Proc. Nati Acad. Sci. USAVol. 78, No. 7, pp. 4194-4198, July 1981Biochemistry

Genes VI, VII, and IX of phage M13 code for minor capsid proteinsof the virion

(differential amino acid labeling/amino-terminal sequence copy numbers/chimeric phages)

Guus F. M. SIMONS, RUUD N. H. KONINGS, AND JOHN G. G. SCHOENMAKERSLaboratory of Molecular Biology, University of Nijmegen, Nijmegen, 6525 ED, The Netherlands

Communicated by Norton D. Zinder, April 3, 1981

ABSTRACT The minor capsid proteins C and D from phageM13 have been characterized by differential amino acid labelingand amino-terminal sequence analysis. We demonstrate that Dprotein (Mr 12,260) is the product of gene VI, whereas the C com-ponent is composed of the products of both gene VII (Mr 3580) andgene IX (Mr 3650). Our data further show that the proteins ofgenesVI, VII, and IX are not subject to proteolytic processing but arepackaged into mature virions as their primary translational prod-ucts. On the basis ofincorporation of specific amino acids, the copynumbers of these proteins in M13 virions could be estimated rel-ative to the number ofA protein molecules. The M13 phage con-tains on the average 5 molecules of A protein, 5 molecules of VIprotein and 3-4 molecules of both VII protein and IX protein.These copy numbers remained unchanged in M13 recombinantphages of up to two times the length of wild-type phages, a factthat indicates that these minor capsid proteins are located at eitherone or both ends of the phage filament.

M13 and its very close relatives fd and fl are filamentous phagesthat infect Escherichia coli cells carrying the F episome (for areview, see ref. 1). The M13 genome consists of a 6407-nucleotide-long single-stranded DNA molecule (2), which isencapsulated in a tubular protein coat about 895 nm long and6 nm wide. Over the years there have been reports which sug-gest that the composition ofthe virion coat is more complex thanoriginally thought (3-5). The major capsid or B protein (Mr5200), which is encoded by gene VIII, is present in about 2700copies, which are arranged in a helix around the DNA (6). Thegene III-encoded minor capsid or A protein (Mr 42,600) is pres-ent as only four or five copies (3, 7), which are located at onetip of the filament (4, 5). Besides these two proteins, two ad-ditional minor capsid proteins of Mr 3500 and 11,500, desig-nated C protein and D protein, respectively, were recentlydiscovered (8). Their genetic origin and their biological functionhave not yet been solved.

Assembly of M13 virions does not occur in the cytoplasm:infectious virus has never been detected within the infected celland the major capsid protein is an integral constituent of thecytoplasmic membrane prior to virus assembly. In the cyto-plasm, progeny viral DNA is covered by the DNA-binding pro-tein encoded by gene V, forming a filamentous nucleoprotein(9). The assembly process is apparently one of exchange of coatprotein for gene V protein as the viral DNA leaves the cell. Theminor coat or A protein has most probably a very early and latefunction. It may lead the DNA into the host cell (10, 11) andits presumed site of DNA replication (12) after attachment ofthe virus to the Fpilus, and there is also evidence which suggeststhat it functions as a cut-off agent in the final stage of the V pro-tein-major coat protein exchange reaction (10). At least four

other M13 genes (I, IV, VI, and VII) are required for virus as-sembly, but their specific role in this process has yet to be as-certained. Further research in this field is hampered by the factthat the products of some genes (VI and VII) have not yet beendetected either in vivo or in vitro (13-15), whereas the productsof genes I and IV are present in the infected cell in very lowamounts (3, 16).

To gain a better understanding of the assembly functions ofthese genes and to find out whether the recently discoveredminor capsid proteins are host-encoded or M13-specified prod-ucts, we have studied these proteins in more detail. In this re-port we demonstrate that D protein is the product of gene VIand that the C protein component is composed of two poly-peptides, one of which is encoded by gene VII and the otherby gene IX.

MATERIALS AND METHODSBacteria and Phages. The E. coli strains KA805 (amber sup-

pressor SuI+) and KA807 (amber suppressor SuIII+) were ob-tained from B. Glickman. M13 and its amber mutant am7H2have been described (17). The recombinant phage fdlO6Sm2(18) originated from H. Schaller. Phages M13S100 and S200,containing the 1.8- and 3.0-kilobase-pair Hae II A fragments ofpBR322 and pBR325, respectively, were constructed as de-scribed in ref. 18.Growth and Purification of Radioactive Phages. Amino acid-

labeled M13 phages were prepared essentially as described (8)with minor modifications. E. coli strain K-38 was grown in 25ml ofM9 minimal medium (19) supplemented with 20 mM glu-cose but no amino acids were added. At 1 x 108 cells per ml,M13 phages were added (multiplicity of infection 20) and thena total of 1.0 mCi of 3H-labeled amino acid or 0.25 mCi of 14C-labeled amino acid was added in four equal portions at 15, 45,75, and 105 min after infection (1 Ci = 3.7 x 10'° becquerels).Labeling with [3S] methionine was performed similarly witha total of0.1 mCi. In all cases amino acids with the highest spe-cific activity currently available were used (Amersham). Aminoacid labeling of M13S100, M13S200, and fdlO6Sm2 was carriedout in the presence of ampicillin (25 ,ug/ml), chloramphenicol(25 ,tg/ml), or streptomycin (10 tkg/ml), respectively. Phageswere isolated after a labeling period of4 hr and further purifiedby two successive precipitations with 5% polyethylene glycol6000 in the presence of 0.1% Sarkosyl, followed by centrifu-gation on CsCl gradients as described (8).

Isolation of Radioactive Phage Proteins. The fractionationofcapsid proteins on NaDodSOJ8 M urea polyacrylamide slabgels and their subsequent electrophoretic elution from gel seg-ments have been described in detail (8). After dialysis of theeluate, the radioactive proteins were precipitated, washed, andlyophilized as described by Shaw et al. (20).

Automated Edman Degradation. Prior to degradation, theprotein samples were subjected to a 3 M HCl treatment as de-

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scribed by Palmiter et al. (21). Thereafter the acid was removedby lyophilization and the sample was then washed three timeswith 1% triethylamine and dried under reduced pressure. Sam-ples were dissolved in 70% (wt/wt) formic acid and 2 mg ofparvalbumin was then added as carrier (22). Automated Edmandegradation was performed on a Beckman 890C sequenator,using a 0.33 M Quadrol program of Hunkapiller and Hood (23).The recovered phenylthiohydantoin amino acids were driedunder nitrogen and dissolved in 200 A.l of methanol, and theirradioactivity was determined in 5 ml of Lumagel (Baker), usinga Packard scintillation spectrometer.

Quantitation of Phage Capsid Proteins. Copy numbers ofcapsid proteins were determined from tritiated amino acid-la-beled phages only. After electrophoretic fractionation of the in-dividual capsid proteins and subsequent fluorography (24), theappropriate gel segments were excised from the dried gel. Thegel segments were completely burned with oxygen in a PackardTri-Carb model 300 sample oxidizer and the radioactivity wasdetermined by adding 10 ml of Lumagel to each [3H]watereluate and scintillation counting. 3H-Labeled standard samples(Packard) were applied for recovery determinations.

RESULTSGenetic origin of D proteinElectrophoretic analysis of the proteins present in 14C-labeledM13 phages showed that the virion coat is composed of themature products of genes Ill and VIII and two additional smallprotein components (Fig. 1). The latter, denoted C protein andD protein, have apparent molecular weights of3500 and 11,500,respectively.

Because conventional ion-exchange and exclusion chromato-graphic methods for the isolation and subsequent characteriza-tion ofC and D proteins were unsuccessful, we have attemptedto characterize these proteins on the basis of their capability toincorporate several representative 3H-labeled amino acids. Inthis approach the established nucleotide sequence ofM13 DNA(2) was used as a guide for these labeling experiments. Fromthese studies it turned out that D protein can be labeled withall amino acids except histidine (Fig. 1). Because the M13 DNAsequence dictated that gene VI codes for a protein of 112 aminoacids in which histidine is absent, the genetic relationship be-tween gene VI and D protein became apparent. On the other

O-_ ----- _ -A- a mm .

C -- _ _..

1 2 3 4 5 6 7 8

FIG. 1. NaDodSO4/urea/polyacrylamide gel electrophoresis ofM13 phage capsid proteins that were labeled with a 14C-labeled aminoacid mix (lane 1), histidine (lane 2), arginine (lane 3), methionine (lane4), proline (lane 5), lysine (lane 6), tryptophan (lane 7), or aspartic acid(lane 8). Individual amino acids were all 3H-labeled. A and B refer tothe gene III-encoded minor capsid protein (Mr 42,600) and the geneVIII-encoded major capsid protein (Mr 5240). Sometimes a protein ag-gregate ofunknown composition that does not enter the separating gelwas noted (lanes 1 and 7; 0, origin), particularly when B protein-la-beled phages were disrupted at high concentrations. A similar phe-nomenon was noted by Grant et al. (25).

hand, gene VIII protein (Mr 5240) also lacks histidine (Fig. 1),and the possibility therefore remained that D protein is not anew capsid protein but merely represents a gene VIII proteindimer. This possibility, however, has been ruled out becauseD protein can be labeled with arginine, an amino acid that isknown to be absent from the major coat protein (Fig. 1).Our initial attempts to characterize D protein by amino acid

analysis were only partly successful. The overall amino acidcomposition of electrophoretically purified, uniformly '4C-la-beled D-protein, as determined with '4C-labeled gene V, VIII,and Ill proteins as internal radioactive standards, did show theabsence of histidine, low percentages of arginine and methio-nine, and relatively high amounts of leucine, as expected forgene VI protein, but also suggested that the protein was stillcontaminated, most probably with major capsid protein (datanot shown).To demonstrate unambiguously that D protein originates

from gene VI, M13 phages labeled with a single radioactiveamino acid were prepared and the electrophoretically isolatedD protein was then subjected to amino-terminal sequence anal-ysis by automated Edman degradation. The results obtained arepresented in Fig. 2. The DNA sequence predicts that gene VIprotein contains a single methionine residue, which is locatedat the amino terminus of this protein. Edman degradation ofDprotein from [3S]methionine-labeled phages, however, did notliberate any radioactivity after the first degradation step (Fig.2), despite the fact that D protein can be labeled with methi-onine (Fig. 1). To find out whether D protein molecules areblocked by a formyl group at their amino terminus, prior to se-quential degradation, the protein was subjected to a HCl treat-ment essentially as described by Palmiter et al. (21). It appearedthat only after such treatment could the amino-terminal se-quence of this protein be determined, strongly suggesting thatthis protein is present in the virion in its N-formylated form.The yield of Edman degradation after this 3 M HCl treatmentwas low and ranged between 20% and 40%. Accordingly, Dprotein from phages that were labeled with proline, glycine, orleucine were treated with HCl and subjected to automated se-quence analysis. With the exception of two degradation steps,the radioactivities obtained after the various sequence runswere found in full accordance with the amino-terminal sequenceof gene VI protein as deduced from the DNA sequence (Fig.3). The exceptions are the positions 3 and 6, where in both caseseither a glycine or a proline residue could be proposed. On thebasis ofrepetitive yield ofEdman sequence analysis, which wasusually about 92%, and the pronounced intensity of proline atposition 6 as compared to proline residues 2 and 8, it is obviousthat we are elucidating the amino-terminal sequence of twodifferent proteins. Proline residues 2 and 8 agree with the VIprotein sequence, whereas a proline at position 6 fits with theamino-terminal sequence ofmature VIII protein. A similar con-clusion emerged from the sequence run of [14C]glycine-labeledD protein. The glycine residue at position 6 belongs to VI pro-tein, and glycine at position 3 fits again with the sequence ofmature VIII protein. From these degradation results we inferthat D protein consists of VI protein but also of VIII proteindimers. We calculated that a residual dimerization of the majorcapsid protein of only 0.2% is sufficient to cause the anomalousdegradation results. Such low values cannot be excluded in theNaDodSOJ8 M urea/polyacrylamide system applied to frac-tionate these phage proteins.

Genetic origin of C proteinAs is shown in Fig. 1, the smallest minor capsid protein C cannotbe labeled with histidine, proline, or lysine. The only M13 geneproducts the sizes of which are =3500 daltons and that lack

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4196 Biochemistry: Simons et al.

1.2 Met Leu 6.0

0.8 '.0

0

I1.0

2.0 0.5

2 4 6 8 10 12 14 2 4 6 8 10 12 14

+ Met ) Leu2.0 2.0

1.0 1.0

E- r a\Tyr Ala a3.0

Z'~~~~~~~~~~~~~~0

t; 1.5 *~s X0t4.5 .......... - - ....... l.

~ Tyr (am 7H2) * Asp

2 4 6 10 12 2 4 6 8 10 12 14sequence cycle

FIG. 2. Amino-terminal sequence anaylsis of: (A) capsid protein D,and (B) capsid protein C, both isolated from M13 phages that were la-beled with a single 'H- or 'C-labeled amino acid as indicated. Thebroken lines indicate the sequence runs of the proteins that were notpretreated with 3 M HCI; the solid lines represent the runs of treatedprotein samples. am7H2 represents the degradation of [5Hltyrosine-labeled C protein from am-7 phages propagated in SuIn' E. coli.

these amino acids are those of gene VII and the recently dis-covered gene IX (17, 26). From the nucleotide sequence ofbothgenes it is also evident that gene VIl contains aspartic acid,which is absent in gene IX, and that tryptophan is present in

1 10A Met-Pro-Val-Leu-Leu-Gly-lle-Pro-Leu-Leu-Leu-Arg-Phe-Leu

1 10B Met-Glu-GIn-Val-Aia-Asp-Phe-Asp-Thr-lle-Tyr-GIn-Ala

1 10

C Met-Ser-Val-Leu-Val-Tyr-Ser-Phe-Ala-Ser-Phe-Val-Leu

FIG. 3. Sequences A, B, and C represent the amino-termini of VI,VII, and IX proteins, respectively, as deduced from the M13 DNA se-quence (2). The amino acids given in boldface are those found afterEdman degradation of C and D proteins that were labeled with singleradioactive amino acids.

gene IX but not in gene VII. C protein can, however, be labeledwith both amino acids (Fig. 1), suggesting that this protein isa mixture of both gene products.To confirm this, automated Edman degradation was also car-

ried out with C protein that was labeled with single amino acids.The results obtained are shown in Fig. 2. Also with this proteinan amino-terminal sequence could be determined only after a3 M HCI treatment. Untreated [3H]tyrosine-labeled C proteinshowed no radioactivity peaks even after 15 degradation steps,whereas treated C protein revealed a peak of great intensity atposition 6 and one of low intensity at position 11 (Fig. 2). Theintensity ofthe latter did not fit the value expected on the basisof the repetitive yield of the Beckman sequencer, an observa-tion that, in turn, strongly suggests that the two tyrosine resi-dues belong to two different proteins. This was also evident fromour observations that at position 6 a tyrosine was found but alsoan aspartic acid residue, and at position 13 both a leucine andan alanine residue were noted. By comparing the positions ofthe various radioactive residues with the amino-terminal se-quence of VII and IX proteins as deduced from the DNA se-quence (Fig. 3), our interpretation ofthese data is that tyrosine-6 belongs to gene IX and tyrosine-il to gene VII. Leucine-4 andleucine-13 are both derived from IX protein. Consistent withthe repetitive yield, alanine-5 and alanine-13 belong to geneVII, whereas the higher intensity of alanine-9 exactly fits withthe gene IX sequence. In a similar way we infer that the asparticacid residues at position 6 and 8 belong to gene VII. We do notyet have a plausible explanation for the occurrence of radioac-tivity at position 1 of the aspartic acid label, because at thatposition the occurrence of methionine was also demonstrated.

That C protein indeed is composed of the proteins encodedby gene VII (33 amino acids) and gene IX (32 amino acids) isfurther supported by the results ofour labeling studies with theM13 phage mutant am7H2. We previously demonstrated (17)that this mutant bears two mutated sites. One is a C -3 T changein gene VII at position 1114 ofthe DNA sequence, which altersthat CAG codon into aTAG codon. The other is a C -- T changeoutside gene VII but in the gene IX sequence (position 1141),where a CGT (Arg) codon is converted into a TGT (Cys) codon.Consequently, gene IX protein encoded by this mutant containsonly one instead oftwo arginine residues. When am7H2 phageswere propagated in SuI+ E. coli cells in the presence of[I4C]arginine and the proteins of the purified phages were ana-lyzed on polyacrylamide gels, the radioactivity contained in theband representing C protein was markedly reduced as com-pared to wild-type phages (Fig. 4).

That VII protein also forms part of the C protein componentis confirmed by our Edman degradation data of am7H2 phageproteins. When this mutant phage was propagated in SuIII'E. coli cells in the presence of [3H]tyrosine, a tyrosine residuewas noted in C protein not only after the sixth but also after thethird degradation step (Fig. 2). Residue 6 reflects gene IX pro-tein, whereas residue 3 corresponds with a tyrosine suppressionat the amber codon in gene VII, namely at position 1114 oftheDNA sequence.

Copy numbers of C and D proteinsThe great abundance of major coat protein as compared to Cand D protein and the well-known tendency of the major coatprotein to aggregate even under strongly denaturing conditionsmake an estimation of their copy numbers in phage particlesvery difficult a priori. Unless certain precautions have beenmade, cross-contamination is expected to occur during the elec-trophoretic separation of these phage proteins. We have cir-cumvented these problems by analyzing the radioactivity dis-tribution of D protein in [3H] arginine-labeled M13 phages in

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0

A

D

C

a b

FIG. 4. NaDodSO48 M urea/polyacrylamide gel electrophoresisof the capsid proteins of M13 wild-type phage (lane a) and am7H2phage(laneb) that were propagated in SuIl E. coli cells in the presenceof[H]arginine. The radioactivities applied were 12,503 and 11,718dpm, respectivley.

order to eliminate any radioactivity contribution of VIII proteindimers to D protein. The ratio of 3H label in IIl protein to the3H label in the D protein band, as estimated by oxidizing thecorresponding gel segments in a Packard sample oxidizer andscintillation counting, was found to be 8.1-9.5 to 1. This ratio,coupled with the known arginine content of III and VI protein,allowed us to calculate the copy numbers of VI protein. Assum-ing 5 copies of III protein (4, 5), a value of 4.7 ± 0.5 was foundfor VI protein in wild-type and am7H2 mutant phages (Table1).

In a similar way we estimated the copy numbers of VII pro-tein from [3H]aspartic acid-labeled phages and of IX proteinfrom [3H]tryptophan-labeled phages. As shown in Table 1, theestimated values were 3.3 and 3.5 copies for VII and IX protein,respectively, which suggests that both proteins are present inphage particles in equimolar amounts. With [3H]valine labeledphages a value of 7.6 and with [3H] arginine-labeled am7H2phages a value of 6.2 was found. Because genes VII and IX inthese phages have identical numbers of valine and arginine res-idues, the latter values represent the sum of VII and IX proteincopies in mature phage particles. They are in fair agreementwith the copy numbers found for these small capsid proteins.

Location of C and D protein in M13 virions

Woolford et al. (4) and also Goldsmith and Konigsberg (5) clearlydemonstrated that the minor capsid protein encoded by geneIII is located at only one tip of the phage filament. To find outwhether the other minor capsid proteins are also located at or

clustered near one tip of the filament, we have estimated thecopy numbers of these proteins in M13 recombinant phages ofvarious lengths. For this purpose we used the helper-indepen-dent phages M13S100, M13S200, and fdl06Sm2, the lengthsof which were 1.2, 1.5, and 2.0 times the length of wild-typephage particles, respectively. Our results obtained with thesephages are presented in Table 1. They show that with increasinglengths of the phage particles the copy numbers do not increasebut remain of the same order of magnitude as that in wild-type

Table 1. Copy numbers of VI, VII, and IX proteins in M13 wild-type and chimeric phages

3H label Copy numbers of proteinsPhage used VI* VIF IXt

Wild type Tryptophan ND - 3.5 (0.5)Wild type Aspartic acid 4.3 (0.7) 3.3 (0.4) -

Wild type Valine ND 3.8 3.8AM7H2 Arginine 5.4 (0.1) 3.1(0.1) 3.1(0.1)Wild type Arginine 4.3 (0.3) 3.8(0.4) 3.8 (0.4)M13S100 Arginine 4.1 3.3 3.3M13S200 Arginine 3.9 (0.3) 3.2 (0.2) 3.2 (0.2)fdlO6Sm2 Arginine 3.2 (0.5) 2.9 (0.3) 2.9 (0.3)* The values given are calculated from the determined amounts of 'Hlabel in theD andIIIprotein bands. Radioactivities were determinedfrom several independent phage preparations and the molar ratiobetween D and III protein was calculated on the basis of the knownnumber of amino acid residues as deduced from the DNA sequence(2). The copy number of VI protein was then derived by multiplyingthe molar ratio by the copy number of IX protein. The latter valueis assumed to be 5 on the basis of data of Woolford etal. (4) and Gold-smith and Konigsberg (5).

t Copy numbers of VII and IX proteins were estimated as describedabove. The values obtained with arginine- and valine-labeled phagesare based on the assumption that these proteins arepresent in virionsin equimolar amounts as evidenced by the experiments with tryp-tophan and aspartic acid. Parentheses indicate SEM. ND, notdetermined.

particles. Our interpretation of these data is that an insertionof C and D protein molecules in a periodical array along thelongitudinal axis ofthe filamentous protein tube is very unlikely.On the basis of the constant ratios of III protein to C and Dprotein in longer filaments, we infer that the minor capsid pro-teins encoded by genes-VI, VII, and IX, although not locatedper se on different ends, are clustered on the tips of the fila-mentous phage particles.

DISCUSSIONDespite our detailed knowledge ofthe molecular biology ofthesingle-stranded filamentous DNA phages, the process of virionassembly on the host cell membrane is still a largely unsolvedproblem. Although there exists indirect evidence that genes VIand VII are involved in this process, further research in this fieldwas greatly hampered by the fact that the products of thesegenes were still completely unknown (3, 13-16). Recently, theiridentification has been greatly facilitated as the expected sizesof these proteins and their primary sequence have been de-duced from nucleotide sequence data (2, 17, 26). Similarly, fromthe DNA sequence the existence of a new gene, gene IX, hasbeen postulated (17, 26). Our data presented here demonstratethat the products ofgenes VI, VII, and IX are minor constituentsof the phage coat, where they are present in only a few copiesper virion. These data are based on direct sequence analysis ofwild-type proteins and were confirmed, where possible, by spe-cific amino acid substitutions to be expected from conditionallylethal phage mutations. An identical conclusion has recentlybeen reached by Lin et al. (27). Their data on fl are in fairagreement with our data on M13, which strengthens the ar-gument for the presence of these minor capsid proteins in allfilamentous phages of this type.

Our Edman degradation studies further demonstrate that theproteins encoded by genes VI, VII, and IX are not subject toproteolytic cleavage at their amino terminus as is the case withIII and VIII protein but are packaged into mature virions astheir primary translational products. An intriguing aspect is thatthese minor capsid proteins can be degraded only after treat-

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4198 Biochemistry: Simons et al.

ment with 3 M HCi, a procedure that characteristically has beenapplied for deformylating the amino termini of proteins. Untilnow we could not exclude the possibility that some obscurecircumstances have shielded a free amino terminus and, con-sequently, simulated the presence ofan N-formyl group in theseproteins. Because such a blocking or shielding was not apparentduring degradation of similarly isolated B protein, our obser-vation argues for an N-formylated status of these three proteinsin M13 virions. With respect to the amino terminus of VII andIX protein, our data are less clear. They suggest but do not provethat these proteins bear a formylmethionine residue. In ac-cordance with this is our finding of a phenylthiohydantoin de-rivative of [asSimethionine after the first degradation step andthe failure to degrade [asS]methionine or [3H]tyrosine-labeledC protein prior to 3 M HCl treatment. Not in accordance is theradioactivity at position 1 in [3H]aspartic acid-labeled protein.A definite conclusion on the amino-terminal status ofthese pro-teins awaits further investigations.

Data are now also available on the location and possible func-tion ofthese three minor capsid proteins. Irn this study evidenceis presented that these proteins, like the III protein, are alsolocated at either one or both ends of the phage filament. Byshearing normal lengthfd and ft phages and treating the mix-ture with anti III protein antibodies, Grant et al. (25) very re-cently provided evidence that D protein is located at the IIIprotein end of the phage and, consequently, that C protein isat the opposite end. Such a clustering ofVI and III proteins andof VII and IX proteins is of great biological importance becausetheir synthesis is under strict control. Genes III and VI belongto one operon (10), whereas genes V and VII form a secondoperon (28). Moreover, recent protein synthesis studies withseveral M13 amber mutants have shown that gene IX proteinsynthesis also is controlled by gene V expression (unpublisheddata). An overall picture emerges that the four minor capsidproteins of the F-specific filamentous phages can be divided intwo functional groups. One consists of the products of one op-eron (III and VI), which trigger the entry process ofphage DNAinto the host cell but also its reversed process-i.e., the finalstages of packaging the DNA when it leaves the cell-whereasthe other group, composed ofproducts ofthe other operon (VIIand IX), most probably directs the very early events of cha-nelling the V protein-DNA complex through the host cell mem-brane. Our primary goal is to study the regulation of synthesisof these minor capsid proteins.

We thank Mrs. Titia Peters and Rosalie Jongbloed for excellent tech-nical assistance, Piet Wietzes and Theo Cuypers for their help duringthe Edman degradation analyses, Dr. Heinz Schaller for his gift offdlO6Sm2, and Dr. Bob Webster for making manuscripts available priorto publication. This work was supported by the Netherlands Foundation

for Chemical Research (SON) with financial aid from the NetherlandsOrganization for the Advancement of Pure Research (ZWO).

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Proc. Natl. Acad. Sci. USA 78 (1981)

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