P. Time required for Drosophila melanogaster males to exhaust the supply of mature sperm. Collecting Net 15, 169 (abstr.). A comparison between the X-ray induced and the spontaneous notches. Genetics 25, 115-116 (abstr.). With Kaufmann, B. P. An opportunity for students of heredity. Amer. Biol. Teacher 2, 216-217. With Kaufmann, B. P. “Drosophila Guide. A Guide to Introductory Studies of the Genetics and Cytology of Drosophila melanogaster.” (2nd Ed., 1941; 3rd Ed., 1943; 4th Ed., 1945; 5th Ed., 1950; 6th Ed., 1957; 7th Ed., 1961.) Carnegie Inst. Washington, Washington, D.C. With Sutton, E. Unequal breaks in two sister chromatids induced by

354

DEMEREC

X-rays in Drosophila melanogaster. Proc. Nat. Acad. Sci. U.S.A. 26, 632536. Genetic behavior of euchromatic segments inserted into heterochromatin. Genetics 25, 618-627. With Kaufmann, B.P., Sutton, E., and Hinton, 0. T. The gene. Carnegie Inst. Washington Yearb. 39,211-217.

1941 With Fano, U. Measurements of the frequency of dominant lethals induced in sperm of Drosophila melanogaster by X-rays. Genetics 26, 151 (abstr.). Genetic changes. J . Appl. Phvs. 12,344-345 (abstr.) . The nature of the gene. I n “Cytology, Genetics, and Evolution,” University of Pennsylvania Bicentennial Conference, pp. 1-11. University of Pennsylvania Press, Philadelphia. The nature of changes in the white-Notch region of the X-chromosome of Drosophila melanogaster. Proc. Int. Congr. Genet. 7th pp. 99103. With Fano, U. Mechanism of the origin of X-ray induced Notch deficiencies in Drosophila melanogaster. Proc. Nat. Acad. Sci. U.S.A. 27, 2631. With Kaufmann, B. P. Time required for Drosophila males to exhaust the supply of mature sperm. Amer. Natur. 75,366-379. Cold Spring Harbor Symposium on Genes and Chromosomes. J . Hered. 32, 391-392. Unstable genes in Drosophila. Cold Spring Harbor Symp. Quant. Biol. 9, 145-149. With Kaufmann, B. P., Sutton, E., and Fano, U. The gene. Carnegie Inst. Washington Yearb. 40,225-234. 1942 With Hollaender, A,, Houlahan, M. B., and Bishop, M. Effect of monochromatic ultraviolet radiation on Drosophila melanogaster. Genetics 27, 139-140 (abstr.) . With Kaufmann, B. P., and Sutton, E. Genetic effects produced by neutrons in Drosophila melanogaster. Genetics 27, 140 (abstr.) . With Kaufmann, B. P. Sperm utilization in Drosophila melanogaster following single and multiple inseminations. Genetics 27, 150 (abstr.). Chromosomal changes in Drosophila melanogaster and their evolutionary significance. Proc. Amer. Sci. Congr., 8th 111,3741.

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With Brehme, K. S. A survey of Malpighian tube color in the eye color mutants of Drosophila melanogaster. Growth 6,351-355. With Kaufmann, B. P. Utilization of sperm by the female Drosophila melanogaster. Amer. Natur. 76, 445-469. With Kaufmann, B. P., Fano, U., Sutton, E., and Sansome, E R. The gene. Carnegie Inst. Washington Yearb. 41,190-199. 1943 With Zamenhof, S. Heavy water and mutations. Genetics 28, 96 abstr.). With Zamenhof, S. Studies on induction of mutations by chemicals. I. Experiments wit.h heavy water (deuterium oxide.) Amer. Natur. 77, 380-384. With Kaufmann, B. P., Fano, U., Sansome, E. R. and Gay, H. The gene. Carnegie Inst. Washington Yearb. 42, 139-147. 1944 With Fano, U. Mutability to bacteriophage-resistance in Escherichia co2i. Rec. Genet. SOC.Amer. 13, 16 (abstr.). With Sansome, E. R., Hollaender, A., and Zimmer, E . Quantitative effects of radiation on mutation production in Neurospora crassa. Rec. Amer. 13, 33 (abstr.). Genet. SOC. With Fano, U. Genetics: physical aspects. I n “Medical Physics” (0. Glasser ed.) , pp. 495-512. Yearbook Publ., Chicago, Illinois. With Fano, U. Frequency of dominant lethals induced by radiation in sperms of Drosophila melanogaster. Genetics 29, 348-360. With Fano, U., and Sansome, E. R. The gene. Carnegie Inst. Washington Yearb. 43, 108-114. 1945 Production of Staphylococcus strains resistant to various concentrations of penicillin. Proc. Nut. Acad. Sci. U.S.A. 31, 16-24. Genetic aspects of changes in Staphylococcus atireus producing strains resistant to various concentrations of penicillin. Ann. Mo. Bot. Gard. 32, 131-138. With Fano, U. Bacteriophage-resistant mutants in Escherichia coli. Genetics 30, 119-136. With Sansome, E. R., and Hollaender, A. Quantitative irradiation experiments with Neurospora crassa. I. Experiments with X-rays. Amer. J . Bot. 32, 218-226. With Hollaender, A., Sansome, E. R., and Zimmer, E. Quantitative irradiation experiments with Neurospora crassa. 11. Ultraviolet irradiation. Amer. J. Bot. 32, 226-235.

356

DEMEREC

With Luria, S. E. The gene. Carnegie Inst. Washington Yearb. 44, 115-12 1. 1946 With Latarjet, R. Mutations in bacteria induced by radiations. Cold Spring Harbor Symp. Quant. Biol. 11,38-50. Induced mutations and possible mechanisms of the transmission. of heredity in Escherichia coli. Proc. Nut. Acad. Sci. U.S.A. 32, 36-46. With Latarjet, R., Luria, S. E., Oakberg, E. F., and Witkin, E. M. The gene. Carnegie Inst. Washington Yearb. 45,143-157. 1947 Resistance to drugs. Zdeas for Teachers (Nassau County Tuberculosis and Public Health Association), 13,2. Mutations in Drosophila induced by a carcinogen. Nature 159,604. Production of mutations in Drosophila by treatment with some carcinogens. Science 105, 634 (abstr.) . With Witkin, E. M., Newcombe, H. B., and Beale, G. H. The gene. Carnegie Inst. Washington Yearb. 46,127-135. Editor. Advan. Genet. 1, 458 pp. 1948 Origin of bacterial resistance to antibiotics. J . Bacteriol. 56,63-74. Induction of mutations in Drosophila by dibenzanthracene. Genetics 33, 337-348. Eighth International Congress of Genetics. Science 108,249-251. Mutations induced by carcinogens. Brit. J . Cancer 2, 114-117. Genetic potencies of carcinogens. Acta 6,247-251. With Wallace, B., and Witkin, E. M. The gene. Carnegie Inst. Washington Yearb. 47, 169-176. Editor. Advan. Genet. 2,373 pp. 1949 Chemical mutagens. Proc. Int. Congr. Genet. 8th (Hereditas, Suppl.) pp. 201-209. Patterns of bacterial resistance to penicillin, aureomycin, and streptomycin. J . Clin. Invest. 28, 891-893. With Wallace, B., Witkin, E. M., and Bertani, G. The gene. Carnegie Inst. Washington Yearb. 48, 156166.

BIBLIOGRAPHY

357

1950 Reaction of populations of unicellular organisms to extreme changes in environment. Amer. ATatur. 84, 5-16. Genetic mechanism controlling bacterial resistance to streptomycin. Trans. 1li.Y. Acad. Sci. Ser. II. 12, 186-188. With Fano, U., and Caspari, E. Genetics. I n “Medical Physics” (0. Glasser ed.), Vol. 11, pp. 365-385. Yearbook Publ., Chicago, Illinois. With Bryson, V. Patterns of resistance to antimicrobial agents. Ann. N.Y. Acad. Sci. 53,283-289. With Witkin, E. M., Catlin, B. W., Flint, J., Belser, W. L., Dissosway, C., Kennedy, F. L., Meyer, N. C., and Schwartz, A. The gene. Carnegie Inst. Washington, Yearb. 49, 144-157. Editor. Advun. Genet. 3,267 pp. Editor. “Biology of Drosophila,” 632 pp. Wiley, New York.

1951 With Bertani, G., and Flint, J. A survey of chemicals for mutagenic action on E. coli. Arner. Natur. 85,119-136. Studies of the streptomycin-resistance system of mutations in E . coli. Genetics 36, 585-597. With Hanson, J. Mutagenic action of manganous chloride. Cold Spring Harbor Symp. Quant. Biol. 16, 215-228. Biochemical aspects of genetics, biochemical society symposia No. 4 (R. T. Williams, ed.). Arch. Biochem. Biophys. 33, 491492 (review). With Witkin, E. M., Beckhorn, E. J., Visconti, N., Flint, J., Cahn, E., Coon, R. C., Dollinger, E. J., Powell, B., and Schwartz, M. Bacterial genetics. Carnegie Inst. Washington Yearb. 50, 181-195. Editor. Advan. Genet. 4, 343 pp.

1952 With Witkin, E. M., Labrum, E. L., Galinsky, I., Hanson, J. F., Monsees, H., and Fetherston, T. N. Bacterial genetics. Carnegie Inst. Washington Yearb. 51, 193-205. With Cahn, E. Studies of mutability in nutritionally deficient strains of Eschenchia coli. I. Genetic analysis of five auxotrophic strains. J . Bactenol. 65, 27-36. Reaction of genes of Escherichia coli to certain mutagens. Symp. SOC. E q .Biol. [No. 7, 1953],43-54. With Labrum, E. L., Galinsky, I., Hammerly, J., Berris, A. M. M.,

358

DEMEREC

Hanson, J., Blomstrand, I., and Demerec. Z. Bacterial genetics. Carnegie Inst. Washington Yearb. 52,210-221. Editor. Advan. Genet. 5, 331 pp. 1954 What makes genes mutate? Proc. Amer. Phil. SOC.98, 318-322. Genetic action of mutagens. Proc. Int. Congr. Genet. 9th (Caryologia, Suppl., 1954) , pp. 201-217. Editor. Advan. Genet. 6,485 pp. 1955 What is a gene?-twenty years later. Amer. Natur. 89,5-20. With Bryson, V. Bacterial resistance. Amer. J . Med. 18, 723-737. With Hammerly, J. Tests of chemicals for mutagenicity. Cancer Res., SUPPI.NO.3,1955, p. 69-75. With Blomstrand I., and Demerec, Z. E. Evidence of complex loci in Salmonella. Proc. Nat. Acad. Sci. U.S.A. 41,359-364. Dva Predavanja o Genetici Mikroorganizma. (Lecture given a t the Yugoslav Academy of Sciences, Zagreb.) Predavanja Odrzana u Jugoslavenskoj Akademiji Znanosti i Umjetnosti 15, 5 3 8 . Genetic basis of acquired drug resistance. Publ. Health Rept. 70,817-821. With Hartman, P. E., Moser, H., Kanazir, D., Demerec, Z. E., FitzGerald, P. L., Glover, S. W., Lahr, E. L., Westover, W. E., and Yura, T. Bacterial genetics-I. Carnegie Inst. Washington Yearb. 54, 219-234. Editor. Advan. Genet. 7,309 pp. 1956 Added comment. I n “Enzymes: Units of Biological Structure and Function” Henry Ford Hospital Symposium, pp. 131-134. Academic Press, New York. With Demerec, Z. E. Analysis of linkage relationships in Salmonella by transduction techniques. Brookhaven Symp. Biol. 8, 75-84. A comparative study of certain gene loci in Salmonella. Cold Spring Harbor Symp. Quant. Biol. 21, 113-120. Terminology and nomenclature. I n “Genetic Studies with Bacteria,” Carnegie Inst. Washington Publ. 612, pp. 1-4, Washington, D.C. With Hartman, Z. Tryptophan mutants in Salmonella typhimurium. I n “Genetic Studies with Bacteria,” Carnegie Inst. Washington Publ. 612, pp. 5-33, Washington, D.C.

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359

With Moser, H., Clowes, R. C., Lahr, E. L., Ozeki, H., and Vielmetter, W. Bacterial genetics. Carnegie Inst. Washington Yearb. 55, 301-3 15. Editor. Advan. Genet. 8, 402 pp. 1957 Structure and arrangement of gene loci. Proc. Int. Genet. Symp. 1956 (Cytologia Suppl.), 20-31. The Biological Laboratory, Cold Spring Harbor. A I B S Bull. 7, 20-21. Genetic aspects of drug resistance. I n “Ciba Found. Symp. Drug Resistance in Micro-organisms 1957” pp. 47-58. With Lahr, E. L., Ozeki, H., Goldman, I., Howarth, S., and Djordjevid, B. Bacterial genetics. Carnegie Inst. Washington Yearb. S6, 368-376. 1958 With Goldman I., and Lahr, E. L. Genetic recombination by transduction in Salmonella. Cold Spring Harbor Symp. Quant. Biol. 23, 59-67. Genetics and antibiotics. I n “The Pasteur Fermentation Centennial 1857-1957,” pp. 141-145. Pfizer, New York. With Lahr, E. L., Miyake, T., Goldman, I., Balbinder, E., Banic, S., Hashimoto, K., Glanville, E. V., and Gross, J. D. Bacterial genetics. Carnegie Inst. Washington Yearb. 57,390-406. Editor. Advan. Genet. 9,294 pp. 1959 Genetic structure of the Salmonella chromosome. Proc. Int. Congr. Genet. 10th I, 55-62. Blakeslee, A. F. Genetics 44, 1-4. Tests for allelism among auxotrophs of Salmonella typhimurium. Genetics 44, 269-278. With Miyake, T. Salmonella-Escherichia hybrids. Nature 183, 1586. With Hartman, P. E. Complex loci in microorganisms. Ann. Rev. Microbiol. 13, 377-406. With Lahr, E. L., Balbinder, E., Miyake, T., Mack, C., Mackay, D., and Ishidsu, J. Bacterial genetics. Carnegie Inst. Washington Yearb. 58, 433-439. With Sams, J. Induction of mutations in individual genes of E . coli by low X-radiation. Int. J . Radiat. Biol. (Suppl.) pp. 283-291. 1960 Frequency of deletions among spontaneous and induced mutations in Salmonella. Proc. Nut. Acad. Sci. U.S.A. 46, 1075-1079.

360

DEM ER EC

With Lahr, E. L., Balbinder, E., Miyake, T., Ishidsu, J., Mizobuchi, K., and Mahler, B. Bacterial genetics. Carnegie Inst. Washington Yearb. 59, 426-441. With Glanville, E. V. Threonine, isoleucine, and isoleucine-valine mutants of Salmonella typhimurium. Genetics 45, 1359-1374. With Miyake, T. Proline mutants of Salmonella typhimurium. Genetics 45, 755-762. 1961 The nature of the gene. Amer. J . Hum. Genet. 13,122-127. Theory of the gene. Brookhaven Lecture Series No. 10, 13 pp. 1962 The fine structure of the gene. In “Molecular Control of Cellular Activity” (J. A. Allen, ed.) pp. 167-177. McGraw-Hill, New York. “Se1fers”-attributed to unequal crossovers in Salmonella. Proc. Nut. Acad. Sci. U.S.A. 48, 1696-1704. Transductants produced by homologous phage in Salmonella. Microbial Genet. Bull. 19, 7. With Mizobuchi, K., and Gillespie, D. H. Cysteine mutants of Salmonella. Genetics 47, 1617-1627. 1963 Selfer mutants of Salmonella typhimurium. Genetics 48, 1519. With Gillespie, D. H., and Mizobuchi, K. Genetic structure of the cysC region of the Salmonella genome. Genetics 48, 997-1009. 1964 Clustering of functionally related genes in Salmonella typhimurium. Proc. Nut. Acad. Sci. U.S.A. 51,1057-1060. Organization of genetic material in Salmonella. “Erwin-Baur-Gedachtnisvorlesungen 111, 1963” Akademie-Verlag, Berlin. Problems of present-day genetics. Proc. XI Int. Congr. Genet. 2: LVII-LXII. Synthesis. Proc. Int. Congr. Genet. 11th 2,51-54. With Ohta, N. Genetic analyses of Salmonella typhimuriumXEscherichia coli hybrids. Proc. Nut. Acad. Sci. U.S.A. 52, 317-323. 1965 Homology and divergence in genetic material of Salmonella typhimurium and Escherichia coli. In “Evolving Genes and Proteins,” (V. Bryson and H. J. Vogel, eds.), pp. 505-510. Academic Press, New York.

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Gene differentiation. Nut. Cancer Inst. Monogr. 18, 15-20. With New, K. Genetic divergence in Salmonella typhimurium, S. Montevideo, and Escherichia coli. Biochem. Biophys. Res. Commun. 18, 5-6. With Sanderson, K. E. The linkage map of Salmonella typhimurium. Genetics 51, 897-913. With Yan, Y. Genetic analysis of pyrimidine mutants of Salmonella typhimurium. Genetics 52, 643-651. 1966 Properties of genes. I n “Heritage from Mendel,” Proc. Mendel Centennial, Genetics SOC.Amer., Ft. Collins, Colorado, 1964. (R. A. Brink, ed.) Univ. Wisconsin Press, Madison. With Adelberg, E. A., Clark, A. J., and Hartman, P. E. A proposal for a uniform nomenclature in bacterial genetics. Genetics 54, 61-76. 1967 With Itikawa, H. “Ditto deletions” in the cysC region of the Salmonella chromosome. Genetics 55, 63-68. With Nishioka, Y. and Eisenstark, A. Genetic analysis of aromatic mutants of Salmonella typhimurium. Genetics S6, 341-351. 1968 With St. Pierre, M. L. Hybrids of enteric bacteria. I. Bacteria hybrid for the his region. Genetics 59, 1-9. With Itikawa, I. Salmonella typhimurium proline mutants. J . Bacteriol. 95, 1189-1190. With Ino, I. Hybrids of enteric bacteria. 11. Bacteria hybrid for the cys-try-pyrF region. Genetics 59, 167-176. With Gillespie, D. Appearance of new cysteine requirements in populations of cysteine requiring Salmonella mutants. Genetics 59, 433-442. With Itikawa, H . Hybrids of enteric bacteria. 111. Bacteria hybrid for the pro-lac region. unpublished.

AUTHOR INDEX Numbers in italics refer to the pages on which the complete references are listed.

A Aastveit, K., 268, 280,890 Abrahamson, S., 247, 251, 271, 290, 296, 301 Achey, P. M., 254, 255, 290, 291 Adams, M. J., 109, 117 Adelberg, E. A., 54, 77, 131, 132, 139 Adhya, S., 46, 49 Adler, D. L., 104, 117 Ahnstrom, G., 274, 291 Alberts, B., 177, 191 Alexander, P., 253, 297 Alford, C. F., 275, 296 Allen, M. K., 331,346,348 Allison, A. C., 173,191 Amagasa, J., 173, 191 Ames, B. N., 2, 3, 9, 18, 19, 22, 23, 25, 26, 27, 30, 31, 32, 33, 119, 129, 130, 136, 139, 148,161 Ames, G. F., 131,136 Anderson, C. W., 21, 31, 176, 191 Anderson, E. S., 112,116 Anderson, T. F., 202, 232 Ando, A., 280, 291 Andreoli, A. J., 177,194 Anker, H. S., 132, 139 Anraku, Y., 176, 177, 181, 196 Antbn, D. N., 3, 31, 130, 136, 139 Appella, E., 27, 33 Arai, T., 56, 79 Arber, W., 56, 57, 76, 201, 232 Arfin, S. M., 132, 136 Armitage, P., 315, 323 Armstrong, F. B., 43, 49, 54, 61, 64, 67, 68, 69, 70, 71, 73, 76, 77, 78 Aronovitch, J., 211, 224, 234, 236 Ashwood-Smith, M. J., 175, 191 Atkins, C. G., 43, 49, 61, 68, 69, 76 Atkins, J. F., 3, 18, 25, 26, 27, 31

Atkinson, D. E., 145,163 Auerbach, C., 240, 250, 251, 291, 298 Ayling, P. D., 149, 154,161 Azoulay, E., 46, 61, 177, 191

B Bach, M. L., 347,347 Baetchke, K. P., 244, 291, 302 Baker, R., 214, 232 Balbinder, E., 44, 49, 123, 125, 137 Baldwin, R. L., 203, 206, 211, 213, 225, 232 Baldwin, W. F., 241, 242, 268, 272, 282, 291, 297 Balish, E., 146, 160,161 Ball, C., 173, 191 Ballentine, J., 186, 192 Ballesteros-Olmo, A., 131, IS8 Barnett, L., 173, 192 Baron, L. S., 41, 42, 49, 60, 54, 55, 56, 59, 61, 62, 63, 75, 76, '77, 78, 108, 112, 114, 116 Barr, D., 22, 34 Barrand, P., 203, 206, 211, 213, 225, 232 Barton, R. W., 131, 138 Batchelor, A. L., 272, 273, 274, 291, 301 Bateman, A. J., 262, 291 Bauchinger, M., 277, 300 Bauerle, R. H., 125, 138, 148, 161 Baumiller, R. C., 284, 291 Bautz, E. K. F., 94,116 Bautz, F. A., 94,116 Beam, C. A., 261,291 Beardmore, J. A., 284, 303 Beatty, A. V., 248, 291 Beatty, J. W., 248, 291 Becker, M. A., 145, 150, 161, 163

363

364

AUTHOR INDEX

Beckwith, J. R., 19, 33, 44, 61, 110, 118,

121,122,137, 139,258,300 Behrman, R. E., 189,191 Bellin, J. S., 173, 177, 179, 183, 191 Bender, M. A., 267,270,291 Ben-Gurion, R., 223,232 Benzer, S., 173, 192 Berberich, M. A., 9, 18, 27, 32, 33 Berenblum, I., 317, 323 Berg, A., 204, 206, 214, 236, 236 Berger, H., 23, 26, 31, 32 Berkowitz, D., 3, 31 Bertani, G., 43, 61, 58, 77, 85, 116, 200, 201, 203, 204, 205, 206, 209, 211, 215, 217, 218, 219, 221, 225, 226, 227, 229, 231, 232, 232, 233, 236, 237, 347, 347 Bertani, L. E., 201, 204, 209, 210, 211, 219, 220, 221, 222, 223, 227, 229, 231, 233, 236 Bessman, M. J., 24,33 Beumer, J., 201,233 Bhattacharyya, P., 177, 191 Bhorjee, J. Singh, 121, 175, 137,198 Bialy, H., 209, 211, 236 Bick, U. A. E., 242,291 Billen, D., 254, 291, 294 Blasi, F., 131, 138 Blatt, J. M., 134, 137 Blaylock, B. G., 284, 291 Blomstrand, I., 2, 31 Blum, H. F., 167,191 Blume, A. J., 44,49, 125, 137 Bock, R. M., 124,137 Bodmer, W. F., 287, 302, 331, 345, 346, 347 Bohme, H., 173, 175, 191 Bolton, E. T., 83, 91, 97, 116, 117 Bonner, D. M., 3, 33 Boon, T., 335,336,343,344,3& Bowman, J. T., 258, 891 Boyce, R. R., 173, 192 Boyer, H. W., 41,43, .@, 57, 77 Boyle, J. M., 246,291 Brammar, W. J., 23, 31 Breed, R. S., 40, 49 Breitman, T. R., 216, 236 Brendel, M., 173, 192 Brenner, D. J., 48, 49, 54, 77, 83, 85, 87, 88, 90, 91, 94, 97, 98, 105, 108, 110, 111, 116

Brenner, M., 9, 27, 51 Brenner, S., 44, 61, 173, 192 Brewen, J. G., 259, 270, 291, 298 Bridges, B. A., 246,292 Brill, W. J., 45,49,60 Brink, R. A., 264, 301 Britten, R. J., 85, 86, 87, 88, 89, 90,116 Brock, R. D., 259, 280, 292 Brodie, A. F., 186, 192, 196 Brooks, K., 223,233 Brooks, M. R., 285,302 Brown, D. M., 173,194 Brown, J. H., 242, 291 Brown, M. S., 173, 184, 187, 192 Browning, R. M., 268,303 Bruce, A. K., 173, 192 Bruni, C. B., 9,27,33 Bruton, C. J., 147,161 Buchanan, J. M., 146,163 Buchert, A. R., 183, 198 Buckton, K. E., 277, 292 Buiatti, M., 268, 292 Bunker, M. C., 242, 894 Burch, P. R. J., 315,316, 318,322,383 Burchard, R. P., 181, 192 Burgi, E., 204, 236 Burnet, M., 316, 323 Burns, R. O.,133, 137 Burrous, J. W., 41,60,64, 78 Butler, M. A., 21, 31 Butler, W., 181, 193 Buttin, G., 173, 192 C

CabreraJuarez, E., 173, 174, 182, 187, 192, 196

Cairns, J., 177, 193 Calberg-Bacq, D. M., 173, 178, 192 Caldecott, R. S., 312,323 Caldwell, I., 253, 297 Calef, E., 227, 233 Calendar, R., 200, 203, 209, 211, 216, 218, 219,225, 226, 233, 234, 236

Calhoun, D. H., 46, 47, 60 Callaghan, M. M., 248, 297 Callahan, R., 111, 125,137 Calvo, J. M., 132, 137 Campbell, A. M., 46,49,200, 234 Campbell, D. A., 263,298

365

AUTHOR INDEX

Cann, H.M., 282,292 Cantoni, G. L., 147,161 Cantor, C. R., 47,60 Carey, W.F.,41, 49, 54, 55, 56, 76, 77 Carleton, B. C.,48, 61, 69, 79 Carrier, W. L., 173, 197, 255, 301 Cater, M. S.,21, 31 Cattoir, H.,86,116 Cauthen, S. E., 146, 150, 157, 161 CavalliSforza, L. L.,282, 892 Cerda-Olmeda, E.,187, 192 Champney, W.S.,127,13Y Chandley, A. C., 262,292 Chandra, P., 173, 179,192 Chapman, A. B., 280, 282, 294, 302 Charamella, L. J., 56, 77 Chase, M., 207, 215,217,234 Chategee, N.K., 280, 292 Chater, K. F., 149, 152, 154, 155, 161, 162

Chien, J. R., 176, 177, 181, 196 Childs, J. D., 144, 145, 146, 149, 153, 162, 166 Choe, B.K., 209,210,226,227,233,234 Chopra, V. L., 173,192 Christensen, J. R.,201, 934 Chung, Y.S.,243,292, 293 Church, K.,263, 292 Citarella, R. V.,85, 87, 90, 91, 97, 98, 105, 108, 110, 116 Clark, A. J., 54, 77, 170, 198, 203, 218, 219, 223, 233, 234 Clark, J. B., 173,198 Clarke, C. H., 42, 49 Cleary, P.,46, 49 Cleaver, J. E.,189, 192, 255, 292 Cline, A. L.,124,137 Clowes, R.C., 149, 162 Coates, J. H.,128,137 Cohen, D., 206,209,211,230,232,234 Cohen, G. N., 122, 126, 137, 139, 144, 153, 159,162,163, 164 Coker, M., 133,137 Cole, L. J., 317,323 Collins, J., 151, 160,166 Colson, A. M., 41,49, 57, 77 Colson, C., 41,49, 57,77 Conen, P.E.,277,278,292 Conger, A. D., 253, 258, 270, 292

Conklin, J. W., 278,503 Conklin, M. E.,279,292 Connelly, C., 221,236 Cordaro, C., 123, 137 Cork, A., 260, 297 Court Brown, W.M., 241, 277, 292, 293 Cousin, D., 210,232,234 Cowie, D. B., 48, 49, 88, 91,94, 97, 116 Cox, B. S., 243, 244, 245, 247, 298 Cox, D. F., 280, 282,292,298 Cox, G. B., 176, 192 Cramp, W.A.,170,192 Crapo, L., 154, 164 Crasemann, J. M., 173, 197 Creighton, T. E.,44, 47, 49, 69, 77 Crenshaw, J. W.,285, 303 Crichton, C. E.,145, 151,I64 Crippa, M.,260, 298 Crowley, C., 311, 313, 314, 328, 524 Curtis, H.J., 270, 292, 308, 310, 311, 312, 313, 314, 316, 317, 318, 319, 321, 322, 323, 324 Curtis, W.,173, 182, 186, 187, 196 Curtiss, R.,56, 77 Czernik, C., 319, 324

D Dalal, F. R., 149,162 Dalebroux, M. A.,280,291 d'dmato, F.,267,293 Danielsohn, P.,178, 196 Datta, N.,112, 113,117 Davidson, N.,87, 118 Davies, D. R.,276,292 Davis, B. D.,126, 137, 146, 162 Davis, P.H., 38, 49 Davis, W.W., 18,'27, 32 Davison, P. F., 173, 179,192,193 Day, A., 267, 302 Dean, C.J., 253, 297 De Cleene, M.,97,117 de Crombrugghe, B.,121, 137 Deknudt, Gh., 242, 271, 296, 2YI Delavier-Klutchko, C., 145, 162 Delbriick, M.,214, 237 De Ley, J., 86,97,116, 117 Dellweg, H.,178, 179, 192, 198 de Lucia, P.,177,195 De Mars, R.I., 173,193

366

AUTHOR INDEX

Demerec, M., 2, 3, 31, 36, 41, 42, 43, 45, 46, 47, 49, 60, 61, 54, 59, 60, 63, 64, 67, 68, 69, 70, 71, 72, 73, 74, 77, 78, 119, 137, 144, 149, 162, 163, 164 Demerec, Z. E., 2, 31 de Nettancourt, D., 275,293 Denhardt, D. T.,84, 116, 219, 234 de Robichon-Szulmajster, H.,142, 162 de Serres, F. J., 257,292, 293,297 DhBrB, C.,180, 193 Dickie, M.M.,282,300 Dickerman, R.C.,274,293 Dillewijn, I., 246, 293 Dobzhansky, T.,39,60 Doctor, B.P.,83, 111, 116 Doll, R., 315, 323 Dolphin, W.D., 185,193 Domon, M., 251,297 Donch, J., 243,293 Donini, B.,267, 293 Donini, P.,173,193 Donnellan, J. E., Jr., 173, 193 Doty, P.,82, 83, 87, 88, 91, 96, 117, 118 Drabble, W.T.,115, 117 Drake, J. W., 24,31 Drapeau, G. R., 48, 6i, 69, 79 Dressler, D. H., 219, 834 Dreyfuss, J., 144, 145, 151, 162 Driedger, A. A.,254,693 Dubinin, N.P.,250,293 Dubnau, D., 46, 60 Duchesne, J., 173, 178,192 Dulbecco, R., 173, 174, 182, 193 Dumitrescu, A., 176, 196 Dworkin, M.,180, 181, 192, 193 Dwyer, S.B.,133,137 Dyer, K.F., 285, 293

182, 187,188,193, 246, 693 Eisenstark, R., 23, 31, 175, 176, 187, 193, 246, 293 Elkind, M. M., 181,193 Ellis, R.J., 144,145, 151, 157,162, 164 Emmer, M.,121, 137 Entner, G., 183,191 Epel, B.L.,181,193 Ephrussi-Taylor, H.,345,348 Epstein, H.T.,173,193 Eron, L.,110,118 Escobar, M.R.,104,116 Espinoza, M.,173,192 Esposito, M.S.,266,293 Evans, A. H.,326, 332, 345, 348 Evans, E. P.,273,274,301 Evans, H.J., 241, 242, 273, 274, 276, 292, 293, 301 Ewing, W. H.,40, 60, 95, 96, 99, 104, 116 Ezekiel, D. H.,127,137

F

Fabre, F., 275,298 Failla, G., 310, 324 Falk, R., 257, 284, 285, 293, 297 Falkow, S., 37, 41, 60, 54, 56, 77, 78, 85, 87, 90, 91, 97, 98, 105, 108, 110, 112, 114, 115,116, 117 Fanning, G. R.,85, 87, 90, 91, 97, 98, 105, 108, 110, 116 Feary, T.W., 46,47,60 Ference, M.,130,138 Ferretti, J. J., 22, 31 Ferron, W. L., 170, 173, 182, 186, 187, 188, 193 Fialkow, P.J., 278, 293 Fink, E., 245,296 E Fink, G. R., 2, 18, 19, 23, 25, 31, 130, 139 Eastburn, J., 215,217,234 Flavin, M., 145,150,162, 163 Echols, H., 213, 234 Fogel, S., 247, 264, 296, 299, 326, 344, Ecochard, R., 275, 293 345, 349 Edgar, R.S.,207,234 Ford, C. E., 273,301 Edsall, P.C.,187,196 Formal, S. B.,41,60 Edwards, P.R., 40,60, 95,96,99,116 Foster, M. A., 146, 150, 156, 157, 161, Ehrenberg, L.,274,291 162, 163, 166 Eisenberg, R.J., 180,193 Eisenstark, A., 23, 31, 54, 57, 59, 63, 67, Fournier, M. J., 83, 111,116 77, 168, 170, 173, 174, 175, 176, 177, Fowler, A. V.,121,137

AUTHOR INDEX

Fox, A. M., 308, 323 Fox, E.,173, 197 Fox, M.S.,331,346, 348 Fraenkel-Conrat, H.,173, 178,197 Franklin, N.C., 21, 31 FrBdBricq, P.,201,226,234 Freifelder, D.,173, 179, 192, 193 Freifelder, D.R.,173, 193 Freundlich, M.,132,133,137 Frey, K.T.,268, 296 Frey, L.,177, 191 Friedman, S.,146, 163 Fritsch, A., 203, 206, 211, 213,225,232 Fujisawa, T.,187, 193 Fuller, M.,313, 314, 324

367

Glickman, B. W.,261, 293 Glover, S. W.,2, 3, 31, 203, 206, 211, 224, 234, 236 Glucksmann, S., 180, 193 Goldberger, R. F., 3, 9, 18, 26, 27, 30, 32, 33, 130, 131, 138, 148, 161 Goldmark, P. J., 169, 170, 185, 194 Goldschmidt, E.P.,21,31 Goldthwait, D. A., 203, 206, 211, 213, 223, 225, 232, 234 Goldthwaite, C.,46, 60 Gollub, E.,126,138 Golub, E.I., 211, 224, 234 Good, J. V.,268,293 Goodgal, S. H., 188,196,331,346,348 Goodman, H. M.,83, 111, 117 Goodwin, T.W.,147,163 G Gopal-Ayengar, A. R.,250, 299 Gordon, M.P.,173,179,196 Gabay, E., 179,196 Gordon, W.G., 183,198 Galivan, J., 146, 150,162 Gorini, L.,160, 163 Ganesan, A. K.,173, 184, 186, 194 Gots, J. S.,2,3,31, 149,162 Ganesan, A. T.,176,177,193 Gough, M.,212, 234 Gardner, C.O.,280,292 GarrickSilversmith, L.,3, 18, 21, 31, 60, Grabner, M.,2, 3, 18, 26, 27, 28, 29, 32 Y7 Graf, U., 242, 293 Garry, B., 2,30, 119,136 Graham, D.E.,175,19Y,245,303 Gartner, T.K., 46,60 Grant, C.J., 259, 293 Gaul, H., 268,290 Grasso, R.J., 57,58,77 Gebhard, K.L.,308,310,323 Gray, T.C., 345,348 Geiduschek, E.P.,179,193 Grayston, M.J., 254,293 Geisselsoder, J., 203,206,234 Greceanu, V.,176, 196 Geissler, E.,223, 234 Geissler, K.,173,194 Greeb, J., 3, 18, 26, 27,31 Gemski, P.,Jr., 41, 42, 49, 60, 54, 59, Green, E. L., 240, 280, 293, 294 61, 63, 77 Greenberg, J., 173, 198, 243, 292, 293 German, J., 255, 301 Greene, A.,21,31 Ghiron, C. A., 178,157 Greene, R. C.,147, 155, 156, 157, 159, 162, 166 Gholson, R.K., 177,194 Giblak, R.E.,249, 303 Greer, S.,173,194 Gibson, F., 176, 192 Gregory, W.C., 268,294 Gibson, Q.H., 145, 150,163 Griffin, A. B.,242,294 Gilbert, W.,120,138, 154, 164 Griffiths, M., 180,197 Gillespie, D.,84, 109, 110, 117 Grogan, C. O.,267,300 Gillespie, D.H.,144, 145, 149,164 164 Gross, T. S., 147, 154, 155, 156, 162 Gillis, M.,97,117 Grossman, L. I., 173, 179, 191, 194, 197 Gingery, R.,213,234 Guerrini, F., 227, 233 Guest, J. R.,48, 61, 69, 79, 146, 163, 166 Gladstone, G. P.,131, 138 Glatzer, L., 54, 64, 67, 69, 70, 71, 73, Gupta, A. K.,268,294 77 Gupta, M.N.,272,294

368

AUTHOR INDEX

Guthrie, J. P., 177,194 Gwinn, D.D.,58,78

H Haapala, D. K., 114, 115,117 Haas, F.,173, 198 Haefner, K.,251, 260, 261, 294 Hagiwara, S.,201, 236 Haig, M. V.,265,294 Hales, H. B.,173,I94 Hall, B. D.,85,118 Hall, C.A., 45,61,71,79 Hall, Z.W.,176, 177, 181,196 Hamada, K.,126,138 Hamm, L.,246, 299 Hamplova, D.,177, 196 Hanawalt, P.,187, 192 Hansen, J. C.,282, 294 Harm, W.,173, 183, 186, 188, 194, 196 Harris, M. A., 260, 296 Harrison, A. P.,173,178,194 Hartley, B.S.,147,161 Hartman, P. E., 2, 3, 9, 18, 19, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 54, 60, 77, 129, 130, 136, 138, 139, 148, 161 Hartman, Z., 2, 3, 18, 26, 27, 28, 29, 31, 32

Hastings, P. J., 337, 341, 342, 348 Hathaway, A.,219,234 Hattman, S.,201,234 Hattori, T.,56,79 Haverstein, G.B.,282,294 Hayashi, S.,262, 294 Hayflick, L.,320, $24 Heath, S.,22, 33 Heddle, J. A., 259, 260, 278, 294 Held, W.A., 125, 126,138 Helinski, D. R., 44, 47, 49, 69, 77 Hellewell, A. B., 244,302 Heneen, W., 278, 298 Herriott, R. M., 173, 187, 188, 192, 196, 346, 348 Hershey, A. D., 204,236 Hertmfan, I. M., 170,194,223,236 Herzenberg, L. A., 2,30 Hewitt, R.,254,291, 294 Heywood, V. H., 38,49 Higa, A., 204,236

Hill, R. F., 173, 174,194 Hiraga, S.,123,124,126,138 Hirono, Y.,279, 294 Hogness, D.S., 204,236 Hollaender, A., 167, 173, 174, 194 Holliday, R.,247,294, 326,342,344,348 Holloway, B.W.,47, 61 Holloway, C. T., 147, 155, 156, 157, 159, 162, 166 Holmes, M. C.,309, 324 Holt, G.G., 277, 298 Hopper, J. E., 269,294 Horii, Z.,170,194 Horodniceanu, T.,176, 196 Hotchkiss, R.D., 326,332,343,345,$48 Hottinguerde-Margerie, H., 275, $98 Houston, E.W.,278,297 Howard, A.,265,294 Howard-Flanders, P.,170, 173, 192, 194, 196, 246, 294 Howarth, S., 144,149,163 Howells, D.J., 147,163 Hoyer, B.H., 88,91,116 Hsia, D. Y.,189, 191 Huennekens, F. M., 146, 150, 162 Hug, O.,260, 297 Humphrey, R.M., 254,296 Humphries, S. K.,157,162? Hurst, D. D.,264, 296, 326, 348 Hushon, J. M.,3,31 Hutchinson, F.,173,194 Hutton, J. J., 203,836

I IAEA, 240,241,268,276,296 Iohikawa, S.,267, 282, 2996 Ihler, G., 110,118 Ikeda, H.,212, 236 Ikenaga, M., 184,.194 Ikushima, T.,267, 296 Imamoto, F.,123, 124, 125, 138, 139 Ingraham, J. L.,129,139 Inman, R. B., 203, 204, 205, 206, 217, 218,236, 236 Ino, I., 36, 42, 60, 54, 59, 60, 63, 67, 71, 72, 78 Inouye, M., 177, 194 Inselberg, J., 21,32 Ippen, K.,110, 118

369

AUTHOR INDEX

Ishihara, T., 277, 278, 296 Ishizaka, S., 251, 297 Itikawa, H., 149, 163 Ito, D., 124, 138 Ito, J., 123, 124, lSS, 139, 1.40 Ito, K., 123, 126, 138 Ito, T., 173, 191, 249, 251, 297, 298

J Jacob, F., 2, 18, 25, SO, 32, 119, 120, 122, 124, 129, 136, 136, lSY, 138, 202, 203, 206, 211, 213, 223, 225, 232, 232, 234, 236 Jacobson, R., 178, 196 Jacoby, G. A,, 160,163 Jagger, J., 173, 182, 183, 184, 185, 186, 187, 194, 196, 196, 197 James, A. P., 250, 260, 261, 296 Janecek, J., 155,166 Janin, J., 144, 159, 163 Jannsen, S., 245, 296 Jenkins, P. G., 115,117 Jensen, R. A., 127, 137 Jesaitis, M. A,, 203, 236 Johnson, D. B., 147, 163 Johnson, E. M., 41, 42, 49, 54, 56, 59, 61, 62, 63, 77, 78, 112, 114, 116 Johnson, H. D., 306,324 Johnson, J. L., 88,9l, llY Johnson, K. E., 85, 87, 90, 91, 97, 98, 105, 108, 110, 116 Jones, D., 37, 40, 42, 60 Jones, L. P., 280,296 Jones-Mortimer, M. C., 145, 151, 157, 163, 164 Jorgensen, G., 254, 292 Joshi, S. N., 268, 296 Joshua, D. C., 250,299 Jukes, T. H., 47,60 Jyssum, K., 175, 196

K Kahan, L., 173, 197 Kaiser, A. D., 204, 236 Kale, P. G., 262, 296 Kamada, N., 278, 296 Kamin, H., 145, 150, 163 Kano, Y., 126, 138

Kaplan, H. S., 188,196 Kaplan, M. M., 145, 150,163 Kaplan, W. D., 276, 299 Kappler, J. W., 173,197 Karlstrom, O., 160, 163 Kashket, E. R., 186, 196 Kato, T., 245, 295 Katzen, H. M., 146, 163 Kaul, B. L., 249, 296 Kellenberger, E., 41, 60 Kellerev, A., 260, 297 Kelly, B., 43, 61, 215, 224, 225, 226, 232, 636, 237 Kelly, B. L., 230, 236 Kelly, E. M., 250, 300 Kerr, C. B., 286, 302 Kerszman, G., 203, 206, 211, 224, 234, 236 Kibler, H. H., 306, 324 Kida, S., 123, 124, 139 Kimball, P. C., 213, 236 Kimball, R. F., 242, 243, 254, i?96 King, B. M., 104, 117 King, J. L., 280,287, 296 Kintner, L. D., 306,324 Kirchner, C. E. J., 19, 26,32 Kiriazis, W. C., 271, 296 Klein, R. M., 187, 196 Klopotowski, T., 2, 18, 19, 23, 25, 31, 32 Kohne, D. E., 83, 85, 86, 87, 88, 89, 90, 110, 111, 116, 117 Kohno, T., 9, 27, 34 Kojima, K. I., 280, 292 Kondo, S., 245, 246,296, 296 Kornberg, A., 332, 334, 348 Kovach, J. S., 130, 131,138 Kowyama, Y., 244,303 Krauss, R. W., 181, 185,193 Kredich, N. M., 145, 150, 151, 161, 163 Krinsky, N. I., 173, 196 Kubitschek, H. E., 173, 175, 196, 198 Kumar, S., 247, 296 Kumatori, T., 277, 278, 296

L LaBrie, D. A., 54, 64, 67, 69, 70, 71, 73, 77, 78 La Brot, K . J., 175, 197, 245, 303

370

AUTHOR INDEX

Lacks, S., 331,346,848 Ladda, R.L., 62,78 Laird, C. D.,48, 60, 84, 94, 98, 103, 117, 173,197,331,345,346,347 Lakchaura, B., 186,196 Lamb, M. T.,274,296 Lamfrom, H., 174, 196 Lamola, A. A., 179,196 Lanier, W. B.,247, 296 Laphisophon, T.,254, 291 Laskowski, W., 245, 296 Latarjet, R.,173,198 Laurent, T.,201, 221, 225, 283 Law, J., 246, 292 Lawley, P.D.,24,32 Lawrence, C. W., 262,296 Lawrence, D. A., 152, 153, 154, 155, 156, 157, 162, 163, 164 Lawton, W. D.,58,78 Lazinyi, A., 249,296 Lazzarini, R.A.,145,163 Leavitt, R.I., 132,138 Leavitt, R.W., 62,78 LeBeau, P.,27,323 Le Bras, G.,144, 153, 159, 164 Lederberg, J., 2, 3, 34, 176, 177, 193 Lederberg, S.,211,236,237 Lee, B. T. O., 46, 60, 244, 899 Lee, L. W.,152,164 Lee, W. R.,275,296 Lefevre, G., 282, 296 Leff, J., 173,196 Lehman, I. R.,176, 177, 181, 185, 196 Leigh, B.,242,248, 296 Leith, J., 312, 324 Lejeune, J., 278, 296 Lemoine, F.,150, 164 Lennox, J. E.,247, 296 LBonard, A., 242, 271, 296, 297 Leroy, P.P.,274,297 Lett, J. T.,253, 697 Levan, A., 278, 298 Lever, J., 131,136 Levin, A. P.,129,138 Levin, W.C., 278,297 Levine, J., 173, 197 Levine, K.,26,32 Levine, M., 212, 223, 234, 236 Levinthal, M., 3, 18,24, 32 Levy, J. A.,209,231,233

Lew, K. K.,27,32,61,78 Lieb, M., 41, 60 Lifschytz, E.,257, 297 Lindahl, G.,207, 209, 210, 211, 212, 213, 214, 215, 216, 219, 220, 221, 225, 226, 233, 236 Lindop, P. J., 317,324 Lindqvist, B. H., 203, 207, 211, 218, 219, 220, 221,224,B4,236,236 Linn, S., 56,76, 201,232 Livingston, R.,178,197 Lockmann, E.R.,245,296 Lohman, P.H.M.; 253,297 Loper, J. C., 2, 3, 18, 25, 26, 27, 28, 29, 31, S2 Lorentz, J. R., 169,198 Loutit, J. S.,47,61 Loviny, T.,153,164 Lubin, A. H., 104,116 Luning, K.G., 285,297 Luria, S. E.,41, 60, 54, 58, Y8, 109, 117, 223, 236 Lyman, J. T., 279, 294 Lynn, S.,169,170,185,194 Lyon, M.F.,270,297 M Ma, T. H., 266,297 Macbean, I. T.,244,299 McCann, K.M., 176,192 McCarthy, B. J., 48, 60, 83, 84, 87, 88, 91, 94, 97, 98, 103, 110, 116, 117 McConaughy, B. L., 48, 60, 84, 87, 94, 98, 103, 117 McGregor, J. F., 272, 298 Mchattie, L., 110, 118 McLaren, A. D.,182,196 McLean, A. S.,241,293 McSheehy, T.W., 274,296 McWhorter, A. C.,104,116 Mager, J., 145,151,164 Magasanik, B.,45,49,60 Magnus, I. A.,173,191 Margolin, P.,148, 161 Maini, M. M., 317,324 Miikela, P.H.,43,46,60 Malamy, M.,25,32 Malcolm, F.E., 206,236 Malina, M. M., 173, 175,198

371

AUTHOR INDEX

Malling, H. V., 257, 297 Mandel, M., 37, 60, 53, 78, 203, 204, 206, 214, 234, 236, 236

Mangan, J., 246, 303 Manikas, G., 268,297 Marchelli, C., 227, 233 Margolies, M. N., 3, 18, 26, 27, 32 Margolin, P., 125, 132, 137, 138 Mamur, J., 37, 46, 60, 54, 77, 82, 83, 87, 88, 91, 96, 117, 118

Martin, M. A., 88, 91, 116 Martin, R. G., 3, 9, 18, 19, 22, 23, 25, 26, 27, 28,29, 30, 31, 32, 33, 148, 161 Marver, D., 9, 32 Masters, B. S. S., 145, 150, 163 Mathews, M. M., 173, 177, 181, 196 Matney, T. S., 21, 31, 177, 194 Matsubara, H., 47,bO Matsudaira, Y., 251, 897 Matsumoto, S., 245, 298 Matsushiro, A., 123, 124, 126, 138, 139 Matsuyama, T., 124, 138 Mays, J. A., 56, 77 Mee, B. J., 46, 60 Mehra, P. N., 265, 297 Meiss, H. K., 45, 60 Meistrich, M. L., 179, 196 Meningmann, H. D., 180,196 Mericle, L. W., 263, 297 Mericle, R. P., 263, 297 Meselson, M., 219, 237 Merz, T., 242, 243, 249,299 Meynell, E., 112, 113,117 Meynell, G. G., 112, 113, 117 Michaelis, G., 25, 32 Middleton, R. B., 54, 55, 56, 58, 67, 69, 71, 78

Milius, P., 173, 197 Milkman, R. D., 287, 297 Milner, L., 157, 164 Miltenberger, H. G., 260, 297 Mingioli, E. S., 146, 162 Mintzer, L., 45, 61 Miovic, M., 203, 212, 236 Misty, K. B., 250, 299 Mitsuhashi, S., 112, 117 Mittler, S., 248, 297 Miyake, T., 41, 45, 60, 61, 54, 55, 56, 57, 78

Miyazawa, Y., 85, 117

Mizobuchi, K., 144, 145, 149, 162, i64 Mojica-a, T., 54, 55, 56, 57, 58, 60, 64, 67, 69, 71, 78

Molino, M., 268, 292 Monod, J., 2, 32, 119, 120, 124, 136,138 Montesi, V., 260, 298 Monty, K. J., 144, 151, 160, 162, 166 Moore, R. L.,110,117 Morris, N. P., 277, 298 Morris, T., 270, 297 Mortimer, R. K., 247,299, 344,345,348 Morton, N. E., 282, 294 Moseley, B. E. B., 253, 298 Mosteller, R. D., 126, 139 Moustaechi, E., 275, 298 Moutschen-Dahmen, J., 274, 291 Moutschen-Dahmen, M., 274, 291 Moyed, H. S., 123,128, 139 Mudd, S. H., 147, 164 Muller-Hill, B., 120, 138, 154, 164 Mullaney, P. D., 282, 298 Munier, R., 126, 139 Munson, R. J., 246,292 Murakami, A., 244,249,298 Murata, M., 285, 302 Murray, E. G. D., 40,49 Murray, M. L., 18,32 Murtie, C. R. K., 186,196

N Nakai, S., 245, 298 Nakamura, A., 115,118 Nakane, K., 3, 18, 24,32 Nakaya, R., 115, 118 Nasim, M. A., 251, 252, 261,298 Nass, G., 134, 139 Natarajan, A. T., 247, 274, 291, ,296 Nayar, G . G., 282, 298 Neary, G . J., 259,300 Neidhardt, F. C., 133, 134, 139 Nester, E. W., 128,137 Neu, H. C., 48, 61 New, K., 36, 42, 49, 54, 67, 77 Newcombe, H. B., 272, 288,298 Newmark, J. F., 173,196 Newton, A., 176, 198 Nichols, W. W., 278, 298 Nikaido, H., 3, 18, 24, 32 Nikaido, K., 3, 18,24, 32

372

AUTHOR INDEX

Nishiyama, I., 269,299 Norman, A., 277, 300 North, D. T., 277,298 Notani, N., 331,346, 348 Novick, A., 320,324 Novick, R. P., 112,118 Nowell, P. C., 277, 298, 317, 323 Nru, V., 265, 299 Nygaard, A. P., 85,118 0

O’Donovan, G. A,, 129,139 Oeschger, N. S., 2, 19, 21, 22, 23, 25, 29, 33 Oftedal, P., 270, 276, 299 O’Hara, J. A., 177,181, 196 Ohara, L., 276,302 Ohta, N., 36, 41, 42, 60, 54, 67, 68, 69, 71, 77 Oishi, M., 169, 185,196 Oka, H., 269, 299 Okada, M., 41, 61, 57, 78 Okada, Y., 173, 179,197 Okun, L., 173, 197 Olivera, B. M., 176, 177, 181, 185, 196 O’Neil, D. M., 75, 78, 79 Onimaru, K., 251, 252, 302 Opree, W., 179, 192 Ordal, E. J., 88,91,117 Orlowa, G. G., 224, 234 Oster, G., 173, 191 Oxender, D. L., 147,164 Ozaki, H., 124, 138 Ozeki, H., 2, 3,31, 149,164

P Paigen, K., 57, 58, 77 Paiva, A. C. M., 183, 196 Paiva, T. B., 183, 196 Palta, H. K., 265, 2yT Papworth, D. G., 273, 274, 301 Pardee, A. B., 122, 139, 150, 164, 180, 193 Parry, J. M., 243, 244, 245, 247, 292 Parsons, P. A., 244,299 Pasten, I., 121, 137 Pasternak, C. A., 145, 151, 157, 162, 163, 164, 166

Paszewski, A., 339, 341, 343, 344, 348 Patil, S. H., 269, 299 Patrick, M. H., 184,194, 196 Patt6, J. C., 144, 153, 159, 164 Pauling, C., 246, 299 Pearce, L. E., 47,61 Peloquin, S. J., 269, 294 Perdue, S. W., 242, 243,264,296 Perlman, R., 121, 137 Person, S., 173, 186, 187,196 Pfahler, P. L., 282, 299 Phang, J. M., 130, 131,138 Phillips, R. J. S., 241, 272, 274, 291, 301 Phillips, S. L., 173, 186, 187, 196 Piatkowska, B., 242, 293 Pichinaty, F., 177, 191 Pietersma, K., 42, 46, 61 Piperno, J., 147, 164 Pittard, J., 127,140, 176,192 Pizer, L. I., 203, 211, 212,236, 237 Ponce-De Leon, M., 173,196 Pond, V., 244, 291,302,303 Potter, V. R., 277, 298 Prembree, T., 242, 243, 249, 299 Preston, R. J., 259, SO0 Pritchard, R. H., 342, 346, 348 Ptashne, M., 120, 139 Puglisi,, P. P., 247, 299 Puig, J., 46,61, 177,191 Purdom, C. E., 265, 274, 296, 299 Puro, J., 262, 299 Pylkas, L., 203, 211,212,236, 237

R Raab, O., 178, 196 Raabe, V. E., 173,194 Rake, A., Ramakrishnan, T., 131, 132, 139 Rana, R. S., 274, 299 Randolph, M. L.,253, 278, 292, 303 Rao, N. S., 250, 299 Raper, J. A., 173,191 Rapetti, L., 180, 193 Ratzkin, B., 132, 136 Ravel, J. M., 152, 164 Rechler, M. M., 9, 27,29, 33 Reed, T. E., 287, 302 Regan, J. D., 255,301 Rennart, 0. M., 132, 139

AUTHOR INDEX

Reshetnikova, V. N., 211, 224, 234 Resnick, M. A., 175, 189, 196, 197, 244, 245,246, 251,299,303 Reynaerts, A., 86, 116 Rich, A., 83, 111, 117 Richaud, F., 144, 159, 164 Riddle, D. L., 22, 33 Riley, M., 46, 50 Riley, P. A., 173, 197 Rilling, H. C., 181, 198 Rillo, F. O., 268, 303 Rinehart, R., 247, 299 Ritchie, D. A., 173, 196, 206, 236 Ritzmann, S. E., 278, 297 Roberts, P. A., 262,299 Rodarte, U.,247, 299 Rogan, E. G., 24,33 Ronnbach, C., 285, 301 Ronayne, M. E., 177, 191 Rorsch, A., 175, 176, 187, 193, 246, 293 RosCn, C. G., 274, 291 Rotblat, J., 317, 324 Roth, J. R., 3, 9, 18, 22, 23, 27, 30, 31, 32, 33, 34, 61, 78, 130, 131, 136, 139, 148, 161 Roth, M. M., 180,196 Rothman-Denes, L., 19,33 Roulland-Dussoix, D., 57,77 Rowbury, R. J., 144, 145, 147, 152, 153, 154, 155, 156, 157, 162, 163, 164 Rownd, R., 41, 60, 88, 108, 115, 116, 117, 118 Ruff, D., 182, 187, 193 Rupert, C. S., 173, 183, 186, 188, 196 Rupp, W. D., 173, 196, 246, 294 Russell, W. L., 250, 300

S Saedler, H., 25, 32 St. Pierre, M. L., 18, 33, 36, 42, 61, 54, 60, 63, 67, i'S, 129, 139 Sakazaki, R., 104,118 Salceda, V. M., 284, 300 Samata, Y., 272, 294 Sand, S. A., 271,272, 300 Sanderson, K. E., 1, 3, 18, 24, 33, 43, 44, 45, 61, 53, 54, 65, 67, 70, 71, 72, 73, 75, 78, 79, 148, 160, 165, 176, 196 Sankaranarayanan, K., 247,248, 285,300

373

Sarabhai, A., 174, 196 Sarvella, P., 267, 300 Sasaki, I., 43, 51, 215, 225, 226, 237 Sasaki, M. S., 277, 300 Sasarman, A., 176, 196 Sastry, X. S., 173, 179,196 Sato, K., 123, 124, 139 Saunders, A. S., 260, 261, 295, 298 Savage, J. R. K., 259, 270, 274, 300 Scarascia Mugnozza, G. T., 267, 293 Schafler, C., 45,51 Schafler, S., 45, 51 Scheid, W., 271, 303 Schildkraut, C. L., 83, 88, 91, 96, 117, 118 Schlager, G., 282, 300 Schlesinger, S., 152, 166 Schmickel, R., 278, 300 Schmid, E., 277,300 Schneider, H., 41, 50 Schnos, M., 203, 204, 205, 206, 218, 235, 236

Scholefield, J., 262, 300 Schroder, J. H., 242, 262, 280, 282, 300 Schwartz, D. O., 19, 33, 258, 300 Scott, D., 242, 243, 273, 274, SO1 Scott, J., 242, 303 Scott, J. R., 212, 236 Seaman, E., 173, 197 Searle, A. G., 240, 241, 272, 273, 291, 301 Sedita, B. A., 254, 296 Seeley, B. A., 247, 301 Sega, G. A., 275, 296 Serman, D., 2, 3, 26, 29, 31 Setlow, J. K., 173, 183, 186, 196, Stelow, R. B., 173, 183, 186, 193, 255, 301 Shapiro, J., 110, 118 Shapiro, S. K., 146, 160, 161 Sharpe, H., 273, 274, 301 Shaver, D. L., 244, 291, 302 Sheppard, D. E., 18, 33, 129, 139, 165 Sheridan, W., 273, 282, 285, 301 Shill, K. L., 264, 301 Shimura, Y., 126, 138 Shive, W., 128, 139, 152, 164 Shock, N. W., 310, 320, 324

297,

274,

197 197,

153,

374

AUTHOR INDEX

Sliugar, D., 182,196 Shuster, 216, 236 Siegel, L.M.,145,150,163 Signer, E. R.,44, 61, 213, 236 Silbert, D.F.,130, 139 Silver, R.P.,114, 115, 117 Silver, S.,177,191 Simon, L.D.,203,206,236 Simon, M. I., 173, 178, 179, 197 Singer, A. C., 280, 301 Singer, B., 173, 178,lR Singh, Bhorjee, J., 121, 175,137,198 Sironi, G.,203, 209, 211, 215, 218, 219, 234, 236, 236 Sistrom, W.R.,177,180, 181,196,197 Six, E. W., 202, 203, 206, 209, 211, 220, 221, 222, 224, 225, 226, 227, 228, 229, 230, 231,232, 233, 236, 236 Skalka, A.,204, 236 Skerman, 97, 116 Skinner, C. G., 128, 139 Skinner, J. S.,308,323 Slater, T.F.,173,197 Sloan, N. H., 127, 1.40 Smarda, J., 173,197 Smith, D. A., 144, 145, 146, 149, 152, 153, 154, 155, 156, 157, 162, 163, 164,

Soyfer, V. N., 250, 293 Spalding, J. F.,285, 302 Sparrow, A. H., 244, 267, 282, 291, 296, 29S, 302, 303 Spencer, H. T., 151, 160,166 Spiegelman, S.,84, 109, 110, 117 Spikes, J. D.,173, 177, 178, 197 Spilman, W. M.,41, 49, 54, 55, 56, 76, 77 Spizek, J., 155,166 Sprinson, 0. B., 126,138 Stadtman, E.R.,144,166 Stafford, R. S., 173, 182, 183, 184, 186, 187, 196 Stahl, F. W., 173,197,326,343, 344,348 Stahl, R. C.,2, 3, 18, 26, 27, 28, 29, 32

Stallions, D. R., 56, 77 Stanier, R. Y.,180,197 Stanisich, V.,47,61 Starlinger, P.,25, 32 Stebbins, G.L.,267, 308 Stedeford, J. B., 170,194 Steers, E. J., Jr., 146,166 Steffensen, D., 275,302 Stein, W.,173, 198 Steinhart, W.L.,346, 348 166 Stern, M. G.,246, 302 Smith, D. W.E., 26,28,33 Stevenson, A. C., 286,302 Smith, H., 203, 212, 236 Stevenson, K. G.,310, 311, 324 Smith, H. H.,271, 272, 279, 292, 294, Steward, D.L.,254,296 300 Stich, H. F.,317,324 Smith, H. S., 211,237 Stone, W.S.,173,198 Smith, H. W.,112,118 Stouthamer, A. H.,42,46, 61 Smith, I.,46, 60 Straight, R., 173, 177, 178, 197 Smith, K. C., 173, 179, 184, 186, 194, Strauss, B. S.,253,302 197 Striebeck, U.,260, 261,294 Smith, N. F.,40,49 Stubbe, H., 241, 902 Smith, 0.H., 125, 126,138 Su Ching-Hsiang, 147, 155, 156, 157, 159, Smith, P.G.,277,292 162, 166 Smith, R.C.,152, 166 Sunshine, M. G., 225, 226,230,236,236 Smith, S. C.,127,140 Surdeanu, M.,176, 196 Sneath, P. H.A., 37, 40, 42, 60 Sutton, H., 181,193 Snow, J. M.,183, 184, 196 Suzuki, D.T.,262,294,300 Snow, R.,245,247, 301 Suzuki, K.,170,194 Sobels, F.H., 240,248,301,302 Sved, J. A.,287, 302 Solomon, H.M.,183,198 Swaminathan, M.S.,268,274,294,299 Somerville, R. L.,44, 47, 49, 69, 77, 123, Swenson, P.A., 183, 197 Syakudo, K.,244, 303 140 Sonea, S., 176, 196 Symonds, N.,246, 291

375

AUTHOR INDEX

Sypherd, P. S., 75, 78, 79 Szegli, G., 176, 196 Szentirmai, A., 133, 1.40 Szentirmai, M., 133, 140 Szilard, L., 310, 320, 324

T Talal, N., 18, 25, 28, 29, 32 Tai, C. C., 173, 175, 180, 198 Takebe, H., 173, 184, 196, 197 Tanemura, S., 22, 34 Tates, A. D., 272, 302 Taylor, A. L., 43, 44, 61, 53, 54, 65, 67, 71, 79, 123, 140, 148, 160, 166 Taylor, B. A., 280, 281,302 Taylor, B. S., 282, 294 Taylor, J. H., 326, 348 Taylor, M. M., 75, 79 Taylor, R. T., 142, 146, 150,166 Tazima, Y., 251, 252, 302 Tajerina, G., 146, 163 Teresa, G. W., 173, 197 Teresa, N. L., 173, 137 Terasima, T., 276, 302 Tesi, R., 268, 292 Tessman, I., 212, 214, 232, 236 Theriot, P. L., 170,194 Thomas, C. A., Jr., 85,117,212,236 Thomas, R., 219, 236 Thompson, K. H., 282, 296 Tietjen, G. L., 285, 302 Tilley, J., 312, 313, 314, 319, 321, 323, 324 Ting, R. C., 109, 117 Tobari, I., 285, 302 Todd, P. W., 244,277, 302 Tomizawa, J., 212, 236 Tomkins, G. M., 145, 150, 151, 161,163 Torheim, B., 201, 221, 225, 233 Touchberry, R. W., 280, 301 Toyama, S., 201, 236 Trainin, N., 317, 323 Traut, H., 241, 271, 273, 282, 302, 303 Tritz, G. J., 177, 194 Trosko, J. E., 189, 192 Trudinger, P. A., 142, 166 Truffa-Bachi, D., 144, 159, 163 Tsugita, A., 173, 179, 197 Tuveson, R. W., 246, 247,296, 303

U Uchino, H., 278, 296 Udhara, K., 173, 179,197 Uetake, H., 201, 236 Umbarger, H. E., 132, 133, 134, 136, 137, ISS, 140, 142, 165 Unterbrink, A. G., 244, 303 Upton, A. C., 278, 303 Uretz, R. B., 173, 197

V Valencia, J. I., 251, 290 van Delden, W., 284,303 van Dillewijn, J., 175, 176, 187, 193 Van Pel, A,, 41, 43, 57, 77 van Rapenbusch, R., 150, 164 Van Sickle, R., 23, 31 Van Vunakis, H., 173, 178, 179, 197 Vasington, F. D., 27, 33 Vencovsky, R., 280, 291 Venema, G., 346, 348 Venema-Schroeder, T., 346, 348 Venetianer, P., 9, 33 Venkov, P., 25, 32 Visconti, N., 214, 237 Voelker, R. A., 267, 303 Vogel, H. J., 3, 33 Voll, M. J., 18, 27,33 von Borstel, R. C., 175, 197, 245, 303 von Lochmann, E. R., 173, 198 Vorhaben, J. E., 145, 150, 163 W Wacker, A,, 173, 179, 191, 192, 198 Wagoner, D. E., 266,303 Wahl, R., 173, 198 Walker, D. H., Jr., 203, 206, 236 Walker, J. C., 175, 198 Walker, S., 267, 302 Walker, T . A,, 46, 47, 60 Wallace, A. T., 268, 303 Wallace, B. J., 127, 140 Waller, J. P., 150, 164 Walsh, M. M., 248,297 Wang, C . C., 176, 198 Wang, J. C., 206, 237 Wang, J. H., 184, 186,198

376

AUTHOR INDEX

Wirdell, I., 282, 301 Waskell, L.A,, 173, 179,196 Watanabe, T.,41, 61, 56, 57, 78, 79, 112, 118, 269, 299 Watkins, D. K., 170,192 Watson, W. A. F., 247, 303 Webb, R. B., 169, 173, 175, 184, 187, 102, 198 Webb, S. J., 173, 180, 187, 198 Webber, B. B.,257,293 Weigle, J. J., 41, 60, 58, 77, 173, 174, 182, 193, 201, 233 Weijer, J., 249, 303 Weil, J., 213, 236 Weil, L., 183, 198 Weinblum, D.,179,198 Weinstock, S.,264, 296 Weisbrot, D.R.,283, 284, 303 Weissbach, A., 216, 236 Weissbach, H.,142, 146, 150, 157, 164, 166 Wendt, L., 177, 191 Werner, M. M.,250, 260, 261, 696 Werthessen, N.T.,310, 324 West, B. J., 273,274,301 Westerman, M.,263, 303 Wetmur, J. G.,87,118 Wheldrake, J. F.,145, 151, 157,163,166 Whitfield, C.D.,146, 157,164,166 Whitfield, H. J., Jr., 3, 18, 22, 23, 25, 26,28,31, 33 Whitfield, V. G.,255, 290 Whitehouse H.L. K., 326, 335, 337, 341, 342, 343, 344,348 Whitney, E.,177, 191 Wijesundera, S.,145, 157, 166 Wilkins, B. M.,246, 294 Willetts, N.S.,170,198 Williams, B., 46, 47,60 Wiman, M., 215,225, 226,837 Wimber, D.E.,263, 296 Winkler, U., 173, 192 Winman, M.,43,61 Winshell, E.B., 48, 61 Wise, J., 173, 182, 186, 187, 196

Wisnieski, B. J., 285,303 Witkin, E.M.,173, 174, 175,198, 246,303 Wohlhieter, J. A., 41, 42, 49, 60, 54, 59, 61,62,63,77, 78 Wolf, B.,219, 237 Wolff, S.,240, 241, 290,303 Wollman, E.L.,202, 203,232, 236 Wood, W. B., 207, 234 Woods, D. D.,144, 145, 146, 150, 153, 156, 157,161,162,163,164,166 Wright, L. G., 181, 198 Wright, M.,173,192 Wu, R.,206, 237 Wulff, D.L.,176,198 Wurgler, F.E.,242,293 wyss, O.,173, 198

Y Yamagata, H., 244, 303 Yamagishi, H.,204, 237 Yamaguchi, H., 248,249,303 Yamamoto, K.,248,249,303 Yamasaki, T.,251, 297 Yankus, C.A.,179,191 Yanofsky, C.,23, 31, 44, 47, 48, 49, 61, 69,77, 79, 123, 124, 126,138,139, 140 Young, M.R.,173,191 Young, S. G.,176, 192 Yourno, J., 9, 18, 22, 27, 32, 33, 34 Yura, T.,2, 3, 31, 123, 124, 126, 138

Z Zabin, I., 121, 137 Zampieri, A., 173, 198 Zetterberg, G.,175,198 Zieve, P.D.,183,198 Zimmering, S.,241, 242, 303 Zinder, N. D.,2, 3, 34, 41, 61, 54, 65, 67, 79, 335, 336, 343, 344, 348 Zito-Bignami, R., 260, 998 Zuletta, M.,251, $90 Zwenigorodsky, V. I., 211, 224, 234

SUBJECT INDEX A

frequency, 212 gene arrangement on chromosome, 2 15-21 7 negative interference, 214 prophage and, 214-215 regulation comparative, 224-225 factors affecting lysogenization, 221-222 functions in multiplication, 220-221 induction, 223-224 split-operon control of int gene, 222 related phages, 201-202, 205-206 replication comparative, 220 gene A and, 219-220 intracellular forms of deoxyribonucleic acid, 217-218 phage and backrial functions needed, 218-219 point of origin and direction of, 218 5-Bromouracil, sensitization with, 179-180

Action spectra, radiation effects, 188 Aging, composite theory, 314-317 nature of mutations, 317-318 evidence for mutation theory, direct, 310-314 indirect, 309-310 factors affecting, 307 life span of dividing cells, 320-322 nature of mutations, 319-320 theories, autoimmune, 308-309 collagen, 308 rate-of-living, 307-308 somatic mutation, 309 Aspartate, methionine synthesis and, 144

B Bacteriophage P2 deoxyrihonucleic acid, 202-204 physical map, 204-205 host bacteria, 201 lysogenic state chromosite preference, 225-230 chromosomal sites, 225 comparative, 231-232 eduction of host cell genes, 230 immunity to superinfection, 230-231 mutational types comparative, 211-212 essential functions, 207-209 lysogeny, 209-211 properties of virus particle, 207 origin of, 201 phage particle, 202 recombination comparative, 217 different mechanisms, 212-214

C

Carcinogenesis, ionizing radiations and, 277-279 Cell membrane, visible and near ultraviolet effects, 176-177 Cell surfaces hybrid, differences in, 55-56 Chromophore light-sensitive, recombination and, 184-185 Cross-reacting material, histidine mutants, 25-26 Cysteine fate of, 147 genetic map location and nature of structural genes, 147-149 377

378

SUBJECT INDEX

abortive transduction analysis, 149-150 synthesis, genes and enzymes involved, 142-147 regulation, 151-152 relation to methionine regulation, 157-158 D Density gradient, nucleic acid reassociation and, 83 Deoxyribonucleic acid male chromosome associated, 59-61 not chromosome associated, 61-62 phage P2, 202-204 intracellular forms, 217-218 physical map, 204-205 transforming, near ultraviolet and visible radiations and, 187-188

E Enterobacteriaceae genetic homology, 35-36 concepts of microbial taxonomy and, 37-39 genetic data and, 39-47 glossary of abbreviations, 36 protein structure and, 4748 intercrosses characterization, 62-65 definitions and abbreviations, 55 homologies between species and, 65-72 mapping and, 72-76 nomenclature, 54 production, 55-59 stability, 59-62 nucleic acids, relationships, 96-104 taxonomy and nomenclature, 95-96 Escherichia coli nucleic acid, divergence of strains, 97-99 relative relatedness to other Enterobacteria, 101-104 specific DNA, detection in heterologous organisms, 105-108

G Genetic material differences, hybrids and, 56 Genetic recombination see Recombination Genetic transfer, microbial systematics and, 40 Genome divergence in specific portions, Escherichia coli specific DNA in other organisms, 105-108 lactose operon in Enterobacteria, 108-110 ribosomal RNA genes, 110-111 transfer RNA genes, 111-112 Growth inhibition, near ultraviolet and visible radiations and, 185-187

H Histidine analogs, as genetic and biochemical probes, 128-131 Histidine operon base substitutions and, 22-23 cross-reacting material, 25-26 deletion mutations, 9, 19-21 frameshift mutations, 21-22 gene-enzyme relationship, 27-28 genetic mapping of, 28-29 intragenic complementation, 26-27 map, 10-19 multiple mutations, 24-25 mutants characterization, 3 isolation, 2-3 ratio of missense to nonsense, 23-24 reversion patterns, 3-9 polarity, 25 screening potential mutagens and, 26 spontaneous mutations, 26 stable mutations, 21 Homocysteine methylation of, 146 synthesis of, 145 Hybrids characterization homology, __ 62-63 normalization, 63-65

379

SUBJECT INDEX

production alteration of restriction, 58-59 cell surface differences, 55-56 genetic differences, 56 restriction effects, 56-58 stability chromosomal male DNA, 59-61 nonchromosomal male DNA, 61-62 use for mapping locating wild-type genes, 75-76 ordering of nearby genes, 72-73 orientation of specific regions, 73-75 Hydroxyapatite, nucleic acid reassociation and, 85-86

I Ionizing radiations biological significance of repair mechanisms, 255-256 carcinogenesis and, 277-279 cell death and, 276-277 cell stage and sensitivity, 241-243 consequences in individuals and populations, 279-280 genetic deaths and sex ratio, 281-283 human implications, 286-288 population fitness, 283-286 quantitative characters, 280-281 dose-rate and fractionation effects, 272-273 dose-response relationships, 270-272 external modification of sensitivity, 247-250 fractionals and repair mechanisms, 250-252 genetic control of sensitivity, 243-245 identification of chromosome carrying a particular gene, 266 insect control and plant breeding, 268-269 ion-density effects, 274-275 measurement of gene length, 266-267 molecular basis of resistance, sensitivity and repair, 252-255 nature of genetic changes, chromosomal aberrations, 258-260 gene conversion and paramutations, 263-264 gene mutations, 256-258

induced recombination, 261-263 lethal sectoring, 260-261 miscellaneous effects, 264-266 repair mechanisms and sensitivity, 245-247 studies on meristems, 267-268 transmutation effects and incorporated radioisotopes, 275-276 Isoleucine analogs, as genetic and biochemical probes, 131-135

L Lactose operon, divergence in Enterobacteria, 108-110 Leucine analogs, as genetic and biochemical probes, 131-135 Linkage maps comparisons, microbial systematics and, 43-47 Lysogeny, phage P2 mutational types, 209-211

M Methionine fate of, 147 genetic map location and nature of structural genes, 147-149 abortive transduction analysis, 149-150 synthesis, enzymes and genes involved, 142-147 regulation, 152-157 relation to cysteine regulation, 157-158 uptake of, 147 Mutagens potential, screening of, 26

N Near ultraviolet radiation cell membrane and, 176-177 comparison to ultraviolet, visible and photodynamic effects, 169-174 effects, significance, 188-189 growth inhibition and, 185-187

380

SUBJECT INDEX

mutation by, 174-176 possible target molecules, 181-183 transforming deoxyribonucleic acid and viruses, 187-188 Nitrocellulose filter, nucleic acid reassociation rate, 83-85 Nucleic acid extrachromosomal, relationships in Enterobacteria, 112-115 factors affecting reaasociation base composition, 87 deoxyribonucleic acid fragment size, 87

incubation temperature, 87-88 ionic strength, 87 nucleic acid concentration and time of incubation, 88-90 purity of preparations, 86-87 reassociation interpretation of data, 90-95 taxonomy and nomenclature, 95-96 reassociation techniques, 82-83 density gradient, 83 hydroxyapatite method, 85-86 nitrocellulose filter and agar methods, 83-35 renaturation rate, 86 relationships among Enterobacteria, 96-97

divergence of strains of Eschem'chia coli, 97-99 relatedness of some strains and Shigella, 99-101 relative relatedness of Escherichia coli to other Enterobacteria, 101-104

Protein structure, microbial systematics and, 4748

R Recombination asymmetric, molecular model, 332-334 bacteriophage P2, 212-217 comparisons and generalizations, 341-345

complexes, later steps in resolution, 337-341

conversions and transformations, 345-347

deviations, nature of, 327 light-sensitive chromophore and, 184-185

microbial systematics and, 4 1 4 3 models biochemical requirements, 329-332 evolution of, 32-29 molecular events leading to resolution, 334-337

Repression, model of, 120-122 Restriction alteration, hybrid production and, 58-59

effects, hybrid production and, 56-58

Ribonucleic acid ribosomal, conservation of genes, 110-111

transfer, conservation of genes, 111-112

S

P Phenylalanine analogs, as genetic and biochemical probes, 127-128 Photodynamic action comparison to ultraviolet, near ultraviolet and visible effects, 169-174 visible and near ultraviolet radiations and, 177-179 Photoreactivation, near ultraviolet and visible radiations and, 183-184

Salmonella typhimurium histidine operon, see Histidine operon Sensitizers, internal, 180-181 Shigella nucleic acids, relatedness to Escherichia, 99-101 Species genetic homologies between reliability of data, 65, 67-72 summary of data, 65, 66-67 0-Succinylhomoserine, synthesis from aspartate, 144

381

SUBJECT INDEX

V

Sulfate, cysteine synthesis from, 144-145

T Tryptophan analogs, as genetic and biochemical probes, 122-126 Tyrosine analogs, as biochemical and genetic probes, 126-127

U Ultraviolet radi,ation comparison to near ultraviolet, visible and photodynamic effects, 169174

Viruses, near ultraviolet and visible radiations and, 187-188 Valine analogs, as genetic and biochemical probes, 131-135 Visible radiation cell membrane and, 176-177 comparison to ultraviolet, near ultraviolet and photodynamic effects, 169-174

effects, significance of, 188-189 growth inhibition and, 185-187 mutation by, 174-176 possible target molecules, 181-183 transforming deoxyribonucleic acid and viruses, 187-188

CUMULATIVE AUTHOR INDEX OF CONTRIBUTORS TO VOLUMES 1-16 E

A Allard, R. W., 14, 55 Ames, Bruce N.,16, 1 Atwood, Sanford S.,1, 1

Edwards, A. W. F., 11, 239 Eicher, Eva M.,15, 175 Eisenstark, A., 16, 167

F

B Babcock, Ernest B., 1, 69 Baker, C. M. Ann, 15, 147 Baker, William K.,14, 133 Barratt, Raymond W., 6, 1 Bertani, Giuseppe, 16, 199 Bertani, L. Elizabeth, 16, 199 Blair, W. Frank, 5, 1 Bodmer, W. F., 11, 1 Bradshaw, A. D., 13, 115 Brenner, Don J., 16, 81 Bryson, Vernon, 7, 1 Buzzati-Traverso, Adriano, 7, 47

Falkow, Stanley, 16, 81 Flor, H. H., 8, 29 Ford, E. B., 5, 43 Foster, Morris, 13, 311 ti

Gardner, Eldon J., 15, 115 Garnjobst, Laura, 6, 1 Glass, Bentley, 6, 95 Gluecksohn-Waelsch, Sdome, 4, 1 Gordon, Myron, 1, 95 Grant, Verne, 8, 55, 89; 12, 281

C

Campbell, Allan M., 11, 101 Carson, Hampton L.,9, 1 Carvalho, A., 4, 127 Caspari, Ernst, 2, 1 Catcheside, D.G., 2, 271 Cleland, Ralph E., 11, 147 Croizier, G., 15, 147 Curtis, H. J., 16, 305

D da Cunha, A. Brito, 7, 93 Dahlberg, Gunnar, 2, 67 d’bmato, Francesco, 8, 1 Davidson, Eric H., 12, 143 Delaporte, Berthe, 3, 1 Dronamraju, K.R.,13, 227 Duvick, Donald H., 13, 1

H Hadorn, Ernst., 4, 53 Hancock, John, 6, 141 Hannah, Aloha, 4, 87; 13, 157 Hartman, Philip W., 16, 1 Hartman, Zlata, 16, 1 Hershey, A. D.,5, 89 Hess, Oswald, 14, 171 Heston, W. E., 2, 99 Hoffmann-Ostenhof, Otto, 8, 1 Hopwood, D. A., 11, 273 Horowitz, N.H., 3, 33 Hotchkiss, Rollin D., 16, 327 Hughes-Schrader, Sally, 2, 127

I Irwin, M. R., 1, 133

383

384

CUMULATIVE AUTHOR INDEX TO VOLUMES 1-16

J Jain, S. K., 14, 55

K Kiifer, Etta, 9, 71, 105 Kerr, Warwick E., 8, 109 Kihlman, B. A., 10, 1 Kikkawa, Hideo, 5, 107 Komai, Taku, 8, 155 Krug, C. A., 4, 127

L Laidlaw, Harry H., Jr., 8, 109 Lanni, Frank, 12, 1 Law, C . N., 13, 57 Levine, Philip, 6, 183 Lewis, D.,6, 235 Lewis, E. B., 3, 73 Lissouba, P., 11, 343 Lush, Jay L., 1, 209

M Magoon, M. L., 10, 217 Mangelsdorf, Paul C., 1, 161 Manwell, C., 15, 147 Matthey, R., 4, 159 Mayr, Ernst, 2, 205 Meyer, Giinther F., 14, 171 Michaelis, P., 6, 287 Middleton, Richard B., 16, 53 Milkman, Rbger, 15, 55 Mojica-a, Tobias, 16, 53 Moore, John A., 7,139 Mosig, Gisela, 15, 1 Mousseau, J., 11, 343

N Nagao, Seijin, 4, 181 Newcombe, Howard B., 16, 239 Newmeyer, Dorothy, 6, 1 0

Oehlkers, Friedrich, 12,329 Owen, A. R. G., 3, 117

P Papazian, Haig P., 9, 41 Parsons, P. A., 11, 1 Perkins, David D., 6, 1 Pontecorvo, G., 5, 141; 9, 71

R Rae, A. L., 8, 189 Ravin, Arnold W., 10, 61 Remington, Charles L., 6, 403 Richey, Frederick D., 3, 159 Richmond, T. R., 4, 213 Rick, Charles M., 8, 267 Riley, Ralph, 13, 57 Riaet, G., 11, 343 Robertson, Alan, 6, 451 Rossignol, J. L., 11, 343

S Sadgopal, Anil, 14, 325 Sanderson, Kenneth E., 16, 35 Sawin, Paul B., 7, 183 Schwemmle, J., 14, 225 Sears, E. R., 2, 239 Sermonti, G., 11, 273 Sheppard, P. M., 10, 165 Shrode, Robert R., 1, 209 Smith, D. A,, 16, 141 Smith, Harold H., 14, 1 Sonneborn, T. M., 1,263 Spencer, Warren P., 1, 359 Stahl, Ruth C., 16, 1 Stebbins, G.Ledyard, Jr., 1, 403; 9, 147 Stephens, S. G., 1, 431; 4, 247 Stratil, A., 15, 147 Suomalainen, Esko, 3, 193 Swaminathan, M. S., 10,217 Saybalski, Waclaw, 7, 1

T Takahashi, Ryuhei, 7,227 Tanaka, Yoshimaro, 5, 239

U Umbarger, H. E., 16, 119

1-16

CUMULATIVE AUTHOR INDEX TO VOLUMES

w Waddington, C. H., 10, 257 Westergaard, M., 9, 217 White, M. J. D., 4,267 Whiting, Anna R., 10, 295; 13, 341

Workman, P. L., 14, 55 Wright, Theodore R. F., 15, 261 Z

Ziegler, Irmgard, 10, 349

385

CUMULATIVE SUBJECT INDEX TO VOLUMES 1-16 A Aging, genetic factors in, 16,305 S-amino acid metabolism, in Escherichia coli and Salmonella typhimurium, 16, 141 Angiosperms, and fungi, comparative incompatibility in, 6, 235 Animals Y-chromosome in, 13, 227 parthenogenesis in, 3, 193 Artificial insemination, and livestock, 6, 451 Ascomycete, fine structure of genes in, 11, 343 Aspergillus nidulana 8-chromosome map of, 9, 105 genetics of, 5, 141 Assimilation, genetic, 10, 257

B Bacteria, cytology of, 3, 1 Bacterial cells, mutagenic and lethal effects of light on, 16, 167 Bacteriophage, inheritance in, 5, 89 Bacteriophage T4,recombination in, 15, 1

Barley, cultivated, origin of, 7, 227 Basidiomycetes, genetics of, 9,41 Bees, genetics of, 8, 109 Biochemical genetics of Bombyx mori, 5, 107,239 of Neurospora, 3, 33 Biochemical probes, metabolite analogs as, 16, 119 Biological coding problem, 12, 1 Biological composition, of taxonomic species, 12, 281 Bombyx mori, biochemical genetics of, 5, 107,239

Breeding corn, 3, 159 of forage crops, 1, 1 C

Cancer, genetics of, 2, 99 Cattle genetics of, 1, 209 monozygotic twins in, 6, 141 Chicken eggs, protein polymorphisms of, 15, 147 Chromosomal polymorphism in the diptera, 7, 93 Y-chromosome in man, animals, and plants, 13,227 in spermatogenesis, 14, 171 Chromosome breakage, biochemical mpects of, 10, 1 8-chromosome map of Aspergillus nidulans, 9, 105 Chromosome pairing, genetic variation in, 13, 57 Chromosome repatterning, and adaptation, 8, 89 Chromosomes of vertebrates, 4, 159 Coccids, cytology of, 2, 127 Coding problem, biological, 12, 1 Coffea,genetics of, 4, 127 Colks (lepidoptera), genetics of, 6,403 Corn, cytoplasmic pollen sterility in, 13, I

Corn breeding, 3, 159 Cotton breeding of American cultivated species, 4, 213 New World, origin of, 1, 431 Crepis,, cytogenetics and speciation in, 1, 69 Cytology of ooccide, 2, 127 Cytoplasmic inheritance, 2, 1 in Epilobium , 6, 287

386

CUMULATIVE SUBJECT INDEX TO VOLUMES

in Streptocarpus, 12, 329 Cytoplasmic pollen sterility in corn, 13, 1

D Differentiation, in monolayer tissue culture cells, 12, 143 Dioecious flowering plants, sex determination in, 9, 217 Diptera, chromosomal polymorphism in, 7, 93 DNA, genetic recombination in, 16, 327 Drosophila Y chromosome in, during spermatogenesis, 14, 171 embryogenesis genetics in, 15, 261 lethal factors in, 4, 53 mutations in wild populations, 1, 359 “obscura group” of, 7,47 tumorous head in, 15, 115 Drosophila melanogaster genetic variation in, 15, 55 heterochromatin in, 4, 87 Drosophila robusta, population genetics of, 9, 1

E Embryogenesis genetics in Drosophila, 15, 261 Enterobacteriaceae genetic homology in, 16, 35,53 molecular relationships among, 16, 81 Epilobium , cytoplasmic inheritance in, 6, 28’/ Episomes, 11, 101 Escherichia coli, s-amino acid metabolism in, 16, 141 Euplotes, genetics of, 1, 263 Evolution, linkage and recombination in, 11, 1 of cultivated barley, 7, 227 of maize, 1, 161 Evolution duplication, significance of, 4, 247

1-16

387

F Fertilization, selective, in Oenothera, 14, 225 Fishes, speciation in, 1, 95 Flax, complementary genic systems in, 8, 29 Forage crops, cytogenetics and breeding, 1, 1 Frogs, abnormal systems in, 7, 139 Fungi, incompatibility in angiosperms and, 6,235 G

Gene flow, changes in human populations due to, 6, 95 Genes, fine structure in ascomycete, 11, 343 Genetic analysis, on mitotic recombination, 9, 71 Genetic assimilation, 10, 257 Genetic code, 14,325 Genetic probes, metabolite analogs 89, 16, 119 Genetic structure of Gilia, 8, 51; Genetic variation in chromosome pairing, 13,57 in Drosophila melanogaster, 15, 55 Genetics, transformation, 10, 61 Genic analysis of characters in rice, 4, 181 Genic systems, in flax and flax rust, 8, 29 Gilia genetic structvve of apecies, 8, 55 taxonomic species, biological composition, 12, 281 Gossypium, cytogenetics and New World cottons, 1, 431

H Habrobracon genetics, 10, 295 Heterochromatin, in Drosophila melanogaster, 4, 87 Homology, genetic, in enterobacteriaceae, 16, 35, 53 Human blood factors, genetics of, 6, 183

388

CUMULATIVE SUBJECT INDEX TO VOLUMES

Human populations genetic changes in, 6, 95 genetics of, 2, 69 Human sex ratio, genetics and, 11, 239 Hybrids, inviability, weakness, and sterility of, 9, 147

I Immunogenetics, 1, 133 Inheritance, in bapteriophage, 5, 89 Inviability of interspecific hybrids, 9, 147

L Ladybeetles, genetics of, 8, 155 Lepidoptera polymorphism genetics of, 5, 43 population genetics study of, 10, 165 Lethal factors in Drosophila, 4, 53 Linkage in evolution, 11, 1 Linkage relationship of characters in rice, 4, 181 Livestock, artificial insemination and, 6, 451

Locus R complex, in Mormoniella vitripennis, 13, 341

M Maize, origin and evolution of, 1, 161 Mammalian pigment genetics, 13. 311 Man, Y-chromosome in, 13, 227 Map construction in Neurospora crassa, 6, 1 Metabolism in plants, 8, 11 Metabolite analogs, as probes, 16, 119 Microbial drug resistance, 7, 1 Mitotic recombination, genetic analysis based on, 9, 71 Molecular relationships among enterobacteriaceae, 16, 81 Monozygotic twins in cattle, 6, 141 Mormoniella vitripennis, Locus R complex in, 13, 341 Mouse, physiological genetics of, 4, 1 Mutations in Drosophila. 1, 359 mapping in histidine operon, 16, 1

1-16

spontaneous, in plants, 8, 1

N Near-ultraviolet light effects of bacterial cells, 16, 167 Neurospora, biochemical genetics of, 3, 33

Neurospora crassa, map construction in, 6, 1 New systematics, bearing on genetical problems, 2, 205 Nicotiana, cytogenetic studies in, 14, 1 Nuclear and cytoplasmic systems in frogs and toads, 7, 139 0

Oenothera, cytogenetics of, 11, 147 selective fertilization in, 14, 225 Ommochrome, genetic aspects, 10,349

P Paramecium, genetics of, 1, 263 Parthenogenesis, in animals, 3, 193 P2 phage genetics, 16, 199 Plants. Y-chromosome in, 13,227 evolutionary phenotypic plasticity in, 13, 115 metabolism and mutations in, 8, 1 Plasticity, phenot,ypic, in plants, 13, 115 Polymorphism genetics in Lepidoptera, 5, 43 Polyploids, classification of, 1, 403 Population dynamics of rodents, 5, 1 Population genetics, of Drosophila robusta, 9, 1 study of lepidoptera, 10, 165 Populations, inbreeding, genetics of, 14, 55

Position effect phenomenon, 3, 73 Potato, cytogenetics of, 10, 217 Protein polymorphisms, of chicken eggs and sera, 15, 147 Pterin pigments, genetic aspects of, 10, 349

1-16

CUMULATIVE SUBJECT INDEX TO VOLUMES

R Rabbit, domestic, genetics of, 7, 183 Radiations genetic effects of, 2, 271 ionizing, 16, 239 Recombination genetic, in DNA, 16, 327 theory of, 3, 117 in bacteriophage T4, 15, 1 in evolution, 1, 1 Resistance, microbial drug, 7, 1 Rice, genic and linkage characters in, 4, 181 Rodents, population dynamics of, 5, 1

S Salmonella, mutations in histidine operon, 16, 1 Salmonella typhimurium, s-amino acid metabolism and, 16, 141 Sera, protein polymorphisms of, 15, 147 Sex determination in dioecious flowering plants, 9, 217 Sheep, genetics of, 8, 189 Speciation, in Crepzs, 1, 69 in fishes, 1, 95 Species intercrosses, homology in, 16, 53 nature of, 2, 205 Spermatogenesis genetic activities of Y chromosome, 14, 171 stages of, 13, 157

389

Sterility cytoplasmic pollen, in corn, 13, 1 of interspecific hybrids, 9, 147 Streptocarpus, cytoplasmic inheritance in, 12, 329 Streptomyces coelicolor, genetics of, 11, 273

T Tissue culture cells, differentiation in, 12, 143 Toads, abnormal systems in, 7, 139 Tomato, cytogenetics of, 8, 267 Transformation, genetics of, 10, 61 Tribolium, genetics of, Supp.. 1 Tumorous head in Drosophilu, 15, 115

V Variegation, position-effect, 14, 133 Vertebrates, chromosomes of, 4, 159 Visible light effects on bacterial cells, 16, 167

W Weakness of interspecific hybrids, 9, 147 Wheats, cytology and genetics of, 2, 239

x X-autosome translocations, in mouse, 15, 175 X chromosome, inactivation of, 15, 175