DOMINANCE IN BACTERIOPHAGE T4D1
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1 DOMINANCE IN BACTERIOPHAGE T4D1 HARRIS BERNSTEIN AND KATHLEEN M. FISHER Department of Genetics, University of California, Dauis, California Received July 18, 1967 HIS paper describes investigations of dominance in phage T4 using conditional lethal alleles of gene 37 in mixed infections with wild type. Previous studies of gene 37 mutant alleles were carried out by EDGAR and LIELAUSIS (1965) who showed that this gene specifies a tail fiber structural component, and by BERNSTEIN, EDGAR and DENHARDT (1965) who performed recombination and complementation analyses. It is shown here that various temperature sensitive (ts) and amber (am) mutants of gene 37 in mixed infections with wild type display different degrees of dominance as measured by burst size. Furthermore the plaque-forming progeny of these infections are shown to be functionally and physically distinguishable from wild-type phage. The implications of these observations with respect to the nature of dominance are discussed. MATERIALS AND METHODS Phage and bacterial strains: The wild-type phage strain employed in these investigations was T4D, and all mutants used were derived from this strain. The designations of the 30 ts mutants and four am mutants studied here are indicated in the genetic map in Figure 1. The origins of the ts and am mutants are described by EDGAR and LIEXAUSIS (1964) and EPSTEIN et al. (1963) respectively. E. coli B/5 and S/6 were used as restrictive hosts for am mutants. E. coli CR63 was used as the permissive host. Media H-broth, EHA-bottom layer agar and EHA-top layer agar were prepared as described in STEINBERG and EDGAR (1962). H-broth was used as the liquid nutrient medium in all experiments Preparation of phage stocks: All phage stocks were clonally derived from single plaques. Stocks of ts mutants were prepared on E. coli S/6 at 25 C according to EDGAR and LIELAUSIS (19H). Stocks of am mutants were prepared similarly except that E. coli CR63 was used as the host and growth allowed to occur at 30 C. Burst size measurements in mixed infections: E. coli B/5 grown to a titer of about 10' cells/ ml. were concentrated by centrifugation and resuspended to give a final titer of 4 x 108 cells/ml. These bacteria were incubated at 30 C and KCN was added to give a final concentration of.wm. After 2.5 minutes an equal volume of phage suspension was added. In general, average multiplicities of about nine mutant and nine wild-type phage per bacterium were employed. In the control infections with wild type alone the average input multiplicity was about 18. Ten minutes after addition of phage, when adsorption was about 97% complete, phage antiserum was added inactivating 99.9% of the remaining unadsorbed phage. Then the infected cells were diluted at least lo4-fold into an aerated growth tube at 37 C. Incubations were carried out at 37 C rather than at the usual restrictive temperature for ts mutants, 41"C, after it was found that wild-type burst size was more reproducible at the lower temperature. Infective centers were measured by This research was Npported by National Science Foundation Grant GBM9 Genetics 58: March 1968
2 308 HARRIS BERNSTEIN AND KATHLEEN M. FISHER plating a one-tenth dilution directly from the growth tube using permissive plating conditions. In experiments with ts mutants the permissive plating condition was incubation at 25 C with E. coli S/6 indicator bacteria. Where amber mutants were employed the permissive plating condition was incubation at 37 C on E. coli CR63 indicator bacteria. Unadsorbed phage were measured by diluting into CHC1, plus 0 to 10 ml H-broth, and then plating out a one-tenth dilution under permissive conditions. To measure the progeny titer another 80 minutes were allowed to elapse, chloroform was added to the growth tube, and a IO4 dilution was plated out under permissive conditions. All burst sizes were calculated as progeny titer over infective center titer. Adsorption experiments: E. coli B/5 grown to log phase were concentrated by centrifugatian and resuspended at 30 C to give a titer of 2 x IO9 cells/ml. KCN was immediately added to give a concentration of.ochm. No aeration was used. After 2.5 minutes an equal volume of a suspension of phage progeny from an am x am+ mixed infection was added. Samples were taken at 15 second intervals and added to chloroform to lyse infected bacteria. Unadsorbed phage were measured by plating on E. coli CR63. A similar procedure was followed far the progeny of ts+ x ts mixed infections except that aeration was used in the adsorption tube, and the unadsorbed phage were titered by plating on E. coli S/6. Heat inactivation experiments: The procedure used here was similar to the one reported by JINKS (1961). Lysates were adjusted to approximately IO3 phage per ml in H-broth. Ten ml aliquots were added to screw-capped test tubes. These were placed in a Precision-Freas water bath adjusted to 60.5"C with a variation of less than k.15"c. The upper level of the lysate in the tube was kept at least inch below the water level in the water bath and aluminum foil was used to cover the water bath in order to minimize temperature fluctuations. Samples were taken at 0, 1, 2, 3 and 4 hours. In each case, four.25 ml samples were plated directly without dilution. E. coli CR63 was used as the indicator bacteria and plates were incubated at 37 C. RESULTS Burst size tests with ts mutants: The mean burst sizes from all 30 different ts+ X ts mixed infections are given in Figure 1. On the abscissa the mutant sites in gene 37 are indicated at distances proportional to the map intervals between nearest neighbor sites as determined by two-factor crosses and corrected as indicated in the legend to Figure 1. The ordinate is in percent of the mean wildtype burst size. For most mixed infections burst sizes were measured several times in separate experiments. The number of independent determinations is given just below each mutant designation. The bar associated with each mean value gives the estimated standard error of the mean. The mean wild-type burst size was 210 phage per infective center. The data in Figure 1 show that the different ts alleles of gene 37 in mixed infections with wild type give mean burst sizes which vary over the wide range from 39% to 119% of the mean wild-type burst size. Mutants which by recombination appeared to be defective at the same site generally gave similar burst sizes in mixed infections with wild type. This would be expected if these mutants represent multiple occurrence of the same mutational event. There is no very striking trend of burst size with position of the mutant site in the genetic map. The average burst size for all ts+ x ts mixed infections was 180 phage per bacterium or 86% of the mean wild-type burst size. Genetically unmixed infections with the 30 ts mutants were carried out at
3 PERCENT w j 2- - I I I 1 I I I I I I I I I FIGURE 1.-Burst sizes from mixed infections of gene 37 mutants and wild type. On the abscissa the mutant sites are indicated at distances proportional to the corrected map intervals between nearest neighbor sites determined by two-factor crosses (BERNSTEIN, EDGAR and DEN- HARDT 1965 and unpublished data). The correction of map intervals for high negative interference was according to FISHER and BERNSTEIN (1965). Parentheses are used to enclose genetic sites whzs? relative order in the map is uncertain. The ordinate is calibrated in per cent of the mean burst size obtained on infection by wild type alone. Data are given for 30 ts x ts+ and 4 am x am+ mixed infections. Error bars indicate the estimated standard error of the mean. The mutant designations are given just below the error bars, and the number of repeated determinations contributing to each measurement is indicated just below the mutant designations. Narrow bars are used for tsf X 2s mixed infections, solid bars for am+ X am mixed infections, and a shaded bar for infections with wild-type alone. The estimated standard error of the mean (G) was calculated according to CROXTON (1959).
4 ~ ~ ~~ 310 HARRIS BERNSTEIN AND KATHLEEN M. FISHER TABLE 1 Burst sizes in genetically unmized infections Estmated. Relative Estimated Relative Number of Absolute standard mean Number of Absolute standard mean replicate mean error of burst replicate mean error of burst determi- bunt the mean size in determi burst the mean size in Mutant nations size G % Mutant nations sue 0, % tsb f tsn f tsb68 2.ll f tsb f tsl f tsb f tsp f tsb k.oo.04 ama481 2,443 f tslllo f tsn f tsll f tsn a tsn f tsn f tsb f tscb f tscb f tsl f tscb f amn f tsb k amn k tsnlo 4.17 f.09.ll tscb f tsa f tsl f tsa f amb f tsl f tsc f tsl f tsa f tsl k C and the mean burst sizes determined. These are listed in Table 1. The average input multiplicity in these infections was about eight. The average burst size for all ts mutants was 3.31 phage per bacterium. Generally mutants defective at the same site gave similar burst sizes in these unmixed infections. Each of the burst sizes can also be expressed as a percentage of the mean burst size obtained with the same mutant in combination with wild-type. These are listed in Table 1 under the heading relative burst sizes. The average relative burst size was 1.6%. That burst sizes from the unmixed ts infections were low compared to the ts xts+ infections indicates that the wide range of burst sizes obtained in the different mixed infections is not due to differences in the residual activity of the mutant proteins. Burst size tests with am mutants: Four different equal input am+ x am mixed infections were carried out using the restrictive host E. coli B/5. Three of the mixed infections were repeated seven times and one was repeated six times. The burst sizes obtained by plating on the permissive host E. coli CR63 are shown in Figure 1. In general, the four amber mutants exerted stronger negative effects than the ts mutants. The mean burst size and the estimated standard error of the mean (both expressed as a percent of the mean wild-type burst size) for each mixed infection are: am+ x amb280 (51 f 9) j am+ x a d481 (32 k 6); am+ x amn52 (19 * 3); am+ x amn91 (22 f 4). In genetically unmixed infections these mutants gave negligible burst sizes (Table 1 ). The two mutants, amn52 and amn91 which give similar mean burst sizes in mixed infections with om+ do not recombine with each other and may be
5 DOMINANCE IN BACTERIOPHAGE 31 1 identical alleles. The relative order of the burst size levels obtained with the four am mutants in mixed infections with am+ can be summarized; amb280 > ama481 > amn52 and amn91. This order was regularly obtained within each of the several independent experiments where comparisons could be made, despite variations in burst sizes between successive experiments. When the progeny of the mixed infections is plated on E, coli S/6 only genotypically wild-type phage can form plaques. On the permissive host E. coli CR63, all phage able to successfully infect, whether genotypically am+ or am, will form plaques. Using these two plating bacteria the proportion of the total progeny that were genotypically wild-type could be determined. This proportion was generally found to approximate the proportion of wild type among the infecting phage which varied between.27 and.71. Adsorption experiments: Progeny of wild type by mutant mixed infections were tested for their ability to adsorb to bacteria. In the case of am+ X am mixed infections the progeny were added to E. coli B/5 and the unadsorbed fraction was plotted against time (Figure 2). Since adsorption occurs quickly the points were all obtained within two minutes. The necessity for rapid manipulation accounts for the scatter in experimental points. The curves in Figure 2 show that the progeny of am+ x am infections adsorb appreciably more slowly to bacteria than do wild-type phage. At two minutes the proportion of unadsorbed progeny from the mixed infections was between 37% and 49%. The proportion of unadsorbed wild type was 16%. A second experiment gave qualitatively similar results. The proportion of unadsorbed progeny from the mixed infections at two minutes was between 28% and 60%. For wild type the proportion was 9%. Since the tail fibers are the adsorption organs of phage T4 these experiments suggest that the progeny of the am+ x am mixed infections are defective in these structures. An experiment was included to test the possibility that the am mutants were releasing, upon lysis of the infected cells, incomplete polypeptides which competed with or inhibited the adsorption of phenotypically wild-type phage. Bacteria separately infected with ama481 and wild type were mixed in equal amounts and then allowed to lyse. The adsorption rate of plaque forming phage was measured. As shown in Figure 2 adsorption occurred at about the same rate as with wild-type phage. The same result was obtained when the experiment was repeated. These results indicate that the slower adsorption of the progeny from the mixed infections is not due to interactions occurring subsequent to lysis. Five different gene 37 ts mutants were also employed in mixed infections with wild type and their progeny tested to determine if they adsorbed normally to bacteria. In each case adsorption was significantly slower than observed with wild-type phage alone. The results of these adsorption experiments with two of the lysates, ts+ x tsn5 and tsf x tsn10, are shown in Figure 3. Results very similar to these were obtained in a replicate experiment with the same mutants. Also, in other experiments, the progeny from the mixed infections ts+ x tsb46, ts+ x tsn31 and ts+ x tsn39 gave similar results to those depicted in Figure 3.
6 312 HARRIS BERNSTEIN AND KATHLEEN M. FISHER I I I I TIME (MINUTES) FIGURE 2.-The proportion of unadsorbed phage (P/Po) was determined at 15 second intervals after addition to E. coli B/5. Four lysates from mixed infections were tested: am+ X ama4.81 0; am+ x amn52 ; am+ X amn91 A; and am+ X amb280 A. Also shown are the curves for an am+ lysate and an equal mixture of am+ infected cells with amam1 infected cells, which were allowed to lyse after mixing W. The input ratios (am+/total) in the am+ X am mixed infections were in the range The burst sizes were in the range 2&50 infectious progeny per infective center. Heat inactivation: When free wild-type phage are heated at 60.5"C in H-broth according to the protocol given in MATERIALS AND METHODS inactivation occurs approximately exponentially over a four hour period. The purpose of the experiments described in this section was to determine whether the plaque forming progeny from am+ x am infections are distinguishable from wild type on the basis of sensitivity to heat inactivation.
7 DOMINANCE IN BACTERIOPHAGE 313 FIGURE 3.-The proportion of unadsorbed phage (P/P,) was determined at 30 second intervals after addition to E. coli B/5. The two lysates depicted here were from the mixed infections: tsf x tsn5 and ts+ x tsnlo A. Also shown is the curve for a ts+ lysate H. The ts+ X ts mixed infections in these experiments were carried out at 42 C with input ratios (&/total) about.5. Burst sizes were about 15 phage per infective center at this higher temperature. The four mutants amb280, amn91, ama481 and a"52 were employed in mixed infections with am+ phage and the resulting progeny heated to 60.5"C. In repeated experiments the proportion surviving after each hour was determined and the average results plotted in Figure 4. The number in parenthesis of 1.1 I I 1 P, TIMEIHOURSI FIGURE 4.-Heat inactivation of lysates at 60.5%. P, =titer at time zero; P, = titer at time i. Each curve represents the average of several experiments. The number in parenthesis associated with each curve indicates the number of inactivation experiments for that mixed infection.
8 314 HARRIS BERNSTEIN AND KATHLEEN M. FISHER associated with each curve indicates the number of inactivation experiments for that mixed infection. These results suggest that in two cases (am+ x ama481 and am+ X amn52) the progeny of the mixed infections are more sensitive to heat than wild type. Generally in repeated inactivation experiments with progeny from the same am X am+ parental combination, several different lysates were used. In preparing these lysates various am+/total phage input ratios were employed. These varied from.07 to.50. At the lower input ratios burst sizes were much reduced. Despite the wide variation in input no systematic correlation was found between input ratio and heat sensitivity of surviving progeny. This implies that the infectious fraction of progeny from the mixed infections has similar distributions of heat sensitivities. Also, the four am mutants were grown separately under restrictive conditions and the infected cells in each case mixed with an equal volume of wild-type infected cells. Lysis was then allowed to occur. The heat sensitivity of the infectious phage in the resulting mixed lysate was in all four cases indistinguishable from the heat sensitivity of wild-type phage. This indicates that heat sensitivity is not acquired after lysis by the interaction of phenotypically am+ phage with polypeptides specified by the am mutant. In order to improve the discrimination between the inactivation curves for different lysates a variation of the procedure given in MATERIALS AND METHODS was tried. Instead of plating directly at room temperature from a heated suspension containing about lo3 phage per ml, a more concentrated suspension at about lo5 phage per ml was heated and a 100-fold dilution made into 0 C broth before plating. Two separate experiments were performed which gave very similar results. The cold shock treatment substantially decreased the survival of the progeny from am+ x am lysates compared to wild-type survival. In both experiments the am+ x ama481 lysate was less sensitive than the other three am+ X am lysates which were roughly equivalent in sensitivity. The results of one of these experiments are given in Figure 5. DISCUSSION Dominance of ts mutants: Twelve of the 30 ts mutants used here had previously been tested and shown to undergo intragenic complementation (BERN- STEIN, EDGAR and DENHARDT 1965). Experiments in other systems indicate that the molecular basis of intragenic complementation is the interaction of mutant polypeptides to form a partially functional mixed aggregate (FINCHAM and CODDINGTON 1963a,b; SCHLESINGER and LEVINTHAL 1963). It is reasonable to infer that in ts+ x ts infections mixed aggregates are also formed. In such aggregates interactions could occur leading to a partial amelioration of the steric defect in the mutant monomers and/or the imposition of steric defects in the wild-type monomer. The mutant polypeptides might also prevent normal association of the wild-type tail fiber components or partially inhibit the action of the wild-type components. Different ts mutants encoding differently altered protein
9 ~ DOMINANCE IN BACTERIOPHAGE 315 Ob. 0, , I 1 I monomers would be expected to display varying degrees of interaction with the wild-type monomers. The above model is in accord with the observations that different ts mutants give varying burst sizes in ts+ x ts infections (Figure l), and also that the progeny from these infections are functionally distinguishable from normal phage by their slower adsorption to bacteria, as might be expected of structurally hybrid phage. BERNSTEIN, EDGAR and DENHARDT (1965) have shown that in mixed infections with different ts mutants defective in gene 37 some degree of positive complementation is usually observed. However in certain cases, especially where one of the ts mutants is leaky, the burst size from the mixed infection is significantly lower than the mean burst size from the individual single infections. It was proposed that these can be considered instances of dominance of a less functional mutant allele over a more functional mutant allele, and could be interpreted in terms of polypeptide interaction. FOLEY, GILES and ROBERTS ( 1965) have studied adenylosuccinase mutants of Aspergillus. Some of these mutants were similar to the ts mutants used here in that they were capable of intragenic complementation. These were tested for dominance to wild type in heterozygous diploids. It was observed that enzyme activity in the heterozygote was reduced between 50% to 10% or less of the homozygous wild type activity, depending on the mutant allele used. In this study, also. it was suggested that the allele specific dominance effects could be explained by polypeptide interactions. Dominance of the am mutants: SARABHAI et al. (1964) have shown that amber mutants defective in gene 23 form incomplete polypeptides. It can be assumed that amber mutants defective in gene 37 have the same effect. In Figure 1 it is
10 316 HARRIS BERNSTEIN AND KATHLEEN M. FISHER shown that the four amber mutants used in these studies are defective at three different widely spaced sites within gene 37. Thus these mutants probably specify polypeptide chains of three different lengths. One possible interpretation of the results reported here is that the incomplete polypeptides specified by the am mutants interact with the homologous wildtype polypeptides to form tail-fibers of mixed constitution. This could lead to a reduction in the efficiency of tail fiber attachment to the plating bacteria used for measuring the burst sizes of plaque forming progeny from the mixed infections. That these plaque forming progeny are distinguishable from wild type by their slower adsorption to bacteria and increased sensitivity to heat inactivation would be expected if they were structural hybrids. The observed differences in burst size from the different mixed infections would be expected if polypeptide fragments of unequal lengths do not interact identically with the homologous wild-type polypeptides. It is also possible that the progeny of the am+ x am infections are defective because they lack a complete set of wild-type components. This would explain the reductions in burst size, the slower adsorption and increased sensitivity to heat of the progeny phage. However this interpretation does not explain the variation in burst size obtained with the different amber mutants, unless it is further postulated that the polypeptide fragments specified by the amber mutants are involved in some way in the formation of progeny phage. It is possible for instance that the fragments interact with the base plate and inhibit the attachment of the wild-type tail fiber polypeptides. The first possibility, that fragmentary polypeptides can hybridize with homologous polypeptides is supported indirectly by evidence from the z gene of the lac operon of E. coli. ULLMAN et al. (1967) have shown that different incomplete polypeptides representing only portions of the z gene product can interact to form aggregates with partially restored function. Generalizations about the mechmzism of dominance: The results reported here suggest that one important determinant of dominance in phage is the interaction between the polypeptide products of homologous genes. The work of SNUSTAD (1966a,b and personal communication) indicates that another important factor is the sufficiency of the level of activity supplied by a single dose of the wild-type gene. In phage it appears that the expression of a single wild-type gene specifying an enzymatic protein is likely to be sufficient for a normal burst size despite possible interactions between mutant and wild-type gene products. On the other hand, the expression of a single gene specifying a structural protein is generally insufficient for a normal burst size. Thus, in phage, dominance is apparently determined by (1) the nature of the interaction between gene products and (2) whether the expression of a single wild-type gene is sufficient for normal function. We believe that these factors are likely to be of general significance as determinants of dominance in other organisms.
11 DOMINANCE IN BACTERIOPHAGE 31 7 We are grateful to PHYLLIS MCVEY for her able technical assistance and to DR. CAROL BERN- STEIN for critically reading the manuscript. We would also like to thank DR. ROBERT S. EDGAR for supplying the phage stocks. SUMMARY In higher organisms, genes with a series of mutant alleles displaying varying degrees of dominance to wild type are common. We were interested in investigating such a gene in phage in order to gain understanding of the molecular basis of allele specific dominance. Gene 37, which determines a structural component of the phage tail fibers, was chosen for study. It was shown that various mixed infections of wild type and a temperature sensitive mutant (ts+ X ts) gave different burst sizes depending on the ts mutant employed in the infection. Furthermore the plaque forming progeny from these infections were distinguishable from wild type by their slower adsorption to bacteria. Mixed infections of wild type and amber mutants (am+ x am) gave generally lower burst sizes than the ts+ x ts infections. The burst size levels achieved also differed depending on the am mutant used. The plaque forming progeny from the am+ x am infections, as in the case of the progeny from the ts+ x ts infections, differed from wild-type phage by their slower adsorption to bacteria. Furthermore these progeny had increased sensitivity to heat inactivation. These results can be interpreted by assuming that the polypeptides specified by the mutant genes interact with the wild-type polypeptides so that aberrant progeny with hybrid tail fibers are formed. It is suggested that interaction between homologous polypeptides may be of general significance as a determinant of dominance in other organisms. LITERATURE CITED BERNSTEIN, H., R. S. EDGAR, and G. H. DENHARDT, 1965 Intragenic complementation among temperature sensitive mutants of bacteriophage T4D. Genetics 51 : CROXTON, F. E., 1959 Elementary statistics. Dover, New York. EDGAR, R. S., and I. LIELAUSIS, 1964 Temperature-sensitive mutants of bacteriophage T4D: their isolation and characterization. Genetics 49 : 64s Serological studies with mutants of phage T4D defective in genes determining tail fiber structure. Genetics 52: EPSTEIN, R. H., A. BOLLE, C. M. STEINBERG, E. KELLENBERGER, E. BOY DE LA TOUR, R. CHEVALLEY, R. S. EDGAR, M. SUSMAN, G. H. DENHARDT, and A. LIELAUSIS, 1963 Physiological studies of conditional lethal mutants of bacteriophage T4D. Cold Spring Harbor Symp. Quant. Biol. 28: FINCHAM, J. R. S., and A. CODDINGTON, 1963a Complementation at the am locus of Neurospora crussa a reaction between different mutant forms of glutamate dehydrogenase. J. Mol. Biol. 6: The mechanism of complementation between am mutants of Neurospora crassa. Cold Spring Harbor Symp. Quant. Biol. 28: FISHER, K. M., and H. BERNSTEIN, 1965 The additivity of intervals in the riia cistron of phage T4D. Genetics 52: FOLEY, J. M., N. H. GILES, and C. F. ROBERTS, 1965 Complementation at the adenylosuccinase locus of Aspergillus nidulans. Genetics 52 :
12 31 8 HARRIS BERNSTEIN AND KATHLEEN M. FISHER JINKS, J. L., 1961 Gene structure and function in the hiii region of bacteriophage T4. Heredity 16: SARABHAI, A. S., A. 0. W. STRE", S. BRENNER, and A. BOLLE, 1964 Co-linearity of the gene with the polypeptide chain. Nature 201: SCHLESINGER, M. J., and C. LEVINTHAL, 1963 Hybrid protein formation of E. coli alkaline phosphatase leading to in vitro complementation. J. Mol. Biol. 7: SNUSTAD, P. D., 1966 Limited genome expression in bacteriophage T4-infected Escherichia coli. I. Demonstration of the effect. 11. Development and examination of a model. Genetics 54: STEINBERG, C. M., and R. S. EDGAR, 1962 A critical test of a current theory of genetic recombination in bacteriophage. Genetics 47: ULLMAN, A., F. JACOB, and J. MONOD, 1967 Characterization by in vitro complementation of a peptide corresponding to an operator-proximal segment of the p-galactosidase structural gene of Escherichia coli. J. Mol. Biol. 24:
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