The Genome of Bacteriophage T4

Anecdotal, Historical and Critical Commentaries on Genetics Edited by James F. Crow and William F. Dove The major task for the elderly is to create meaning out of what they have done with their lives. Take what they tell you with a grain of salt. Anonymous Old Sage I was asked to share my reflections about an article that appeared over 40 years ago in the 1963 Cold Spring Harbor Symposium for Quantitative Biology. The article (Epstein et al. 1963) described the phenotypes of 130 conditional-lethal mutants of T4, 69 amber mutants, and 61 temperature-sensitive (ts) lethal mutants and presented a genetic map of these mutations. The mutations were located in 47 different genes, 35 of which appeared to be involved in morphogenesis of the phage particles. When these morphogenesis mutants were grown under restrictive conditions, most of the infective processes appeared to continue normally; there was nuclear breakdown followed by rapid DNA synthesis and cell lysis, but no viable phage were released. Only phage components were produced: heads, or tails, or heads and tails, or particles lacking tail fibers. The locations of these genes are not random on the linkage map. Genes with similar phenotypes are clustered. Mutants defective in each of the other 12 genes showed aberrations in DNA synthesis. They also showed gross defects in cell lysis and phage component production as well. These genes also showed clustering in the genome. We argued that a significant fraction of the essential genes of T4 had been identified. When I received the invitation to write this Perspectives article, I had almost nothing at hand except my memories. I could not find any of my reprints, and my science files had long since disappeared. I retired in 1990, and for the last 14 years I have engaged almost entirely in nonscientific matters. I recall with some embarrassment that 20 or so years ago the Genetics Society of America asked that I give my papers to the University Library for safekeeping. I then looked at my old notebooks from the 1950s and 1960s—loose-leaf binders with the pages falling out, many undated, just lists of numbers. They were total gibberish to me. I hid them away then and they eventually disappeared. To write this retrospective, I contacted Bill Dove, one of the Perspectives editors, and he sent me a copy of the Cold Spring Harbor article. I was relieved to find that I could still understand the material. My friend and colleague for 50 years, Frank Stahl, also came to my aid and sent me copies of my grant reports for 1961–1963. He also sent me our correspondence from the 1950s and 1960s. It has been claimed that one of the tasks for the elderly is to create meaning out of what they have done with their lives. I can certainly see that urge now in me, and so I have approached the preparation of this article with considerable interest. I have sifted through the few documents made available to me, and I have dug into my memories and the memories of those who collaborated with me in this work. In 1960, when Dick Epstein and I started to work with conditional-lethal mutants, our knowledge of the genome of T4 was quite sketchy. Until 1960 we had been living with the view that T4 had three linkage groups, perhaps three or more DNA molecules, which duplicated, recombined, and somehow assorted themselves properly into phage heads at maturation. Figure 1 The historical map of T4. The linkage map, composed of three presumably independent linkage groups, shows the disposition of available genetic markers as of 1959. The map has been extracted from a grant report for 1961 submitted by Max Delbrück and Bob Edgar to the National Foundation for Infantile Paralysis. is the “historical” T4 linkage map I drew for our grant report in 1961. It consisted basically of the three linkage groups presented by Doermann and Hill (1953), and most of the mutations were plaque morphology mutations whose phenotypic basis was not known. The lone exception on the map was the gene coding for the phage endolysin, gene e, discovered by George Streisinger in 1959. In 1960, Streisinger and Bruce demonstrated that all the genes of T4 formed one linkage group by establishing the linkage II-I-III. In 1961 I collaborated with George (Streisinger et al. 1964) to demonstrate that there was linkage between linkage groups II and III, establishing that the T4 genetic map was circular. At about the same time we learned that the DNA of T4 was just one single molecule and not many small ones. When I left the University of California—Santa Cruz in 1990 and cleared out my office, I had saved a few scientific books. One was Phage and the Origins of Molecular Biology, a collection of articles honoring Max Delbrück's 60th birthday in 1966. I read again my article in the book (Edgar 1966) about the discovery of conditional lethals. It described how Dick Epstein had discovered the amber mutants in early 1960 or late 1959 and how, during the summer of 1960 at Cold Spring Harbor, Alan Campbell and I had come up with the strategy of looking for conditional lethals. It also describes how I had started to look for temperature-sensitive lethal mutations in the fall of 1960. Both Alan Campbell (1993) and Millard Susman (1995) have written Perspectives articles for Genetics that mention that encounter. Frank Stahl's 1995 Perspectives article for Genetics about the ambers is detailed and excellent. Curiously, my article for the Delbrück Festschrift, probably written in 1965, made no mention of the 1963 Cold Spring Harbor article that was the first publication about conditional lethals in T4 and their usefulness for the study of morphogenesis. Millard Susman and Charley Steinberg were Max Delbrück's last phage graduate students, and both were authors on the Cold Spring Harbor article. They helped Dick and me with the conditional-lethal work in many ways. Charley died in 1999, and a Perspectives article about him appeared in Genetics (Wu and Lindahl 2001). Recently Millard wrote to me about his experiences working with me and Dick and Charley: One evening, Dick, Charley, and I did 100 crosses to map a bunch of amber mutants. Dick prepared everything. He made all the mixtures of parental phages, labeled all the plates (I think), and measured the broth in all the dilution tubes. Charley and I were just extra hands. It was exciting. Dick started a new cross every 30 sec, and we did the standard assays of unadsorbed phage, IC's, and such. At the end, we had huge piles of plates, all of which Dick counted. At the end, we all believed that we had completed the experiment without any mistakes. That experiment, done in the quiet of the evening hours at Caltech, remains in my memory as one of the glorious events in my life as a biologist. When I wrote a ‘Perspectives’ article on the Cold Spring Harbor Phage Course, I wrote that I remembered that day when you told me, on the beach at the end of Bungtown Road, at Cold Spring Harbor, that you were planning a new initiative when we returned to Caltech. You were going to try to isolate temperature-sensitive mutations, and you were doing it because the Horowitz and Leupold experiment (Horowitz and Leupold 1951) suggested that such mutations could be found in many genes that encoded essential enzymes. You asked me not to tell Charley what you were planning, because he would think of a million reasons not to search for such mutations. I have no doubt that my memory is clear on all of that. Your logic was unassailable, and I admired your plan to look for ts mutants. And I understood why you might be fearful of Charley's skepticism, because, as much as I loved Charley, I was permanently humbled by him. As best I can recall, when I returned to Caltech in the fall of 1960, Dick Epstein was already in the process of moving to the University of California—Los Angeles, where he continued his work of isolating, mapping, and characterizing amber mutants. While there, he collaborated with others at the Massachusetts Institute of Technology on the first paper on the ambers, indicating that one of the amber mutants (gene 42 am N122) was defective for production of the phage-specific deoxycytidylate hydroxymethylase (Wiberg et al. 1962). Meanwhile, I started the search for temperature-sensitive mutants. My first search was, as I remember, successful, and only then did I tell Charley. His response was, yes, he had thought of looking for them but doubted very much that any would be stable enough to work with. Like Millard, I, too, was a bit intimidated by Charley! I spent the autumn of 1960 happily isolating and mapping ts mutants. During the winter of 1960–1961, Eduard Kellenberger, a leading electron microscopist from Geneva, came to Caltech for a time and helped us get our gene physiology studies started. Eduard had been a student of Jean Weigle's while Jean was still a physics professor in Geneva. Jean had retired and moved to Pasadena, California, and was working on phage in Max's lab when I first met him in the late 1950s. It was during Eduard's visit that we did most of the physiological experiments on the mutants. For those cells that went through the infective process normally except for failing to release live phage, we used electron microscopy to look in cell lysates for the presence of phage particles. I remember the thrill of seeing on the microscope screen for the first time, not phage particles, but component parts only. In some mutant lysates, we saw only heads, in others only tails, and in still others, only heads and tails but unassembled. Eduard has recently written me: I was not familiarized with your beautiful system, before I arrived in Caltech. I participated a little bit on the early screens of mutants: I introduced the optical microscopy of nuclear breakdown (with the method of equalizing refractive indexes) and the chemistry of DNA synthesis. The method of studying in situ lysates by electron microscopy and of agar-filtration I had developed earlier; I think that at Caltech we used mainly the second method for studying lysates as a whole. I had been proposed as a candidate for a position as Electron Microscopist at Caltech but I declined. I guess that Max Delbrück and Jean Weigle considered your work so important that they decided to invite me for a visit of a few months to help you in electron microscopy. I also taught Alex Lielausis in the of that your studies could continue I In I that the would have out quite had Eduard not Caltech when he He not only us up to the physiology experiments on the ts mutants and to continue them he but also Dick to him in where the work on the amber mutants continued with I I was the only phage working at the time to have been as a as a or In when I was an there was no such as was and there was Both of these consisted almost of and I as a and loved I my first in while I did and while in graduate but just the not the I remember that there was only one on and that was entirely on The was and the paper and was just the As a of my I that I was very doing what but very doing what with and In the summer of or I remember very by at a on the of at Cold Spring to me that the of was with chemistry and that I he was of The of by studies those of and of et al. with the system, were just about Eduard had not Caltech, I I would have my lab on We would have and of ts mutants, and others would have the would have written a article much the Cold Spring Harbor article. these Frank Your about your one of is My was in Biology, and it was of the I only enough and to the very for a my in Biology, since did not grant and I my was not of the the fall of Dick had moved to Geneva, and the work on the ambers was again at At the end of I drew the map of T4 in Figure map of the of historical markers with amber and temperature-sensitive mutants. map was for a grant report for 1961 submitted by Max Delbrück and Bob Edgar to the National Foundation for Infantile Paralysis. for our grant Dick Epstein had by then amber mutants, and I had ts mutations. of the genes both amber and ts mutations. We had also the between these conditional lethals and the plaque morphology mutations which the genetic map had been to all the ts mutant phenotypes had been a time, the more mutants we the new genes we We to genes into of these were very the time we out of we had ts mutants and had genes (Edgar and Lielausis We into an map of the amber and ts mutations (Edgar et al. Frank developed a mapping to for and we this to our mapping to create a genetic map that we was with the et al. We also had for 20 of the genes et al. These us some about the of the I think it was in the fall of that I to to write the Cold Spring Harbor article with Dick and We to our and write the article. As I recall, I spent about a in Geneva. I remember that as my out of the I could see the on and that in my in I had the the and of the time with these and our work had made me my The summer of 1963 and it was time to this paper on our In those an paper at the Cold Spring Harbor Symposium was on the It was Dick the The for me about the work was the of genes that we found that were involved in the of the phage particles. At the time it was believed that the phage perhaps only three to we had found 35 genes just for the of the me It demonstrated the of to were had already been in the work of and and others in the genetic of in by we could genetic to the of such as phage, similar to those of the this work was Bill and I used these to work out some of the phage in (Edgar and and I was about with and years our article was I was the National of in Molecular Biology. The his and of the method of lethal for the of the genetic of morphogenesis at the When I was told of the my first was I it have been to Dick Epstein and me and probably Eduard as well. it was for our work presented at the Cold Spring Harbor Symposium in I told our that I could not the it was to both Dick Epstein and me. I was into the of the of He to me to the I was in of him at the time, I to my that we the or I would not also and me to the few Max Delbrück told me he had received a from the of the National of The because of my they had decided not to give an that It out no such was Max loved at when he made I and the my I decided to get I would I on a I could but I loved to a Frank that I the to Dick, but I not remember I In a Dick I that you were in such an in to the and am very by your of Frank is about your the with me. I was very clear from your correspondence what the was all but I was to get the The last map we was in a article (Edgar and Epstein that time we had amber and ts mutants and The map is in Figure The map of the genes of by conditional-lethal mutations. map was in for a article (Edgar and Epstein The map was by the mapping developed by et al. genes whose was by mapping are by The locations of genes for which no were available are by The defective phenotypes are in For some genes many mutations were and so the of the gene could be from These genes are by in Figure this we that the genes we had about of the genetic map. While this article, I to about the of knowledge of the T4 genome. I found the map of T4 in Figure The map of T4 as of was from et al. and is at on the It is from et al. of the T4 genome was in by et al. The DNA was completed only recently to the of working with It is that genes are into the genome. The and of genes have been by by the of gene The authors of the article et al. Only of the T4 genes are under standard 30 to mutants in a few other genes very small under standard of these genes are much the T4 they almost of the genome. They genes that of the and of the and most of the and of the phage of these genes were first by the of amber or temperature-sensitive conditional-lethal mutations and were before their were I in that we had what we had We had most of the essential genes of the phage, and these genes about the genome. mapping well. In the article I wrote for the Delbrück in 1965, I claimed that amber mutants were to in 1995 Frank claimed that they were for I found by the and made with him 40 I had discovered that he was, in a with me et al. the of the ambers, this is what wrote me: In 1960 when I was a graduate student at I was with Charley Steinberg a and Dick Epstein I was doing my on and Charley and Dick were working on phage T4 with Bob who was then an At the time Charley, Dick and were all and were in the of working late into the but time to see a One evening, I was with my and to the of where the phage lab was with the of my to to a they were into a phage mutant and suggested I help them My help consisted of a to then it to phage from on one and then other which had been with some time I asked them to what they were As I remember I thought their to look for conditional lethal mutants and I no remember what their was, and probably understand it at the In any they were about the and we as of a that the experiment did work out, they would mutants my In years Charley remembered it in the was and they to me in a as the of the book Charley's memory was that they to any mutants they found me as an to help them The day it was clear that they had found the new mutant they had decided to amber mutants. I had my I had most of phage I had and so I had nothing to the The of the amber mutants as mutants was not months but with the mutants of Bob phage to all the essential phage genes, work was much to the genes and a few other with Millard he to see that there is still over the of the mutations. At the of I am not prepared to tell you that I am that my memory of events 40 years ago is but I I was when into the lab to invite Dick and Charley to to a with him. At that is what I have been in my for the 35 years or to my of the with the made to was that the mutants would be for not for his I remember that I was when I Dick tell his of the in a Dick has written in to me, is about the of the When Frank was his article, Charley and I presented different of the Charley and I the but I was that my I that Charley was that the mutants his was And Dick has the last Millard Susman made in his correspondence with me was the was a to my and I to out that you and Dick were with your mutants, them with who to on the genetic of phage That to me a of scientific to your the with of the at the of such and are of the of their I guess these mutants are I not but in old we Dick and I and our mutants to who asked for was to each I a for a mutant to a of what was going to be made of that did not my it as that the of the was on and were in other they were not As Frank has out to me, was thought by some to be one of Max's to phage I have thought a about the of the between Dick and me. we or He had found the amber mutants. I was to look for the temperature-sensitive mutants in because of the ambers were his to and as he saw We about our and our and were already old and The end was a study that we for most of the time, of In the I to I the because for the was the same as had been for T4. In he told me that he had spent a working that with T4 just a few years before our work with conditional lethals. He had that small plaque mutants would out to be lethal mutations that he could much as we had used conditional lethals. He then to and the because of and small of cells The genome of the is a much genome that of T4, but still small for a the had to a each lab working on a of the through lab and came to up was to understand the To be successful, we were on their with other many of us had already the of and on this of working in the under our We the a because so many of the early had started with T4, Bill Bob and Dick I from my the of with a memory for In the every fall the at Caltech a In to my Bill asked me I would be his I to while Bill was the He had been on the and was then the Pasadena, California, I was We had been in the lab working on phage in so why not be on the He told me we would I just as as I could and then out of his We made it all the to the were to be Max Delbrück and Max was quite as a and was almost as as He was also graduate student and working on T4 Bill is of is and very but try as he might he had The day of the and Max a at am from He had the when asked him about his to the he that mainly he was to his because he had a very important up in the Bill and I arrived at the with our of to find the so with and we had a time That day Max and could no And we we I can still see over the and Max in the with a of on his Bill and I were The of T4 had his day on I am very to Frank for me with and I to Dick Eduard Kellenberger, and Millard of the article, for of this article and for and memories that they with me. I also them and for to from their