TEMPERATURE-SENSITIVE MUTANTS OF BACTERIOPHAGE T4D: THEIR ISOLATION AND GENETIC CHARACTERIZATION1

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1 TEMPERATURE-SENSITIVE MUTANTS OF BACTERIOPHAGE T4D: THEIR ISOLATION AND GENETIC CHARACTERIZATION1 R. S. EDGAR AND I. LIELAUSIS Division of Biology, California Institute of Technology, Pasadena Received November 18, 1963 ENETIC studies with bacteriophage T4 have contributed greatly to our present knowledge of gene structure and mutation. This is partly due to the suitability of this organism for genetic investigations. However, physiological genetic studies with T4 have been hampered by a lack of suitable mutations which can be used to study the genetic control of phage growth and structure. The most useful mutations for such studies are conditional lethals, i.e., mutations which produce a lethal effect only under conditions which the experimenter can control. Such mutations have been used in physiological studies of the temperate phage lambda. JACOB, FUERST and WOLLMAN (1957) studied a number of defective mutants of lambda. These mutants can be propagated in lysogenic bacteria as prophage, but after induction of the lysogenic bacteria, vegetative growth of the defective phage is abortive due to the inability of the mutant gene to carry out its function successfully. These mutations are widely distributed over the genome of lambda and affect a variety of functions of the phage. CAMPBELL (1961), also using phage lambda, has isolated a number of sensitive mutants. Some of these mutants are host-defective mutants in the sense that they propagate in one host bacterium but not in another. He has also isolated mutants which do not grow at a high temperature (temperature-sensitive mutants), and mutants which do not grow at an extreme ph (ph-sensitive mutants). EPSTEIN, BOLLE and STEINBERG ( 1964) report on work with host-defective mutants of bacteriophage T4D. Temperature-sensitive mutations have long been recognized as a general class of mutations which can occur in many different genes controlling a variety of different functions (HOROWITZ 1950,1951). It is the purpose of the present paper to report on the isolation and genetic characterization of temperature-sensitive mutants of bacteriophage T4D. The mutants described herein are characterized by the fact that they can propagate at low temperature, but, unlike the wild-type phage, they are unable to grow at high temperature. It will be shown that these mutations are widely distributed over the genome of the phage. MATERrALS AND METHODS Phage strains: Wild-type strain T4D, temperature-sensitive mutants derived from this strain, and various standard plaque morphology mutants of this strain were used. 1 This investigation was aided by a grant from the &National Foundation and by a grant from the Public Health Service (RG-6965). Genetics 49: April 1964

2 650 R. S. EDGAR AND I. LIELAUSIS Bacterial strains: Escherichia coli strain B or B/5,1 was used as host for crosses and for preparation of lysates for isolation of mutants. E. coli strain S/6 or S/6/5,1 was used for preparation of stocks of phage and as plating indicator. Medim H broth was used as a growth medium for phage and bacteria. T broth was used as dihting fluid. EHA bottom agar and EHA top agar were used as media for plates. The recipes for these media are given by STEINBERG and EDGAR (1962). Preparation of plating bacteria: An overnight aerated culture of S/6 was diluted by a factor of 100, grown with aeration for 2.5 hr at 30 C, and concentrated 10 times by centrifugation. Preparations were used for 3 or 4 dsays and refrigerated when not in use. Cross procedure: E. coli B or B/5,1 bacteria were grown to a concentration of aborut 4 X IO7 cells/ml, concentrated by centrifugation, and adjusted to 4 x 10s cells/ml. KCN was added to the suspension of cells to a final concentration of M. The bacteria were added to an equal volume of phage mixture containing 3 x 109 particles/ml of each parental type. This reaction mixture was agitated gently at 30 C by hand for 10 min, at which time a 40,000-fold dilution into a growth tube was made. This growth tube was incubated at 30 C or 25 C without aeration. Within 10 min after dilution, samples were taken from the growth tube for measurements of infective centers 'and unadsorbed phage (the chlorosform method was used to measure unadsorbed phage, S~CHAUD and KELLENBERCER 1956). At 90 min chlorodorm was added to the growth tube and platings from dilutions of this tube were made for examination of progeny phage. 'Criteria for acceptable crosses are those of DOERMANN and HILL (1953). Preparation of phage stocks: An overnight aerated culture of S/6 was diluted 500.fold in 25 ml of H broth, and aerated at 30 C for 2.5 hr. This culture was then inoculated with a whole 4-hr plaque from a plate incubated at 30 C. For the preparation of ts mutant stocks, S/6 cultures were inoculated with la 5- or 6-hr plaque from a plate incubated at 25'C and the inoculated culture was continuously aerated at 25'C until it cleared, or for 16 hr (overnight.) Chloroform was then added 'and the stock filtered through a Mandler candle filter. Such stocks have titers of 1010 to 3 x 1011 particles/ml and are stable when stored at 4 C. Preparation of 2-amimpurine (ZAP) treated phage: To log-phage B bacteria, grown in T broth to 1 x 108 cells/ml, 1 mg/ml of 2-aminopurine nitrate (2AP), B grade (California Corporation for Biochemical Research) and 2 x 109 T4D wild-type phage were added. The reaction mixture was aerated at 30T for 60. min and then lysed with chloroform and diluted. A dilution of this lysate was used as a source of mutants. Preparation of 5-bromodeoxyuridine (5BUDR) treated phage: The same procedure was used for the preparation of SBUDR phage as was used for 2AP phage, except that H broth rather than T broth was used for bacterial growth medium. The 5BUDR was obtained from Cyclo Chemical Corporation. Preparation of nitrous acid (HNO,) treated phage: Mutants were isolated from a sample of treated phage kindly given to us by R. H. EPSTEIN. Mutant nomenclature: All temperature-sensitive mutants are identified by the symbol ts which indicates the nature of the mutant phenotype. To avoid possible confusiomn with mutants which may be isolated in other laboratories, each mutant name is prefixed with the letters ED to indicate that the mutants were isolated in our laboratory. Individual mutants are identified by a number prefixed with a letter which indicates the particular mutagen used or method of isolation. Mutants described in this paper come from one of six different isolation series. One spontaneous mutant (designated S) has been isolated. Randomly isolated mutants have come from phage treated with 2-aminopurine (series A), nitrous acid (series N), 5-bromodeoxyuridine (series B). Two series (G and L) involve special selection procedures which are described subsequently. Mutants of series G and L were induced with 5-bromodeoxyuridine. As an example of this nomenclature system, mutant ts EDN42 is a temperature-sensitive mutant (ts) isolated by us (ED). It comes from isolation series N and thus is a random isolate from a stock of phage treated with nitrous acid. Since the nomenclature is uniform throughout this paper, in referring to particular mutants the prefix ts ED will not be used here.

3 PHAGE TEMPERATURE MUTANTS 65 1 RESULTS Isolation of mutants. Series A, N, and B: T4D wild-type phage form plaques with no differences in efficiency of plating (e.0.p.) at temperatures ranging from 25 C to 42 C. Above about 42 C the growth of the plating bacteria, S/6 is impaired and the e.0.p. of the phage decreases. (Most commercial incubators are difficult to maintain at temperatures as high as 42 C. Actual incubator temperatures used in these experiments were approximately 42 C f 2 C.) Temperature-sensitive mutants of the A and N series were isolated in the following manner. Samples of mutagen-treated phage were plated at a density of about 200 plaques per plate and incubated at 25 C overnight. All plaques well separated from one another were stabbed with a sterile pin and transferred to pairs of plates seeded with S/6. The first plate of the pair of identical plates was incubated at 42 C, the second at 25 C. A small fraction of the mutagen-treated phage plaques failed to produce lysis upon transfer to 42 C but did cause lysis upon transfer to 25 C. In these cases a sample from the 25 C spot was picked and retested by diluting and plating the phage at both the high and the low temperatures. Most of the clones which behaved in this manner turned out to be mutants which could form plaques at 25 C but which produce no plaques at 42 C. The frequency of such mutants was comparable to the frequency of r mutants in the lysates (approximately 1 to 2 percent in the 2AP and HNO, lysates). In this manner, 72 mutants (31 A and 41 N) were isolated and stocks from single plaques of each of them were prepared. (Mutants which grow well at 42 C but not at 25 C have been isolated by this technique but have not been studied.) Ninety B mutants were isolated by a modification of this procedure. Since genetic analysis of the A and N mutants showed that several double mutants had been isolated, a concentration of 5BUDR sufficient to induce only 0.5 percent r mutants was used (about 4 pg/ml). For the isolation of the mutants, plates of the mutagen-treated phage were incubated at 25 C for 5 to 6 hr followed by overnight incubation at 42 C. Reconstruction experiments show that under these conditions ts mutants make small sharp plaques, whereas wild-type phage make large plaques. Only small sharp plaques were tested by the stab method. Five percent to 20 percent of the small plaque-forming phage were found to be ts mutants. All high temperature stab plates were incubated at 37 C rather than 42 C in an attempt to recover mutants with greater temperature sensitivity (i.e., mutants which would not produce plaques at either 37 C or 42 C). Isolation of mutants. Series G and L: As will be shown later, although the mutants isolated by random means are widely distributed over the genome of the phage, there are large areas barren of such mutants. One such region is between rll and r48 loci, a region in which are located what we chose to call early mutants. Physiological studies of mutants located in this region will be reported in a later publication. These early mutants are arrested in development at an early stage of growth by incubation of the infected bacteria at high temperature. The mutant-infected cells do not lyse at the elevated temperature and if, after incu-

4 652 R. S. EDGAR AND I. LIELAUSIS bation at the elevated temperature for 30 to 60 min, they are transferred to 25 C, the complexes resume growth with the eventual lysis and the production of phage progeny. However, most other mutants ( late mutants) are insensitive to temperature during the early stages of growth, but if kept at the elevated temperature, lyse at the time characteristic of the wild-type phage with the production of no viable progeny. The G and L series of mutants represent attempts to enrich the population of mutagen-treated phage with respect to the early mutants, by taking advantage of the above described differences between early and late mutants. Mutants of the G series were isolated by DR. 5. GURDON in the following manner. A phage stock was treated with SBUDR in the manner described above. Cells were infected with a multiplicity of about one phage per bacterium. The cells were incubated at 39.5 C in the presence of antiphage antiserum (k = 10 in the growth tube). Infective centers were assayed at different times and were found to fall in titer after 30 min incubation. Platings at 25 C were made at various times after 55 min for surviving infective centers. From these plates, mutants were isolated by the method used for the A and N mutants. The rationale behind this method of isolating mutants was as follows. Wild-type infected cells should lyse at about 25 min and the progeny phage should be destroyed by the antiserum. Cells infected with late mutants should also lyse at this time. However, cells infected by early mutants should be arrested in growth at the high temperature, but should resume growth after dilution and plating and form plaques. The dilution and plating reduce the concentration of antiserum to a point where its effects are neglible. In this manner some 62 G mutants were isolated. These mutants constituted about 4 percent of the surviving infective centers. Thirty-nine of the G mutants recovered appear to be identical mutations at a site in a late gene. We speculate that these mutants are members of a jackpot, or fortuitously large clone of spontaneous mutants, already present in the wildtype stock before the selective procedures were instigated. To insure against such a happening in the L series, a stock of phage was used which had been propagated at 39.5 C to ensure that ts mutants arising during growth could not multiply ap preciably. This stock was then grown in the presence of SBUDR and then cycled once at a multiplicity of about two phage per bacterium to allow segregation of mutational heterozygotes. The next step consisted of infecting bacteria at low multiplicity (0.3) and incubating the complexes at low temperature (to permit early mutants to initiate growth). The complexes were then transferred to 39.5 C at 20 minutes, or just before maturation ensues. The cells were then allowed to lyse and mutants were isolated from the progeny in the manner used for the B mutants. The sequence of low multiplicity growth cycles (the steps after treatment with SBUDR) was repeated a number of times to reduce the chance of repeated isolation of members of the same mutant clone. No more than ten mutants were picked from any one sequence. This overall selection procedure in principle should select against late mutants but not against early mutants. The frequency of ts mutants among the phage produced by this procedure was between 0.1 and 1 percent.

5 PHAGE TEMPERATURE MUTANTS 653 Table 1 summarizes information about the various isolation series. Characterization of mutants: The A and N mutants are very heterogeneous with regard to temperature-sensitivity. All mutants form plaques at 25 "C and 30"C, although some produce plaques smaller than wild type at both temperatures. About one half of the mutants form no plaques at either 37 C or 42 C. A few form plaques at 37 C and very minute plaques at 42 C ("leaky mutants"). Due to greater selectivity in the isolation procedure, the majority of the B and L mutants do not form plaques at 37 C. The production of minute plaques at 42 C by the leaky mutants is not reproducible, probably due to small temperature fluctuations during the early stages of incubation. However, plaques produced by leaky mutants are invariably extremely small at high temperatures, whereas plaques produced by wild-type phage are large. It appears that the e.0.p. of is mutants remains the same with increased temperature of incubation, but the plaques get smaller until a temperature is reached at which plaques can no longer be discerned. The burst size at various temperatures of bacteria infected with many of the mutants has also been measured. Table 2 gives results with a few mutants selected to give an indication of the degree of heterogeneity found in temperature sensitivity. In general, the burst sizes of the mutants are low (less than 1) at 39.5"C, and normal (about 250) at 25 C. However, a few mutants have low burst sizes even at 25 C. The burst size of wild type is imparied only at temperatures above about 40 C. Most mutant stocks produce a number of large plaques if concentrated samples are plated and incubated at the high temperature. These plaques are produced by revertants which are able to form normal plaques at the elevated temperature. However, the frequency of revertants measured in this way is only an upper limit of the true reversion frequency in a stock, since it is probable that some growth of the mutant phage takes place on the plate before temperature equilibration is reached. Thus revertants can be formed on the plate and contribute to the number of revertants one scores. Stocks in which the measured frequency of revertants was greater than about were discarded. Thermostability of mutant particles: The inability of the mutants to form TABLE 1 Techniques used for differeni isolation series Series Temperature, of growth before treatment with mutagen- A 30 C N 30 C B 30 C G 30 C L 39.5% Incubation temperature for mutant Type qf Percent Mutagen Enrichment+ isolationz plaques picked r mutantss 2AP 0 25 "C random - 2% HNO, 0 25 C random - 1% 5BUDR 0 25 '-2" C minute - 0.5% 5BUDR + 25 "C random 5EUDR + 25 C42"C minute - 0.5% * Temperature at which wild-type stock was made before it was treated with mutagen. f The enrichment techniques used to increase the fraction of recovered early mutants are described in the text. $ Temperature at which plates were incubated for isolation of mutants. 25 C420C plates were incubated at 25'C for 5 to 6 hr followed by incubation at 42 C for 14 to 18 hr. S Percent of r mutants in phage population from which ts mutants were isolated.

6 654 R. S. EDGAR AND I. LIELAUSIS TABLE 2 Temperature sensitiuity of some ts mutants Burst size' Plaque fonning ability+ 25 (: 33 C 39.5 C xoc jnoc 3 7 ~ : 42 C Wild type so B B39 29% BO NI N (+) N <O.Ol minute * Bacteria were infected with a multiplicity of about five in nr KCN. Unadsorbed phage were neutralized with anti-phage antiserum. Complexes were diluted lo')-fold, incubated at the appropriate temperature and lysed at 90 min. with CHC nieans plaques produced on plates incubated at indicated teniperature with efficiency of plating comparable to that of 25OC plates; 0 indicates no plaques. plaques at the elevated temperature shows that the growth of the mutant is impaired by the high temperature. Experiments were performed to see whether or not the mutant phage particles, produced at low temperature, are more readily inactivated by heat than are particles of wild-type genotype. Mixtures of each of the A and N mutants with phage of genotype r48 as ts+ control were prepared and inactivated to 10 percent survival at 45 C. The ratio of r to r+ before and after inactivation was measured. The vast majority of the mutants appeared to be inactivated at the same rate as the wild-type phage, judging from the unaltered r/r+ ratio after inactivation. Several of the 72 mutants did show a significant change in the r/r+ ratio after heat inactivation, indicating that the temperaturesensitive mutant was somewhat more sensitive than r48 (ts+) to heat inactivation. However, the differences were very slight, at most a factor of two in slope. Since the half-life of wild-type T4D is about 8 hr at 45"C, the slightly greater heat sensitivity of mature particles of these few mutants cannot account for their inability to form plaques at 42 C. Some of the mutants were also tested at 70 C and again no great differences between mutants and wild type were found with regard to thermostability of the particles. Thus, in all cases, it seems that the step which is temperature sensitive is at a stage before mature particles are completed. Complementation experiments: As will be shown later, the ts mutations are widely distributed over the genome of the phage. To reduce the number of crosses required to map the mutations, complementation tests were first performed so that the mutations could be assigned to genes affecting independent functions. Mutations in the same gene are clustered together in the genome and thus initial mapping can be done with representatives from each gene. The results of the complementation tests are complex and require special consideration. These results are reported in a companion paper ( EDGAR, DENHARDT and EPSTEIN 1964). Suffice it to say here that on the basis of our complementation test we can, with

7 PHAGE TEMPERATURE MUTANTS 655 reasonable assurance, assign each of our 382 ts mutations to one of 37 genes. The distribution of the various mutations among the genes is given in Table 3. Mapping: Crosses between ts mutants were performed in the manner described in MATERIALS and METHODS. Samples from the growth tube, after lysis, were TABLE 3 Distribution of mutations among genes Number of mutants from each isolation series Gene name A N B G 1, Total mutants A A Q* A A B N I 3 A N A N A NU) A N A N L N NI N L N a 0 2 L A N N N A G34-t L A L L $ BllO L L L Total mutants $ Number of genes * Thirty-nine of these mutants probably represent members of the same clone t This gene has not been mapped t Included i~ one spontaneous mutant

8 656 R. S. EDGAR AND I. LIELAUSIS plated at 25 C to assay total progeny, and at 42 C to measure wild-type recombinant progeny. In all cases, over 200 recombinant and 400 total progeny plaques were scored. In many cases, parallel platings at comparable dilutions were made of the parental mixture to insure that the recombinants measured were not present in the input and do not arise on the plate. Recombination values are calculated as twice the percentage of tsf progeny. Most of our mapping involved the use of one selected mutant from each gene. Crosses were also performed between IS mutants and various r mutants, In these crosses, the frequency of recombination was determined as twice the titer of r+ which form plaques at 42 C to the total titer measured at 25 C. Also used in the crosses was a temperature-sensitive lysozyme mutant of T4D, ec103, obtained from DR. G. STREISINGER. No attempt has been made to demonstrate the presence of double mutailts in the cross progeny. It is assumed that the double-mutant recombinants form plaques at 25 C but not at 42 C. If double-mutant recombinants were more temperature-sensitive than either mutant alone and did not form plaques even at 25 C, the calculated recombination values would be in error, but for short distances would not result in misordering of the mutations. In crossing the various mutants, it was discovered that a few of the ts mutants were double ts mutants. These were discovered by the abnormal behavior of the mutant strains in crosses. It is probable that most double mutants are revealed by the mapping procedure, but some double mutants assigned to a particular functional group but not used in crosses would not be recognized as double mutants. The double mutants which have been recognized as such have been rejected from this analysis. A small number of the ts mutant stocks appear to have, in addition to the ts mutation, a minute plaque morphology mutation usually distantly linked to the ts mutation. Thus in some ts x ts crosses, large and small plaques are found on the high-temperature recombinant plates. The method of isolation of the ts mutants in some series (picking small plaques from plates incubated at 25 C then transferred to 42 C) may tend to select for such double mutants. In all cases studied, the minute phenotype appears to be insensitive to the temperature of incubation. Some of our mapping data are presented in Figures 1 and 2. These results show that the ts mutations are widely distributed over the genome. Since we have data from over 800 crosses, not all cross data are presented in Figures 1 and 2. However, the data not presented are consistent with the order of the mutations given in the figures. Most of the data not presented are from crosses involving either very closely linked or very distant mutations. Some of the data from crosses between mutations within the same gene are presented in Figures l and 2 and give at least some indication of the length of the gene within which these mutations lie. Some of the genes are quite long, e.g. gene A44. Recombination experiments in T4 do not show good additivity. This is due to negative interference and to the fact that recombination values have a large coefficient of variation (EDGAR 1958). Thus ordering of the mutations by data from two-factor crosses is somewhat uncertain, especially if the elementary intervals

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10 658 R. S. EDGAR AND I. LIELAUSIS are unequal. The maps shown in Figures 1 and 2 are the best fit to the data and probably contain very few inversions of order, since most of the elementary intervals are approximately equal. A map of the ts mutations is given in Figure 3. The overall results give independent confirmation of the circularity of the genetic map ( STREISINGER, EDGAR and DENHARDT 1964) since even the greatest gap in the map, 30 percent recombination, is a clear indication of linkage. This map, however, does not give a true representation in physical terms of the relative distances apart of the various mutations. This is because recombination distances are not strictly additive, even for moderately short distances within a gene (EDGAR, FEYNMAN, KLEIN, LIELAUSIS and STEINBERG 1962). There are two sources of negative interference which produce this lack of additivity-high negative interference ( CHASE and DOERMANN 1958) which has its effects in small regions-and low negative interference, which affects linkage at greater distances. Thus, recombination distances are not proportional to physical distances. We are now attempting to devise a mapping function which permits the transformation of recombination distances into map distances such that these map distances are additive over the whole range of 1 r 40 A41 4r N3 4A3 FIGURE 3.-Map of ts mutations of T4D. The map summarizes the data presented in Figures 1 and 2. The mutations are located at distances proportional to recombination frequencies observed in crosses of adjacent markers. Mutations in the same gene are indicated by the inner lines. The scale is 10 of arc = 15 percent recombination,

11 PHAGE TEMPERATURE MUTANTS 659 measurable recombination frequencies. It is hoped that such a transformation will give a map which better reflects relative physical distances between mutations than does the map presented here. Distribution of mutations: It can be seen from Table 3 that there is a very large spread in the distribution of mutations among genes. Some genes contain only a few mutations, while others have more than 40. On the basis of the information given here, one cannot tell if this heterogeneity in distribution reflects differences in size or in mutability of the various genes. The longest gene (A44) has many but not the most mutations. A more compelling consideration concerning the distribution of the mutations is presented in Table 4. The genome is arbitrarily divided into three segments defined by the r loci. The table shows that the frequency of mutations in the three segments is not proportional to the map length of the three segments. Particularly striking is the fact that although segment rzz-r48 probably contains about 25 percent of the genetic material, only about 3 percent of the mutants from the random isolation series fall in this region. The table also shows that the enrichment techniques did in fact result in an increased yield of mutations located in this particular segment of the map. This result shows that ts mutations do occur in genes in this area, but are not picked up in the random isolation series. This may come about because this region has a lower ts mutability than the rest of the genome, or perhaps because the so-called random isolation procedure in some way tends to select against mutations in this region. It would be interesting to know if new genes will be uncovered if more mutants are isolated, and to what extent the results presented here indicate a saturation of the map with regard to ts mutations. It is not possible to calculate the number of as yet LLundiscovered genes from the distribution of known ones, because the distribution of mutations among genes is clearly not random. However, one can get some notion of the degree of saturation of the system (but not the genome) from the proportion of genes which contain mutations from two independent iso- TABLE 4 Effect of mode of isolation on the distribution of mutants Percent mutations in segments r48-r67 (Number of mutants) Percent mutations in segment r67-rii (Number of mutants) Percent mutations in segment rii-r48 (Number of mutants) Total number of mutants Random series A N B Enrichment series Relative G G L length: % (14) (14) (27) * G series with the suspected clone of 38 mutants discarded. t The relative lengths of the various segments are determined from Figure 3 and thus are not too reliable, since this map 1s based on uncorrected recombination data.

12 660 R. S. EDGAR AND I. LIEUUSIS lation series. In general, if T represents the total number of genes from which mutation series A and mutation series B are random samples, then T = A X B/ AB where A is the total number of genes in which A mutations are found, B the total number of genes in which B mutations are located and AB is the number of genes in which both A and B mutations are located. Pair-wise comparisons of the three random isolation series (A, N and B) give values for T of 37,31 and 28, that is, about 32 genes. This suggests that with the random isolation technique roughly 32 genes would eventually be found. In fact, from the A, N and B series, 29 genes have already been identified. This calculation suggests that the random isolation method has yielded close to the maximum number of genes it can yield. In support of this contention is the fact that of the 90 B mutants isolated, only three mutations were located in genes not already represented by A and N mutants. However, the enrichment techniques did result in the isolation of mutants located in genes not previously represented in the random isolation series. Thus it would appear that ts mutations in other as yet undiscovered genes may be found if suitable selective techniques are devised. Further support for this notion comes from the fact that ts mutations have been found to occur in the lysozyme gene ( STREISINGER, MUKAI, DREYER, MILLER and HORIUCHI 1961 ), although none has been uncovered in any of our isolation series. DISCUSSION The results presented here show that temperature-sensitive mutants of T4D can be readily isolated. The majority of the mutants are sensitive to temperature only during growth; the mature particles are as stable, or almost as stable, as nonmutant particles to thermal inactivation. This result is somewhat surprising, since it was expected that these temperature-sensitive strains would be mutant in phenotype because of the production of altered proteins more sensitive to thermal denaturation than the corresponding protein from the wild-type strain. Thus, it might be expected that such mutations, in genes controlling proteins present in the mature particle, would cause the phage particle, as well as its synthesis, to be sensitive to temperature. Studies to be presented elsewhere indicate that at least some of the ts mutations are in genes which control the structural proteins of the phage particle. In spite of this, the temperature sensitivity of the mutants appears to manifest itself primarily during growth. This result could be explained in a number of ways. Modified proteins could be thermo-labile only during formation of tertiary structure, or only before assembly into finished particles. The temperature-sensitive step could be at some stage of gene action prior to the formation of the polypeptide chain. However, the nature of the alteration in gene product induced by the temperature-sensitive mutations described here is yet to be determined. The ts mutations are widely distributed over the genome of the phage. However, it is clear that the mutations thus far found are not distributed uniformly over the genome. There are long segments in which ts mutations have not been found with the techniques presented here, while there are other regions densely

13 PHAGE TEMPERATURE MUTANTS 661 populated with mutations. However, we have shown that modifications of the isolation procedures do yield ts mutations located in otherwise sparsely populated segments of the genome. Thus, it would appear probable that many of the blank segments are regions of low detectable mutability rather than regions in which ts mutations cannot occur. It is hoped that various selective techniques will permit the isolation of ts mutations in additional genes and that eventually the majority of the genes of the phage can be identified. In principle, it would be expected that most of the genes of the phage would mutate to temperature sensitivity since it is probable that the conditions needed for a gene to produce ts mutations are that (1 ) the gene must perform some function essential for phage growth (the mutations are in fact temperature-sensitive lethals) and (2) the gene must be able to mutate at some point in its structure to give a temperature-sensitive gene product. We are indebted to DR. R. H. EPSTEIN for his example and inspiration. We wish to thank DR. JOHN GURDON for the mutants he isolated and for permission to describe the manner used to obtain them. SUMMARY The isolation of temperature-sensitive mutants of bacteriophage T4D is described. These mutants form plaques at 25"C, but unlike the wild-type strain, cannot form plaques at 42 C. It is shown that the temperature sensitivity of 72 independently isolated mutants is not accounted for by the inactivation of the phage particles but must be due to a temperature-sensitive stage during growth. It is shown that the 382 mutations studied are located in 37 genes widely scattered over the genome of T4D. The distribution of the mutations over the genome and among genes is not random. CAMPBELL, A., 1961 LITERATURE CITED Sensitive mutants of bacteriophage A. Virology 14: CHASE, M., and A. H. DOERMANN, 1958 High negative interference over short segments of the genetic structure of bacteriophage T4. Genetics 43: DOERMANN, A. H., and M. B. HILL, 1953 Genetic structure of bacteriopage T4 as described by recombination studies of factors influencing plaque morphology. Genetics 38: EDGAR, R. S., 1958 Mapping experiments with rzz and h mutants of bacteriophage T4D. Virology 6: EDGAR, R. S., 'R. P. FEYNMAN, S. KLEIN, I. LIELAUSIS, and C. M. STEINBERG, 1962 Mapping experiments with r mutants of bacteriophage T4D. Genetics 47: 17%186. EDGAR, R. S., G. H. DENHARDT, and R. H. EPSTEIN, A comparative genetic study of conditional lethal mutations of bacteriophage T4D. Genetics 49 : EPSTEIN, R. H., A. BOLLE, and C. M. STEINBERG, 1964 Amber mutations of bacteriophage T4D: their isolation and genetic characterization. (In preparation). HOROWITZ, N. H., 1950 Biochemical genetics of Neurospora. Advan. Genet. 3: Some recent studies bearing on the one gene-one enzyme hypothesis. Cold Spring Harbor Symp. Quant. Bid. 16:

14 662 R. S. EDGAR AND I. LIELAUSIS JACOB, F., C. FUERST, and E. WOLLMAN, 1957 Ann. Inst. Pasteur 93: Recherches sur les bactkries lysogknes dkfectives. S~CHAUD, J., and E. KELLENBERGER, 1%6 Lyse prkcoce, provoquke par le chloroform. chez les bactkries infectkes par du bactkriophage. Ann, Inst. Pasteur 90: STEINBERG, C. M., and R. S. EDGAR, 1962 A critical test of a current theory of genetic recombination in bacteriophage. Genetics 47: STREISINGER, G., F. MUKAI, W. J. DREYER, B. MILLER, and S. HORIUCHI, 1961 Mutations affecting the lysozyme of phage T4. Cold Spring Harbor Symp. Quant. Biol. 26: STREISINGER, G., R. S. EDGAR, and G. H. DENHARDT, 1964 The gross structure of the genome of phage T4. I. The circularity of the linkage map. (In preparation).