this possibility is suggested by the finding that the synthesis likely to function as critical timing elements in the establishment Head

Transcription

1 Proc. Nat. Acad. Sci. USA Vol. 68, No. 9, pp , September 1971 Establishment and Maintenance of Repression by Bacteriophage Lambda: Proteins (E. coli/lysogeny versus lysis/cy- mutants/dna binding) The Role of the ci, cit, and ciii HARRISON ECHOLS AND LINDA GREEN Department of Molecular Biology, University of California, Berkeley, Calif Communicated by A. D. Kaiser, July 8, 1971 ABSTRACT To define the events necessary for the establishment and maintenance of repression in a X- infected cell, we have studied the requirements for efficient synthesis of the ci protein ("X-repressor"). Three classes of X mutants defective in the establishment of repression are also defective in the appearance of ci protein activity at the normal time. Two of these mutational classes (ciiand ciii-) probably result from inactivation of X-specified proteins, but the third class (cy-) may involve a structural defect. We conclude that at least three regulatory elements are likely to be required for the normal turn-on of ci protein synthesis in an infected nonlysogenic cell: cli and ciii proteins and an "active" y-region of X DNA. From these and other results, the complete role of cii and ciii proteins in the establishment of repression may involve a bifunctional regulatory activity: positive regulation of the ci gene and negative regulation of late genes. A possible molecular model for cii and ciii action is discussed. Since 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 increase of ci protein activity is substantially greater than after infection of a nonlysogen. From these and other results, the ci protein may also have a bifunctional regulatory activity: positive regulation of the ci gene and negative regulation of early lytic genes. After infection by the temperature phage X, the establishment of lysogeny requires two functionally separate events: a repression of the capacity of the phage DNA for lytic growth, and a site-specific genetic recombination event that integrates the viral DNA into the host DNA at a specific site. The maintenance of the lysogenic state is inherently a simpler phenomenon than its establishment; the repression of lytic capacity must simply be maintained, and no recombination events are involved. The maintenance of repression for phage X is probably accomplished by one phage protein-the ci protein (or "Xrepressor")-which acts to repress RNA synthesis from critical early genes required for viral DNA replication and lytic gene activation (Fig. 1, see also refs. 1 and 2). The establishment of repression for phage X involves a more complex regulatory situation, because at least one early viral protein (the product of the int gene) is required to catalye the integrative recombination essential for stable lysogeny. Thus, the phage must allow expression of at least some early viral genes and yet impose repression before an irreversible commitment to lytic growth. Two general mechanisms have been considered that might provide for the "properly timed" establishment of repression. One involves a repression of viral functions, in addition to that 2190 provided by the ci protein; for example, an inhibition of synthesis of late proteins might delay the late stage of lytic development until cl-mediated repression can take over (3). Another plausible mechanism involves timing the establishment of repression by the time of synthesis of ci protein; this possibility is suggested by the finding that the synthesis of ci protein is subject to regulation (4). Since active cli and cii genes are essential for the effective establishment of lysogeny in an infected cell, but not for the maintenance of lysogeny (5), the cii and ciii proteins are likely to function as critical timing elements in the establishment of repression. As a result of previous experimental efforts to understand the role of the cii and ciii genes, we provided indirect evidence that the cii and ciii proteins perform both "activities" indicated above: an inhibition of Head Tail -0 int cm N cicdhop Q Recomb Reg DNA Lysis * A^0 FIG. 1. Genetic and functional map of X DNA. Genes with related function exhibit extensive clustering along the X DNA. molecule. This is indicated on the diagram by "Head" for genes concerned with the structure of the phage head; "Tail" for genes concerned with tail structure; "Recomb" for genes involved with general and site-specific recombination; "Reg" for genes exerting a regulatory function in lytic development or lysogeny; "DNA". for genes specifying replication proteins; and "Lysis" for genes concerned with cell lysis. Specific genes of the "regulation region"-ciii, N, ci, cii-are indicated above the "X DNA", as are the integrative recombination gene int, the DNA replication genes OP, and the late regulator gene, Q. Approximate DNA regions transcribed and the direction of transcription during the different stages of lytic growth are also. shown: v represents immediate early RNA synthesis, performed solely by the host transcription machinery; -_ represents delayed early RNA synthesis, dependent on N protein; s. represents late RNA synthesis, dependent on Q protein (see refs. 23, 33, 34 for recent detailed reviews). The ci protein blocks immediate early RNA synthesis; this provides for complete repression of lytic genes, because delayed early and late RNA. synthesis is dependent upon N protein (see refs. 1, 2 for recent detailed reviews). Mutations affecting the establishment of repression have been located in the chi and clii genes and in the "y-region" of X DNA between the ci and cli genes (5, 13)-see Results section.

2 Proc. Nat. Acad. Sci. USA 68 (1971) synthesis of late viral proteins and an activation of synthesis of ci protein (6). This report provides much more substantial 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 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 been derived from other types of experiments by Eisen and Pereira da Silva (personal communication), and by Kourilsky (9) Ṡince the cii and ciii genes are repressed under conditions of stable lysogeny (10), the maintenance of ci protein synthesis would be expected to occur by a different mechanism. Our results suggest that the ci protein activates its own synthesis in a lysogen. This conclusion agrees with that derived from RNA synthesis experiments (11, 12) and from other measurements of ci protein (7). MATERIALS AND METHODS Bacteriophage and bacteria The Escherichia coli strains used were W3102su- and W3104. The X mutations were the following: the ci Amber-mutation c114, the cli mutations cii68 and cii28(amber), the ciii mutations c11167 and c (Amber), and the cy mutations cy42 and cy2001. For the genetic characteriation of the ci, cli, clii, and cy mutations, see refs. (5) and (13). Phage growth Phage stocks were prepared by lytic growth in W3104 bacteria; the growth medium was T-broth liquid (per liter: 10 g of Difco Tryptone, 5 g NaCl) or T-broth agar (T-broth containing 1.2% Difco Bacto-agar for the underlayer and 0.7% agar for the soft-agar overlayer). Phage were concentrated and partially purified by precipitation with polyethylene glycol (10% polyethylene glycol-0.5 Ml NaCl), and phage stocks were stored in "adsorption buffer" (0.01 M MIgSO M Tris HCl, ph 7.4) containing 0.01% gelatin. In experiments to measure the synthesis of ci protein, W3102 cells were grown at 370C in "supplemented T-broth" (T-broth + 0.2% maltose % yeast extract) to an A59o of about 2, centrifuged, and resuspended in adsorption buffer at an A590 of 4. To this cell suspension, phage were added, at a multiplicity of 5 or 10, for an adsorption period of 20 min at room temperature. The infected cells were then diluted 10-fold into supplemented T-broth (prewarmed to 370C) and aerated at 370C until the time at which the level of ci protein was to be measured; the culture was then chilled and extracts were prepared. Assay of ci protein by DNA-binding activity The ci protein binds tightly and specifically to X DNA (14). This DNA-binding activity can be made the basis for a simple "'membrane filter" assay for the ci protein; the DNA-protein complex adheres to a membrane filter, but free DNA does not. Excess nonspecific DNA can be added to compete for the binding of proteins that associate with DNA, but lack the specificity of the ci protein for X DNA. The membranefilter assay has been used extensively in studies of the lac repressor (15, 16) and has been used as an assay to guide the purification of the ci protein (17, 18). In the experiments reported here, we have used the membrane-filter assay to C3 > 0 2 cam 0 0o L Phage Lambda Repression 2191 MINUTES AFTER INFECTION FIG. 2. Kinetics of production of active ci protein. Infection was at a multiplicity of 5 phage per bacterium. At the specified times cells were chilled, centrifuged, and extracts were prepared. DNA-binding activity was measured by the retention of 32P-labeled X (or Ximm434) DNA on a nitrocellulose filter in the presence of excess unlabeled "chicken-blood DNA". DNAbinding activity is expressed as DNA-binding units/109 cells represents ci + infection, assayed with XDNA; represents ci- infection, assayed with XDNA; -A-A- represents ci+ infection, assayed with Ximm434 DNA. measure the level of ci protein in crude extracts. The critical control experiments are presented in Fig. 2 (Results). Extracts for assay of ci protein were prepared as follows. Infected cells (about 6 X 1010) were collected by centrifugation and suspended in 3 ml of "lysis buffer" [0.05 M Trist HCl (ph 7.9)-2 mm EDTA-0.45 M NH4Cl-14%(w/w) sucrose]. The resultant cell suspension was centrifuged, and the pellet was froen and stored at -20 C. For the preparation of lysates, the froen cells were suspended in 1.5 ml of lysis buffer and lysoyme was added to a concentration of 330,jg/ml. After incubation for 5 min at 370C, the suspension was chilled, and MgCl2 and 2-mercaptoethanol were added to 50 mm and 14 mm, respectively. Lysis was completed by the addition of the nonionic detergent Brij-58 to a concentration of 0.5%. After 30 min at 0WC, the NH4Cl concentration was increased to 1.0 M and the lysate was centrifuged for 2 hr at 100,000 X g. The supernatant fraction was dialyed into a buffer containing 0.01 M Tris HCl (ph 7.4)-0.01 AI MgCl M NH4CI--0.1 mm dithiotreitol. The dialyed extract was clarified by centrifugation for 15 min at 7500 rpm, and the supernatant fraction ("crude extract") was stored at 4VC. The DNA-binding activity of the ci protein was quite stable in extracts prepared and stored as described above-generally less than 50% of the activity was lost after several weeks of storage. The DNA-binding assay was essentially that described previously (18). An aliquot of crude extract was mixed with: 0.25 ml of "binding buffer" (0.01 M Tris HCl (ph 7.4) \I magnesium acetate-0.02 M KCl-0.25 mm EDTA- 14 mm 2-mercaptoethanol), 40,gg of "chiclen-blood DNA" (CalBiochem), and 0.5 jig of 32P-labeled X DNA, in a final volume of 0.35 ml. After incubation for 5 min at 0WC, 0.1 ml

3 2192 Biochemistry: Echols and Green of the mixture was added to a membrane filter (Schleicher and Schuell B6, 25 mm); the filter was washed with 0.4 ml of binding buffer, dried, and the retained radioactivity was determined by liquid scintillation counting. One unit of DNAbinding activity is defined as the quantity sufficient to retain 0.5,ug of X DNA on the filter. The retention of X DNA on the filter was approximately proportional to added extract in the range from 0.1 to 0.5 DNA-binding units, and assays were routinely performed in this range. RESULTS Kinetics of appearance of ci protein activity in infected cells As a first step in exploring the establishment of cl-mediated repression in infected cells, we performed experiments designed to measure the kinetics of synthesis of ci protein. The assay for ci protein was specific binding to X DNA, as judged by retention of X DNA to a membrane filter. Fig. 2 shows the results of DNA-binding experiments for extracts prepared at different times after infection. The appearance of X-specific DNA-binding activity begins about 6-9 min after infection with normal X (ci+cii+ciii+) phage. We attribute this DNAbinding activity to the ci protein because of two control experiments, also shown in Fig. 2. First, substantial DNA-binding activity is not found after infection by a ci- mutant. Second, the X-specific DNA-binding activity found after ci+ infection is not demonstrable if X imm434 DNA is used for the assay instead of X DNA; X imm434 DNA lacks the binding sites for the X ci protein (14) (see Fig. 1) and therefore should not be retained on the filter in the presence of the X ci protein. The experiments presented in Fig. 2 thus show that the DNA-binding assay can be used to measure ci protein activity in extracts prepared from infected cells. The detayedappearance of ci protein activity is consistent with the con- TABLE 1. Effect -of c11, cii -, and cy mutations on ci protein activity X-specific DNA-binding activity Infecting phage 12 min 18 min c cm cii68 < cii ciii ciii cy cy Infection was at a multiplicity of 5 phage per bacterium. At either 12 min or 18 min after infection, cells were chilled, centrifuged, and extracts were prepared. DNA-binding activity was measured by the retention of 32P-labeled X DNA on a nitrocellulose filter in the presence of excess unlabeled "chicken-blood DNA." DNA-binding activity is expressed as DNA-binding units/109 cells. The designation "X-specific DNA-binding activity" means that the binding activity of an uninfected extract (0.1) has been subtracted from the binding activity of the extract prepared from infected cells. The c+, cil4, cii68, ciii67, and cy42 infections were repeated two or more times with similar results. cept that the time of rapid synthesis of ci protein is of critical importance in the properly timed establishment of repression. Effect of cl-, c11l-, and cy - mutations on the appearance of ci protein activity From the results presented in Fig. 2, it should be possible to investigate regulation of ci protein synthesis through a study of the effect of various regulatory mutations on the appearance of X-specific DNA-binding activity. Besides ci-, three classes of repression-defective mutants are known: cii-, ciii-, and cy-. As judged by complementation for lysogeny, the cil- and ciii- mutations inactivate cytoplasmic products (5); these cytoplasmic gene products are probably proteins since nonsense mutations exist in both the cii and ciii genes. The nature of the cy- mutations is less clear. cy- mutations have been located between the ci and cii genes, (Fig. 1); cy- mutant phage fail to give effective complementation for lysogeny after mixed infection with cimutants, suggesting that cy- mutations might affect a site concerned with ci protein synthesis (13). The effect of cii-, ciii-, and cy- mutations on the appearance of ci protein activity is shown in Table 1; all three classes of mutant phage are defective. We conclude that the cli and ciii proteins are probably necessary for effective synthesis of the ci protein and that the "y region" of X DNA between the ci and cii genes may also be important for the synthesis of ci protein. Complementation between repression-defective mutants-cis-dominance of cy- In an attempt to establish more clearly the role of the v- region of X DNA in the synthesis of ci protein, we compared cy- and cil- or ciii- mutants in their ability to complement with4i- fo- the-p dtion- 4. el pretii. The- results' are shown in Table 2. As expected for phage with mutations that inactivate a protein gene product, ci- and cii - or ciii- mutants exhibit trans complementation for the appearance of ci protein activity (lines 3 and 4) (the most likely anticipated level would be half the c+ case (line 1) because only half as many ci+ genes are present). In contrast, ci- and cy- phage TABLE 2. Comnplementation between cii-, ciii-, and cymtutants for ci protein activity Infecting phage Proc. Nat. Acad. Sci. USA 68 (1971) X-specific DNA-binding activity c+ 3.8 ci- 0.1 cl- and cii cl- and ciii- 1.6 ci- and cy- 0.1 c+andci-cii 2.1 c+ and ci-ciii- 2.2 c+ and cl-cy- 2.9 Infection was at a total multiplicity of 10 phage per bacterium (5 of each phage type for mixed infections). 12 min after infection, cells were chilled, centrifuged, and extracts were prepared and assayed for DNA-binding activity as in Table 1. Each extract was also assayed with Ximm434 DNA to provide assurance that the "complementation activity" showed the proper specificity; only units were assayable with Ximm434 DNA. The complete experiment was performed twice, with similar results.

4 Proc. Nat. Acad. Sci. USA 68 (1971) 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 synthesis. We conclude that the y-region of X DNA probably exerts an important structural role in the synthesis of ci protein. Effect of cl-mediated repression on the appearance of ci protein activity Since synthesis of the cii and ciii proteins is repressed in a lysogen, the problem remains as to how the phage provides for continued synthesis of ci protein once cl-mediated repression is- established.- There-are two-obvious- possibilities: ci protein is synthesied at a low, constitutive rate in the absence of cil/cili activation-a rate sufficient to provide for continued iepression-or ci protein activates its own synthesis. To investigate those possibilities, we sought to compare the cli-ciil-activated and "constitutive" rates of ci protein synthesis to the rate found in a lysogen. To try to estimate the constitutive rate of synthesis of ci protein, we used infection of a nonlysogen by ci- phage. To eliminate gene-dosage effects because of replication, we used phage unable to replicate effectively because of a mutation in the P gene (3, 19, 20). Fig. 3A compares ci protein activity after infection of a nonlysogen and a lysogen by P-c+ phage. Fig. 3B makes the same comparison under conditions where cil-cili-activation is blocked by mutation. ci protein activity after cii- infection of a nonlysogen is clearly lower than that found after infection of a lysogen. Similar results to those in Fig. 3B were obtained after infection of a lysogen carrying a cii- prophage instead of c+. The experiments of Fig. 3B are consistent with two possible explanations: the ci protein activates-its own synthesis in a lysogen, or, the synthesis of ci protein after cil- infection of a nonlysogen is not in the "constitutive" rate, but is negatively regulated by another X protein. We favor the possibility that ci activates its own synthesis because the only known negative regulator of ci function-the cro gene product-does not exert a demonstrable effect on synthesis of ci protein until about 20 min after infection (7) (see Discussion). The results of Fig. 3A indicate that the rate of cil-cimiactivated synthesis of ci protein is substantially greater than the presumptive ci-activated rate found in a lysogen. The biological relevance of the rapid cii-cili-activated synthesis is presumably to allow the synthesis of early phage proteins (see Introduction) and yet to permit the synthesis of ci protein to catch up with the multiple copies of the replicating viral DNA. The results of Fig. 3A also suggest that c1i-ci1- activated synthesis of ci protein ceases at later times, since the rate of appearance of ci protein activity slows markedly. DISCUSSION The role of the cii and ciii proteins in the establishment of repression As noted in the Introduction, the regulatory problem faced by a X phage desirous of provoking lysogeny is to provide for an effective transition from initiated lytic development to the cl-mediated repression of lytic genes that maintains the stable lysogenic state. The phage must provide for sufficient synthesis of int protein to achieve a high probability of integration, but it must also stop lytic development before lysis renders synthesis of int protein irrelevant. 5 '1-4 I-. KE0. 0.! u Phage Lambda Repression MINUTES AFTER INFECTION FIG. 3. Rate of appearance of ci protein after infection of lysogenic and nonlysogenic cells. Infection by P c+ and P-cIIphage was at a multiplicity of about 10 phage per bacterium, and ci protein was assayed by DNA-binding as in Fig. 2. For this figure, the "ci protein activity" represents the excess of binding to X DNA over that found for Ximm434 DNA (about 0.1 unit) represents ci protein activity after infection of W3102; represents ci protein activity after infection of the lysogenicstrain W3112(X+). The results reported here suggest that the turn-on of synthesis of ci protein at the "proper" time is a critical factor in the establishment of repression. We have shown that three classes of mutants defective in the establishment of repression are also defective in the normal appearance of ci protein activity. Complementation tests indicate that two of these mutational classes (cii- and ciii-) result from inactivation of a gene product, but the third class (cy-) may involve a structural defect. Thus, at least three regulatory elements are likely to be required for the normal turn-on of ci protein synthesis in an infected cell: cli and ciii proteins, and an "active" y-region of X DNA. Previous experiments (6) have indicated that the cii and cmii proteins also inhibit synthesis of late phage proteins, since ci -ci - or ci -c11 - mutants commence the synthesis of lysoyme at an earlier time than ci-. Recent experiments have shown that the cy- mutation also results in an advanced synthesis of lysoyme (Court, unpublished). The complete role of the cii and ciii proteins in the establishment of repression thus may involve a bifunctional regulatory activity: positive regulation of the ci gene and negative regulation of late genes. Based upon our rather limited present state of knowledge, a plausible molecular mechanism is the following (see Fig. 4). The cii and ciii proteins form a regulatory oligomer*, which acts at a site in the y-region of X DANA to provide for activation of I-strand transcription from the ci gene and for inhibition of r-strand transcription in the opposite direction. The repression of r-strand transcription from the ciiopq region of X DNA might inhibit the synthesis of late proteins in two possible ways: suboptimal synthesis of Q protein, which is * St-rack and Ziegler (32) have also suggested that the cii and ciii proteins may act as an oligomer, based upon the existence of mutations that probably are located in the cii gene, but which complement as if cii- and.ciii-. We have isolated similar mutations and shown that they also complement as cii-ciii- in the.assayloril.pratein activity (Mantei,<Court, Greenr and FxLs,7.- unpublished).

5 2194 Biochemistry: Echols and Green 2- strand r-strand - 40liiN ci vcro li 0 cm N -. ci x Y. c.ci.0 P ci protein possible binding site binding sites for cil/cl proteins FIG. 4. Possible mechanism for the action of cii and ciii proteins. The cil-clil regulatory oligomer acts at a site in the y-region of X DNA to provide for activation of i-strand transcription toward the ci gene (e ---- and inhibition of r-strand transcription toward the chop genes (-A) (see text). Once the supply of ci protein becomes sufficient, lytic genes are completely repressed by the ability of the ci protein to inhibit immediate early RNA synthesis, represented by (It) (see Fig. 1.) The binding sites for the ci protein are located to the left and right of the ci gene, very close to the initiation sites for immediate early RNA. The division of the area between the ci and chi genes into x- and y-regions is based upon the end of the region of nonhomology between phages X and 434 (imm-region); x is inside the immregion, y is outside (19). The terms x and y therefore do not denote A genes. The cro gene is within the x-region. needed to activate late-gene transcription (21, 22), or failure of normal rates of initiation of RNA synthesis for late genes in the absence of transcription to the initiation site for late RNA synthesis from the ciiopq genes to the left (see refs. 12 and for a discussion of such mechanisms). The existence of another regulatory gene-cro-complicates the definition of events during the establishment of repression. 'Physiological experiments have indicated that the product of the cro gene (see Fig. 4) antagonies the synthesis or activity -of the ci protein (26-29). Reichardt and Kaiser (7) have presented evidence that the cro product turns off synthesis of.ci protein at later times. The physiological function of cro might be to prevent cii- ciii-mediated repression from channeling all infected cells to lysogeny. Alternatively (or in addition), the physiological role of the cro gene might be to provide for a rapid release of repression when the lysogenic cell is induced to lytic growth. The role of the ci protein in the establishment and maintenance of repression Once the supply of active ci protein becomes sufficient for the number of A DNA molecules, complete repression should occur rapidly. As noted in the Introduction, the ci protein will block synthesis of new N protein and DNA-replication proteins. In addition, N and replication proteins cannot act effectively, even if present, on a X DNA molecule to which the,ci protein is bound (23, 30, 31). From our experiments and -those of others (7, 11, 12), the maintenance of ci protein synthesis once repression is established appears to result from 4'self-activation" by the ci protein. Thus, the ci protein is likely to have a bifunctional regulatory activity: positive regulation of the ci gene and negative regulation of early lytic genes. Clearly, the patterns of regulation for even so simple a creature as phage can be relatively complex. Proc. Nat. Acad. Sci. USA 68 (1971) We thank Joseph Ferreti, Dale Kaiser, Ethan Signer, and Rene Thomas for phage stocks and Don Court, Harvey Eisen, Philippe Kourilsky, Margaret Lieb, and Lou Reichardt for useful discussion and communication of unpublished results. This research was supported in part by U. S. Public Health Service grant GM Ptashne, M., in The Bacteriophage X, ed. A. D. Hershey (Cold Spring Harbor, in press, 1971). 2. Echols, H., Annu. Rev. Biochem., 40, 827 (1971). 3. Joyner, A., L. N. Isaacs, H. Echols, and W. S. Sly, J. Mol. Biol., 19, 174 (1966). 4. Eisen, H. A., L. Pereira da Silva, and F. Jacob, C.R. Acad. Sci., Paris, 266, 1176 (1968). 5. Kaiser, A. D., Virology, 3, 42 (1957). 6. McMacken, R., N. Mantei, B. 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Fuerst, L. Siminovitch, R. Thomas, L. Lambert, L. Pereira da Silva, and F. Jacob, Virology, 30, 224 (1966). 20. Ogawa, T., and J.-I. Tomiawa, J. Mol. Biol., 38, 217 (1968). 21. Dove, W. F., J. Mol. Biol., 19, 187 (1966). 22. Skalka, A., B. Butler, and H. Echols, Proc. Nat. Acad. Sci. USA, 58, 576 (1967). 23. Echols, H., in The Bacteriophage X, ed. A. D. Hershey (Cold Spring Harbor, in press, 1971). 24. Butler, B., and H. Echols, Virology, 40, 212 (1970). 25. Herskowit, I., and E. R. Signer, J. Mol. Biol., 47, 545 (1970). 26. Oppenheim, A. B., Z. Neubauer, and E. Calef, Nature, 226, 31 (1970). 27. Eisen, H. A., P. Brachet, L. Pereira da Silva, and F. Jacob, Proc. Nat. Acad. Sci. USA, 66, 855 (1970). 28. Sly, W. S., K. Rabideau, and A. Kolber, in The Bacteriophage A, ed. A. D. Hershey (Cold Spring Harbor, in press, 1971). 29. Spiegelman, W. G., Virology, 43, 16 (1971). 30. Thomas, R., and L. E. Bertani, Virology, 24, 241 (1964). 31. Luati, D., J. Mol. Biol., 49, 515 (1970). 32. Strack, H. B., and R. Ziegler, Mol. Gen. Genet., 106, 80 (1969). 33. Calendar, R., Annu. Rev. Microbiol., 24, 241 (1970). 34. Thomas, R., Curr. Top. Microbiol. Immunol., in press (1971).