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Repressor synthesis in vivo after infection of E. coli by a prm− mutant of bacteriophage λ
VIROLOGY
63,273~277(1975)
SHORT Repressor
COMMUNICATIONS
Synthesis
by a P&
in Viva After
Mutant
Infection
of Bacteriophage
GARY N. GUSSIN’ AND KWANG-MU
of E. co/i X
YEN
Department of Zoology, University of Iowa, Iowa City, Iowa AND
LOUIS F. REICHARDT’ Department of Molecular Biology, University of Geneva, Geneva, Switzerland Accepted September 9, 1974 Genetic-complementation experiments have shown previously that the mutation prmlls, which maps near the right operator in phage lambda, inactivates the prm promoter but not the pr. promoter for c1 (repressor) gene expression. Direct assays for repressor synthesis in vivo confirm these conclusions: first, Xp,,,,, does not synthesize repressor after infection of an immune lysogen, when prm should normally be active; second, activation of pr. by cI1 and ~111proteins after infection of nonlysogenic cells is not affected by thep,,,,, mutation. Unexpectedly, however, Xp,,,,, does not shut off repressor synthesis at the normal time after infection of a nonlysogen, so that, in fact; repressor levels late in infection with hp,,,,, are actually greater than those observed after wild-type infection. Since cro gene product is required to shut off repressor synthesis under these conditions, we speculate that the prmlls mutation may also alter a cro target site near the right operator.
thus ultimately turns off c1 transcription from pre. A second promoter for c1 transcription, prm (the maintenance promoter) is functional in the presence of active repressor. Transcription from this promoter-apparently at a lower rate than from pre (3)-is necessary to maintain repressor levels in an immune lysogen and may actually require the presence of repressor as a positive regulator (see Refs. 7-9, for conflicting views). Transcription from both promoters can be prevented by the action of the X cro product. By binding near pL and pR (10-12, 16), cro product blocks cI1 and ~111gene expression; since cI1 and ~111proteins are unstable (9, 12), this ultimately causes a ‘To whom all communications should be ad- shut-off of p,,-promoted transcription of c1. In addition, cro product directly blocks c1 dressed. transcription initiated at prm (13-16) prob*Present address, Department of Neurobiology, ably by binding near this promoter (Fig. 1). Harvard Medical School, Boston, Massachusetts.
The X phage repressor promotes lysogenization by binding at two operators, OR,and oL (Fig. l), thereby blocking transcription initiated at two promoters, pR and pL, and directly or indirectly preventing expression of almost all other X genes (see Refs. 1, 2). Synthesis of the repressor is regulated in a complex way; transcription of the c1 gene, which codes for the repressor, can be initiated at either of two promoters depending on which phage gene products are present (3, 4). After infection of a sensitive cell (no repressor present), c1 transcription is initiated at pre (the establishment promoter) and requires the products of genes cII and cIII (3-5). The action of repressor at oRand oL blocks expression of cI1 and ~111(6) and
273 Copyright 0 1975by AcademicPress,Inc. All rights of reproductionin any form reserved.
274
SHORT
COMMUNICATIONS
FIG. 1. The regulatory region of X. Repressor encoded by the c1 gene, acts at operators 9. and os to prevent transcription of major early messages initiated at pt and ps, respectively (I, 22). The synthesis of c1 mRNA, which also includes the message for the rer gene (17, 20), proceeds from left to right on the X map (21) and can be initiated at either of two promoters, prmor pre (3, 4). The products of genes cI1 and cIII, which are required for initiation of c1 transcription at pre (3-5), are not expressed by the prophage (6). Expression of the c1 gene in a lysogen requires the continued presence of repressor (15, 22), perhaps as a positive regulator of transcription (3, 7-9), and is initiated at prm. Cro product binds near ot to turn off major leftward transcription of the N operon (10, 11, 16, 23) and perhaps near os to turn off rightward transcription from pR (II, 16, 24). In addition, cro product can prevent leftward transcription from prm (9, 13, 15, 16). Although P~,,,,~~ maps to the left of known mutations in os (17), it is possible that pm , oa, pR, and the putative cro binding site overlap. Wavy lines indicate X messenger RNA’s. Shaded segments represent control sites in h DNA.
tally to bind radioactive X DNA to Millipore filters. The second assay measures the ability of repressor antigen in crude extracts to compete with purified IzsIrepressor for binding sites on antirepressor antibodies. Both assays have been described previously (3, 18, 19). Repressor synthesis after infection of a Xc1857 lysogen by either X or Ap,,,,, was monitored using the DNA binding assay (Fig. 2). Normally, superinfection of a lysogen stimulates repressor synthesis by a factor of two to four under the conditions used for this experiment. However, the ~I857 temperature-sensitive repressor binds poorly to X DNA in vitro (3, 26), so binding activity attributable to the preex25
0
10
20
30
40
50
Finally, it has not been excluded that high Time After lnfechon lmtn) levels of cro product might also directly FIG. 2. Repressor synthesis after infection of E. inhibit c1 transcription by preventing a coli K12 (XcI857). After infection of strain W3102 fraction of the transcription complexes (XcI857) either with X or Ap,,,,,, cultures were samformed at pre from progressing through (oR, pled at the indicated times, pelleted, sonicated, and assayed for repressor in the DNA binding assay (3,18, pR) and into ~1. Previously, we described a mutant, 19). Cells were grown at 30’ in TY Medium (0.2% prmlls that possessesgenetic properties ex- Maltose, 0.01% yeast extract, 0.25% NaCl, 1.0% pected of ap,,- mutant (17). Here we show Bacto-tryptone) to a titer of 2 or 3 x 108/ml, chilled, that the effects of prmlls on repressor syn- pelleted, and resuspended in one-tenth vol of 0.01 M pH 7.4, 0.01 M MgSO,. Phage were added at a thesis in uiuo are consistent with its genetic Tris, multiplicity of about five per cell and allowed to properties. Xp,,,,, fails to synthesize re- adsorb for 30 min at 0”. At time zero, cultures were pressor on infection of a lysogen, when only rediluted lo-fold into fresh TY Medium and aeration the prm pathway for c1transcription should was begun at 30”. In this particular experiment, cells be active, but produces high levels of were infected at a titer of 3.2 x 10Vml with 6.2 phage repressor on infection of a sensitive cell, per cell. Binding activity is corrected for nonspecific binding by subtracting activity for 8zP-Ximm21 DNA when the pre pathway should predominate. Moreover, repressor assays have revealed binding to filters. One unit of binding activity is an unexpected additional effect of prmIls. defined as activity necessary to bind 33.3 ng of S2P-h On infection of a sensitive cell by Aprmlls, DNA (one-third of the DNA present) to a Millipore after 7 min incubation at 30”. Protein in crude repressor synthesis continues for a much filter extracts was assayed by the method of Lowry et al. longer time than it does after wild-type (25). (x), infection with X wild type; (0), infection infection. (0) and dotted line represent steadywith ~~~~~~~~ Assays of repressor synthesis after phage state level of repressor binding activity in the unininfection are of two types. One assay is fected lysogen, sampled in parallel with infected based on the ability of repressor specifi- cultures at 30 min.
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isting ~I857 repressor is very low in these infections.g Thus, the level of repressor binding activity detected after superinfection of the mutant lysogen by wild-type X is about five times that detected in the uninfected lysogen. In contrast, Xp,,,,, does not raise the level of repressor binding activity after it infects the same lysogen. In other words, AP,, , Is synthesizes no detectable repressor on infection of lysogenic cells, in which the prm pathway should normally be active. (The effect ofp,,I1s cannot be due to the production of an inhibitor of repressor synthesis since Ap,,,,, can form double lysogens with cI+ pr,,,+ phage (17). In addition, the level of repressor produced by the prophage is unaffected by superinfection with prmlla, at least in the experiment shown in Fig. 2.) In the experiment illustrated in Fig. 3, the appearance of repressor after infection of nonlysogenic cells by X or Ap,,,,, was assayed using the antigen-competition assay. As expected, cI1 and ~111proteins can promote efficient p,,-directed repressor synthesis by either phage. There is little difference in repressor synthesis for the first 30 min after infection by the two phages at 30”. However, after 30 min, repressor synthesis begins to level off in cells infected with wild-type phage, but continues at a high rate in cells infected with AP,, 1l8. In some experiments, the DNA binding assay has also been used, with essentially identical results. Previously (17), genetic evidence showed that prmlle exerts a &-specific effect on expression of both c1 and rex, a gene that is cotranscribed with ~1. This cI- phenotype of prmlla was shown, however, to be restricted to situations (6) in which ~11and ~111 are not expressed. On infection of SPreviously it was reported (3, 26) that ~1857 (temperature-sensitive) repressor is unable to bind X DNA in vitro. However, the results in Fig. 2 indicate an appreciable level of binding of ~I857 repressor in our experiments, under assay conditions identical to those used previously (3). We consistently observed binding of ~1857 repressor even during in oitro incubation at 30”; this binding was estimated to be 20-30s as efficient as binding of wild-type repressor.
0 Time After Infectnon knin)
FIG. 3. Repressor synthesis after infection of nonlysogenic E. coli K12. After infection of strain W3102, either with X or Aprmlls,at 30”, cultures were sampled at the indicated times, pelleted, sonicated, and assayed for repressor antigen in the radioimmune competition assay (3). The infection protocol was similar to that described in the legend to Fig. 2. In this particular experiment, cells were infected at a titer of 1.6 x lOa cells/ml with 6.4 phage per cell. (O), infection with X wild-type; (x), infection with Xp,,,,,. Repressor levels obtained in Iowa City (this Figure) are always two to three times as great as those observed in Geneva (see Table 1) or at Stanford (Ref. 3). We have no explanation for these differences.
sensitive cells, prmlls does complement, for example, the pre- mutant, Xcy42. Direct repressor assays confirm the basic conclusions deduced from genetic evidence: APnnll8 is unable to synthesize repressor after infection of a lambda lysogen, when the prm pathway for repressor synthesis should normally be active, but does direct repressor synthesis after infection of sensitive cells, when the pre pathway should be active. These results together with genetic evidence strongly support the hypothesis that prmlls alters the maintenance promoter prm. Results of genetic-complementation tests (17) have shown that this mutation is dominant in cis and recessive in tram, and thus behaves as if it alters a site at which repressor synthesis is controlled. The repressor assays reported here show that the &-specific effect of prmlls on repressor and rex gene expression can not be due to a polar mutation in ~1,because such a mutation would have to affect both the establishment and maintenance pathways of repressor synthesis. Nor can prmlls be a c1 mutation that reduces the binding affinity
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There are several possible explanations for the intriguing observation that Aprmlls does not turn off repressor synthesis during infection of sensitive cells (Fig. 3). Since Infecting Assay used Repressor Ratio” phage” activityb the kinetics of repressor synthesis are very similar to kinetics observed on infection by x Antigen-compe654 kg/g a prm+ cro- phage (3), Ap,,,,, might contition 0.21 tain a second mutation in the cro gene. h DNA binding 140 units/mg Although we have no genetic evidence to Antigen-compe954 fig/g UP,,,,, rule out this possibility, it seem unlikely 0.20 tition since preliminary experiments (data not DNA binding 166 unitslmg AP,,,,, shown) indicate that the failure of Ap,,,,, a Cells were infected as described in the legend to to turn off repressor synthesis is cis-speFigs. 2 and 3. In this particular experiment, E. cob cific. Coinfection with a cro+ phage does W3102 was infected at a density of 2.3 x lOa ml, at a not result in normal shut-off of repressor multiplicity of 4.3 phage per cell. Infected cultures synthesis. In addition, whereas cro- muwere sampled at 25 min after infection. Comparison of tants containing a temperature-sensitive c1 these data with the 25-min points in Fig. 3 indicates mutation fail to form plaques at temperaan approximate 2-fold difference between repressor tures above 40” (13, 23), Xc1857 prmlls antigen detected in this experiment performed in plates well at these temperatures. Geneva, and that illustrated in Fig. 3, performed in A second explanation of the effect of Iowa. prmlls on the turn-off of repressor synthesis bAntigen assay units: pg repressor antigen/gram soluble cell protein; DNA binding assay units are might be that prmlIs alters a cro target defined in legend to Fig. 2. site which overlaps prm. In this case, rec Binding unita/10m3 pg repressor antigen. pressor synthesis could continue either beof the CI protein for DNA to the extent that cause of continued expression of ~11, or the low repressor level maintained in a because cro product fails to interfere with the progress of transcription complexes lysogen becomes ineffective in maintaining immunity. Complementation experiments initiated at pre. Proof that prmlls actually (17) have already shown that prmlls pre- did alter a cro binding site would provide vents cis expression of the rex gene even in specific evidence, previously unavailable, the presence of repressor supplied by an- for the location of one cro binding site near other phage. prrn and 0,. Several alternative explanations for the The ratio of DNA-binding activity to activity in the antigen competition assay cro- phenotype of Ap,,,,, can be imagined, is virtually the same for wild-type and particularly those involving undetected prmlls repressors (Table 1). This result secondary mutations. Experiments indemonstrates that the prmlls mutation does tended to test the various possibilities are not reduce the affinity with which prmlls in progress. repressor binds to DNA in vitro. FurtherACKNOWLEDGMENTS more, genetic experiments showed that prmlls does not reduce the efficiency with This work was supported by USPHS Grant AI-10019 and by a Grant from the Swiss National which wild-type repressor blocks rightward transcription from pR by binding at oR(17). Fund to H. Eisen. L. R. was a recipient of a fellowship Therefore, it is unlikely that prmlls pre- from the Jane Coffin Childs Memorial Fund for vents repressor from maintaining c1expres- Medical Research. We are indebted to Ms. Kathleen sion (7-9) either by affecting repressor Williams for excellent technical assistance and to Dr. Eisen for support of the work done in Geneva. itself or by altering a putative target site for its regulation of c1 transcription.’ REFERENCES TABLE
1
COMPARISON OFDNA-BINDING
ACTIVITIES OF WILD-TYPE AND prmlls REPRESSOR
‘This conclusion is confirmed by unpublished experiments of S. Flashman (personal communication), showing that prmlls does not affect weak repressor binding (27) at 0s.
I. PTASHNE, M., and HOPKINS, N., Proc. Nat. Acad. Sci. USA 60, 1282 (1968). 2. THOMAS, R., in “The Bacteriophage Lambda” (A. D. Hershey, ed.) p. 211, Cold Spring Harbor
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SHORT COMMUNICATIONS Laboratory, Cold Spring Harbor, NY, 1971. 3. REICHARDT, L., and KAISER, A. D., Proc. Nat. Acad. Sci. USA 68,2185 (1971). 4. STEIGELMAN, W. G., REICHARDT, L. F., YANIV, M., HEINEMANN, S. F., KAISER, A. D., and EISEN, H.,
15. CASTELLAZZI, M., BRACHET, P., and EISEN, H., Mol. Gen. Genet. 117, 211 (1972). 16. REICHARDT, L., submitted toJ. Mol. Biol. (1974b). 17. YEN, K.-M., and GUSSIN, G., Virology 56, 300
Proc. Nat. Acad. Sci. USA 69, 3156 (1972). 5. ECHOLS, H., and GREEN, L., Proc. Nat. Acad. Sci. USA 68, 2190 (1971). 6. BODE, V. C., and KAISER, A. D., Virology 25, 111
18. ORDAL, G., in “The Bacteriophage Lambda” (A.
(1973).
19.
(1965). 7. HEINEMANN, S., and SPIEGELMAN, W., Proc. Nat. Acad. Sci. USA 67, 1122 (1970). 8. HAYES, S., and SZYBALSKI, W., Mol. Gen. Genet. 126, 275 (1973). 9. REICHARDT, L., submitted toJ. Mol. Biol. (1974a). 10. SLY, W. S., RABIDEAU, K., and KOLBER, K., in “The Bacteriophage Lambda” (A. D. Hershey, ed.), p. 575, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1971. 11. ECHOLS, H., GREEN, L., OPPENHEIM, A. B., OPPENHEIM, A., and HONIGMAN, A., J. Mol. Biol. 80, 203 (1973). 12. REICHARDT, L., Ph.D. Thesis, Stanford Univ., 1972. 13. EISEN, H., BRACHET, P., PEREIRA DA SILVA, L., and JACOB, F., Proc. Nat. Acad. Sci. USA 66, 855 (1970). 14. NEUBAUER, Z., and CALEF, E., J. Mol. Biol. 51, 1 (1970).
20. 21. 22.
23.
24. 25.
26. 27.
D. Hershey, ed.), p. 565, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1971. RIGGS, A. D., and BOURGEOIS,S., J. Mol. Biol. 34, 361 (1968). ASTRACHAN, L., and MILLER, J. F., J. Viral. 9, 510 (1972). TAYLOR, K., HRADECNA, Z., and SZYBALSKI, W., Proc. Nat. Acad. Sci. USA 57, 1618 (1967). EISEN, H., PEREIRA DA SILVA, L., and JACOB, F., C. R. Acad. Sci. Ser. D 266, 1176 (1968). FRANKLIN, N., in “The Bacteriophage Lambda” (A. D. Hershey, ed.), p. 621, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1971. BERG, D., submitted to Virology (1974). LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). SHINEGAWA, H., and ITOH, T., Mol. Gen. Geqet. 126, 103 (1973). MANIATIS, T., PTASHNE, M., and MALJRER,R., Cold Spring
(1973).
Harbor
Symp.
Quant.
Biol.
38, 857
63,273~277(1975)
SHORT Repressor
COMMUNICATIONS
Synthesis
by a P&
in Viva After
Mutant
Infection
of Bacteriophage
GARY N. GUSSIN’ AND KWANG-MU
of E. co/i X
YEN
Department of Zoology, University of Iowa, Iowa City, Iowa AND
LOUIS F. REICHARDT’ Department of Molecular Biology, University of Geneva, Geneva, Switzerland Accepted September 9, 1974 Genetic-complementation experiments have shown previously that the mutation prmlls, which maps near the right operator in phage lambda, inactivates the prm promoter but not the pr. promoter for c1 (repressor) gene expression. Direct assays for repressor synthesis in vivo confirm these conclusions: first, Xp,,,,, does not synthesize repressor after infection of an immune lysogen, when prm should normally be active; second, activation of pr. by cI1 and ~111proteins after infection of nonlysogenic cells is not affected by thep,,,,, mutation. Unexpectedly, however, Xp,,,,, does not shut off repressor synthesis at the normal time after infection of a nonlysogen, so that, in fact; repressor levels late in infection with hp,,,,, are actually greater than those observed after wild-type infection. Since cro gene product is required to shut off repressor synthesis under these conditions, we speculate that the prmlls mutation may also alter a cro target site near the right operator.
thus ultimately turns off c1 transcription from pre. A second promoter for c1 transcription, prm (the maintenance promoter) is functional in the presence of active repressor. Transcription from this promoter-apparently at a lower rate than from pre (3)-is necessary to maintain repressor levels in an immune lysogen and may actually require the presence of repressor as a positive regulator (see Refs. 7-9, for conflicting views). Transcription from both promoters can be prevented by the action of the X cro product. By binding near pL and pR (10-12, 16), cro product blocks cI1 and ~111gene expression; since cI1 and ~111proteins are unstable (9, 12), this ultimately causes a ‘To whom all communications should be ad- shut-off of p,,-promoted transcription of c1. In addition, cro product directly blocks c1 dressed. transcription initiated at prm (13-16) prob*Present address, Department of Neurobiology, ably by binding near this promoter (Fig. 1). Harvard Medical School, Boston, Massachusetts.
The X phage repressor promotes lysogenization by binding at two operators, OR,and oL (Fig. l), thereby blocking transcription initiated at two promoters, pR and pL, and directly or indirectly preventing expression of almost all other X genes (see Refs. 1, 2). Synthesis of the repressor is regulated in a complex way; transcription of the c1 gene, which codes for the repressor, can be initiated at either of two promoters depending on which phage gene products are present (3, 4). After infection of a sensitive cell (no repressor present), c1 transcription is initiated at pre (the establishment promoter) and requires the products of genes cII and cIII (3-5). The action of repressor at oRand oL blocks expression of cI1 and ~111(6) and
273 Copyright 0 1975by AcademicPress,Inc. All rights of reproductionin any form reserved.
274
SHORT
COMMUNICATIONS
FIG. 1. The regulatory region of X. Repressor encoded by the c1 gene, acts at operators 9. and os to prevent transcription of major early messages initiated at pt and ps, respectively (I, 22). The synthesis of c1 mRNA, which also includes the message for the rer gene (17, 20), proceeds from left to right on the X map (21) and can be initiated at either of two promoters, prmor pre (3, 4). The products of genes cI1 and cIII, which are required for initiation of c1 transcription at pre (3-5), are not expressed by the prophage (6). Expression of the c1 gene in a lysogen requires the continued presence of repressor (15, 22), perhaps as a positive regulator of transcription (3, 7-9), and is initiated at prm. Cro product binds near ot to turn off major leftward transcription of the N operon (10, 11, 16, 23) and perhaps near os to turn off rightward transcription from pR (II, 16, 24). In addition, cro product can prevent leftward transcription from prm (9, 13, 15, 16). Although P~,,,,~~ maps to the left of known mutations in os (17), it is possible that pm , oa, pR, and the putative cro binding site overlap. Wavy lines indicate X messenger RNA’s. Shaded segments represent control sites in h DNA.
tally to bind radioactive X DNA to Millipore filters. The second assay measures the ability of repressor antigen in crude extracts to compete with purified IzsIrepressor for binding sites on antirepressor antibodies. Both assays have been described previously (3, 18, 19). Repressor synthesis after infection of a Xc1857 lysogen by either X or Ap,,,,, was monitored using the DNA binding assay (Fig. 2). Normally, superinfection of a lysogen stimulates repressor synthesis by a factor of two to four under the conditions used for this experiment. However, the ~I857 temperature-sensitive repressor binds poorly to X DNA in vitro (3, 26), so binding activity attributable to the preex25
0
10
20
30
40
50
Finally, it has not been excluded that high Time After lnfechon lmtn) levels of cro product might also directly FIG. 2. Repressor synthesis after infection of E. inhibit c1 transcription by preventing a coli K12 (XcI857). After infection of strain W3102 fraction of the transcription complexes (XcI857) either with X or Ap,,,,,, cultures were samformed at pre from progressing through (oR, pled at the indicated times, pelleted, sonicated, and assayed for repressor in the DNA binding assay (3,18, pR) and into ~1. Previously, we described a mutant, 19). Cells were grown at 30’ in TY Medium (0.2% prmlls that possessesgenetic properties ex- Maltose, 0.01% yeast extract, 0.25% NaCl, 1.0% pected of ap,,- mutant (17). Here we show Bacto-tryptone) to a titer of 2 or 3 x 108/ml, chilled, that the effects of prmlls on repressor syn- pelleted, and resuspended in one-tenth vol of 0.01 M pH 7.4, 0.01 M MgSO,. Phage were added at a thesis in uiuo are consistent with its genetic Tris, multiplicity of about five per cell and allowed to properties. Xp,,,,, fails to synthesize re- adsorb for 30 min at 0”. At time zero, cultures were pressor on infection of a lysogen, when only rediluted lo-fold into fresh TY Medium and aeration the prm pathway for c1transcription should was begun at 30”. In this particular experiment, cells be active, but produces high levels of were infected at a titer of 3.2 x 10Vml with 6.2 phage repressor on infection of a sensitive cell, per cell. Binding activity is corrected for nonspecific binding by subtracting activity for 8zP-Ximm21 DNA when the pre pathway should predominate. Moreover, repressor assays have revealed binding to filters. One unit of binding activity is an unexpected additional effect of prmIls. defined as activity necessary to bind 33.3 ng of S2P-h On infection of a sensitive cell by Aprmlls, DNA (one-third of the DNA present) to a Millipore after 7 min incubation at 30”. Protein in crude repressor synthesis continues for a much filter extracts was assayed by the method of Lowry et al. longer time than it does after wild-type (25). (x), infection with X wild type; (0), infection infection. (0) and dotted line represent steadywith ~~~~~~~~ Assays of repressor synthesis after phage state level of repressor binding activity in the unininfection are of two types. One assay is fected lysogen, sampled in parallel with infected based on the ability of repressor specifi- cultures at 30 min.
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isting ~I857 repressor is very low in these infections.g Thus, the level of repressor binding activity detected after superinfection of the mutant lysogen by wild-type X is about five times that detected in the uninfected lysogen. In contrast, Xp,,,,, does not raise the level of repressor binding activity after it infects the same lysogen. In other words, AP,, , Is synthesizes no detectable repressor on infection of lysogenic cells, in which the prm pathway should normally be active. (The effect ofp,,I1s cannot be due to the production of an inhibitor of repressor synthesis since Ap,,,,, can form double lysogens with cI+ pr,,,+ phage (17). In addition, the level of repressor produced by the prophage is unaffected by superinfection with prmlla, at least in the experiment shown in Fig. 2.) In the experiment illustrated in Fig. 3, the appearance of repressor after infection of nonlysogenic cells by X or Ap,,,,, was assayed using the antigen-competition assay. As expected, cI1 and ~111proteins can promote efficient p,,-directed repressor synthesis by either phage. There is little difference in repressor synthesis for the first 30 min after infection by the two phages at 30”. However, after 30 min, repressor synthesis begins to level off in cells infected with wild-type phage, but continues at a high rate in cells infected with AP,, 1l8. In some experiments, the DNA binding assay has also been used, with essentially identical results. Previously (17), genetic evidence showed that prmlle exerts a &-specific effect on expression of both c1 and rex, a gene that is cotranscribed with ~1. This cI- phenotype of prmlla was shown, however, to be restricted to situations (6) in which ~11and ~111 are not expressed. On infection of SPreviously it was reported (3, 26) that ~1857 (temperature-sensitive) repressor is unable to bind X DNA in vitro. However, the results in Fig. 2 indicate an appreciable level of binding of ~I857 repressor in our experiments, under assay conditions identical to those used previously (3). We consistently observed binding of ~1857 repressor even during in oitro incubation at 30”; this binding was estimated to be 20-30s as efficient as binding of wild-type repressor.
0 Time After Infectnon knin)
FIG. 3. Repressor synthesis after infection of nonlysogenic E. coli K12. After infection of strain W3102, either with X or Aprmlls,at 30”, cultures were sampled at the indicated times, pelleted, sonicated, and assayed for repressor antigen in the radioimmune competition assay (3). The infection protocol was similar to that described in the legend to Fig. 2. In this particular experiment, cells were infected at a titer of 1.6 x lOa cells/ml with 6.4 phage per cell. (O), infection with X wild-type; (x), infection with Xp,,,,,. Repressor levels obtained in Iowa City (this Figure) are always two to three times as great as those observed in Geneva (see Table 1) or at Stanford (Ref. 3). We have no explanation for these differences.
sensitive cells, prmlls does complement, for example, the pre- mutant, Xcy42. Direct repressor assays confirm the basic conclusions deduced from genetic evidence: APnnll8 is unable to synthesize repressor after infection of a lambda lysogen, when the prm pathway for repressor synthesis should normally be active, but does direct repressor synthesis after infection of sensitive cells, when the pre pathway should be active. These results together with genetic evidence strongly support the hypothesis that prmlls alters the maintenance promoter prm. Results of genetic-complementation tests (17) have shown that this mutation is dominant in cis and recessive in tram, and thus behaves as if it alters a site at which repressor synthesis is controlled. The repressor assays reported here show that the &-specific effect of prmlls on repressor and rex gene expression can not be due to a polar mutation in ~1,because such a mutation would have to affect both the establishment and maintenance pathways of repressor synthesis. Nor can prmlls be a c1 mutation that reduces the binding affinity
276
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COMMUNICATIONS
There are several possible explanations for the intriguing observation that Aprmlls does not turn off repressor synthesis during infection of sensitive cells (Fig. 3). Since Infecting Assay used Repressor Ratio” phage” activityb the kinetics of repressor synthesis are very similar to kinetics observed on infection by x Antigen-compe654 kg/g a prm+ cro- phage (3), Ap,,,,, might contition 0.21 tain a second mutation in the cro gene. h DNA binding 140 units/mg Although we have no genetic evidence to Antigen-compe954 fig/g UP,,,,, rule out this possibility, it seem unlikely 0.20 tition since preliminary experiments (data not DNA binding 166 unitslmg AP,,,,, shown) indicate that the failure of Ap,,,,, a Cells were infected as described in the legend to to turn off repressor synthesis is cis-speFigs. 2 and 3. In this particular experiment, E. cob cific. Coinfection with a cro+ phage does W3102 was infected at a density of 2.3 x lOa ml, at a not result in normal shut-off of repressor multiplicity of 4.3 phage per cell. Infected cultures synthesis. In addition, whereas cro- muwere sampled at 25 min after infection. Comparison of tants containing a temperature-sensitive c1 these data with the 25-min points in Fig. 3 indicates mutation fail to form plaques at temperaan approximate 2-fold difference between repressor tures above 40” (13, 23), Xc1857 prmlls antigen detected in this experiment performed in plates well at these temperatures. Geneva, and that illustrated in Fig. 3, performed in A second explanation of the effect of Iowa. prmlls on the turn-off of repressor synthesis bAntigen assay units: pg repressor antigen/gram soluble cell protein; DNA binding assay units are might be that prmlIs alters a cro target defined in legend to Fig. 2. site which overlaps prm. In this case, rec Binding unita/10m3 pg repressor antigen. pressor synthesis could continue either beof the CI protein for DNA to the extent that cause of continued expression of ~11, or the low repressor level maintained in a because cro product fails to interfere with the progress of transcription complexes lysogen becomes ineffective in maintaining immunity. Complementation experiments initiated at pre. Proof that prmlls actually (17) have already shown that prmlls pre- did alter a cro binding site would provide vents cis expression of the rex gene even in specific evidence, previously unavailable, the presence of repressor supplied by an- for the location of one cro binding site near other phage. prrn and 0,. Several alternative explanations for the The ratio of DNA-binding activity to activity in the antigen competition assay cro- phenotype of Ap,,,,, can be imagined, is virtually the same for wild-type and particularly those involving undetected prmlls repressors (Table 1). This result secondary mutations. Experiments indemonstrates that the prmlls mutation does tended to test the various possibilities are not reduce the affinity with which prmlls in progress. repressor binds to DNA in vitro. FurtherACKNOWLEDGMENTS more, genetic experiments showed that prmlls does not reduce the efficiency with This work was supported by USPHS Grant AI-10019 and by a Grant from the Swiss National which wild-type repressor blocks rightward transcription from pR by binding at oR(17). Fund to H. Eisen. L. R. was a recipient of a fellowship Therefore, it is unlikely that prmlls pre- from the Jane Coffin Childs Memorial Fund for vents repressor from maintaining c1expres- Medical Research. We are indebted to Ms. Kathleen sion (7-9) either by affecting repressor Williams for excellent technical assistance and to Dr. Eisen for support of the work done in Geneva. itself or by altering a putative target site for its regulation of c1 transcription.’ REFERENCES TABLE
1
COMPARISON OFDNA-BINDING
ACTIVITIES OF WILD-TYPE AND prmlls REPRESSOR
‘This conclusion is confirmed by unpublished experiments of S. Flashman (personal communication), showing that prmlls does not affect weak repressor binding (27) at 0s.
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