E mentation tests between the members of many pairs of amber and temperature-sensitive

Transcription

1 INTERGENIC CIS-TRANS POSITION EFFECTS IN BACTERIOPHAGE T4 J FRANKLIN W. STAHL, NOREEN E. MURRAY,3 ATSUO NAKATA AND JEAN M. CRASEMANN Medical Research Council Unit for Molecular Biology, Hills Road, Cambridge, England, and Instituie of Molecular Biology, University of Oregon, Eugene Received February 28,1966 PSTEIN et al. (1963) have defined the genes of T4 by means of comple- E mentation tests between the members of many pairs of amber and temperature-sensitive mutations. In tests involving at least one amber mutant, the results are clear cut-one observes either no complementation or complementation sufficient to produce a yield of phage comparable to that obtained in infections by wild-type T4 (EDGAR, ~NHARDT, and EPSTEIN 1964; EPSTEIN and EDGAR, personal communication; STAHL and MURRAY 1966). However, we chanced to observe (STAHL and MURRAY 1966) a few cases of rather poor complementation in certain control experiments between T4 amber mutants which were considered to be in different genes. For two of the more striking cases we constructed the double amber type in order to conduct a proper cis-trans test (LEWIS 1951, and see BENZER 1955). In both cases the phage yields from cells mixedly infected by wild-type and double amber particles were greater than the yields from cells mixedly infected by each of the single amber types. Either of the two nontrivial standard explanations seemed plausible to us-(1) The product of one or both of the genes involved is unstable except in combination with the other; unless the genes are translated in the immediate neighborhood of each other, one or both of the products decays partially before combining. (2) The translation and/or the transcription of one or the other of the two complementing wild-type genes is being partially depressed by the amber mutation cis to it; i.e., one or the other of the two amber mutants is a polar mutant (see NEWTON et al. 1965). In this paper we give details of two cases of cis-trans position effect with T4 amber mutants as well as experiments which demonstrate that the effect is better explained by alternative (2). Our methodology is simple and generalizable. If operons with defined limits exist in T4, our method should permit their identification as well as define direction in which each is polarized. MATERIALS AND METHODS Complementation tests: Complementation tests between amber (am) mutants were performed Supported in part by a research grant from the National Science Foundation (GB-294). Send requests for reprints to Institute of Molecular Biology, University of Oregon, Eugene, Oregon. On loan from the Botany School, Cambridge University. Supported by a NATO Fellowship from the Department of Scientific and Industrial Research (Great Britain). Genetics 54: July 1966.

2 224 F. w. STAHL et al. as described by STAHL and MURRAY (1966). Briefly, about five particles of each of two genotypes were adsorbed to cells of a bacterial strain in which am mutants cannot grow (restrictive cells). The infected culture was then diluted and the total phage yield measured following cell lysis. Complementation tests involving temperature-sensitive (ts) mutants were performed similarly but at 42 C. Preparation of phage stmks: Stocks of am mutants were prepared as described by STAHL and MURRAY (1966). Stocks of ts mutants were prepared on strain BB at 30 C. Bacterial strains: The following E. coli strains were used for the purposes indicated: Strain Obtained from Properties Use BB J. W. DRAKE Restrictive for Host in all experiments ambers B... Restrictive For isolating am+ revertants CR63 Permissive for Plating bacterium in ambers most experiments; preparation of am stocks Bw Restrictive Preparation of wildtype phage stock 011' Permissive Plating bacterium in double layer platings (see RESULTS) Phage strains: The following phage strains were used: Phage T4D wild type am NI20 am N58 am B252 am S29 amam4 am B270 am NI31 ts B30 IS A20 ts N30 ts A82 Obtained from R. s. EnGm R. S. EDGAR R. S. EDGAR R. S. EnGm Number of the gene in which the mutation resides (see EPSTEIN et al. 1963) Double mutants were derived from crosses of the appropriate single mutants described above. None of the am mutants used can propagate by itself on our host strain, BB. Amber and ts mutants will be referred to by the number of the gene in which the mutation resides, am 27, for instance, being used to designate the strain carrying the mutation am N RESULTS The basic cis-trans experiment: Our basic test involves the comparison of the yields per input bacterium from two sets of mixedly infected bacteria. One set

3 POSITION EFFECT IN PHAGE 225 (the trans test) is infected with about five particles each of two am strains mutant in different genes. The other set (cis test) is infected with about five particles each of wild type and of the double mutant which combines the two relevant am mutations. The existence of a cis-trans position effect is concluded when the trans test gives substantially-fewer viable particles per cell than does the cis test. In most cases, along with each cis-trans comparison, we conducted infections with the following pairs of phages: (1 ) Wild type plus each am mutant. When compared with the yield from cells infected by wild type only, these infections measured the extent to which each of the ambers reduced the total yield. (2) The double amber plus each single amber. This test confirms the genotype of the double amber and sets an upper limit to the degree of growth of each of the ambers (its leakiness ) in strain BB. (3) An am mutant in the remote gene #1 plus each single am mutant. This test demonstrates that each of the relevant single am stocks does participate satisfactorily in mixed infections. A given cis-trans test and the relevant tests (1) to (3) as well as the measurement of yield from cells infected only by wild-type phage were all performed simultaneously using a single bacterial culture. The tests analogous to (3), but using the double rather than the single ambers, were performed on different days as part of the experiments reported in Table 3. The satisfactory reproducibility of our yield measurements permits the inclusion of these data in Table 1 which gives results of basic cis-trans tests involving the neighboring genes 27 and 51 and the neighboring genes 34 and 35 respectively. In the case of genes 27 and 51, the yields when the wild-type alleles were in the trans position were only one tenth of the yields obtained when they were cis to each other. For genes 34 and 35 the analogous figure was one fifth. The successful participation of our single and double am TABLE la Cis-trans complementation tests inuoluing amber mutants in genes 27 and 51 Yield/input bacterium Test Puroose of test Exwriment 1 ExDeriment x wild cis-trans comparison 27 x x wild to assess partial dominance x wild toeach am mutant wild x wild x X x 1 51 x 1 to confirm genotype of double amber and check leakage to confirm participation of each of the single am mutants X 1 From Experiment 1 and 2 of Table 3 to confirm participation of the double amber

4 226 F. w. STAHL et al. TABLE Ib Cis-trans complementation tests inuoluing amber mutants in genes 34 and 35 Test Purpose of test x wild cis-trans comparison 34 x 35 - Yield/input bacterium Experiment 1 Experiment x wild to assess partial dominance x wild of each am mutant wild x wild x x XI 35 x x 1 to confirm genotype of double <I <I am and check leakage <I <I to confirm participation of each of the single am mutants From Experiment 1 and 2 of Table 3 to confirm participation of the double amber In all tests, the nominal multiplicity of infection of each infecting type was five. Measurements of input phage and bacteria and of unadsorbed phage verified the acceptability of each experiment. Adsorbtions occurred in M KCN. Yields were measured after chloroforming a dilute suspension of the complexes following 70 minutes of development. The yields reported each contain a small correction for unadsorbed input phage. strains was further verified by an examination of the constitution of lysates produced by coinfection of BB with the am strain and wild-type T4. The lysates examined were from Experiment 1 in Table la. Wild-type and am particles in the lysates were distinguished by a double-layer plating method devised by R. H. EPSTEIN (personal communication). The coinfection involving wild type and am 27 (see MATERIALS AND METHODS for mutant nomenclature convention) gave 46% am particles; that involving wild type and am 51 gave 39% am particles, while that involving wild type and the double amber likewise gave 39% am particles in the lysate. The purpose of the cis test is to show that the low yield encountered in a trans test is not merely the consequence of the partial dominance of one or the other of the two am mutations (or of the combination of the two). The ability of the test to reveal this dominance is shown by data from tests involving am 22 (Table 2). It can be seen that this amber strongly depresses the yield in coinfection with wild-type phage and that both the trans and the cis tests with other ambers give correspondingly low yields. Recessiveness of the trans-configuration: Two alternative classes of explanations for the small burst sizes observed with the trans tests involving gene pairs and suggest themselves. (1) One or the other (or perhaps both) of the amber stocks is producing subnormal amounts (or activities) of the protein product of the wild-type gene for which the other stock is amber. (2) The combination of the two amber phage stocks used in the trans test somehow interferes

5 POSITION EFFECT IN PHAGE 227 TABLE 2 Cis-trans complementation tests involving a partially dominant amber mutation in gene 22 For procedures. see Table 1 Test Yield/mput bacterium x wild x x wild x wild 271 wild x wild x 22 < x 26 < x wild x x wild see above 51 x wild 432 wild x wild see above x 22 <I x 51 <1 in an active way with the production of phage; for instance, the combination of stocks may induce lysis from without. Alternatives (1 ) and (2) are distinguished by a demonstration that the trans-configuration is recessive in determining a low yield. The essence of the demonstration is that the yield in the trans tests can be elevated to that of the cis test by coinfxtion with a phage which is amber in a remote gene and wild type for the pai of genes in question. The results of these experiments are presented in Table 3. TABLE 3 Recessiveness of the small yield in the trans-complementation tests involving gene pairs and Test x wild 27 x x wild x 1 27 x 51 x x wild 34 x x wild x 1 34 x 35 x 1 Yield/input bacterium - Purpose of test Experiment 1 Evperiment 2 Basic cis-trans comparison Removal of cis-trans effect by coinfection with an amber in a remote gene Experiment 3 Experiment 4 Basic cis-trans comparison Removal of cis-trans effect by coinfection with an amber in a remote gene The nominal multiplicity of infection of each parent was fire. For procedures, see Table 1

6 228 F. w. STAHL et al. Determination of the direction of the cis-trans effect: (a) ts x am complementation tests: Complementation tests between ts and am mutants have the possibility of determining which of the two am strains is responsible or the small yields in the trans test. If only one of the two stocks is responsible, and if the ts stocks themselves have normal rates of production for the nonmutant neighboring gene, then one of the two possible intergenic ts x am complementation tests will produce a large yield while the other one will give a yield as small as that observed in the am x am tests. The am strain involved in the latter ts x am test is the stock which underproduces the product of the neighboring gene. The results of these tests with relevant controls (Table 4) show that am 51 underproduces the product of gene 27 and that am 34 underproduces the product of gene 35. (b) Depmdence of burst size on relatiue multiplicity of infection in the trans TABLE 4 Complementation between ts and am mutants Yield/input bacterium Test Purpose of test Experiment 1 Experiment 2 a. Genes 27 and 51 am x wild am 27 x am 51 ts 27 x ts 51 ts 27 x am 51 ts 51 x am 27 Cis- trans comparisions ts27 x wild ts51 x wild wild x wild To assess partial dominance 36 4a of each ts mutant am 27 x ts 27 <I <I am51 x ts51 To confirm genotypes <I <I am 27 x am and check leakage <I <I am 51 x am <I <I b. Genes 34 and 35 am x wild am 34 x am 35 ts 34 x ts 35 ts 34 x am 35 ts 35 x am 34 Cis-trans comparisons ts 34 x wild ts 35 x wild wild x wild To assess partial domi name of each ts mutant am 34 x ts 34 <I <I am 35 x ts 35 To confirm genotypes <I <I am 34 x am and check leakage <I <I am 35 x am <I <I Tests were conducted as usual except that incubation of the complexes after dilution out of KCN was at 42.

7 POSITION EFFECT IN PHAGE 229 test: The ts x am tests indicate that am 51 and am 34 underproduce the products of genes 27 and 35 respectively. This conclusion predicts that yields in the trans tests should be improved under conditions of unequal input. More precisely, input ratios of 27/51 and 35/34 which are less than one should give better complementation than ratios which are greater than one. Such tests are reported in Figure 1. The total multiplicities of infection were held constant at ten while the nominal relative multiplicities varied from 1/9 to 9. The results show that the maximal yields per productive cell are obtained when am 51 is in excess of am 27 and when am 34 is in excess of am 35. That these results reflect relationships between members of the pairs am 51-am 27 and am 34-am 35 rather than 500 /U..h I 10 I I 0.1 I 10 Input 27/51 50 A I I 10 Input 35/34 FIGURE 1.-Burst size as a function of relative multiplicity of infection of two ambers. Infections of BB were made with varying numbers of ambers 27 and 51 (la) and ambers 34 and 35 (lb) respectively. The total input was nominally ten phage per cell; the relative inputs were determined from assays of the diluted phage stocks used for making the infections. Adsorbtion of phage to cells was never less than 80% of the input. The number of productive complexes was determined by plating before lysis on CR63 after serum treatment to inactivate unadsorbed phage. Total yields were determined as usual. Burst size is total yieldbprcductive complexes. Results of two independ?nt experiments are presented for each mutant combination.

8 230 F. w. STAHL et al $ = 50 z 4 n I 10 Input 35/51.c Q 10 I I I I 10 Input 27/34 FIGURE 2.-Burst size as a function of relative multiplicity of infection of two ambers. The procedures were as described for Figure 1 except that the complementing ambers were nonneighbors. Results of two independent experiments are presented for each mutant combination. intrinsic properties of one or the other member of the pair is demonstrated by the trans tests of am 34-am 27 and am 51-am 35. In these latter cases maximal yields are obtained *when approximately equal multiplicities of infection are used (Figure 2). Properties of am+ reuertants: None of the experiments described to this point identifies the source of the depressed gene activity in our am strains. The underproduction of gene 35 product by our am 34 strain, for instance, may mean only that this am strain carries an additional mutation in gene 35 which results in gene product of lowered activity. To test this possibility, we isolated am+ revertants from am 34 and am 51 respectively. We then compared each of these revertants with wild-type T4 with respect to the yield produced in mixed infections with the neighboring am mutant strain. We repeated the cis-trans tests at the same time to provide a further point of comparison. The results of these tests (Table 5 ) show that reversion of each amber to an+ restored the full level of activity of its neighboring gene.

9 POSITION EFFECT IN PHAGE 23 1 TABLE 5 Properties of revertants of ambers 51 and 34.rest Experiment 1 Experiment 2 27 x x 51 revertant x wild x 51 revertant x wild Expeiinient 3 Experiment 3 35 x x 34revertant x wild x 34 revertant x wild Tests were carried out as usual The revertants were picked from stocks of amber 51 and 34 respectively following plating of the stocks on E coli strain I3 On the basis of this result, we conclude that the am mutants per se in genes 51 and 34 depress the levels of activity of genes 27 and 35 respectively. DISCUSSION In the introduction two plausible explanations for the intergenic cis-trans position effects were proposed. Prior to discussing their relative merits, however, we may properly inquire whether the mutations involved are really in two different genes (i.e., cistrons). The operational definition of a cistron (BENZER 1957) in this case is somewhat ambiguous. Intermediate degrees of complementation could imply rather good intracistronic complementation or rather poor intercistronic complementation. In the case of phage, complementation tests have an additional ambiguity. Intracistronic recombination early enough in the latent period might produce wild-type alleles which make sufficient protein to result in a (small) phage yield. From our data alone, it is not possible to construct a tight argument that our cases of poor complementation represent bad intercistronic complementation. We shall forego the possible plausible arguments in favor of arguments which depend on information from other experiments. Intracistronic complementation between ambers appears impossible in view of the chain-terminating nature of am mutations ( SARABHAI, STRETTON, BRENNER, and BOLLE 1964). As a general explanation recombination is ruled out by observations (EDGAR and WOOD 1966) that ambers in cistrons 34 and 35 complement in uitro. We may now turn to the two possible explanations for poor intercistronic complementation with some amber mutants. The assumption that poor complementation results from one of the two am mutants being a polar mutant makes a strong prediction regarding the outcome of the experiments with the ts mutants. It predicts that one of the ts x am complementations should be good while the other should be as poor as the am x am case.

10 232 F. w. STAHL et al. This prediction is fulfilled. Only with ad hoc postulates can we fit these results with the unstable product explanation for the cis-trans effect. The experiments and considerations described render the following statements likely to be true: (1) Some pairs of neighboring genes are cotranscribed in T4. (2) The direction of transcription and translation of each of the late genes 51, 27,34 and 35 is clockwise on the conventional map (EPSTEIN et al. 1963). The first of these two conclusions is compatible with the observations of ASANO (1965) on the molecular weight distribution of T2 messenger RNA. The second conclusion may become of interest when analogous information for a large number of the T4 genes becomes available. RICHARD RUSSELL and GEORGE STREISINGER suggested important controls. R. S. EDGAR and generously provided the T4 mutants employed. The invaluable moderating influence of M. M. STAHL and GEORGE STREISINGER is gratefully acknowledged. SUMMARY We have demonstrated intercistronic cis-trans position effects between the members of two pairs of amber mutants in T4. In each case we conclude that one of the two ambers is a polar mutant. ASANO, K., 1965 LITERATURE CITED Size heterogeneity of T2 messenger RNA. J. Mol. Biol. 14: BENZER, S., 1955 Fine structure of a genetic region in bacteriophage. Proc. Natl. Acad. Sci. U.S. 41: The elementary units of heredity. pp The Chemical Basis of Heredity. Edited by W. D. MCELR~Y and B. GLASS. Johns Hopkins Press, Baltimore. EDGAR, R. S., G. H. DENHARDT, and R. H. EPSTEIN, A comparative genetic study of conditional lethal mutations of bacteriophage T4D. Genetics 49: EDGAR, R. S., and W. B. WOOD, 1966 Morphogenesis of bacteriophage T4 in extracts of mutantinfected cells. Proc. Natl. Acad. Sci. U.S. 55: EPSTEIN, R. H., A. BOLLE, C. M. STEINBERG, E. KELLENBERGER, E. BOY DE LA TOUR, R. CHEVALLEY, R. S. EDGAR, M. SUSMAN, G. H. DENWARDT, and A. LIELAUSIS, 1963 Physiological studies of conditional lethal mutations of bacteriophage T4D. Cold Spring Harbor Symp. Quant. Biol. 28: LEWIS, E. B., 1951 Pseudoallelism and gene evolution. Cold Spring Harbor Symp. Quant. Biol. 16: NEWTON, W. A., J. R. BECKWITH, D. ZIPSER, and, 1965 Nonsense mutants and polarity in the Lac operon of Escherichia coli. J. Mol. Biol. 14: SARABHAI, A. S., A. 0. W. STRE,, and A. BOLLE, CO-linearity of the gene with the polypeptide chain. Nature 201 : STAHL, F. W., and N. E. MURRAY, 1966 The evolution of gene clusters and genetic circularity in microorganism. Genetics 53 :