Proc. Nat. Acad. Sci. USA Vol. 68, No. 9, pp. 2190-2194, September 1971

Establishment and Maintenance of Repression by Lambda: The Role of the ci, cit, and ciII Proteins (E. coli/lysogeny versus lysis/cy- mutants/DNA binding)

HARRISON ECHOLS AND LINDA GREEN Department of , University of California, Berkeley, Calif. 94720 Communicated by A. D. Kaiser, July 8, 1971

ABSTRACT To define the events necessary for the provided by the cI protein; for example, an inhibition of establishment and maintenance of repression in a X- of late proteins might delay the late stage of lytic infected cell, we have studied the requirements for efficient synthesis synthesis of the cI protein ("X-repressor"). Three classes development until cl-mediated repression can take over (3). of X mutants defective in the establishment of repression Another plausible mechanism involves timing the estab- are also defective in the appearance of cI protein activity lishment of repression by the time of synthesis of cI protein; at the normal time. Two of these mutational classes (cII- this possibility is suggested by the finding that the syn- and cIII-) probably result from inactivation of X-specified proteins, but the third class (cy-) may involve a structural thesis of cI protein is subject to regulation (4). defect. We conclude that at least three regulatory elements Since active clI and cII genes are essential for the effective are likely to be required for the normal turn-on of cI pro- establishment of lysogeny in an infected cell, but not for the tein synthesis in an infected nonlysogenic cell: clI and maintenance of lysogeny (5), the cII and cIII proteins are cIII proteins and an "active" y-region of X DNA. From likely to function as critical timing elements in the establish- these and other results, the complete role of cII and cIII proteins in the establishment of repression may involve a ment of repression. As a result of previous experimental ef- bifunctional regulatory activity: positive regulation of the forts to understand the role of the cII and cIII genes, we cI gene and negative regulation of late genes. A possible provided indirect evidence that the cII and cIII proteins molecular model for cII and cIII action is discussed. Since perform both "activities" indicated above: an inhibition of the cII and cIII genes are repressed by the cI protein under conditions of stable lysogeny, a separate mechanism is required for the maintenance of cI protein synthesis. After infection of a lysogen by c11- phage, the rate of in- crease of cI protein activity is substantially greater than after infection of a nonlysogen. From these and other int cM N cIcdHOP Q results, the cI protein may also have a bifunctional regu- Head Tail Recomb Reg DNA Lysis latory activity: positive regulation of the cI gene and * A^0 negative regulation of early lytic genes. ______-0 After infection by the temperature phage X, the establishment FIG. 1. Genetic and functional map of X DNA. Genes with of lysogeny requires two functionally separate events: a related function exhibit extensive clustering along the X DNA. repression of the capacity of the phage DNA for lytic growth, molecule. This is indicated on the diagram by "Head" for genes recombination event that concerned with the structure of the phage head; "Tail" for genes and a site-specific genetic integrates "Recomb" involved with DNA at a site. The concerned with tail structure; for genes the viral DNA into the host specific general and site-specific recombination; "Reg" for genes exerting maintenance of the lysogenic state is inherently a simpler a regulatory function in lytic development or lysogeny; "DNA". phenomenon than its establishment; the repression of lytic for genes specifying replication proteins; and "Lysis" for genes capacity must simply be maintained, and no recombination concerned with cell lysis. Specific genes of the "regulation re- events are involved. gion"-cIII, N, cI, cII-are indicated above the "X DNA", as The maintenance of repression for phage X is probably ac- are the integrative recombination gene int, the DNA replication complished by one phage protein-the ci protein (or "X- genes OP, and the late regulator gene, Q. repressor")-which acts to repress RNA synthesis from critical Approximate DNA regions transcribed and the direction of early genes required for viral DNA replication and lytic gene transcription during the different stages of lytic growth are also. activation (Fig. 1, see also refs. 1 and 2). The establishment of shown: v represents immediate early RNA synthesis, per- for X a more sit- formed solely by the host transcription machinery; -_ rep- repression phage involves complex regulatory RNA on N at least one viral resents delayed early synthesis, dependent protein; uation, because early protein (the product - - - -s. represents late RNA synthesis, dependent on Q protein (see of the int gene) is required to catalyze the integrative recom- refs. 23, 33, 34 for recent detailed reviews). The cI protein blocks bination essential for stable lysogeny. Thus, the phage must immediate early RNA synthesis; this provides for complete allow expression of at least some early viral genes and yet repression of lytic genes, because delayed early and late RNA. impose repression before an irreversible commitment to synthesis is dependent upon N protein (see refs. 1, 2 for recent lytic growth. detailed reviews). Mutations affecting the establishment of Two general mechanisms have been considered that might repression have been located in the cHI and clII genes and in the provide for the "properly timed" establishment of repression. "y-region" of X DNA between the cI and clI genes (5, 13)-see One involves a repression of viral functions, in addition to that Results section. 2190 Downloaded by guest on September 28, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Phage Lambda Repression 2191

synthesis of late viral proteins and an activation of syn- thesis of cI protein (6). This report provides much more sub- stantial evidence for the activation of cI protein synthesis by direct assay of the levels of cI protein in extracts from infected cells. The results reported here also suggest that the site of action of the clI and cIII proteins may be between the cI and C3 cli genes. Similar results and conclusions with a different assay > for cI protein are reported in the accompanying paper by Reichardt and Kaiser (7). Conclusions similar to ours have 0 been derived from other types of experiments by Eisen and z 2 Pereira da Silva (personal communication), and by Kourilsky z (9). z Since the cII and cIII genes are repressed under conditions cam of stable lysogeny (10), the maintenance of cI protein syn- thesis would be expected to occur by a different mechanism. Our results suggest that the cI protein activates its own syn- 0 L thesis in a lysogen. This conclusion agrees with that derived from RNA synthesis experiments (11, 12) and from other 0o measurements of cI protein (7). MINUTES AFTER INFECTION MATERIALS AND METHODS FIG. 2. Kinetics of production of active cI protein. Infection Bacteriophage and bacteria was at a multiplicity of 5 phage per bacterium. At the The Escherichia coli strains used were W3104. specified times cells were chilled, centrifuged, and extracts were W3102su- and prepared. DNA-binding activity was measured by the retention The X mutations were the following: the ci Amber-mutation of 32P-labeled X (or Ximm434) DNA on a nitrocellulose filter in c114, the clI mutations cII68 and cII28(Amber), the cIII mu- the presence of excess unlabeled "chicken-blood DNA". DNA- tations c11167 and c111611 (Amber), and the cy mutations cy42 binding activity is expressed as DNA-binding units/109 cells. and cy2001. For the genetic characterization of the cI, clI, -0-0- represents cI + infection, assayed with XDNA; -0-0- repre- clII, and cy mutations, see refs. (5) and (13). sents cI- infection, assayed with XDNA; -A-A- represents cI+ Phage growth infection, assayed with Ximm434 DNA. Phage stocks were prepared by lytic growth in W3104 bacteria; the growth medium was T-broth liquid (per liter: measure the level of cI protein in crude extracts. The critical 10 g of Difco Tryptone, 5 g NaCl) or T-broth agar (T-broth control experiments are presented in Fig. 2 (Results). containing 1.2% Difco Bacto-agar for the underlayer and Extracts for assay of cI protein were prepared as follows. 0.7% agar for the soft-agar overlayer). Phage were con- Infected cells (about 6 X 1010) were collected by centrifuga- centrated and partially purified by precipitation with poly- tion and suspended in 3 ml of "lysis buffer" [0.05 M Trist ethylene glycol (10% polyethylene glycol-0.5 Ml NaCl), and HCl (pH 7.9)-2 mM EDTA-0.45 M NH4Cl-14%(w/w) phage stocks were stored in "adsorption buffer" (0.01 M sucrose]. The resultant cell suspension was centrifuged, and MIgSO4-0.01 M Tris HCl, pH 7.4) containing 0.01% gelatin. the pellet was frozen and stored at -20°C. For the prepara- In experiments to measure the synthesis of cI protein, tion of lysates, the frozen cells were suspended in 1.5 ml of W3102 cells were grown at 370C in "supplemented T-broth" lysis buffer and lysozyme was added to a concentration of 330 (T-broth + 0.2% maltose + 0.01% yeast extract) to an A59o ,jg/ml. After incubation for 5 min at 370C, the suspension of about 2, centrifuged, and resuspended in adsorption buffer was chilled, and MgCl2 and 2-mercaptoethanol were added to at an A590 of 4. To this cell suspension, phage were added, at a 50 mM and 14 mM, respectively. Lysis was completed by the multiplicity of 5 or 10, for an adsorption period of 20 min addition of the nonionic detergent Brij-58 to a concentration at room temperature. The infected cells were then diluted of 0.5%. After 30 min at 0WC, the NH4Cl concentration was 10-fold into supplemented T-broth (prewarmed to 370C) increased to 1.0 M and the lysate was centrifuged for 2 hr and aerated at 370C until the time at which the level of cI at 100,000 X g. The supernatant fraction was dialyzed protein was to be measured; the culture was then chilled and into a buffer containing 0.01 M Tris HCl (pH 7.4)-0.01 AI extracts were prepared. MgCl1-0.02 M NH4CI--0.1 mM dithiotreitol. The dialyzed extract was clarified by centrifugation for 15 min at 7500 Assay of cI protein by DNA-binding activity rpm, and the supernatant fraction ("crude extract") was The cI protein binds tightly and specifically to X DNA (14). stored at 4VC. The DNA-binding activity of the cI protein This DNA-binding activity can be made the basis for a simple was quite stable in extracts prepared and stored as described "'membrane filter" assay for the cI protein; the DNA-protein above-generally less than 50% of the activity was lost after complex adheres to a membrane filter, but free DNA does several weeks of storage. not. Excess nonspecific DNA can be added to compete for The DNA-binding assay was essentially that described the binding of proteins that associate with DNA, but lack previously (18). An aliquot of crude extract was mixed with: the specificity of the cI protein for X DNA. The membrane- 0.25 ml of "binding buffer" (0.01 M Tris HCl (pH 7.4)- filter assay has been used extensively in studies of the lac 0.01 1\I magnesium acetate-0.02 M KCl-0.25 mM EDTA- repressor (15, 16) and has been used as an assay to guide the 14 mM 2-mercaptoethanol), 40,gg of "chiclen-blood DNA" purification of the cI protein (17, 18). In the experiments re- (CalBiochem), and 0.5 jig of 32P-labeled X DNA, in a final ported here, we have used the membrane-filter assay to volume of 0.35 ml. After incubation for 5 min at 0WC, 0.1 ml Downloaded by guest on September 28, 2021 2192 : Echols and Green Proc. Nat. Acad. Sci. USA 68 (1971)

of the mixture was added to a membrane filter (Schleicher cept that the time of rapid synthesis of cI protein is of critical and Schuell B6, 25 mm); the filter was washed with 0.4 ml importance in the properly timed establishment of repression. of binding buffer, dried, and the retained radioactivity was determined by liquid scintillation counting. One unit of DNA- Effect of cl-, c11l-, and cy - mutations on the appearance of cI protein activity binding activity is defined as the quantity sufficient to retain 0.5 ,ug of X DNA on the filter. The retention of X DNA on From the results presented in Fig. 2, it should be possible the filter was approximately proportional to added extract to investigate regulation of cI protein synthesis through a in the range from 0.1 to 0.5 DNA-binding units, and assays study of the effect of various regulatory mutations on the were routinely performed in this range. appearance of X-specific DNA-binding activity. Besides cI-, three classes of repression-defective mutants are known: RESULTS cII-, cIII-, and cy-. As judged by complementation for Kinetics of appearance of cI protein activity in lysogeny, the cIl- and cIII- mutations inactivate cyto- infected cells plasmic products (5); these cytoplasmic gene products are As a first step in exploring the establishment of cl-mediated probably proteins since nonsense mutations exist in both the repression in infected cells, we performed experiments de- cII and cIII genes. The nature of the cy- mutations is less signed to measure the kinetics of synthesis of cI protein. The clear. cy- mutations have been located between the cI and assay for ci protein was specific binding to X DNA, as judged cII genes, (Fig. 1); cy- mutant phage fail to give effective by retention of X DNA to a membrane filter. Fig. 2 shows the complementation for lysogeny after mixed infection with cI- results of DNA-binding experiments for extracts prepared at mutants, suggesting that cy- mutations might affect a site different times after infection. The appearance of X-specific concerned with cI protein synthesis (13). DNA-binding activity begins about 6-9 min after infection The effect of cII-, cIII-, and cy- mutations on the ap- with normal X (cI+cII+cIII+) phage. We attribute this DNA- pearance of cI protein activity is shown in Table 1; all three binding activity to the cI protein because of two control ex- classes of mutant phage are defective. We conclude that the periments, also shown in Fig. 2. First, substantial DNA-bind- clI and cIII proteins are probably necessary for effective syn- ing activity is not found after infection by a cI- mutant. thesis of the cI protein and that the "y region" of X DNA Second, the X-specific DNA-binding activity found after between the cI and cII genes may also be important for the cI+ infection is not demonstrable if X imm434 DNA is used synthesis of cI protein. for the assay instead of X DNA; X imm434 DNA lacks the Complementation between repression-defective binding sites for the X cI protein (14) (see Fig. 1) and there- mutants-cis-dominance of cy- fore should not be retained on the filter in the presence of the an to more of v- XcI protein. In attempt establish clearly the role the The experiments presented in Fig. 2 thus show that the region of X DNA in the synthesis of cI protein, we compared DNA-binding assay can be used to measure cI protein ac- cy- and cIL- or cIII- mutants in their ability to complement tivity in extracts prepared from infected cells. The deTayed- with4I- fo- the-p dtion- 4. el pretii. The-results' are appearance of cI protein activity is consistent with the con- shown in Table 2. As expected for phage with mutations that inactivate a protein gene product, cI- and cII - or cIII- mut- ants exhibit trans complementation for the appearance of cI TABLE 1. Effect -of c11, cII -, and cy mutations on cI protein activity (lines 3 and 4) (the most likely anticipated protein activity level would be half the c+ case (line 1) because only half as many cI+ genes are present). In contrast, cI- and cy- phage X-specific DNA-binding activity TABLE 2. Comnplementation between cII-, cIII-, and cy- Infecting phage 12 min 18 min mtutants for ci protein activity c+ 2.9 5.0 X-specific cM14 0.2 0.3 DNA-binding cII68 <0.1 0.1 Infecting phage activity cII28 0.1 0.1 cIII67 0.2 0.6 c+ 3.8 cIII611 0.3 0.3 cI- 0.1 cy42 0.1 0.2 cl- and cII- 1. 8 cy2001 0.1 0.2 cl- and cIII- 1.6 cI- and cy- 0.1 Infection was at a multiplicity of 5 phage per bacterium. At c+andcI-cII 2.1 either 12 min or 18 min after infection, cells were chilled, centri- c+ and cI-cIII- 2.2 fuged, and extracts were prepared. DNA-binding activity was c+ and cl-cy- 2.9 measured by the retention of 32P-labeled X DNA on a nitro- cellulose filter in the presence of excess unlabeled "chicken-blood Infection was at a total multiplicity of 10 phage per bacterium DNA." DNA-binding activity is expressed as DNA-binding (5 of each phage type for mixed infections). 12 min after infection, units/109 cells. The designation "X-specific DNA-binding cells were chilled, centrifuged, and extracts were prepared and as- activity" means that the binding activity of an uninfected ex- sayed for DNA-binding activity as in Table 1. Each extract was tract (0.1) has been subtracted from the binding activity of the also assayed with Ximm434 DNA to provide assurance that the extract prepared from infected cells. The c+, cIl4, cII68, cIII67, "complementation activity" showed the proper specificity; only and cy42 infections were repeated two or more times with similar 0.1-0.2 units were assayable with Ximm434 DNA. The complete results. experiment was performed twice, with similar results. Downloaded by guest on September 28, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Phage Lambda Repression 2193

do not show effective trans complementation (line 5), even though cI -cy- is recessive to cI +cy+ (line 8). The cis-dominant behavior of the cy- mutation is most simply interpreted as mutational inactivation of a site necessary for cI protein I-. 5 synthesis. We conclude that the y-region of X DNA probably '1- exerts an important structural role in the synthesis of cI 4 protein. z Effect of cl-mediated repression on the appearance of cI 0. protein activity KE 0.! u Since synthesis of the cII and cIII proteins is repressed in a lysogen, the problem remains as to how the phage provides 20 0 10 for continued synthesis of cI protein once cl-mediated repres- MINUTES AFTER INFECTION sion is- established.- There-are two-obvious- possibilities: ci protein is synthesized at a low, constitutive rate in the ab- FIG. 3. Rate of appearance of cI protein after infection of sence of cIl/cIlI activation-a rate sufficient to provide for lysogenic and nonlysogenic cells. Infection by P c+ and P-cII- continued iepression-or cI protein activates its own synthesis. phage was at a multiplicity of about 10 phage per bacterium, and To investigate those possibilities, we sought to compare the cI protein was assayed by DNA-binding as in Fig. 2. For this cli-cIIl-activated and "constitutive" rates of cI protein figure, the "cI protein activity" represents the excess of binding to X DNA over that found for Ximm434 DNA (about 0.1 unit). synthesis to the rate found in a lysogen. -0-0- represents cI protein activity after infection of W3102; To try to estimate the constitutive rate of synthesis of -0-0- represents cI protein activity after infection of the lyso- cI protein, we used infection of a nonlysogen by cI- phage. genicstrain W3112(X+). To eliminate gene-dosage effects because of replication, we used phage unable to replicate effectively because of a muta- tion in the P gene (3, 19, 20). Fig. 3A compares cI protein The results reported here suggest that the turn-on of activity after infection of a nonlysogen and a lysogen by synthesis of cI protein at the "proper" time is a critical factor P-c+ phage. Fig. 3B makes the same comparison under con- in the establishment of repression. We have shown that three ditions where cIl-cIlI-activation is blocked by mutation. classes of mutants defective in the establishment of repres- cI protein activity after cII- infection of a nonlysogen sion are also defective in the normal appearance of cI protein is clearly lower than that found after infection of a lysogen. activity. Complementation tests indicate that two of these Similar results to those in Fig. 3B were obtained after in- mutational classes (cII- and cIII-) result from inactivation fection of a lysogen carrying a cII- prophage instead of c+. of a gene product, but the third class (cy-) may involve a The experiments of Fig. 3B are consistent with two possible structural defect. Thus, at least three regulatory elements are explanations: the cI protein activates-its own synthesis in a likely to be required for the normal turn-on of cI protein syn- lysogen, or, the synthesis of cI protein after cIL- infection of a thesis in an infected cell: clI and cIII proteins, and an "active" nonlysogen is not in the "constitutive" rate, but is negatively y-region of X DNA. regulated by another X protein. We favor the possibility that Previous experiments (6) have indicated that the cII and cI activates its own synthesis because the only known nega- cMII proteins also inhibit synthesis of late phage proteins, tive regulator of cI function-the cro gene product-does not since cI -cI - or cI-c11 - mutants commence the synthesis of exert a demonstrable effect on synthesis of cI protein until lysozyme at an earlier time than cI-. Recent experiments about 20 min after infection (7) (see Discussion). have shown that the cy- mutation also results in an advanced The results of Fig. 3A indicate that the rate of cIl-cIMI- synthesis of lysozyme (Court, unpublished). The complete activated synthesis of cI protein is substantially greater than role of the cII and cIII proteins in the establishment of repres- the presumptive cI-activated rate found in a lysogen. The sion thus may involve a bifunctional regulatory activity: biological relevance of the rapid cII-cIlI-activated synthesis positive regulation of the cI gene and negative regulation of is presumably to allow the synthesis of early phage proteins late genes. (see Introduction) and yet to permit the synthesis of cI pro- Based upon our rather limited present state of knowledge, tein to catch up with the multiple copies of the replicating viral a plausible molecular mechanism is the following (see Fig. 4). DNA. The results of Fig. 3A also suggest that c1I-cI1- The cII and cIII proteins form a regulatory oligomer*, which activated synthesis of cI protein ceases at later times, since acts at a site in the y-region of X DANA to provide for activa- the rate of appearance of cI protein activity slows markedly. tion of I-strand transcription from the cI gene and for inhibi- DISCUSSION tion of r-strand transcription in the opposite direction. The The role of the cII and cIII proteins in the establishment repression of r-strand transcription from the cIIOPQ region of repression of X DNA might inhibit the synthesis of late proteins in two As noted in the Introduction, the regulatory problem faced possible ways: suboptimal synthesis of Q protein, which is by a X phage desirous of provoking lysogeny is to provide for an effective transition from initiated lytic development to * St-rack and Ziegler (32) have also suggested that the cII and the cl-mediated repression of lytic genes that maintains the cIII proteins may act as an oligomer, based upon the existence of stable lysogenic state. The phage must provide for sufficient mutations that probably are located in the cII gene, but which synthesis of int protein to achieve a high probability of in- complement as if cII- and.cIII-. We have isolated similar muta- tions and shown that they also complement as cII-cIII- in the tegration, but it must also stop lytic development before .assayloriL.pratein activity (Mantei,

- We thank Joseph Ferreti, Dale Kaiser, Ethan Signer, and 40liiN ci vcrox li 0 2- strand cM N -. cI x Y. c.CI .0 P Rene Thomas for phage stocks and Don Court, Harvey Eisen, r-strand Philippe Kourilsky, Margaret Lieb, and Lou Reichardt for useful discussion and communication of unpublished results. cI protein possible binding site This research was supported in part by U. S. Public Health binding sites for cIl/cl proteins Service grant GM 17078. FIG. 4. Possible mechanism for the action of cII and cIII 1. Ptashne, M., in The Bacteriophage X, ed. A. D. Hershey proteins. The cIL-cLIL regulatory oligomer acts at a site in the (Cold Spring Harbor, in press, 1971). y-region of X DNA to provide for activation of i-strand trans- 2. Echols, H., Annu. Rev. Biochem., 40, 827 (1971). cription toward the cI gene (e ---- and inhibition of r-strand 3. Joyner, A., L. N. Isaacs, H. Echols, and W. S. Sly, J. Mol. transcription toward the chOP genes (-A) (see text). Once the Biol., 19, 174 (1966). supply of cI protein becomes sufficient, lytic genes are completely 4. Eisen, H. A., L. Pereira da Silva, and F. Jacob, C.R. Acad. Sci., Paris, 266, 1176 (1968). repressed by the ability of the cI protein to inhibit immediate 5. Kaiser, A. D., Virology, 3, 42 (1957). early RNA synthesis, represented by (It) (see Fig. 1.) The bind- 6. McMacken, R., N. Mantei, B. Butler, A. Joyner, and H. ing sites for the ci protein are located to the left and right of the ci Echols, J. Mol. Biol., 49, 639 (1970). gene, very close to the initiation sites for immediate early RNA. 7. Reichardt, L. B., and A. D. Kaiser, Proc. Nat. Acad. Sci. The division of the area between the ci and cHI genes into x- USA, 68, 2185 (1971). and y-regions is based upon the end of the region of nonhomology 8. Deleted in proof. between phages X and 434 (imm-region); x is inside the imm- 9. Kourilsky, P., Virology, in press (1971). region, y is outside (19). The terms x and y therefore do not denote 10. Bode, V. C., and A. D. Kaiser, Virology, 25, 111 (1965). A genes. The cro gene is within the x-region. 11. Heinemann, S. F., and W. G. Spiegelman, Proc. Nat. Acad. Sci. USA, 67,1122 (1970). needed to activate late-gene transcription (21, 22), or fail- 12. Kourilsky, P., M-F. Bourguignon, M. Bouquet, and F. Gros, Cold Spring Harbor Symp. Quant. Biol., 35, 305 ure of normal rates of initiation of RNA synthesis for late (1970). genes in the absence of transcription to the initiation site for 13. Brachet, P., and R. Thomas, Mutat. Res., 7, 257 (1969). late RNA synthesis from the cIIOPQ genes to the left (see 14. Ptashne, M., Nature, 214, 232 (1967). refs. 12 and 23-25 for a discussion of such mechanisms). 15. Riggs, A. D., and S. Bourgeois, J. Mol. Biol., 34, 361 (1968). The existence of another regulatory gene-cro-complicates 16. Bourgeois, S., Meth. Enzymol., 12C, in press (1971). the definition of events during the establishment of repression. 17. Chadwick, P., N. Hopkins, V. Pirrotta, R. Steinberg, and the of M. Ptashne, Cold Spring Harbor Symp. Quant. Biol., 35, 'Physiological experiments have indicated that product 283 (1970). the cro gene (see Fig. 4) antagonizes the synthesis or activity 18. Wu, A. M., S. Ghosh, M. Willard, J. Davison, and H. -of the cI protein (26-29). Reichardt and Kaiser (7) have pre- Echols, in The Bacteriophage X, ed. A. D. Hershey (Cold sented evidence that the cro product turns off synthesis of Spring Harbor, in press, 1971). .cI protein at later times. The physiological function of cro 19. Eisen, H. A., C. R. Fuerst, L. Siminovitch, R. Thomas, L. might be to prevent cII- cIII-mediated repression from chan- Lambert, L. Pereira da Silva, and F. Jacob, Virology, 30, 224 in (1966). neling all infected cells to lysogeny. Alternatively (or 20. Ogawa, T., and J.-I. Tomizawa, J. Mol. Biol., 38, 217 (1968). addition), the physiological role of the cro gene might be to 21. Dove, W. F., J. Mol. Biol., 19, 187 (1966). provide for a rapid release of repression when the lysogenic 22. Skalka, A., B. Butler, and H. Echols, Proc. Nat. Acad. Sci. cell is induced to lytic growth. USA, 58, 576 (1967). 23. Echols, H., in The Bacteriophage X, ed. A. D. Hershey (Cold The role of the cI protein in the establishment and Spring Harbor, in press, 1971). maintenance of repression 24. Butler, B., and H. Echols, Virology, 40, 212 (1970). Once the supply of active cI protein becomes sufficient for the 25. Herskowitz, I., and E. R. Signer, J. Mol. Biol., 47, 545 number of A DNA molecules, complete repression should occur (1970). rapidly. As noted in the Introduction, the cI protein will 26. Oppenheim, A. B., Z. Neubauer, and E. Calef, Nature, 226, block synthesis of new N protein and DNA-replication pro- 31 (1970). 27. Eisen, H. A., P. Brachet, L. Pereira da Silva, and F. Jacob, teins. In addition, N and replication proteins cannot act Proc. Nat. Acad. Sci. USA, 66, 855 (1970). effectively, even if present, on a X DNA molecule to which the 28. Sly, W. S., K. Rabideau, and A. Kolber, in The Bacteri- ,cI protein is bound (23, 30, 31). From our experiments and ophage A, ed. A. D. Hershey (Cold Spring Harbor, in press, -those of others (7, 11, 12), the maintenance of cI protein syn- 1971). thesis once repression is established appears to result from 29. Spiegelman, W. G., Virology, 43, 16 (1971). 4'self-activation" by the cI protein. Thus, the cI protein is 30. Thomas, R., and L. E. Bertani, Virology, 24, 241 (1964). J. Mol. likely to have a bifunctional regulatory activity: positive 31. Luzzati, D., Biol., 49, 515 (1970). 32. Strack, H. B., and R. Ziegler, Mol. Gen. Genet., 106, 80 regulation of the cI gene and negative regulation of early (1969). lytic genes. 33. Calendar, R., Annu. Rev. Microbiol., 24, 241 (1970). Clearly, the patterns of regulation for even so simple a 34. Thomas, R., Curr. Top. Microbiol. Immunol., in press creature as phage can be relatively complex. (1971). Downloaded by guest on September 28, 2021