Proc. Natl. Acad. Sci. USA Vol. 73, No. 7, pp. 2249-2253, July 1976

Purification and properties of a DNA-binding protein with characteristics expected for the Cro protein of X, a repressor essential for lytic growth (bacteriophage X cro gene/promoter-operator DNA) ATIS FOLKMANIS, YOSHINORI TAKEDA, JOSEF SIMUTH, GARY GUSSIN*, AND Department of , University of California, Berkeley, Calif. 94720, and * Department of Zoology, University of Iowa, Iowa City, Iowa 52242 Communicated by A. D. Kaiser, April 14, 1976

ABSTRACT The Cro protein specified by bacteriophage X viously (8), X DNA was labeled with P2p by growth of phage in is a repressor essential for normal lytic growth of the virus, thus low phosphate medium containing 5 ,Ci/ml of UP-labeled having a physiological role distinct from that of cI, the repressor that maintains lyso We have purified a X-specific DNA- inorganic phosphate. Phage were purified by precipitation with binding protein witheny.the requirements for synthesis and bio- polyethylene glycol and centrifugation to equilibrium in a CsCI chemical activities expected for Cro protein from studies in vivo. density gradient (2-3 times). DNA was extracted with redis- As isolated, the protein appears to be a dimer of molecular tilled phenol, the phenol was removed by extraction with ether, weight approximately 18,000 with DNA-binding properties that and the DNA was dialyzed into and stored in 10mM Tris-HCl, are very similar, but not identical, to those of the cI protein. We 0.2 mM EDTA, at pH 7.3. infer that bacteriophage X uses the same regulatory region of DNA for two different DNA-binding repressor proteins with DNA-Binding Assay. The DNA-binding assay used to seek subtle differences in binding activity specialized for different the activity of Cro protein has been described previously (7, 8). physiological roles. The assay measures retention of X [32P]DNA on a nitrocellulose filter (B-6, Schleicher and Schuell) by virtue of its tight binding The temperate bacteriophage X specifies two different repressor to a specific DNA-binding protein; an excess (100-fold) of un- proteins, cI and Cro, which play a major role in regulation of labeled "chicken blood" DNA (Calbiochem) is added to com- viral development. Each appears to have a specialized physi- pete for the binding of proteins that associate with DNA but ological role for one of the two possible life styles that the phage lack specificity for X DNA (22). The assay mixture contained: can pursue: lysogeny or lysis. The cI protein maintains lysogeny 0.2 ,ug of X [32PJDNA, 20 ,g of "chicken blood" DNA, 10mM through a repression of transcription of the genes essential for Tris'HCI at pH 7.3, 20 mM KCI, 10 mM MgCl2, 0.2 mM di- the earliest stage of lytic development; the Cro protein poten- thiothreitol, and 0.2 mM EDTA, in a total volume of 0.1 ml. tiates lytic growth through a repression of the early gene region One unit of DNA-binding activity is defined as the quantity during the late stage of lytic development (1-4) (A. Folkmanis, sufficient to retain 1 Mug of X DNA on the filter. The binding P. Mellon, H. Echols, and A. Skalka, manuscript in preparation). values are corrected for a "background" of DNA that is retained In spite of their different physiological functions, the bio- on the filter in the absence of binding proteins; this varies from chemical activities of the two proteins have been inferred from 5 to 15% with DNA preparation. in vivo experiments to be quite similar: inhibition of tran- Purification of Presumptive Cro Protein. Thirty liters of scription originating at the early promoters PL and PR (Fig. 1); C600 Su- cells were grown in a broth medium (1% Difco however, cI appears to stimulate transcription from the cI gene tryptone, 0.5% Difco yeast extract, 0.5% NaCI, 0.2% maltose) promoter PM during the maintenance of lysogeny, whereas Cro in three carboys. At a density of 4 X 108 cell per ml, cells were probably shuts off this transcription (see refs. 1-4 for a more infected with cIaml4Nam53vlvS phage at a multiplicity of detailed discussion). 10 phage per cell; 25 min after infection, crushed ice was added The cI protein has been purified and extensively character- and the cells were harvested by centrifugation in a Spinco 170 ized in vitro for both specific binding to X DNA and capacity continuous flow rotor. The cells were resuspended in 340 ml to repress RNA synthesis (5-9). The action of the Cro protein of 10% sucrose, 0.05 M Tris-HCI at pH 7.3, and were quick has previously been defined only through an analysis in vio. frozen in a dry ice-alcohol bath. The frozen cells were placed This report describes the purification of a DNA-binding protein in an ice bath for 20 min and then held at 200 until melted. At with properties expected for Cro protein and a partial charac- this time 34 ml of a solution of 0.25 M Tris.HCI at pH 7.3, 0.001 terization of its binding to the operator region that regulates M EDTA, 2 mg/ml of lysozyme and 18 ml of 2 M KC1 were early X genes. added and the mixture was kept at 00 for 40 min. The mixture was then made 0.01 M in MgCl2, the temperature was raised MATERIALS AND METHODS to 32', 0.5 mg of DNAse I was added, and after the addition of Bacteriophage and Bacteria. The Escherichia coli strains the DNAse had decreased the viscosity (about 5 min), the used and their relevant genetic characteristics were: W3350 Su- mixture was placed in an ice bath. Then 36 ml of 2 M KCI was (10), C600 Su+ (10), and C600 Su- (11). The X phage mutations added and the lysate was centrifuged at 25,000 rpm for 4 hr in used were: cI gene mutations cIts857 (12), cltsAt2 (13), clam 14 a Spinco 30 rotor. The supernatant fraction was then dialyzed (36); N gene mutations Nam53 and Nam7 (10); cro gene in Spectrapor no. 1 membrane tubing (Spectrum Medical In- mutations fedl (13) tof2 and tof6 (14); operator region point dustries) for 4 hr with one change against buffer A [10 mM mutations v2, vl, v3 (15, 16), vS326 (17), and prmll6 (18); and KPO4 at pH 6.35, 0.2 mM EDTA, 0.2 mM dithiothreitol, 5% operator region substitution mutations bio24-5 (19), imm434 (vol/vol) glycerol] with 0.1 M KC1. The dialyzed extract was (20), and imm2l (21). then applied to a 2.5 X 10 cm column of phosphocellulose Preparation of Phage DNA. Essentially as described pre- (Whatman P11) at a rate of 1.5 ml/min. The column was 2249 Downloaded by guest on September 26, 2021 2250 Biochemistry: Folkmanis et al. Proc. Natl. Acad. Sci. USA 73 (1976) 40 cro

Recomb. genes clII N Ci cro cII Repin. genes

-0. ci PM c 0 FIG. 1. The activity of cI and Cro inferred from experiments in co vivo. To maintain the repression essential for the B 20 lysogeny, cI protein z acts at OL and OR to repress leftward and rightward transcription in- 0 itiated at the early promoter sites PL and PR, respectively; the cI protein probably also activates leftward transcription of the cI gene 0N initiated at the maintenance promoter PM. The repression activity of cI keeps the entire phage genome repressed because the N gene product is required as a positive regulator of other genes. The Cro protein acts to turn off (or turn down) the expression of early genes during the late stage of lytic development, presumably through its capacity to act at OL and OR to repress early gene transcription; the Cro protein probably also represses transcription of the cI gene ini- 10 15 tiated at PM. After infection the initial transcription of the cI gene Tube Number occurs through activation by cII and c1II at a site to the right of the FIG. 2. cro gene. (See refs. 1-4.) Repin., replication; Recomb., recombination. Sedimentation properties of Cro activity. Material frac- tionated on phosphocellulose was sedimented in a 10-30% (vol/vol) glycerol gradient containing 0.4 M KCI at 50,000 rpm for 24 hr. washed with buffer A plus 0.1 M KCI and then eluted with a Fractions were collected and aliquots were assayed for DNA-binding 180 ml linear gradient (0.1 M to 1.0 M KCl) in buffer A. activity as described in Materials and Methods. Sedimentation was The X-specific DNA-binding activity eluted at from right to left. Arrows denote the position of marker proteins (B 0.35-0.45 M = bovine serum albumin, 0 = ovalbumin, C = chymotrypsin, and M KCI. These fractions were concentrated by precipitation with = myoglobin) ofmolecular weights 65,000,45,000,25,000, and 17,000, ammonium sulfate (80% saturation), and half of this sample was respectively. (0), DNA-binding activity for X DNA; (0), DNA-binding fractionated on a 2.5 X 81 cm column of Sephadex G-75 activity for Ximm434 DNA. (Pharmacia) in buffer A plus 0.4 M KCI (see Fig. 2 in Results). The Sephadex fractions with X-specific DNA-binding activity were diluted with buffer A to 0.1 M KCI (determined by con- would reflect Cro activity. We also included the operator region ductivity) and refractionated on a 2 X 3 cm phosphocellulose mutations vlv3 to try to reduce any self-repression by Cro column. The X-specific DNA-binding activity was eluted with protein (25, 26). a 20 ml linear gradient (0.1-1.0 M KCI) in buffer A. The peak Although we were unable to detect any X-specific DNA- fractions were stored in 50% glycerol at -20°. The purification binding activity in-crude extracts, we were able to find such procedure was later modified by the use of a X DNA-cellulose activity after phosphocellulose chromatography of the extract. column to replace the second phosphocellulose column. The The X-specific DNA-binding activity could be distinguished remaining half of the ammonium sulfate fraction was dialyzed from cI protein by its low molecular weight, as judged by ve- against buffer B (identical to buffer A except that 10 mM locity sedimentation in a glycerol gradient (Fig. 2). The mo- Tris-HCl at pH 7.3 was used in place of KPO4) with 0.4 M NaCI lecular weight of the X-specific DNA-binding activity can be and was fractionated on the same Sephadex G-75 column noted estimated as about 15,000-20,000, whereas the cI protein has above. Fractions with X-specific DNA-binding activity were a monomer molecular weight of 30,000, and sediments under diluted 1:3 with buffer B and were applied to 1.5 X 5 cm col- these conditions as a dimer of 60,000 (5, 8). To eliminate the umn of X DNA cellulose (23) (0.5 mg DNA per ml of column possibility that the 15,000-20,000 molecular weight protein is volume). The X-specific DNA-binding activity was eluted with an active fragment of cI protein produced by the nonsense a 40 ml linear gradient (0.2-1.4 M NaCl) in buffer B. mutation, rather than the product of the cro gene, a similar experiment was done with material prepared after infection RESULTS by N-cIts phage; again only the 15,000-20,000 molecular weight X-specific DNA-binding activity was found (data not Purification of a DNA-Binding Protein with Properties shown). To provide additional evidence that the A-specific Expected for Cro Protein. The strategy that we employed to DNA-binding activity is the product of the cro gene, we have assay for Cro protein was to seek a DNA-binding activity spe- studied the binding properties of the glycerol gradient fraction cific for X DNA but not for Ximm434 DNA. Since Ximm434 prepared after infection by phage carrying missense mutations DNA differs from X only in a relatively small region of inho- in the cro gene. For two cro mutants (fedI and tof6) we failed mology that includes the cro gene and the postulated binding to find significant levels of A-specific DNA-binding activity; sites for Cro protein (1-4, 24), we expected that such an assay for a third mutant (tof2), the binding activity was altered in would eliminate most other DNA-binding proteins (X- or its response to changes in temperature and ionic strength. The host-specified). The main problem anticipated with this ap- results with tof2 are summarized in Table 1 and will be re- proach is the binding activity of cI protein, which is known to ported in detail elsewhere. exhibit the same X versus Aimm434 specificity (5-9). In an ef- The phosphocellulose and glycerol gradient purifications fort to eliminate cI activity we looked for DNA-binding activity yielded material with a considerable amount of nonspecific after infection by X phage with nonsense mutations in the N and DNA-binding activity, as judged by the X versus Ximm434 cI genes. Since N protein is needed indirectly for cI gene ex- criterion. An extensive purification of the phosphocellulose pression (and maximal expression of all other X genes except material with elimination of most of this nonspecific binding cro) (1-4), and since any cI protein produced should be inactive, was achieved by fractionation on Sephadex G-75 (Table 2, Fig. we expected that any specific DNA-binding activity observed 3). The elution volume for the A-specific DNA-binding activity Downloaded by guest on September 26, 2021 Biochemistry: Folkmanis et al. Proc. Natl. Acad. Sci. USA 73 (1976) 2251 Table 1. Altered binding properties of Cro protein specified by a mutant cro gene I Relative X-specific DNA-binding activity b Binding condition Wild type Mutant 0 U - d e 25- 200, 0.02 M KCI 100 100 C' 20, 0.17MKCl 41 <10 I20 - 370, 0.02 M KCl 71 10 c 15 0 DNA-binding assays were carried out as described in Materials .0 I z and Methods except that the concentration of A [32P]DNA was 8 a jsg/ml and the reaction mixture was 160 ul. Each entry in the table is derived from an average of three 50 ul samples filtered sepa- rately. To derive data for binding activity, the retention 0l A-specific 0 0.2 0.4 0.6 0.8 1.0 of Aimm434 DNA has been subtracted from the A DNA value. 1.2 1.4 1.6 The wild-type and mutant (tof2) proteins were purified by phos- Relative mobility phocellulose chromatography and glycerol gradient sedimentation FIG. 4. Electrophoresis of denatured protein in sodium dodecyl as described in Materials and Methods. The two protein prepara- sulfate gel and assay for activity of reconstituted protein. Material tions were adjusted to have approximately the same levels of chromatographed on DNA-cellulose was subjected to polyacrylamide A-specific DNA-binding activity (20%) at 200 and 0.02 M KCl. gel electrophoresis in sodium dodecyl sulfate (28). One gel was stained with Coomassie brilliant blue (a); another gel run concurrently was sliced, and protein was eluted, renatured, and assayed for binding to (2.0 X void volume) is that expected for a molecular weight of A DNA (0) or Aimm434 DNA (W) (b). 15,000-20,000, as judged by marker proteins run in the same column. The Sephadex material was either refractionated on X-specific DNA-binding activity. described above and the phosphocellulose or purified further on DNA-cellulose (Table DNA-binding specificity described below, we believe that the 2), and this material was used for further characterization. protein that we have purified is the Cro protein of phage X and To test the purity of the DNA-binding activity and to de- we will refer to it as Cro in the following text. termine the monomeric molecular weight, we subjected the DNA-Binding Specificity. The available evidence from DNA-cellulose fraction to acrylamide gel electrophoresis under experiments in tvo suggests that Cro protein acts at or near the denaturing conditions in sodium dodecyl sulfate. After staining, operator sites used by cI protein to control the early X promoter one major band was found, representing 75% of the total protein sites. Specifically, the v2 mutation in the left-side operator site (Fig. 4a). From the migration of this species with respect to OL reduces Cro activity (31), and the vlvS mutations in the marker proteins, we estimate a molecular weight of 9000. A gel right-side operator site OR appear to impair, although not run in parallel was sliced, and the denatured protein was eluted, eliminate, Cro activity (25, 32). To investigate DNA-binding renatured (29), and assayed for X-specific DNA-binding activity specificity in vitro, we have studied binding by Cro and cI (Fig. 4b). The peak of DNA-binding activity coincided with proteins to A DNA carrying operator region mutations. We first the position of the major band in the stained gel. From the es- compared binding by Cro and cI proteins to X+ and Xv2vlvS timated molecular weight of the native and denatured proteins, DNA. we conclude that the native protein is probably a dimer of The results (Fig. 5) show that both Cro and cI bind less ef- identical subunits. The monomer molecular weight of about fectively to the mutant DNA than to the wild type; however, 9000 is consistent with that expected for Cro protein on the basis as noted previously (8), the residual cl-binding is extremely of mRNA size (30), gene size (14), and preliminary experiments dependent on ionic strength, whereas Cro-binding is not. Thus with labeled X proteins (Y. Takeda, unpublished data). Cro and cI appear to recognize the same region of A DNA, but Because of the genetic and biochemical properties of the differ in the details of the protein-DNA interaction.

0 To

z

-0 0-

10 20 30 40 50 60 Tube Number FIG. 3. Gel filtration of Cro activity. Material chromatographed on phosphocellulose was fractionated on Sephadex G-75 and assayed for DNA-binding activity (see Materials and Methods). (-), DNA-binding activity for A DNA; (1), DNA-binding activity for Aimm434 DNA; (o), A280- Downloaded by guest on September 26, 2021 2252 Biochemistry: Folkmanis et al. Proc. Nati. Acad. Sci. USA 73 (1976)

Table 2. Purification of Cro protein DNA- binding Specific activity Volume Protein activity (units/mg) Fraction (ml) (mg/ml) (units) of protein) I Lysate 375 9.9 II Phosphocellulose 34 1.2 486 12 III Ammonium sulfate 4 10 416 10 IV SephadexG-75 44 0.013 244 427 Va Phosphocellulose 6 0.029 133 756 Vb DNA-cellulose 5 0.020 115 1150* Assays and fractionation were carried out as described in Materials and Methods. Protein was determined by the method of Lowry et al. (27) or by A280. The Sephadex G-75 material was rechromatographed on either phosphocellulose (Va) or DNA-cellulose (Vb). (See Materials and Methods.) * Five-fold higher activity was found for this highly purified fraction in the absence of the excess unlabeled chicken blood DNA; thus the specific activities quoted appear to be underestimates because of nonspecific binding. A low level of residual binding to Ximm434 DNA was also found in this most purified fraction, probably also reflecting the nonspecific binding phenomenon.

In a second series of experiments, we focused on the right-side DISCUSSION operator region by the use of DNA that carried a deletion of the We have extensively purified a protein that has the genetic and left-side operator region (OL) and contained various point biochemical attributes expected for the Cro protein of phage mutations in the right-side operator region (OR) (Table 3). Two X. The evidence that the X-specific DNA-binding protein that concentrations of Cro and cI protein are given for wild-type we have purified is Cro and not cI (or some other X protein) can DNA (lines 1 and 2) to demonstrate proportionality of the assay be summarized as follows: (a) the activity is found under con- (the remainder of the table was done with the concentrations ditions in which Cro is the only X protein expected in substantial used for the data of line 1). The results given in Table 3 reveal quantity and in which active cI should explicitly not be made some similarities as well as clear differences in the binding (N-cI- phage); (b) either the activity is not found after infec- specificity of Cro and cI. Both cI and Cro-binding are severely tion by cro- phage or the protein exhibits altered binding impaired by the vlv3 combination (line 5) that affects both the properties; (c) the binding specificity is that expected for Cro ORl and oR2 sites of the reiterated operator regions (9, 3, 34). protein, and there are small but unambiguous differences from However, the vS mutation in the "strong-binding" site ORl is the binding specificity of cI protein, although both are affected sufficient to eliminate cI-binding under these conditions, but by some of the same operator region point mutations (Fig. 4 and has no strong effect on Cro-binding (line 6). The prm 16 Table 2); (d) antibody to cI has no effect on the binding activity mutation, located between the probable OR2 and ORS sites (35), that we have purified at a 5-fold excess over that required to seriously impairs neither Cro nor cI binding (line 7) [the prm116 eliminate the binding activity of cI (A. Folmanis, unpublished mutation appears to be in the promoter PM used for cI gene data); (e) Cro activity is not retained by DEAE-cellulose, transcription during the maintenance of lysogeny (18)]. From whereas cI protein is bound (A. Folkmanis, unpublished data the results of Table 3, we infer that Cro normally binds to the OR region, but has a slightly different recognition specificity than that of cI. The basis for the reduction in binding by both Table 3. Effect of operator region mutations on binding Cro and cI observed for the OLVOR+ DNA compared to wild activity of Cro protein than to type is not clear; it may reflect weaker binding to OR+ Percent DNA bound OL+ DNA added Cro cI

60 X+(OL OR) 56 44 0 X[1/2 conc.] 29 20 OLVORV 5 2 24 24 4 QLVoR+ C~~~~~ OL'VORulv3 7 <1 OLVORvS326 21 1 co oLVoRprmll6 19 19 z DNA-binding assays were carried out as described in Materials and Methods. The KCl concentration was 0.1 M. The deletion of both the left and right-side operator regions (OLVORV) was the imm434 substitution. The deletion of the left-side operator region (oLVoR+) was the bio24-5 substitution. This substitution was combined with the point mutations vlv3, vS326, and prmll6 of the 0.02 0.05 0.10 0.15 0.20 right-side operator region for the data of the last three lines. The [KCI], M level of DNA-binding activity used (except for line 2) was ap- proximately 70% of the maximum for each protein with wild-type FIG. 5. DNA-binding activity of Cro and cI for A+ and v2vlv3 DNA. This experiment has been performed with three separately DNA. Assays were carried out as described in Materials and Methods. prepared DNA samples of each genotype, with substantially the (- *), Cro protein and A+ DNA; (13 a), cI and A+ DNA; (40 -- - same results (in one experiment the binding of vS DNA by Cro *), Cro and Xv2vlv3 DNA; (O --- o), cI and Xv2vlv3 DNA. was about 25% lower than that given above). Downloaded by guest on September 26, 2021 Biochemistry: Folkmanis et al. Proc. Natl. Acad. Sci. USA 73 (1976) 2253 and refs. 5 and 8); (f) the molecular weight of the native and 10. Campbell, A. (1961) Virology 14,22-32. denatured DNA-binding protein are both substantially less than 11. Court, D., Green, L. & Echols, H. (1975) Virology 63, 484- the minimum (monomer) molecular weight of cI (Fig. 2 and 491. ref. 5). From the sum of these observations, we conclude that 12. Sussman, R. & Jacob, F. (1962) C. R. Hebd. Seances Acad. Sci. we are studying Gro 254, 1517-1519. activity. 13. Franklin, N. (1971) in The Bacteiphage Lambda, ed. Hershey, Although the cI and Cro proteins clearly recognize the same A. D. (Cold Spring Harbor Laboratory, Cold Spring Harbor, region of X DNA, the precise relationship of the two binding N.Y.), pp. 621-638. activities remains to be established. This relationship is par- 14. Takeda, Y., Ogata, K. & Matsubara, K. (1975) Virology 65, ticularly interesting because Cro and cI carry out totally dif- 385-391. ferent roles in viral development in vivo (lytic development 15. Jacob, F. & Wollman, E. L. (1954) Ann. Inst. Pasteur Paris 87, versus maintenance of lysogeny) and cannot substitute for each 653-673. other under normal conditions (refs. 1-4; A. Folkmanis, P. 16. Hopkins, N. & Ptashne, M. (1971) in The Bacteriophage Lambda, Mellon, H. Echols, and A. Skalka, manuscript in preparation). ed. Hershey, A. D. (Cold Spring Harbor Laboratory, Cold Spring We would like to know whether Cro binds in the same se- Harbor, N.Y.), pp. 571-574. 17. Ordal, G. & Kaiser, A. D. (1973) J. Mol. Biol. 79,704-722. quential fashion to reiterated regions of X DNA as does cI (9), 18. Yen, K.-M. & Gussin, G. N. (1973) Virology 56,300-312. whether the recognition sequences are identical or only over- 19. Kayajanian, G. (1970) Virology 41, 170-174. lapping, whether the recognition mechanisms employed by the 20. Kaiser, A. D. & Jacob, F. (1957) Virology 4,509-521. two proteins are closely similar, and whether there is genetic 21. Liedke-Kulke, M. & Kaiser, A. D. (1967) Virology 32, 465- homology between the cro and cI genes that provides for similar 474. structural features of the two proteins. 22. Riggs, A. D., Suzuki, H. & Bourgeois, S. (1970) J. Mol. Biol. 48, 6783. We thank Stephen Chung for purified cI protein, Louis Reichardt 23. Alberts, B., & Herrick, G. (1971) in Methods in Enzymology, eds. and Dale Kaiser for cI antibody, and Barbara Meyer for the commu- Grossman, L. & Moldave, K. (Academic Press, New York), Vol. nication of unpublished results. This investigation was supported in 21, pp. 198-217. part by Public Health Service Research Grant GM 17078 from the 24. Pero, J. (1971) in The Bacteriophage Lambda, ed. Hershey, A.. National Institute of General Medical Sciences. A.F. is a Postdoctoral D. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), Fellow of the National Cancer Institute and J.S. was a visiting scientist pp.599-608. from the Slovak Academy of Sciences, sponsored by the U.S. National 25. Matsubara, K. (1974) J. Virol. 13, 03-607. Academy of Sciences. 26. Berg, D. (1974) Virology 62,224-233. 27. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 1. Echols, H. (1972) Annu. Rev. Genet. 6,157-190. *28. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 4406- 2. Herskowitz, I. (1973) Annu. Rev. Genet. 7, 289-324. 4412. 3. Echols, H. (1974) Biochimie 56,1491-1496. 29. Weber, K. & Kuter, 0. (1971) J. Biol. Chem. 246,4504-4509. 4. Reichardt, L. (1975) J. Mol. Biol. 93, 267-288, 289-30. 30. Roberts, J. W. (1970) Cold Spring Harbor Symp. Quant. Biol. 5. Chadwick, P., Pirrotta, V., Steinberg, R. A., Hopkins, N. & 35,121-126. Ptashne, M. (1970) Cold Spring Harbor Symp. Quant. Biol. 35, 31. Sly, W. S., Rabideau, K. & Kolber, A. (1971) in The Bacteriophage 283-294. Lambda, ed. Hershey, A. D. (Cold Spring Harbor Laboratory, 6. Steinberg, R. A. & Ptashne, M. (1971) Nature New Biol. 230, Cold Spring Harbor, N.Y.), pp. 575-588. 276-280. 32. Echols, H., Green, L., Oppenheim, A. B., Oppenheim, A. & 7. Wu, A. M., Ghosh, S., Willard, M., Davison, J. & Echols, H. (1971) Honigman, A. (1973) J. Mol. Biol. 80,203-216. in The Bacteriophage Lambda, ed. Hershey, A. D. (Cold Spring 33. Maniatis, T., Ptashne, M., Backman, K., Kleid, D., Flashman, S., Harbor Laboratory, Cold Spring Harbor, N.Y.), pp. 589-598. Jeffrey, A. & Maurer, R. (1975) Cell 5, 109-113. 8. Wu, A. M., Ghosh, S., Echols, H. & Spiegelman, W. G. (1972) J. 34. Walz, A. & Pirrotta, V. (1975) Nature 254, 118-121. Mol. Biol. 67,407-421. 35. Meyer, B. J., Kleid, D. G. & Ptashne, M. (1975) Proc. Natl. Acad. 9. Maniatis, T. & Ptashne, M. (1973) Proc. Nati. Acad. Sd. USA 70, Sci. USA 72,4785-4789. 1531-1535. 36. Lieb, M. (1976) Mol. Gen. Genet., in press. Downloaded by guest on September 26, 2021