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Identification of early proteins coded by bacteriophage P22
VIROLOGY
68, 525-533 (1975)
Identification
of Early Proteins KENNETH
Department
LEW’
of Biology, Massachusetts
Coded
by Bacteriophage
SHERWOOD
AND Institute
of Technology,
P22
CASJENSZ Cambridge,
Massachusetts
02139
Accepted June 25,1975 Twelve early proteins coded by phage P22 have been resolved by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis of labeled infected cell lysates. Four early gene products, those of genes 12, 17, int, and erf, was identified by their absence in amber mutant-infected lysates. Several additional nonessential proteins were identified by their absence from lysates of cells infected with a deletion mutant, bp5, which is missing a portion of the early region of the chromosome. Mutations in genes 12 and 23 prevent normal expression of the phage-coded late proteins, and a mutation in gene 24 prevents expression of early and late protein synthesis. These last findings support the notion that P22 protein synthesis is controlled by two sequentially acting positive regulation elements, the products of nenes24 and23, which serve functions analogous to the gene N and Q proteins of phageh. INTRODUCTION
The development of bacteriophage P22, like that of other complex, double-stranded DNA phages, has been found to proceed through two distinct phases (Levine, 1972; Calendar, 1970). During the first phase, a set of proteins, called the “early proteins” is produced. These proteins are involved in DNA replication and recombination (Botstein and Levine, 1968; Botstein and Matz, 1970; Levine and Schott, 1972) and the exclusion of superinfecting phage (Susskind et al., 1971). If the lytic pathway is chosen, a second phase of protein synthesis ensues in which the “late proteins” are synthesized. The late proteins are responsible for the cutting and encapsidation of the mature phage chromosome and assembly of the phage particle (Botstein et al., 1973; King et al., 1973; Tye et al., 1974b). The genes coding for the early and late proteins map in two separate clusters (Fig. 1) (Botsteinet al., 1972). 1 Present address: Department of Biological Chemistry, Harvard Medical School, Boston, Mass. 02139. p Present address: Department of Microbiology, University of Utah Medical Center, Salt Lake City, Utah 84132. 525
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
This sequential order of gene expression appears to be regulated by positive controlling elements that are P22 gene products. A functional gene 24 protein is necessary for the expression of the early genes (Hilliker, 1974; results presented below), and it could function in a manner analogous to the phage AN gene protein (N gene function reviewed by Echols (1971)). The product of gene 23 is required for late protein synthesis (Botstein et al., 1973; Hilliker, 1974) and appears to be analogous to the A gene Q product (Herskowitz and Signer, 1970a,b; Echols, 1971). In this report we have studied the synthesis of the early and late proteins of P22 by using sodium dodecyl sulfate (SDS)polyacrylamide-gel electrophoresis. We have identified a number of the early gene products in the electrophoretic band pattern by observing that single protein bands are missing after infection by polypeptide chain termination (amber) mutants in the various cistrons and have confirmed directly the requirements of the gene 24 and 23 products for early and late protein synthesis, respectively. The late gene protein products have previously been identified in this manner by Botstein et al. (1973) and Casjens and King (1974).
526
LEW AND
FIG. 1. Genetic map of phage P22 (from Botstein et al. (1972), Hilliker and Botstein (1975) and Botstein, personal communication.) MATERIALS
AND
METHODS
Bacterial strains. The bacterial host used in all experiments was strain TA1530 (sup, uurB-) described by Botstein et al. (1973), nonpermissive for amber mutants. Phage stocks were prepared on the permissive (su+am19) host DB74 (Botstein and Matz, 1970). Phage strains. For the experiments described here, representative amber alleles were chosen for each of the early genes in which amber mutations have been isolated. The actual strains used were as follows:5-amN114 cl-7 (Botstein et al., 1973); 12-amH80 (Kolstad and Prell, 1969); 17-amH339 (gift of Dr. David Botstein); 18-amH100 cl-7 (gift of Dr. S. Hilliker); 23-amH791 cl-7 (Kolstad and Prell, 1969); 23-amS4 cl-7 (Hilliker and Botstein, 1975); int-am37 (gift of Dr. S. Hilliker); erf-aml2B cl-7 (Dr. K. Lew, unpublished). The deletion mutant bp5 was isolated by Chan et al. (1972) and physically characterized by Tye et al. (1974a). The clear plaque mutation cl -7 was described by Levine and Curtiss (1961); this mutation did not cause obvious systematic changes in the electrophoretic band pattern. Analysis of phage proteins in infected cells. Cells were grown, irradiated with ultraviolet light to suppress host protein synthesis, infected (multiplicity = 7) and
CASJENS
labeled with “C-labeled mixed amino acids (New England Nuclear) as has been previously described (Botstein et al., 1973). In experiments where infected cells were pulse labeled, radioactive amino acids were added at the times indicated in the text; “early-labeled” infected cells were kept in the presence of radioactive amino acids from 0 to 10 min after infection; “late-labeled” infected cells had the labeled amino acids added 15 min after infection and were harvested at 30 min. After the labeling period, excess salt-free casein hydrolysate was added, and the cells were pelleted rapidly (2 min at 10,000 g). The pellet was resuspended in SDS containing electrophoresis sample buffer, boiled for 1 min, and applied to discontinuous SDSpolyacrylamide electrophoresis slab-gels. The electrophoresis was carried out as described by O’Farrell et al. (1973). The resulting gels were dried according to the method of Maize1 (1971), and autoradiograms were prepared on Kodak singlecoated blue-sensitive X-ray film (Kodak SB54). RESULTS
We studied the proteins synthesized after infection of Salmonella typhimurium by bacteriophage P22. The infected cells were labeled with “C-amino acids and the resulting labeled proteins were separated by polyacrylamide-gel electrophoresis in the presence of SDS. Since normal P22+ infection does not efficiently shut off host protein synthesis, the method of Hendrix (1971) as applied to the P22 infected system by Botstein et al. (1973) was used to depress host-directed protein synthesis. The host cells (strain TA1530) were irradiated with 2500 erg/mm” of ultraviolet light before infection with the desired phage strain; this causes a substantial decrease in host protein synthesis, while leaving the host relatively competent to synthesize phage-specific proteins after infection. Kinetics of Protein Synthesis after P22 Infection Figure 2 shows the kinetics with which the various proteins in the electrophoretic band pattern are labeled. Cells were in-
IDENTIFICATION
OF EARLY
527
PROTEINS
TIME AFTER INFECTION(MIN) 5
10
15
20
25
30
35
40
45
- gP1 _
_ -_L7
gP12-
AleLI
*-
lY9
gp16 EP2
--
- -
gP5 gP20 gp8
gpint pE40pE32, pE30pE27gperfpE21pE20’ pE17’
gp26
EP17’
-
PX gP3
pa -
FIG. 2. Time course of wild-type P22-infected Salmonella cells. Irradiated cells infected with P22+ (prepared as in Materials and Methods) were labeled with %-labeled amino acids for 5-min pulses ending at the times indicated above the gel tracks. Immediately after the labeling period the cells were pelleted and dissolved in hot SDS. The samples were subjected to SDS-gel electrophoresis and autoradiography according to the procedures described in Materials and Methods. The early proteins are named on the left and the late proteins are named on the right side of the figure.
fected with wild-type P22 and pulse labeled with 14C-amino acids for 5 min ending at the times indicated. It can be seen that a set of proteins, called early proteins, are synthesized between 0 and 20 min, and synthesis of a second set of proteins, the late proteins, begins at about 20 min under our conditions. The late proteins are labeled according to their genetic identification (Botstein et al., 1973; Casjens and King, 19741, and the early proteins are labeled3 according to their identification below. The late proteins all appear simultaneously and appear to increase in parallel. The product of gene 8 has previously been found not to increase in parallel with 3 The polypeptide gene product of a cistron X is called gpX. Genetically unidentified proteins have been named p (protein) E (early) followed by the molecular weight, e.g., pE32 (after the nomenclature of Casjens and King (1975a)).
the other late proteins but to turn off soon after its synthesis begins (King and Casjens, 1974). This effect is much less pronounced in irradiated cells than in nonirradiated cells and may reflect the general low level of protein synthesis in irradiated cells; this is discussed further in Casjens and King, (1975b). The early proteins, on the other hand, do not all have similar kinetics of appearance. All of the early proteins except pE27 appear immediately after infection and are labeled during the 0-5-min pulse of radioactive amino acids; pE27 synthesis is delayed and begins between 5 and 10 min after infection. It is evident from Fig. 2 that, after reaching a maximal rate of synthesis at about lo-15 min after infection, the early protein synthesis rate decreases rapidly, presumably by an active shut-off mechanism, even under our conditions.
This turn-off does not occur simultaneously for al1 of the early proteins: pE30 synthesis decreases very rapidly after 5 min; gp12 synthesis decreases after about 10 min; and synthesis of the others, with the possible exception of gp int, declines after 15 min with gp erf, gp17 and pE27 decreasing more rapidly than the remaining proteins. Genetic Identification
of Early Proteins
We have identified some of the early proteins genetically with the use of amber and deletion mutations. Since amber mutations cause the premature termination, in a nonpermissive host, of the growing polypeptide produced by the gene in which it is located (Sarahbai et al., 19641, the mutant proteins are smaller than the wild-type gene products. Since SDS-polyacrylamide
gels separate proteins on the basis of size (Shapiro et al., 1967; Weber and Osborn, 1969), an amber mutation will cause the gene product affected to migrate more rapidly. Our gels are calibrated to the molecular weights of the P22 late gene products that were determined by Botstein et al. (1973). Figure 3 shows an experiment where S. typhimurium was infected by amber mutants in a number of the early genes. Amber mutations in gene 18 or gene 23 did not alter the early band-pattern, suggesting that their gene products are present in very small amounts or they comigrate with one of the other major early gene products. 15% acrylamide gels gave similar results (not shown), suggesting that our failure to identify these proteins is not simply explained by postulating that they have a low molecular
FIG. 3. Identification of P22-coded early proteins. 5’. typhimurium was infected with various mutants of P22 (extract description in Materials and Methods), and cells were labeled with W-labeled amino acids for the first 10 min of infection. The labeled cells were pelleted and resuspended in hot SDS buffer. The samples were applied to SDS gels for electrophoresis and subsequently were autoradiographed (see Materials and Methods).
IDENTIFICATION
OF EARLY
weight. Infection by an amber 12 mutant phage, unable to replicate DNA, causes the disappearance of a band of 54,000 molecular weight and the appearance of a new protein of a slightly smaller size, presumably the amber fragment. Several other amber 12 mutants showed neither the 54,000-MW band nor the putative amber fragment. Amber mutations in gene int, 17 or erf caused the disappearance of proteins of MW 46,000, 14,000 or 23,000, respectively. The int gene protein functions in the integration process (Smith and Levine, 1967), the 17 gene protein in allowing growth of P22 on a fels-1 lysogen (Susskind and Botstein, personal communication), and the erf gene protein in recombination (Botstein and Matz, 1970), respectively. The int gene protein appears to be among the latest of the early proteins to be turned off, if it is turned off at all. This is interesting in view of the observation of Smith and Levine (1967) that integration occurs very late after infection, and Shimada and Campbell’s discovery (1974) of a separate promotor for the int gene of phage A, which is thought to be analogous to the P22 int gene; see below. Eight early proteins were not affected by the various early amber mutations tested. Analysis of the proteins produced by the deletion mutation, bp5 (Tye et al., 1974a), showed that six of these proteins, pE40, pE32, pE21, pE20, pE17 and pE8, as well as g-p int were not produced. Presumably the first six are products of nonessential genes, since the deletion mutant, bp5, is viable. No amber mutations have been isolated in these genes. The bp5 deletion is missing about 3.8 x 10” daltons of DNA, 14% of the chromosome (Tye et al., 1974a), and the combined molecular weights of the missing proteins require 3.3 x lo6 daltons of DNA for their structural genes. Thus, it is possible that the actual structural genes of these proteins are deleted in the mutant, although it is also possible that some positive controlling element for the synthesis of these proteins is missing rather than the actual structural genes. Chan et al. (1972) have presented genetic evidence that the int gene and the integration site (att) are deleted in the bp5 mutant. The appearance of early proteins pE32, pE30
529
PROTEINS
and pE27 was somewhat variable and was not affected reproducibly by any of the mutants tested. This could be due to the prophage repressor or other controlling elements; this is currently under study. Table 1 summarizes the early proteins and their genetic identification. Control of Protein
Synthesis
The kinetics of synthesis suggests that synthesis of the proteins made after P22 infection is carefully regulated. This has been further shown by the genetic analysis of Hilliker and Botstein (1975) who showed that gene 24 product is required for the synthesis of a number of early functions. Botstein et al. (1973) and Hilliker (1974) showed that gene23 product is required for late protein synthesis. We have confirmed these results directly, by analyzing the proteins made by amber mutants in genes 23 and%. Figures 3 and 4 show the results of such infections: The 23 pattern showed no difference from the wild-type early protein pattern, even at late times. The 24 infection failed to produce normal amounts of early proteins; densitometry of these gels (results not shown) showed less than onetenth of the normal incorporation of label by the 2& phage. In some experiments gp17 seems to be made somewhat more efficiently than other early proteins under 24 conditions, but this effect was not reTABLE IDENTIFICATION
Early protein
gpl2 gp int pE40 pE32 pE30 pE27 gp erf pE21 pE20 pE17 gpl7 PEG
Gene
12 int ? ? ? ?
erf ? ? ? 17 ?
I
OF EARLY
Missing W
PROTEINS
in
No Yes (?I Yes Yes C?) No (?) No (?) No Yes Yes Yes No Yes
Approximate molecUlX
weight ( x lo-:!)
54 46 40 32 30 27 23 21 20 17 14 8
530
LEW
AND
producible. At later times some late proteins were produced by the 24- infected cells but in amounts well below normal. An amber mutation in the DNA synthesis gene, 12, also blocked the appearance of normal amounts of late proteins, although a small amount of gp5 (the major late protein) was made.
Comparison with Phage L Phage L is closely related to P22 in that it has similar morphology, serological properties, and sufficient homology to allow some genetic recombination between them (Bezdek and Amati, 1968; Favre et al., 1968; Chan and Botstein, 1972). In Fig. 5 we have compared the proteins made after infection by the two phages at early and late times. It can be seen that there is some similarity in the early band-patterns of the two phages, including gp 12, pE20, pE17 and possibly pE27 and gp erf. In these cases proteins of nearly identical molecular weights are made by both phages. The homology of gp12 is difficult to assess
r’
1 E
CASJENS
because the major late protein gp5 runs in the same position in the gel. The late proteins coded by the two phages are very similar; among the P22 late proteins separated in this experiment all have analogs of similar size in the phage L band-pattern, and phage L produces proteins of MW 78,000 and 21,000 which do not have obvious P22 analogs. From these results it is likely that P22 and L phage particles are assembled in similar fashion. DISCUSSION
We have separated about 12 polypeptides coded by the phage P22 early genes by polyacrylamide gel electrophoretic analysis of the proteins made soon after infection. Amber mutations have previously been isolated in four essential early genes, 12, 18, 23 and 24 (Botstein et al., 1972; Hilliker, 1974). Only one protein product of these essential genes, gp12 that is required for DNA replication, could be identified by its absence in amber mutant-infected cells. Its molecular weight, as estimated by elec-
23-
24-
12-
18-
gp12,gp5gp9-. pE40’
gp erfgPl7-
FIG. 4. Control of P22 protein synthesis. S. typhimurium was infected with wild-type phage and various mutants and labeled with W-labeled amino acids at late times (15-30 min after infection) when wild-type phage would normally be producing late proteins. The labeled infected cells were subjected to SDSpolyacrylamide-gel electrophoresis and autoradiography as described in Materials and Methods. The dose of ultraviolet irradiation for the 12- and 18- infections was about two-thirds the standard dose (see Materials and Methods), hence the presence of a background of host bands.
IDENTIFICATION
P22
1
OF EARLY
PROTEINS
531
teins account for about 65% of the coding capacity of the P22 genome. The products of all the known late genes except 13 and 19 have been identified in the electrophoretie gel-patterns; however there are a number of genetically defined early and control genes, cl, c2, c3,6,21,18,23,24, sieA, sieB, mnt and ant (Botstein et al., 1972; Levine, 1972; Botstein et al., 1975), whose protein products have not yet been visualized in this manner. It is supposed that these gene products are present in very low amounts in the infected cell extracts; this may be due to a low rate of synthesis or rapid turnover in the cell or possibly even rapid degradation after lysis. It is also possible that the synthesis of some gene products is affected by the heavy ultraviolet irradiation of the host cells before infection. Experiments with unirradiated cells have shown that this is EARLY LATE not likely to be the case with any of the FIG. 5. Comparison of the proteins coded by major proteins (Botstein et al., 1973; J. phages P22 and L. Cells were infected with P22+ or King and our unpublished results). L+ phage and labeled at early (O-10 min) or late Mutations in three cistrons, 12,23 and (15-30 mini times after infection. The labeled infected cells were subjected to SDS-polyacryl24, have pleiotropic effects on the expresamide-gel electrophoresis and autoradiography as sion of the viral proteins. The gene 12 described in Materials and Methods. product is required for viral DNA synthesis (Botstein and Levine, 1968). It is possitrophoretic migration rate in SDS gels, is ble that the effect of the 12- mutation on 54,000. The products of three nonessential late protein synthesis reflects a requireearly genes, gp erf, gp int, and gp17, were ment for normal DNA replication rather identified as proteins of MW 23,000, than a direct effect of gp12 on control of late protein synthesis. The analogy with 46,000, and 14,000, respectively. A deletion that mutation isolated by Tye et al. (1974b), phage A makes the interpretation gp12 acts indirectly to promote late protein which deletes about 14% of the chromoIt is generally some near the attachment site, causes the synthesis more likely. thought that the only requirement for late loss of seven proteins from the gel pattern. A protein synthesis is the gene Q product, These must be the products of previously unidentified nonessential genes since the and that the 0 and P products are required deletion phage is viable. Only two early only secondarily to promote DNA replicaproteins consistently seen in the gel pat- tion and thereby provide sufficient gene tern (pE27 and pE30) were not altered by copies to give high levels of late transcripany of the mutants tested. The sum of the tion (see Echols (1971) and Herskowitz and estimated molecular weights of the 12 Signer (1970a,b) for discussion of A gene expression). However, P22, LB- phage, also early proteins is 331,000. The combined unable to replicate DNA (Botstein and Levpreviously determined molecular weights of the late proteins (Botstein et al., 1973; ine, 19681, do produce some late proteins during nonpermissive infection; it is not Casjens and King, 1974) is 574,000. Thus, yet clear whether this is due to “leakiness” 17-18 x lo6 daltons of double-stranded of the 18- mutation or a difference between DNA are required to code for the synthesis P22 and h. of the observed phage-specific proteins. We have confirmed the observation of Since the molecular weight of P22 DNA is 27 x lo6 (Rhoades et al., 19681, these pro- Botstein et al. (1973) that in the absence of
P22
1
532
LEW AND
the gene 23 product, early proteins are produced but no late proteins are synthesized, suggesting that gp23 may be a positive regulatory element for the late genes. Mutants in gene 24 fail to produce normal amounts of early proteins or late proteins (about one-tenth of normal incorporation of radioactive amino acids into early proteins occurs), showing directly the gp24 is required for early gene expression. The 24- mutation used is known to be quite “leaky” in streptomycin-sensitive hosts (Hilliker and Botstein, 1975). Since the host used in these experiments was streptomycin sensitive this leakiness could explain the observation that the 24- phage produced one-tenth the normal level of early proteins rather than no early proteins. Botstein et al. (1972) have drawn attention to the observation that the early regions of the P22 and A genetic maps are very similar and have suggested that the control of development of the two phages might be analogous. Furthermore, it has been shown that P22 and A share about 18% homology by solution hybridization (Cowie and Szafranski, 1967) and that this homology resides in the early region of the A chromosome (Shalka and Hanson, 1972; Botstein and Herskowitz, 1974). Our results lend support to this hypothesis in several ways. First, mutations in genes23 and24 affect phage-specific protein synthesis in ways similar to those found by Hendrix (1971) for h genes Q and N, respectively. These genes are also genetically analogous (Botstein et al., 1972; Hilliker, 1974; Hilliker and Botstein, 1975). Second, the products of the int and erf genes, which occupy positions on the genetic map approximately equivalent to the A int and red genes, produce proteins of molecular weights similar to the A int and red proteins (Hendrix, 1971). Chan and Botstein (1972) found that a number of P22 gene functions could be rescued from phage L, suggesting that the two phages are very closely related. Our comparison of the two phages confirms this suggestion. Phage L produces several early proteins with molecular weights similar to P22 early proteins: pE20, pE17 and,
CASJENS
perhaps, gp12, gp erf, and pE27. The late proteins of the two phages were very similar. There were proteins of similar size for those gene products involved in DNA encapsidation (genes 1,2,5 and8), phage tail formation (gene 9), and DNA injection function (genes 16 and20). It is clear that there has been considerable conservation of homology between the essential genes of P22 and L. ACKNOWLEDGMENTS We thank Jonathan King and David Botstein, in whose laboratories this work was carried out, for help and advice during the course of the work and preparation of the manuscript and S. Hilliker for discussion of her unpublished results. Support came from NIH Grant No. GM17,980 to J. King, American Cancer Society Grant No. VCIBC to D. Botstein and a Helen Hay Whitney fellowship to S. Casjens. REFERENCES M., and AMATI, P. (1968,. Properties of P22 and a related Salmonella typhimurium phage. I. General features and host specificity. Virology 31, 272-278. BOTSTEIN, D., CHAN, R., and WADDELL, C. 119721. Genetics of bacteriophage PZ2. II. Gene order and gene function. Virology 19, 268-282. BOTSTEIN, D., and HERSKOWITZ, I. (19741. Properties of hybrids between Salmonella phage P22 and coliphage lambda. Nature ilondon) 251, 584-589. BOTSTEIN, D., and LEVINE, M. (1968). Synthesis and maturation of phage P22 DNA. II. Properties of temperature-sensitive phage mutants defective in DNA metabolism. J. Mol. Biol. 34, 643-654. BOTSTEIN, D., LEW, K., JARVIK, J., and SWANSON, C., JR. (1975). Role of the antirepressor in the bipartite control of repression and immunity by bacteriophage P22. J. Mol. Biol. 91, 439-462. BOTSTEIN, D., and MATZ, M. 11970). A recombination function essential to the growth of bacteriophage P22. J. Mol. Biol. 54, 417-440. BOTSTEIN, D., WADDELL, C., and KING, J. 11973). Mechanism of head assembly and DNA encapsulation in Salmonella phage P22. I. Genes, proteins, structures and DNA maturation. J. Mol. Biol. 80, 669-695. CALENDAR, R. (1970). The regulation of phage development. Annu. Reu. Microbial. 25, 241-296. CASJENS, S., and KING, J. (1974). P22 Morphogenesis. I: Catalytic scaffolding protein in capsid assembly. J. Supramol. Strut. 2, 202-224. CASJENS, S., and KING, J. (1975a). Virus assembly. Annu. Reu. Biochem., in press. CASTENS, S., and KING, J. (1975b). Control of scaffolding protein synthesis during bacteriophage P22 development. In preparation. BEZDEK,
IDENTIFICATION
OF EARLY
CHAN, R., and BOTSTEIN, D. (1972). Geneticsofbacteriophage P22. I. Isolation of prophage deletions which affect immunity to superinfection. Virology 49,257-267. CHAN, R., BOTSTEIN, D., WATANABE, T., and OGATA, Y. (1972). Specialized transduction of tetracycline resistance by phage P22 in Salmonella typhimurium. Virology 50, 883-898. COWIE, D., and SZAFRANSKI, P. (1967). Thermal chromatography of DNA-DNA reactions. Biophys. J. 7,567-584. DOVE, W. (1966). Action of the lambda chromosome. I. Control of functions in late bacteriophage development. J. Mol. Biol. 19, 187-201. ECHOLS, H. 11971). Regulation of lytic development. In “The Bacteriophage Lambda” (A. D. Hershey, ed.), pp. 247-270. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. FAVRE, R., AMATI, P., and BEZDEK, M. (1968). Properties of P22 and a related Salmonella typhimurium phage. II. Effect of H markers on infection of strain 1559. Virology 35, 238-247. HENDRIX, R. (1971). Identification of proteins coded in phage lambda. In “The Bacteriophage Lambda” (A. D. Hershey, ed.), pp. 355-370. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. HERSKOWITZ, I., and SIGNER, E. (1970a). A site essential for expression of all late genes of phage lambda. J. Mol. Biol. 47, 545-556. HERSKOWIT~, I., and SIGNER, E. (1970b). Control of transcription from the r strand of bacteriophage lambda. Cold Spring Harbor Symp. Quant. Biol. 35,355-368. HILLIKER, S., and BOTSTEIN, D. (1975). An early regulatory gene of Salmonella phage P22 analogous to gene N of coliphage A. Virology 00, OOO000. HILLIKER, S. (1974). “Specificity of Regulatory Elements in Temperate Bacteriophages.” Doctoral thesis, Massachusetts Institute of Technology, Cambridge, Mass. KING, J., and CASJENS, S. (1974). Catalytic head assembling protein in virus morphogenesis. Nature (London) 251, 112-119. KING, J., LENK, E., and BOTSTEIN, D. (1973). Mechanism of head assembly and DNA encapsulation in Salmonella phage P22. II. Morphogenetic path-
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way. J. Mol. Biol. 80, 669-695. KOLSTAD, R., and PRELL, H. (1969). An amber map of Salmonella phage P22. Mol. Gen. Genet. 104,339350. LEVINE, M. (1972). Replication and lysogeny with phage P22 in Salmonella typhimurium. CUFF. Top. Microbial. Immunol. 58, 135-156. LEVINE, M., and CURTISS, R. (1961). Genetic line structure of the C region and the linkage map of phage P22. Genetics 46, 1573-1580. LEVINE, M., and SCHOTT, C. (1971). Mutations of phage P22 affecting phage DNA synthesis and lysogenization. J. Mol. Biol. 62, 53-64. MAIZEL, J. V., JR. (1971). Polyacrylamide gel electrophoresis of viral proteins. Methods Viral. 5, 179246. O’FARRELL, P., GOLD, L., and HUANG, W. (1973). Identification of prereplicative T4 proteins. J. Biol. Chem. 248, 5499-5503. SARAHBAI, A., SHETTON, A., BRENNER, S., and BOLLE, A. (1964). Colinearity of the gene with the polypeptide chain. Nature (London) 201, 13-15. SHAPIRO, A., VIAUELA, E., and MAIZEL, J. V., JR. (1967). Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. Biophys. Res. Commun. 28, 815-820. SHIMADA, K., and CAMPBELL, A. (1974). Int-constitutive mutants of bacteriophage lambda. Proc. Nat. Acad. Sci. USA 71, 237-241. SHALKA, A., and HANSON, P. (1972). Comparisons of the distributions of nucleotides and common sequences in DNA from selected bacteriophages. J. Viral. 9, 583-593. SMITH, H., and LEVINE, M. (1967). A phage P22 gene controlling integration of prophage. Virology 13, 207-216. TYE, B., CHAN, R., and BOTSTEIN, D. (1974a). Packaging of an oversize transducing genome by Salmonella phage P22. J. Mol. Biol. 85, 485-500. TYE, B., HUBERMAN, J., and BOTSTEIN, D. (1974b). Non-random circular permutation of phage P22 DNA. J. Mol. Biol. 85, 501-532. WEBER, K., and OSBORN, M. (1969). The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406-4412.
68, 525-533 (1975)
Identification
of Early Proteins KENNETH
Department
LEW’
of Biology, Massachusetts
Coded
by Bacteriophage
SHERWOOD
AND Institute
of Technology,
P22
CASJENSZ Cambridge,
Massachusetts
02139
Accepted June 25,1975 Twelve early proteins coded by phage P22 have been resolved by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis of labeled infected cell lysates. Four early gene products, those of genes 12, 17, int, and erf, was identified by their absence in amber mutant-infected lysates. Several additional nonessential proteins were identified by their absence from lysates of cells infected with a deletion mutant, bp5, which is missing a portion of the early region of the chromosome. Mutations in genes 12 and 23 prevent normal expression of the phage-coded late proteins, and a mutation in gene 24 prevents expression of early and late protein synthesis. These last findings support the notion that P22 protein synthesis is controlled by two sequentially acting positive regulation elements, the products of nenes24 and23, which serve functions analogous to the gene N and Q proteins of phageh. INTRODUCTION
The development of bacteriophage P22, like that of other complex, double-stranded DNA phages, has been found to proceed through two distinct phases (Levine, 1972; Calendar, 1970). During the first phase, a set of proteins, called the “early proteins” is produced. These proteins are involved in DNA replication and recombination (Botstein and Levine, 1968; Botstein and Matz, 1970; Levine and Schott, 1972) and the exclusion of superinfecting phage (Susskind et al., 1971). If the lytic pathway is chosen, a second phase of protein synthesis ensues in which the “late proteins” are synthesized. The late proteins are responsible for the cutting and encapsidation of the mature phage chromosome and assembly of the phage particle (Botstein et al., 1973; King et al., 1973; Tye et al., 1974b). The genes coding for the early and late proteins map in two separate clusters (Fig. 1) (Botsteinet al., 1972). 1 Present address: Department of Biological Chemistry, Harvard Medical School, Boston, Mass. 02139. p Present address: Department of Microbiology, University of Utah Medical Center, Salt Lake City, Utah 84132. 525
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
This sequential order of gene expression appears to be regulated by positive controlling elements that are P22 gene products. A functional gene 24 protein is necessary for the expression of the early genes (Hilliker, 1974; results presented below), and it could function in a manner analogous to the phage AN gene protein (N gene function reviewed by Echols (1971)). The product of gene 23 is required for late protein synthesis (Botstein et al., 1973; Hilliker, 1974) and appears to be analogous to the A gene Q product (Herskowitz and Signer, 1970a,b; Echols, 1971). In this report we have studied the synthesis of the early and late proteins of P22 by using sodium dodecyl sulfate (SDS)polyacrylamide-gel electrophoresis. We have identified a number of the early gene products in the electrophoretic band pattern by observing that single protein bands are missing after infection by polypeptide chain termination (amber) mutants in the various cistrons and have confirmed directly the requirements of the gene 24 and 23 products for early and late protein synthesis, respectively. The late gene protein products have previously been identified in this manner by Botstein et al. (1973) and Casjens and King (1974).
526
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FIG. 1. Genetic map of phage P22 (from Botstein et al. (1972), Hilliker and Botstein (1975) and Botstein, personal communication.) MATERIALS
AND
METHODS
Bacterial strains. The bacterial host used in all experiments was strain TA1530 (sup, uurB-) described by Botstein et al. (1973), nonpermissive for amber mutants. Phage stocks were prepared on the permissive (su+am19) host DB74 (Botstein and Matz, 1970). Phage strains. For the experiments described here, representative amber alleles were chosen for each of the early genes in which amber mutations have been isolated. The actual strains used were as follows:5-amN114 cl-7 (Botstein et al., 1973); 12-amH80 (Kolstad and Prell, 1969); 17-amH339 (gift of Dr. David Botstein); 18-amH100 cl-7 (gift of Dr. S. Hilliker); 23-amH791 cl-7 (Kolstad and Prell, 1969); 23-amS4 cl-7 (Hilliker and Botstein, 1975); int-am37 (gift of Dr. S. Hilliker); erf-aml2B cl-7 (Dr. K. Lew, unpublished). The deletion mutant bp5 was isolated by Chan et al. (1972) and physically characterized by Tye et al. (1974a). The clear plaque mutation cl -7 was described by Levine and Curtiss (1961); this mutation did not cause obvious systematic changes in the electrophoretic band pattern. Analysis of phage proteins in infected cells. Cells were grown, irradiated with ultraviolet light to suppress host protein synthesis, infected (multiplicity = 7) and
CASJENS
labeled with “C-labeled mixed amino acids (New England Nuclear) as has been previously described (Botstein et al., 1973). In experiments where infected cells were pulse labeled, radioactive amino acids were added at the times indicated in the text; “early-labeled” infected cells were kept in the presence of radioactive amino acids from 0 to 10 min after infection; “late-labeled” infected cells had the labeled amino acids added 15 min after infection and were harvested at 30 min. After the labeling period, excess salt-free casein hydrolysate was added, and the cells were pelleted rapidly (2 min at 10,000 g). The pellet was resuspended in SDS containing electrophoresis sample buffer, boiled for 1 min, and applied to discontinuous SDSpolyacrylamide electrophoresis slab-gels. The electrophoresis was carried out as described by O’Farrell et al. (1973). The resulting gels were dried according to the method of Maize1 (1971), and autoradiograms were prepared on Kodak singlecoated blue-sensitive X-ray film (Kodak SB54). RESULTS
We studied the proteins synthesized after infection of Salmonella typhimurium by bacteriophage P22. The infected cells were labeled with “C-amino acids and the resulting labeled proteins were separated by polyacrylamide-gel electrophoresis in the presence of SDS. Since normal P22+ infection does not efficiently shut off host protein synthesis, the method of Hendrix (1971) as applied to the P22 infected system by Botstein et al. (1973) was used to depress host-directed protein synthesis. The host cells (strain TA1530) were irradiated with 2500 erg/mm” of ultraviolet light before infection with the desired phage strain; this causes a substantial decrease in host protein synthesis, while leaving the host relatively competent to synthesize phage-specific proteins after infection. Kinetics of Protein Synthesis after P22 Infection Figure 2 shows the kinetics with which the various proteins in the electrophoretic band pattern are labeled. Cells were in-
IDENTIFICATION
OF EARLY
527
PROTEINS
TIME AFTER INFECTION(MIN) 5
10
15
20
25
30
35
40
45
- gP1 _
_ -_L7
gP12-
AleLI
*-
lY9
gp16 EP2
--
- -
gP5 gP20 gp8
gpint pE40pE32, pE30pE27gperfpE21pE20’ pE17’
gp26
EP17’
-
PX gP3
pa -
FIG. 2. Time course of wild-type P22-infected Salmonella cells. Irradiated cells infected with P22+ (prepared as in Materials and Methods) were labeled with %-labeled amino acids for 5-min pulses ending at the times indicated above the gel tracks. Immediately after the labeling period the cells were pelleted and dissolved in hot SDS. The samples were subjected to SDS-gel electrophoresis and autoradiography according to the procedures described in Materials and Methods. The early proteins are named on the left and the late proteins are named on the right side of the figure.
fected with wild-type P22 and pulse labeled with 14C-amino acids for 5 min ending at the times indicated. It can be seen that a set of proteins, called early proteins, are synthesized between 0 and 20 min, and synthesis of a second set of proteins, the late proteins, begins at about 20 min under our conditions. The late proteins are labeled according to their genetic identification (Botstein et al., 1973; Casjens and King, 19741, and the early proteins are labeled3 according to their identification below. The late proteins all appear simultaneously and appear to increase in parallel. The product of gene 8 has previously been found not to increase in parallel with 3 The polypeptide gene product of a cistron X is called gpX. Genetically unidentified proteins have been named p (protein) E (early) followed by the molecular weight, e.g., pE32 (after the nomenclature of Casjens and King (1975a)).
the other late proteins but to turn off soon after its synthesis begins (King and Casjens, 1974). This effect is much less pronounced in irradiated cells than in nonirradiated cells and may reflect the general low level of protein synthesis in irradiated cells; this is discussed further in Casjens and King, (1975b). The early proteins, on the other hand, do not all have similar kinetics of appearance. All of the early proteins except pE27 appear immediately after infection and are labeled during the 0-5-min pulse of radioactive amino acids; pE27 synthesis is delayed and begins between 5 and 10 min after infection. It is evident from Fig. 2 that, after reaching a maximal rate of synthesis at about lo-15 min after infection, the early protein synthesis rate decreases rapidly, presumably by an active shut-off mechanism, even under our conditions.
This turn-off does not occur simultaneously for al1 of the early proteins: pE30 synthesis decreases very rapidly after 5 min; gp12 synthesis decreases after about 10 min; and synthesis of the others, with the possible exception of gp int, declines after 15 min with gp erf, gp17 and pE27 decreasing more rapidly than the remaining proteins. Genetic Identification
of Early Proteins
We have identified some of the early proteins genetically with the use of amber and deletion mutations. Since amber mutations cause the premature termination, in a nonpermissive host, of the growing polypeptide produced by the gene in which it is located (Sarahbai et al., 19641, the mutant proteins are smaller than the wild-type gene products. Since SDS-polyacrylamide
gels separate proteins on the basis of size (Shapiro et al., 1967; Weber and Osborn, 1969), an amber mutation will cause the gene product affected to migrate more rapidly. Our gels are calibrated to the molecular weights of the P22 late gene products that were determined by Botstein et al. (1973). Figure 3 shows an experiment where S. typhimurium was infected by amber mutants in a number of the early genes. Amber mutations in gene 18 or gene 23 did not alter the early band-pattern, suggesting that their gene products are present in very small amounts or they comigrate with one of the other major early gene products. 15% acrylamide gels gave similar results (not shown), suggesting that our failure to identify these proteins is not simply explained by postulating that they have a low molecular
FIG. 3. Identification of P22-coded early proteins. 5’. typhimurium was infected with various mutants of P22 (extract description in Materials and Methods), and cells were labeled with W-labeled amino acids for the first 10 min of infection. The labeled cells were pelleted and resuspended in hot SDS buffer. The samples were applied to SDS gels for electrophoresis and subsequently were autoradiographed (see Materials and Methods).
IDENTIFICATION
OF EARLY
weight. Infection by an amber 12 mutant phage, unable to replicate DNA, causes the disappearance of a band of 54,000 molecular weight and the appearance of a new protein of a slightly smaller size, presumably the amber fragment. Several other amber 12 mutants showed neither the 54,000-MW band nor the putative amber fragment. Amber mutations in gene int, 17 or erf caused the disappearance of proteins of MW 46,000, 14,000 or 23,000, respectively. The int gene protein functions in the integration process (Smith and Levine, 1967), the 17 gene protein in allowing growth of P22 on a fels-1 lysogen (Susskind and Botstein, personal communication), and the erf gene protein in recombination (Botstein and Matz, 1970), respectively. The int gene protein appears to be among the latest of the early proteins to be turned off, if it is turned off at all. This is interesting in view of the observation of Smith and Levine (1967) that integration occurs very late after infection, and Shimada and Campbell’s discovery (1974) of a separate promotor for the int gene of phage A, which is thought to be analogous to the P22 int gene; see below. Eight early proteins were not affected by the various early amber mutations tested. Analysis of the proteins produced by the deletion mutation, bp5 (Tye et al., 1974a), showed that six of these proteins, pE40, pE32, pE21, pE20, pE17 and pE8, as well as g-p int were not produced. Presumably the first six are products of nonessential genes, since the deletion mutant, bp5, is viable. No amber mutations have been isolated in these genes. The bp5 deletion is missing about 3.8 x 10” daltons of DNA, 14% of the chromosome (Tye et al., 1974a), and the combined molecular weights of the missing proteins require 3.3 x lo6 daltons of DNA for their structural genes. Thus, it is possible that the actual structural genes of these proteins are deleted in the mutant, although it is also possible that some positive controlling element for the synthesis of these proteins is missing rather than the actual structural genes. Chan et al. (1972) have presented genetic evidence that the int gene and the integration site (att) are deleted in the bp5 mutant. The appearance of early proteins pE32, pE30
529
PROTEINS
and pE27 was somewhat variable and was not affected reproducibly by any of the mutants tested. This could be due to the prophage repressor or other controlling elements; this is currently under study. Table 1 summarizes the early proteins and their genetic identification. Control of Protein
Synthesis
The kinetics of synthesis suggests that synthesis of the proteins made after P22 infection is carefully regulated. This has been further shown by the genetic analysis of Hilliker and Botstein (1975) who showed that gene 24 product is required for the synthesis of a number of early functions. Botstein et al. (1973) and Hilliker (1974) showed that gene23 product is required for late protein synthesis. We have confirmed these results directly, by analyzing the proteins made by amber mutants in genes 23 and%. Figures 3 and 4 show the results of such infections: The 23 pattern showed no difference from the wild-type early protein pattern, even at late times. The 24 infection failed to produce normal amounts of early proteins; densitometry of these gels (results not shown) showed less than onetenth of the normal incorporation of label by the 2& phage. In some experiments gp17 seems to be made somewhat more efficiently than other early proteins under 24 conditions, but this effect was not reTABLE IDENTIFICATION
Early protein
gpl2 gp int pE40 pE32 pE30 pE27 gp erf pE21 pE20 pE17 gpl7 PEG
Gene
12 int ? ? ? ?
erf ? ? ? 17 ?
I
OF EARLY
Missing W
PROTEINS
in
No Yes (?I Yes Yes C?) No (?) No (?) No Yes Yes Yes No Yes
Approximate molecUlX
weight ( x lo-:!)
54 46 40 32 30 27 23 21 20 17 14 8
530
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AND
producible. At later times some late proteins were produced by the 24- infected cells but in amounts well below normal. An amber mutation in the DNA synthesis gene, 12, also blocked the appearance of normal amounts of late proteins, although a small amount of gp5 (the major late protein) was made.
Comparison with Phage L Phage L is closely related to P22 in that it has similar morphology, serological properties, and sufficient homology to allow some genetic recombination between them (Bezdek and Amati, 1968; Favre et al., 1968; Chan and Botstein, 1972). In Fig. 5 we have compared the proteins made after infection by the two phages at early and late times. It can be seen that there is some similarity in the early band-patterns of the two phages, including gp 12, pE20, pE17 and possibly pE27 and gp erf. In these cases proteins of nearly identical molecular weights are made by both phages. The homology of gp12 is difficult to assess
r’
1 E
CASJENS
because the major late protein gp5 runs in the same position in the gel. The late proteins coded by the two phages are very similar; among the P22 late proteins separated in this experiment all have analogs of similar size in the phage L band-pattern, and phage L produces proteins of MW 78,000 and 21,000 which do not have obvious P22 analogs. From these results it is likely that P22 and L phage particles are assembled in similar fashion. DISCUSSION
We have separated about 12 polypeptides coded by the phage P22 early genes by polyacrylamide gel electrophoretic analysis of the proteins made soon after infection. Amber mutations have previously been isolated in four essential early genes, 12, 18, 23 and 24 (Botstein et al., 1972; Hilliker, 1974). Only one protein product of these essential genes, gp12 that is required for DNA replication, could be identified by its absence in amber mutant-infected cells. Its molecular weight, as estimated by elec-
23-
24-
12-
18-
gp12,gp5gp9-. pE40’
gp erfgPl7-
FIG. 4. Control of P22 protein synthesis. S. typhimurium was infected with wild-type phage and various mutants and labeled with W-labeled amino acids at late times (15-30 min after infection) when wild-type phage would normally be producing late proteins. The labeled infected cells were subjected to SDSpolyacrylamide-gel electrophoresis and autoradiography as described in Materials and Methods. The dose of ultraviolet irradiation for the 12- and 18- infections was about two-thirds the standard dose (see Materials and Methods), hence the presence of a background of host bands.
IDENTIFICATION
P22
1
OF EARLY
PROTEINS
531
teins account for about 65% of the coding capacity of the P22 genome. The products of all the known late genes except 13 and 19 have been identified in the electrophoretie gel-patterns; however there are a number of genetically defined early and control genes, cl, c2, c3,6,21,18,23,24, sieA, sieB, mnt and ant (Botstein et al., 1972; Levine, 1972; Botstein et al., 1975), whose protein products have not yet been visualized in this manner. It is supposed that these gene products are present in very low amounts in the infected cell extracts; this may be due to a low rate of synthesis or rapid turnover in the cell or possibly even rapid degradation after lysis. It is also possible that the synthesis of some gene products is affected by the heavy ultraviolet irradiation of the host cells before infection. Experiments with unirradiated cells have shown that this is EARLY LATE not likely to be the case with any of the FIG. 5. Comparison of the proteins coded by major proteins (Botstein et al., 1973; J. phages P22 and L. Cells were infected with P22+ or King and our unpublished results). L+ phage and labeled at early (O-10 min) or late Mutations in three cistrons, 12,23 and (15-30 mini times after infection. The labeled infected cells were subjected to SDS-polyacryl24, have pleiotropic effects on the expresamide-gel electrophoresis and autoradiography as sion of the viral proteins. The gene 12 described in Materials and Methods. product is required for viral DNA synthesis (Botstein and Levine, 1968). It is possitrophoretic migration rate in SDS gels, is ble that the effect of the 12- mutation on 54,000. The products of three nonessential late protein synthesis reflects a requireearly genes, gp erf, gp int, and gp17, were ment for normal DNA replication rather identified as proteins of MW 23,000, than a direct effect of gp12 on control of late protein synthesis. The analogy with 46,000, and 14,000, respectively. A deletion that mutation isolated by Tye et al. (1974b), phage A makes the interpretation gp12 acts indirectly to promote late protein which deletes about 14% of the chromoIt is generally some near the attachment site, causes the synthesis more likely. thought that the only requirement for late loss of seven proteins from the gel pattern. A protein synthesis is the gene Q product, These must be the products of previously unidentified nonessential genes since the and that the 0 and P products are required deletion phage is viable. Only two early only secondarily to promote DNA replicaproteins consistently seen in the gel pat- tion and thereby provide sufficient gene tern (pE27 and pE30) were not altered by copies to give high levels of late transcripany of the mutants tested. The sum of the tion (see Echols (1971) and Herskowitz and estimated molecular weights of the 12 Signer (1970a,b) for discussion of A gene expression). However, P22, LB- phage, also early proteins is 331,000. The combined unable to replicate DNA (Botstein and Levpreviously determined molecular weights of the late proteins (Botstein et al., 1973; ine, 19681, do produce some late proteins during nonpermissive infection; it is not Casjens and King, 1974) is 574,000. Thus, yet clear whether this is due to “leakiness” 17-18 x lo6 daltons of double-stranded of the 18- mutation or a difference between DNA are required to code for the synthesis P22 and h. of the observed phage-specific proteins. We have confirmed the observation of Since the molecular weight of P22 DNA is 27 x lo6 (Rhoades et al., 19681, these pro- Botstein et al. (1973) that in the absence of
P22
1
532
LEW AND
the gene 23 product, early proteins are produced but no late proteins are synthesized, suggesting that gp23 may be a positive regulatory element for the late genes. Mutants in gene 24 fail to produce normal amounts of early proteins or late proteins (about one-tenth of normal incorporation of radioactive amino acids into early proteins occurs), showing directly the gp24 is required for early gene expression. The 24- mutation used is known to be quite “leaky” in streptomycin-sensitive hosts (Hilliker and Botstein, 1975). Since the host used in these experiments was streptomycin sensitive this leakiness could explain the observation that the 24- phage produced one-tenth the normal level of early proteins rather than no early proteins. Botstein et al. (1972) have drawn attention to the observation that the early regions of the P22 and A genetic maps are very similar and have suggested that the control of development of the two phages might be analogous. Furthermore, it has been shown that P22 and A share about 18% homology by solution hybridization (Cowie and Szafranski, 1967) and that this homology resides in the early region of the A chromosome (Shalka and Hanson, 1972; Botstein and Herskowitz, 1974). Our results lend support to this hypothesis in several ways. First, mutations in genes23 and24 affect phage-specific protein synthesis in ways similar to those found by Hendrix (1971) for h genes Q and N, respectively. These genes are also genetically analogous (Botstein et al., 1972; Hilliker, 1974; Hilliker and Botstein, 1975). Second, the products of the int and erf genes, which occupy positions on the genetic map approximately equivalent to the A int and red genes, produce proteins of molecular weights similar to the A int and red proteins (Hendrix, 1971). Chan and Botstein (1972) found that a number of P22 gene functions could be rescued from phage L, suggesting that the two phages are very closely related. Our comparison of the two phages confirms this suggestion. Phage L produces several early proteins with molecular weights similar to P22 early proteins: pE20, pE17 and,
CASJENS
perhaps, gp12, gp erf, and pE27. The late proteins of the two phages were very similar. There were proteins of similar size for those gene products involved in DNA encapsidation (genes 1,2,5 and8), phage tail formation (gene 9), and DNA injection function (genes 16 and20). It is clear that there has been considerable conservation of homology between the essential genes of P22 and L. ACKNOWLEDGMENTS We thank Jonathan King and David Botstein, in whose laboratories this work was carried out, for help and advice during the course of the work and preparation of the manuscript and S. Hilliker for discussion of her unpublished results. Support came from NIH Grant No. GM17,980 to J. King, American Cancer Society Grant No. VCIBC to D. Botstein and a Helen Hay Whitney fellowship to S. Casjens. REFERENCES M., and AMATI, P. (1968,. Properties of P22 and a related Salmonella typhimurium phage. I. General features and host specificity. Virology 31, 272-278. BOTSTEIN, D., CHAN, R., and WADDELL, C. 119721. Genetics of bacteriophage PZ2. II. Gene order and gene function. Virology 19, 268-282. BOTSTEIN, D., and HERSKOWITZ, I. (19741. Properties of hybrids between Salmonella phage P22 and coliphage lambda. Nature ilondon) 251, 584-589. BOTSTEIN, D., and LEVINE, M. (1968). Synthesis and maturation of phage P22 DNA. II. Properties of temperature-sensitive phage mutants defective in DNA metabolism. J. Mol. Biol. 34, 643-654. BOTSTEIN, D., LEW, K., JARVIK, J., and SWANSON, C., JR. (1975). Role of the antirepressor in the bipartite control of repression and immunity by bacteriophage P22. J. Mol. Biol. 91, 439-462. BOTSTEIN, D., and MATZ, M. 11970). A recombination function essential to the growth of bacteriophage P22. J. Mol. Biol. 54, 417-440. BOTSTEIN, D., WADDELL, C., and KING, J. 11973). Mechanism of head assembly and DNA encapsulation in Salmonella phage P22. I. Genes, proteins, structures and DNA maturation. J. Mol. Biol. 80, 669-695. CALENDAR, R. (1970). The regulation of phage development. Annu. Reu. Microbial. 25, 241-296. CASJENS, S., and KING, J. (1974). P22 Morphogenesis. I: Catalytic scaffolding protein in capsid assembly. J. Supramol. Strut. 2, 202-224. CASJENS, S., and KING, J. (1975a). Virus assembly. Annu. Reu. Biochem., in press. CASTENS, S., and KING, J. (1975b). Control of scaffolding protein synthesis during bacteriophage P22 development. In preparation. BEZDEK,
IDENTIFICATION
OF EARLY
CHAN, R., and BOTSTEIN, D. (1972). Geneticsofbacteriophage P22. I. Isolation of prophage deletions which affect immunity to superinfection. Virology 49,257-267. CHAN, R., BOTSTEIN, D., WATANABE, T., and OGATA, Y. (1972). Specialized transduction of tetracycline resistance by phage P22 in Salmonella typhimurium. Virology 50, 883-898. COWIE, D., and SZAFRANSKI, P. (1967). Thermal chromatography of DNA-DNA reactions. Biophys. J. 7,567-584. DOVE, W. (1966). Action of the lambda chromosome. I. Control of functions in late bacteriophage development. J. Mol. Biol. 19, 187-201. ECHOLS, H. 11971). Regulation of lytic development. In “The Bacteriophage Lambda” (A. D. Hershey, ed.), pp. 247-270. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. FAVRE, R., AMATI, P., and BEZDEK, M. (1968). Properties of P22 and a related Salmonella typhimurium phage. II. Effect of H markers on infection of strain 1559. Virology 35, 238-247. HENDRIX, R. (1971). Identification of proteins coded in phage lambda. In “The Bacteriophage Lambda” (A. D. Hershey, ed.), pp. 355-370. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. HERSKOWITZ, I., and SIGNER, E. (1970a). A site essential for expression of all late genes of phage lambda. J. Mol. Biol. 47, 545-556. HERSKOWIT~, I., and SIGNER, E. (1970b). Control of transcription from the r strand of bacteriophage lambda. Cold Spring Harbor Symp. Quant. Biol. 35,355-368. HILLIKER, S., and BOTSTEIN, D. (1975). An early regulatory gene of Salmonella phage P22 analogous to gene N of coliphage A. Virology 00, OOO000. HILLIKER, S. (1974). “Specificity of Regulatory Elements in Temperate Bacteriophages.” Doctoral thesis, Massachusetts Institute of Technology, Cambridge, Mass. KING, J., and CASJENS, S. (1974). Catalytic head assembling protein in virus morphogenesis. Nature (London) 251, 112-119. KING, J., LENK, E., and BOTSTEIN, D. (1973). Mechanism of head assembly and DNA encapsulation in Salmonella phage P22. II. Morphogenetic path-
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way. J. Mol. Biol. 80, 669-695. KOLSTAD, R., and PRELL, H. (1969). An amber map of Salmonella phage P22. Mol. Gen. Genet. 104,339350. LEVINE, M. (1972). Replication and lysogeny with phage P22 in Salmonella typhimurium. CUFF. Top. Microbial. Immunol. 58, 135-156. LEVINE, M., and CURTISS, R. (1961). Genetic line structure of the C region and the linkage map of phage P22. Genetics 46, 1573-1580. LEVINE, M., and SCHOTT, C. (1971). Mutations of phage P22 affecting phage DNA synthesis and lysogenization. J. Mol. Biol. 62, 53-64. MAIZEL, J. V., JR. (1971). Polyacrylamide gel electrophoresis of viral proteins. Methods Viral. 5, 179246. O’FARRELL, P., GOLD, L., and HUANG, W. (1973). Identification of prereplicative T4 proteins. J. Biol. Chem. 248, 5499-5503. SARAHBAI, A., SHETTON, A., BRENNER, S., and BOLLE, A. (1964). Colinearity of the gene with the polypeptide chain. Nature (London) 201, 13-15. SHAPIRO, A., VIAUELA, E., and MAIZEL, J. V., JR. (1967). Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. Biophys. Res. Commun. 28, 815-820. SHIMADA, K., and CAMPBELL, A. (1974). Int-constitutive mutants of bacteriophage lambda. Proc. Nat. Acad. Sci. USA 71, 237-241. SHALKA, A., and HANSON, P. (1972). Comparisons of the distributions of nucleotides and common sequences in DNA from selected bacteriophages. J. Viral. 9, 583-593. SMITH, H., and LEVINE, M. (1967). A phage P22 gene controlling integration of prophage. Virology 13, 207-216. TYE, B., CHAN, R., and BOTSTEIN, D. (1974a). Packaging of an oversize transducing genome by Salmonella phage P22. J. Mol. Biol. 85, 485-500. TYE, B., HUBERMAN, J., and BOTSTEIN, D. (1974b). Non-random circular permutation of phage P22 DNA. J. Mol. Biol. 85, 501-532. WEBER, K., and OSBORN, M. (1969). The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406-4412.