Vol. 53, No. 1JOURNAL OF VIROLOGY, Jan. 1985, p. 174-1790022-538X/85/010174-06$02.00/0Copyright © 1985, American Society for Microbiology

Assembly-Controlled Autogenous Modulation of Bacteriophage P22Scaffolding Protein Gene Expression

SHERWOOD CASJENS,1* MARK B. ADAMS,' CAROL HALL,2t AND JONATHAN KING2

Department of Cellular, Viral and Molecular Biology, University of Utah Medical Center, Salt Lake City, Utah 84132,1and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 012392

Received 27 February 1984/Accepted 26 September 1984

In the assembly of bacteriophage P22, precursor particles containing two major proteins, the gene 5 coatprotein and the gene 8 scaffolding protein, package the DNA molecule. During the encapsidation reaction allof the scaffolding protein molecules are released intact and subsequently participate in further rounds of DNAencapsidation. We have previously shown that even though it lies in the center of the late region of the geneticmap, the scaffolding protein gene is not always expressed coordinately with the remainder of the late proteinsand that some feature of the phage assembly process affects its expression. We present here in vivo experimentswhich show that there is an inverse correlation between the amount of unassembled scaffolding protein and therate of scaffolding protein synthesis and that long amber fragments of the scaffolding protein can turn down thesynthesis of intact scaffolding protein in trans. These results support a model for scaffolding protein regulationin which the feature of the assembly process which modulates the rate of scaffolding protein synthesis is theamount of unassembled scaffolding protein itself.

The notion that proteins involved in the assembly ofmacromolecular structures can turn off their own synthesiswhen not assembled is attractive, since excess unassembledprotein subunits can simply eliminate the production ofadditional subunits when the structure (or a sufficient numberof structures) is completed, and production remains off untilassembly begins again. In this report, we present evidencethat a bacteriophage P22 assembly protein, the scaffoldingprotein, can depress its own synthesis in vivo when unas-sembled.The genetics and molecular biology of bacteriophage P22

have been intensively studied (reviewed in reference 27).The switch to late protein synthesis is controlled by the gene23 product. This regulation is thought to occur by an RNApolymerase antitermination mechanism in which the gene 23protein causes readthrough at a termination site(s) a shortdistance downstream from the late promoter, Plate (2, 18, 24,25, 27). The existence of a single late promoter in P22 has notbeen proven directly, but studies with polar insertion ele-ments (28) and measurements of the temporal length of thelag before exponential decay of individual gene mRNAsafter the addition of rifampin (4) strongly support this idea.There are 15 known genes in the late operon; 2 are requiredfor lysis of the infected cell, and 13 are required for DNApackaging and assembly of progeny phage particles.The pathway by which the protein products of the 13

morphogenetic late genes assemble into infectious phage hasalso been extensively investigated (reviewed in reference10). As is the case with many other double-stranded DNAbacteriophages, a protein particle called a prohead is assem-bled first, which is subsequently filled with DNA (15, 22).The prohead contains ca. 420 molecules of coat protein (gp5[gpX refers to the protein product of gene XA) on theexterior, 200 to 300 molecules of scaffolding protein (gp8) inthe interior, and 10 to 30 molecules each of gpl, gp7, gpl6,and gp2O (3, 7, 15, 23). The prohead encapsidates DNA in a

* Corresponding author.t Present address: Department of Biological Sciences, Stanford

University, Stanford, CA 94305.

complex reaction with the following features: one phagelength ofDNA (43,500 base pairs) is cut from the replicatingconcatameric DNA (3), the products of genes 2 and 3 mustact but are not present in the final phage particle (3, 5, 21),and all of the molecules of scaffolding protein (gp8) exitintact from the structure. These scaffolding protein mole-cules can then recycle by reacting with newly synthesizedcoat protein, gpl, gp7, gpl6, and gp2O to form new proheads(13). Thus, the scaffolding protein in effect catalyzes theassembly of proheads and the encapsidation of DNA, eventhough 200 to 300 molecules are required for a singlecatalytic event.

Although it lies in a nearly central position in the lateoperon and is not synthesized in the absence of the gene 23product, the scaffolding protein is not always synthesizedcoordinately with the other late proteins (14). Mutants whichaccumulate proheads (lacking gene 1, 2, or 3 function)synthesize scaffolding protein at a higher rate relative to theother late proteins than wild-type phage or phage defectivein any of the other late functions, and gene dosage experi-ments have shown that this regulation results in the mainte-nance of coat and scaffolding protein subunits in the properratio for prohead assembly. This regulatory circuit couplesscaffolding protein synthesis to the process of phage morpho-genesis.

In this report we present in vivo experiments whichsupport the view that the feature of the assembly processwhich is important in this modulation is the state of assemblyof the scaffolding protein itself, and that unassembled itnegatively regulates the expression of its own gene.

MATERIALS AND METHODS

Phage and bacterial strains. The bacterial strains used werethe su+ strain DB7004 and su- strains DB21 and DB7000from the collection of D. Botstein (22, 29). All phage strainscarried the 13-amHlOl mutation to prevent lysis of infectedcells and the cl-7 mutation to ensure that only lytic infectionsoccurred. The other nonsense alleles used were 3-amN6,5-amN114, 8-amH202, and 8-amN123 (3). The 8-tsN102

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ASSEMBLY CONTROLLED AUTOGENOUS REGULATION 175

and csRN26D mutations were described by Jarvik and Bot-stein (12).

Analysis of infected cell extracts. The detailed proceduresused in the preparation of labeled and unlabeled cell ex-tracts, sodium dodecyl sulfate (SDS)-discontinuous slab gelelectrophoresis, and quantitation of proteins in these gelshave been described previously (14). Specific strains, times,etc., are given in the text.Measurement of amounts of assembled and unassembled

scaffolding protein. Nonpermissive cells (DB7000) were in-fected at 2 x 108 cell per ml and grown in LB broth (6) at30°C for the times indicated. The cells were chilled andconcentrated 40-fold in LB broth by low-speed centrifuga-tion, lysed by shaking with chloroform, and DNase I (Wor-thington Diagnostics) treated to reduce viscosity, and thecell debris was removed by centrifugation at 10,000 rpm for5 min in a Sorvall SS-34 rotor. The supernatant was layeredon a sucrose-cesium chloride step gradient as previouslydescribed (5), containing steps of 20% sucrose, a CsCldensity of 1.4 g/cm3, and a CsCl density of 1.6 g/cm3, andcentrifuged at 45,000 rpm for 45 min in a Spinco SW50.1rotor. Unassembled proteins remain at the top of the gradi-ent, proheads and empty heads collect in a sharp band on topof the CsCl step with a density of 1.4 g/cm3, and phagecollect on the more dense CsCl step. These three fractionswere collected and dialyzed against SDS sample buffer (20).The three fractions were then diluted to equal total volumeswith SDS sample buffer, and equal volumes were analyzedby SDS-polyacrylamide gel electrophoresis. Protein bandsin the gels were quantitated by Coomassie brilliant bluestaining as previously described (14). Samples taken frompositions between the three fractions described above con-tained very little scaffolding protein.

RESULTS

Our previous results have shown that there is a correlationbetween those amber mutants which accumulate proheadsand do not recycle scaffolding protein and those whichsynthesize high amounts of scaffolding protein (14). Themost obvious possibility for the mechanism of the regulationof the synthesis of scaffolding protein is that unassembled(recycling) scaffolding protein turns down the rate of synthe-sis of additional scaffolding protein. We present here exper-iments designed to test this hypothesis in vivo.

Levels of unassembled scaffolding protein. The above modelpredicts that the amount of free, unassembled scaffoldingprotein present in the infected cell should be inverselyrelated to the rate of synthesis of scaffolding protein. Wild-type P22- and 2--, 3--, and 5--infected cells were analyzedfor amounts of assembled and unassembled scaffolding pro-tein as described above. The results of such an experiment at80 min after infection are shown in Table 1. There is aninverse relationship between the total amount of scaffoldingprotein made and the amount of scaffolding protein unas-sembled in cells infected by various mutant phage. We havepreviously shown that scaffolding protein is stable in theseextracts, so the amount accumulated reflects the overall rateof synthesis (5, 14).

It is also informative to consider the synthesis of scaf-folding protein with time in a wild-type infection. We havepreviously shown that in such an infection, scaffoldingprotein synthesis begins at a relatively high rate coordinatelywith the other late proteins, but its rate of synthesis slowsdown significantly before those of the other late proteins

TABLE 1. Amounts of unassembled gp8 in mutant-infected cellsa

Mutant gp8 gp8 in Total gp8bunassembled proheads

Wild type 1.5 3.8 6.05- 3.9 <0. 1 4.22- <0.5 20.5 23.23- <0.4 25.2 27.3

a The amounts of gp8 were measured at 80 min after infection as describedin the text and are given in arbitrary units per infected cell.

b The total gp8 was determined by gel electrophoresis of the wholeinfectecd cell extract (14) in parallel with the analysis of the amounts ofproheads and unassembled. In general, ca. 10% of the gp8 was lost in the celldebris and unlysed cells.

(14). Figure 1 shows the levels of assembled and unas-sembled scaffolding protein at various times after infection.The unassembled scaffolding protein concentration in-creases with time during infection. Since the only sediment-able form of scaffolding protein is the prohead structure (5),sedimentable scaffolding protein was used as a measure ofthe relative number of proheads in the infected cell. Thus,the rate of synthesis of scaffolding protein is constant or

PHAGE gp5

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MINUTES AFTER INFECTIONFIG. 1. Levels of assembled and unassembled scaffolding pro-

tein after infection. Cells were infected with P22 cl-7 13-amHlOl,and at various times after infection, portions were removed and theamounts of assembled and unassembled scaffolding protein were

measured as described in the text.

PROHEAD gp8

/* UNASSEMBLED gp8I I---

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176 CASJENS ET AL.

cn~ w

we4200 /Lg1F tU5 |/ \

-2300 /gp57LL~ <~~~~~~~a gp8 g5~T200- 2IFOCo ( N

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20 30 40 50 60 70 20 30 40 50 60 70 20 30 40 50 60 70TIME AFTER INFECTION (MIN)

FIG. 2. Synthesis of a scaffolding protein amber fragment. DB21 su- cells were infected at 30'C, pulse-labeled at 4 min with '4C-labeledmixed amino acids, and harvested by freezing in a dry ice-acetone bath, and the proteins separated and quantitated as described previously(14). The top panels show the rates of incorporation at various times after infection, and the bottom panels show this data corrected to molaramounts and normalized to the amount of gpl, whose synthesis is not affected by the mutations being compared, Symbols: 0, coat proteinor its amber fragment; 0, scaffolding protein or its amber fragment; A, gene 1 protein; F0, viable phage. The mutations of primary interestare given in each panel; all phages also carried the cl-7 and 13PamHlOl alleles.

decreasing between 40 and 90 min after infection and isinversely related to the amount of unassembled scaffoldingprotein, rather than proportional to the number of proheadsin the infected cell (which might have been thought to turnon scaffolding protein synthesis).

Synthesis of scaffolding protein amber fragments. We pre-viously reported that those amber fragments of scaffoldingprotein which can be visualized in SDS-polyacrylamide gelsare not overproduced, as might be expected if scaffoldingprotein negatively regulates its own synthesis (14). Figure 2shows that upon closer inspection, one of these amberfragments is in fact synthesized at a consistently lower ratein P22 8-amH1348-infected cells than the scaffolding proteinis synthesized in 5--infected cells, even though the amberfragment was shown to be stable (14). Although this effect isnot large, it has been very reproducible. We have also foundthat other point mutations available in gene 8 do not cause

overproduction of the scaffolding protein when comparedwith the wild-type. Double-mutant phage carrying 8-tsN102or 8-csRN26D (blocked at nonpermissive temperatures inDNA packaging and prohead assembly, respectively; datanot shown) and an amber mutation in gene 5 (amN114) toblock any structure formation accumulate scaffolding pro-tein to the same level as the 5-8+ control at both permissiveand nonpermissive temperatures (data not shown). Theseresults can be explained in terms of the autoregulatoryhypothesis if the assumption is made that the amber (N-ter-minal and unassembled) fragments have regulatory activity.If this regulatory activity were somewhat stronger than thewild-type protein, it would explain why the amber fragmentis made at a lower level than that of unassembled wild-typeprotein in parallel infected cells. The ts and cs mutationstested apparently affect only the assembly function of thescaffolding protein.

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ASSEMBLY CONTROLLED AUTOGENOUS REGULATION 177

Long scaffolding protein amber fragments have regulatoryactivity. To test more directly the hypothesis that the longamber fragments of scaffolding protein can modulate thelevel of expression of the scaffolding protein gene, weperformed several mixed infections which demonstrate thatthe amber fragments can depress the expression of a func-tional scaffolding protein gene in trans. Because of interpre-tational difficulties when phage assembly and scaffoldingprotein recycling are taking place, we chose to perform theexperiments either in a 3- or 5- mutational background. Thefirst of these experiments is shown in Fig. 3 and 4. Thisexperiment compares the effect of relatively long gp8 amberfragments with a short fragment on the expression of afunctional gene 8. The two mutations used which generatelong amber fragments, amH202 and amH1348, producefragments with molecular weights of 21,000 and 12,000,respectively (14). The molecular weight of intact scaffoldingprotein is ca. 40,000 (3, 31). The mutation amN123 mapsnear the N terminus of the gene by deletion mapping (E.

Fraction 8+ Infecting Phage

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FIG. 3. Autoradiogram of a polycarylamide slab gel of extractsof 3-8+-3-8- mixedly infected cells. Cells were infected andpulse-labeled for 2 min with 'IC-amino acids. Cells were harvestedat the times indicated, and the proteins were displayed in a 12 to22% linear gradient polyacrylamide gel as described in the text. Thisautoradiogram is somewhat overexposed to make the protein bandsmore visible. The fraction of 3-8+ infecting phage is given above thelanes, and the labeling time after infection is indicated below (lanesa, 40 min; lanes b, 50 min; lanes c, 60 min).

Wyckoff, personal communication), and no amN123 amberfragment has been seen even in high-percentage or gradientacrylamide gels (data not shown). Nonpermissive cells wereinfected at various ratios and constant multiplicity (20 or 30)with a phage carrying an intact (8+) scaffolding protein geneand another phage carrying an amber mutation in the scaf-folding protein gene. Both phage carried a 3- mutation(amN6). The mutation in gene 3 blocks assembly at the DNApackaging step and assures that proheads accumulate andscaffolding protein is not recycling. Nearly all (>95%) of thescaffolding protein made in these infections is present inproheads of prohead-like structures (data not shown). Par-allel cultures infected at different ratios of input parentalphage were pulse-labeled with "C-amino acids to measure

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FIG. 4. Scaffolding protein synthesis in 3-8+-3-8- mixedly in-fected cells. Nonpermissive infected cells were pulse-labeled for 2min with "C-amino acids in minimal medium (A) or grown in LBbroth (B). The proteins were displayed in polyacrylamide slab gelsand quantitated by autoradiography (A) or Coomassie brilliant bluestaining (B) as described in the text. Each panel shows the combinedresults of several identical experiments. In (A) the total multiplicityof infection was 30, and samples were analyzed at 50 min afterinfection. In (B) the multiplicity of infection was 20, and sampleswere analyzed at 120 min after infection. In both cases, infectingparental phage carried the mutations cl-7, 13-amHlOl, and3-amN6, and the fraction of 8+ infecting phage is given on theabcissa. Symbols: 0, scaffolding protein synthesis in the 3-8+-3-8-amN123 mixedly infected cells; 0, scaffolding protein synthe-sis in the 8+3--3-8-amH1348 mixedly infected cells; U, the rate ofsynthesis of the amH1348 amber fragment.

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178 CASJENS ET AL.

rates of synthesis. SDS-polyacrylamide gels were run todisplay the total proteins in each of the cultures. One suchgel is shown in Fig. 3. Each of the protein bands of interestwas quantitated, and the results are shown in Fig. 4A. Theconinfecting gene 8 amH1348 fragment caused a depressionof the rate of synthesis of complete scaffolding proteincompared with that produced by the 8-amN123 mutant. Asthe fraction of the amH1348 parent increases, there is alinear increase in the amber fragment synthesis rate. Quali-tatively similar results were obtained when the total accu-mulated scaffolding protein was measured, even though thetwo types of experiments were done under very differentgrowth conditions. The results of one such experiment areshown in Fig. 4B. An identical experiment, in which the8-amH202 allele replaced amH1348, gave very similar re-sults.To more firmly rule out other mechanisms, such as amber

fragment poisoning of assembly which in turn modulates thesynthesis of scaffolding protein, a similar mixed infectionexperiment was performed in which both of the parentscarry an amber mutation in the coat protein gene. In thisinfection no scaffolding protein is in the assembled state, andthe long amber fragment effect seen in the previous experi-ment should be present only if the fragment itself is directlyresponsible for the effect. The results of such an experimentare shown in Fig. 5. The total scaffolding protein accumu-lated by 80 or 125 min after infection shows that the mixedinfection which produces a long amber fragment alwayssynthesizes substantially less complete scaffolding proteinthan the infection in which one of the parents makes a shortamber fragment. In this experiment we included as a controlthe 8-amN123 8-amH202 double mutant, which shouldproduce only the short amber fragment, even though itcarries both mutations. It behaves in this analysis indistin-guishably from the amN123 phage. Since the only differencebetween the amN123 mixed infections and the amH1348 oramH202 mixed infections is the mutation in gene 8, weconclude that the long amber fragments must be directlyresponsible for depressing the synthesis of scaffolding pro-tein. One feature of the results in Fig. 5 deserves furthercomment-the fact that the amH202 curve is approximatelylinear and the amNl23 curve is bowed upwards. The amH202curve is expected to be linear, since both the intact proteinand the amber fragment are unassembled and have regula-tory activity in this experiment. The upward bowing of theamN123 curve is explained if one notes that in the 5-8+case, the scaffolding protein gene is fully "repressed." Sincethe short fragment has no regulatory activity, as the fractionof 8+ parental phage decreases, the functional 8 genespresent have the capacity to produce more scaffoldingprotein until a concentration sufficient to depress expressionis reached.

DISCUSSIONThe phage P22 scaffolding protein gene is under the

positive transcriptional control of the 24 and 23 gene prod-ucts, since it is not made when either protein is defective (3,18, 27). However, we previously noted that the expressionof the scaffolding protein gene does not always parallel theexpression of the remainder of the late genes. More specif-ically, about three- to fivefold more scaffolding protein ismade during infections mutationally blocked in a step whichcauses proheads to accumulate, whereas the amounts of theother late proteins made do not change (14). These studiesindicated that the rate of synthesis of scaffolding protein ismodulated in some way by the phage assembly process.

The experiments presented here show that (i) the amountof unassembled scaffolding protein in wild-type P22-infectedcells at various times after infection or in various P22mutant-infected cells is inversely related to the rate ofsynthesis of scaffolding protein, and (ii) long amber frag-ments of the scaffolding protein have the ability to depresssynthesis of complete scaffolding protein, even in the ab-sence of prohead assembly. In addition, in experimentsreported elsewhere, we have shown that scaffolding proteinis the only phage-coded protein required for this regulationto occur in vitro (30). We feel that these facts combine toprovide a compelling argument for a control mechanism inwhich the feature of the assembly process which is critical inthe modulation of the rate of scaffolding protein synthesis isthe state of assembly of the scaffolding protein present.According to this model, the scaffolding protein itself de-presses the synthesis of additional scaffolding protein whenit is unassembled, but not when it is assembled into pro-heads. Thus, any excess of scaffolding protein over theavailable coat protein will depress additional scaffoldingprotein synthesis.

80 min0

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were separated in polyacrylamide slab gels and visualized bystainling with Coomassie brilliant blue, and the proteins. of interestwere quantitated as described in the text. Both of the infectingparental phage carried the mutations cl-7, 13-amHl01, and5-amll 4. One of the parents was 8+' and one was 8-; the fraction ofthe 8+ infection phage is given on the abcissa. Symbols: O, thes'caffolding protein accumulated in 5-8+-5-8-amN123-infected cells;*, the scaffolding protein accumulated in 5-8+-5-8-amH202-infected cells; O, the scaffolding protein accumulated in 5-8+-5-8-amH202 8-amN123-infected cells.

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ASSEMBLY CONTROLLED AUTOGENOUS REGULATION 179

The synthesis of this catalytic morphogenetic protein isregulated by its own state of association with other proteins,and thus by the assembly process itself. Although theunassembled coat protein of the RNA bacteriophages acts asa translational repressor of the phage-coded RNA replicase(17), and the Escherichia coli RNA polymerase a and 'subunit genes may be regulated in such a way that theholoenzyme and the subassembly a2p depress synthesis (butfree subunits do not [9, 11, 26]), P22 scaffolding protein andE. coli ribosomal proteins (19) represent the current, clearestexamples of proteins which participate in a multicomponentassembly process, whose synthesis is directly regulated bythe assembly process itself. In both these cases, the indica-tor is the state of assembly of the protein itself, andregulation is autogenous and negative. This mode of regula-tion is attractive and may be used in other more complexassembly systems and organisms to regulate the synthesis ofstructural proteins or catalytic proteins required in largeamounts (1, 8, 16).The fact that some amber fragments of the scaffolding

protein have regulatory activity suggests that the N-terminalportion of the protein contains the regulatory activity, andsince the active amber fragment of the 8-amH1348 has anapparent molecular weight of only ca. 12,000, the regulatorysite must reside in the N-terminal one-third of the 40,000-dalton scaffolding protein. Genetic mapping of these ambermutations strongly supports the idea that these fragments arein fact N terminal and not "restart" fragments (L. Wyckoff,unpublished data). This amber fragment does not appear tobe able to assemble into proheads (L. Hall and J. King,unpublished data), suggesting that the assembly function ofthe protein requires the C-terminal portion of the scaffoldingprotein.

ACKNOWLEDGMENTSThis work was supported by Public Health Service grant GM17980

(to J.K.) and GM21975 (to S.C.) from the National Institutes ofHealth, as well as National Science Foundation grant PCM8017177(to S.C.).We thank D. Botstein, E. Jackson, A. Poteete, M. Fuller, F.

Winston, and E. Wyckoff for strains, helpful discussions, and accessto unpublished data.

LITERATURE CITED1. Ben-Ze'ev, A., S. Farmer, and S. Penman. 1979. Mechanisms

regulating tubulin synthesis in cultured mammalian cells. Cell17:319-325.

2. Botstein, D., and I. Herskowitz. 1974. Properties of hybridsbetween Salmonella phage P22 and coliphage lambda. Nature(London) 251:584-589.

3. Botstein, D., C. Waddell, and J. King. 1973. Mechanism of headassembly and DNA encapsulation in Salmonella phage P22. 1.Genes, proteins, structures and DNA maturation. J. Mol. Biol.80:669-695.

4. Casjens, S., and M. Adams. 1984. Posttranscriptional modula-tion of bacteriophage P22 scaffolding protein gene expression. J.Virol. 53:185-191.

5. Casjens, S., and J. King. 1974. P22 morphogenesis. I. Catalyticscaffolding protein in capsid assembly. J. Supramol. Struct.2:202-224.

6. Chan, R., and D. Botstein. 1972. Genetics of bacteriophage P22.I. Isolation of prophage deletions which affect immunity tosuperinfection. Virology 49:257-276.

7. Ernshaw, W., S. Casjens, and S. Harrison. 1976. Assembly ofthe head of bacteriophage P22: X-ray diffraction from heads,

proheads and related structures. J. Mol. Biol. 104:387-410.8. Elgin, S., and H. Weintraub. 1975. Chromosomal proteins and

chromatin structure. Annu. Rev. Biochem. 44:725-774.9. Fukuda, R., M. Taketo, and A. Ishihama. 1978. Autogenous

regulation of RNA polymerase beta subunit synthesis in vitro. J.Biol. Chem. 253:4501-4504.

10. Georgopoulos, C., K. Tilly, and S. Casjens. 1983. Lambdoidphage head assembly, p. 279-304. In R. Hendrix, J. Roberts, F.Stahl, and R. Weisberg (ed.), Lambda II. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.

11. Glass, R. 1977. Identification of an amber fragment of the betasubunit of Escherichia coli RNA polymerase: a yardstick formeasuring controls on RNA polymerase subunit synthesis. Mol.Gen. Genet. 155:83-88.

12. Jarvik, J., and D. Botstein. 1975. Conditional-lethal mutationsthat supress genetic defects in morphogenesis by altering struc-tural proteins. Proc. Natl. Acad. Sci. U.S.A. 72:2738-2742.

13. King, J., and S. Casjens. 1974. Catalytic head assemblingprotein in virus morphogenesis. Nature (London) 251:112-119.

14. King, J., C. Hall, and S. Casjens. 1978. Control of the synthesisof phage P22 scaffolding protein is coupled to capsid assembly.Cell 15:551-560.

15. King, J., E. Lenk, and D. Botstein. 1973. Mechanism of headassembly and DNA encapsulation in Salmonella phage P22. II.Morphogenetic pathway. J. Mol. Biol. 80:697-731.

16. Komeda, Y., and T. lino. 1979. Regulation of expression of theflagellin gene (hag) in Escherichia coli K-12: analysis of hag-lacgene fusions. J. Bacteriol. 139:721-729.

17. Kozak, M., and D. Nathans. 1972. Translation of the genome ofa ribonucleic acid bacteriophage. Bacteriol. Rev. 36:109-157.

18. Lew, K., and S. Casjens. 1975. Identification of early proteinscoded by bacteriophage P22. Virology 68:525-533.

19. Nomura, M., S. Jinks-Robertson, and A. Miura. 1982. Regula-tion of ribosome biosynthesis in Escherichia coli, p. 91-104. InM. Grunberg-Manago and B. Safer (ed.), Interaction of transla-tion and transcriptional controls in the regulation of geneexpression. Elsevier Science Publishing Co., Inc., New York.

20. O'Farrell, P., L. Gold, and W. Huang. 1973. The identificationof prereplicative bacteriophage T4 proteins. J. Biol. Chem.248:5499-5501.

21. Poteete, A., and D. Botstein. 1979. Purification and properties ofproteins essential to DNA encapsulation by phage P22. Virology95:565-573.

22. Poteete, A., V. Jarvick, and D. Botstein. 1979. Encapsulation ofphage P22 DNA in vitro. Virology 95:550-564.

23. Poteete, A., and J. King. 1977. Function of two new genes inSalmonella phage P22 assembly. Virology 76:725-739.

24. Roberts, J. 1975. Transcription termination and late control inphage lambda. Proc. Natl. Acad. Sci. U.S.A. 72:3300-3304.

25. Roberts, J., C. Roberts, S. Hilliker, and D. Botstein. 1976.Transcription termination and regulation in bacteriophages P22and lambda, p. 707-718. In R. Losick and M. Chamberlin (ed.),RNA polymerase. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

26. Scaife, J. 1976. Bacterial RNA polymerases: the genetics andcontrol of their synthesis, p. 207-225. In R. Losick and M.Chamberlin (ed.), RNA polymerase. Cold Spring Harbor Lab-oratory, Cold Spring Harbor, N.Y.

27. Suskind, M. M., and D. Botstein. 1978. Molecular genetics ofbacteriophage P22. Microbiol. Rev. 42:385-413.

28. Weinstock, G., P. Riggs, and D. Botstein. 1980. Genetics ofbacteriophage P22. 1II. The late operon. Virology 106:82-91.

29. Winston, F., D. Botstein, and J. H. Miller. 1979. Characteriza-tion of amber and ochre suppressors in Salmonella typhimur-ium. J. Bacteriol. 137:433-439.

30. Wyckoff, E., and S. Casjens. 1984. Autoregulation of the bac-teriophage P22 scaffolding protein gene. J. Virol. 53:192-197.

31. Youderian, P., and M. Susskind. 1980. Identification of theproducts of bacteriophage P22 genes, including a new late gene.Virology 107:258-269.

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