Proc. Natl. Acad. Sci. USAVol. 81, pp. 1639-1643, March 1984Biochemistry

Purification in a functional form of the terminal protein of Bacillussubtilis phage 429

(initiation of replication/DNA-linked protein/radioimmunoassay)

IGNACIO PRIETO, Jose M. LAZARO, JUAN A. GARCfA, Jost M. HERMOSO, AND MARGARITA SALAS

Centro de Biologia Molecular Consejo Superior de Investigaciones Cientificas-Universidad Aut6noma de Madrid, Canto Blanco, Madrid-34, Spain

Communicated by Luis F. Leloir, November 4, 1983

ABSTRACT Phage 429 terminal protein, p3, essentiallypure, was isolated in a denatured form from viral particles,and anti-p3 antiserum was obtained. A radioimmunoassay todetect and quantitate protein p3 was developed. By using thisassay, native protein p3 was highly purified from Escherichiacoli cells harboring a gene 3-containing recombinant plasmid.After three purification steps, the protein was more than 96%pure; its amino acid composition was very similar to that de-duced from the nucleotide sequence of gene 3. The purifiedprotein was active in the formation of the covalent p3-dAMPinitiation complex when supplemented with extracts of B. sub-tilis infected with a sus mutant of 4)29 in gene 3. No DNA poly-merase or ATPase activities were present in the final prepara-tion of protein p3.

DNA polymerases cannot initiate new DNA chains, andthey require a primer that supplies a free 3' hydroxyl groupfor elongation. In the case of a linear DNA that does notform covalently closed circles, concatemers, or terminalhairpin structures, the question of how the 5' ends areprimed arises. A novel mechanism of priming by a deoxynu-cleotide covalently linked to a protein has recently beenshown to occur in phage 429 and in adenoviruses.The Bacillus subtilis phage 429 has a linear DNA, 18,000

base pairs long (1), with the product of gene 3, protein p3,covalently linked to the 5' ends (2-5). 429 DNA replicationstarts at both ends and proceeds by strand displacement (6-8). The new mechanism for the initiation of replication pos-tulates that a free molecule of the terminal protein reactswith the dNTP corresponding to the 5' termini and, by for-mation of a protein-dNMP covalent complex, provides the 3'hydroxyl group needed for elongation (6, 7, 9). In support ofthis model, extracts of 429-infected B. subtilis react withdATP in the presence of 429 DNA-protein p3 complex astemplate, and a covalent complex protein p3-dAMP isformed that is used for elongation (10-12). Similarly, a cova-lent complex between the 80-kilodalton (kDa) precursor ofthe adenovirus terminal protein and dCMP, the terminal nu-cleotide at both 5' ends of adenovirus DNA, is formed invitro (13-17). Because no complex is formed when extractsof B. subtilis infected with either gene 2- or gene 3-deficient429 mutants are substituted for the wild-type 429-infectedextracts, both the product of gene 2 and free protein p3 ap-pear to be required along with the 429 DNA-protein p3 tem-plate for initiation of 429 DNA replication (18).To confirm the requirement of free protein p3 for initiation

complex formation, we undertook the purification of the na-tive protein from extracts of Escherichia coli transformedwith a plasmid harboring the 429 gene 3. In these extractsprotein p3 accounts for about 3% of the total de novo proteinsynthesis (19). In this paper we describe the purification to

virtual homogeneity of native protein p3 active in initiationcomplex formation. A preliminary account of some of thesefindings has been presented (20).

MATERIALS AND METHODSMaterials. [a-32P]dATP (410 Ci/mmol; 1 Ci = 37 GBq), [-

32P]ATP (3000 Ci/mmol), [3H]dTTP (43 Ci/mmol), [3H]ura-cil (25 Ci/mmol), [35S]sulfate (25-40 Ci/mg), and 1251 (14.9mCi/pug) were obtained from the Radiochemical Centre. Mi-crococcal nuclease was from Worthington; Staphylococcusaureus protein A, from Pharmacia; fungal proteinase K,electrophoretically purified, from Merck; DEAE-32 cellu-lose and phosphocellulose P11, from Whatman; and NonidetP-40, Tween-20, Sepharose 2B, polyethyleneimine, ATP,and deoxyribonucleoside 5'-triphosphates, from Sigma; Poly-(dA)-(dT)12_18, from P-L Biochemicals; and polystyrene chlo-ride plates, from Limbro Division, Flow Laboratories.Phage 429 mutant susl4(1242) labeled with [35S]sulfate (21)or [3H]uracil (2), 429 DNA-protein p3 complex for the initia-tion reaction (10), and proteinase K-treated 429 DNA (22)were prepared as described. Single-stranded M13 DNA andreplicative form I (RFI) were obtained from L. del Rio andA. Talavera, respectively. E. coli N99XC1857, harboringplasmids with gene 3 inserted in the correct (pKC30A1) oropposite (pKC30B1) orientation for transcription, were asdescribed by Garcia et al. (19). The phage 429 mutantsus3(91) was from the collection of Moreno et al. (23), andthe delayed lysis mutant susl4(1242) was from Jimenez et al.(24). Phage 4)29 12- particles containing all the structuralproteins except the neck appendages, pl2*, were obtainedfrom J. L. Carrascosa. B. subtilis HA101(59)F lacking DNApolymerase I activity (25), obtained from N. Cozzarelli, andB. subtilis 11ONA try- spoA- (23) were used as su- bacteria.B. subtilis 168 M099 spoA- [Met- Thr-]+ su+3 (23) was usedas su+ bacteria.

Assay for Formation of the Initiation Complex. Extracts ofB. subtilis infected with the )29 mutant sus3(91) were pre-pared as described by Shih et al. (11). The incubation mix-ture for the initiation reaction contained (final volume, 0.05ml) 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM ATP,0.25 ,uM [a-32P]dATP (5 ,uCi), 0.4-0.6 ,g of 429 DNA-pro-tein p3 complex, an extract of sus3-infected B. subtilis (33 ugof protein), and an amount of p3-containing fraction giving alinear response for p3-dAMP complex formation. After incu-bation for 20 min at 30°C, the samples were processed, treat-ed with micrococcal nuclease, and subjected to NaDodSO4/PAGE as described (10). The gels were dried and autoradio-graphed with intensifying screens at -70°C. Quantitationwas done by excising the band from the gel and counting theCerenkov radiation or by densitometry measurements of theautoradiographs by scanning with an Optronics digital mi-crodensitometer, model PDP 11/45, with a 100-,m square

Abbreviations: kDa, kilodalton(s); RFI, replicative form I.

1639

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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raster. The values obtained were processed in a digital mi-crocomputer.Other Assays and Procedures. Protein concentration was

determined by the method of Bradford (26) and DNA by themethod of Kapuscinski and Skoczylas (27). Electrophoresiswas carried out in slab gels containing 10-20% acrylamidegradients (28). After electrophoresis the proteins werestained as described by Fairbanks et al. (29). The stainedgels were photographed on Kodak plus-X film PXP-120,copied on Kodalith Ortofilm type 3, and analyzed by densi-tometry as described above.The incubation for DNA polymerase assay (0.025 ml) con-

tained 50 mM Tris HCl (pH 7.5), 10 mM MgCl2, 1 mM ATP,1 mM spermidine, 1 mM dithiothreitol, 5% glycerol (vol/vol), 1 ,uM [3H]dTTP (1 ,uCi), and 0.25 ,ug of poly(dA)-(dT)12_18 as template; 4-90 ng of purified protein p3 wasused. After incubation for 15 min at 30°C, the trichloroaceticacid-insoluble radioactivity was determined.ATPase activity was assayed after the release of Pi from

[y32P]ATP as described (30). Purified protein p3 at 4-45 ngin a final volume of 0.05 ml was used in the assay.Nuclease activity was assayed in the same buffer used for

initiation complex formation with M13 DNA, single-strand-ed or RF I, as well as 429 DNA. Nine nanograms of purifiedprotein p3 used in a final volume of 0.025 ml. After incuba-tion for 20 min at 30°C, the samples were analyzed by elec-trophoresis in gels containing 1% agarose in 0.1 M Tris bo-rate, pH 8.3/2 mM EDTA (31). (

RESULTSIsolation of Protein p3 from Phage 429 Particles. Fig. 1

shows the isolation of 429 DNA-protein complex by Sepha-rose 2B~chromatography of disrupted phage particles doublylabeled with 3H in the DNA and with 35S in the protein. An

I 00W(,_ . 84

7(1

500k-,I

r-

excluded peak containing both 3H and 35S radioactivity canbe observed. The 35S present in the DNA peak amounts toabout 0.3% of the total. The protein present in the 4)29 DNA-protein complex, after treatment with piperidine to hydro-lyze the DNA-protein p3 linkage, migrated into a NaDod-S04/polyacrylamide gel as a single band with the same mo-bility as that of protein p3 (Fig. 1 Inset, lane a). No proteinband migrated into the gel when the piperidine treatment wasomitted (results not shown), indicating that protein p3 wascovalently linked to the DNA. The absence of other labeledprotein bands in the gel indicates that protein p3 is highlypurified. Densitometry of different preparations of proteinp3 showed a degree of purity of about 95%. One mg of 429DNA-protein p3 complex yielded about 2 ,ug of protein p3.RIA. Protein p3 isolated from phage 4)29 particles as indi-

cated above was used to prepare anti-p3 antiserum after re-moval of the NaDodSO4 by filtration in a small Sephadex G-25 column. A highly specific RIA was developed to quanti-tate protein p3. Fig. 2A shows the results obtained withdifferent amounts of protein p3 isolated from phage particleswith anti-p3 antiserum or with nonimmune serum, both dilut-ed 1:200. When using 10 ng of protein, the binding with non-immune serum was only 2% of that obtained with anti-p3antiserum. When the anti-p3 antiserum was diluted 1000-fold, 30% of the maximal binding obtained with the 1:200diluted serum was still found (data not shown). Fig. 2Bshows the effect of increasing concentrations of protein p3purified from E. coli transformed cells as described below.As little as 100 pg of protein p3 could be detected by thisassay.

Purification of Native Protein p3. To study the role of pro-tein p3 in the initiation of 029 DNA replication, a functionalprotein is required. Because protein p3 isolated from phage429 particles is obtained under denaturing conditions, cell

1~~~~~~~~~1

l\ le),-0<I. v~~~~~~~~~~~~~~~~~~-

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iL

() (1 30 4(

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FIG. 1. Isolation of the 429 DNA-protein p3 complex from phage particles. Phage 429 mutant susl4(1242), doubly labeled in the protein andDNA moieties with 35S- and 3H, respectively, [18.4 x 101 cpm of 35S, 244,000 cpm of 3H, and 0.75 mg of DNA in 3 ml of disruption buffercontaining 10 mM Tris HCl (pH 7.5), 0.1 M NaCl, 5% 2-mercaptoethanol, and 2% NaDodSO4] was heated at 95°C for 7.5 min and passedthrough a column of Sepharose 2B-300 (1.6 cm x 46 cm) equilibrated in disruption buffer containing 1% 2-mercaptoethanol. A sample of 0.05 mlfrom each fraction was used to determine radioactivity. 0, 35S; 0, 3H. (Inset) The excluded peak, containing both 35S and 3H, was precipitatedwith 10o trichloroacetic acid. The pellet was washed with 80% ethanol, dissolved in 1 ml of 0.5 M piperidine, and incubated for 2 hr at 37°C tohydrolyze the DNA-protein p3 linkage. The protein was precipitated with 15% trichloroacetic acid, washed twice with 80% ethanol, anddissolved in disruption buffer. Lanes: a, sample subjected to NaDodSO4/PAGE as described (10); b, structural proteins of 3"S-labeled phage429 mutant susl4(1242). After electrophoresis the gel was dried and fluorography was carried out as described by Chamberlain (32). Sizes areshown in kDa.

1640 Biochemistry: Prieto et aL

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am- 27 --s&& "I p3

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3

I

x

0

x

a

eW"

Protein p3, ng

FIG. 2. RIA of protein p3 was carried out as described byNowinski et al. (33) with the following modifications. The samplewas placed in plates of polystyrene chloride in 0.2 M sodium carbon-ate (pH 9.6; final volume, 0.05 ml) and incubated overnight at 37°C.The liquid was removed from the plate, 0.05 ml of anti-p3 antiserumdiluted 1:200 in phosphate-buffered saline (8.1 mM Na2HPO4/1.47mM KH2PO4, pH 7.4/0.137 M NaCl/2.68 mM KCl) containing0.05% Tween 20 was added, and the plates were incubated for 1 hr at37°C. The plates were washed four times with the above buffer andincubated for 1 hr at 370C with 125I-labeled protein A (60,000 cpm) in0.05 ml of the same buffer. The plates were washed as before, andthe bound material was extracted with 0.08 ml of 2 M NaOH andassayed. (A) Denatured protein p3 isolated from 429 particles. (B)Purified native protein p3. *, Anti-p3 antiserum; o, nonimmune se-rum.

extracts containing free native protein p3 would be a suitablesource for purification of this protein. As the amount of freeprotein p3 present in 429-infected B. subtilis is low and can-not be detected by the RIA (data not shown), we used ex-tracts of E. coli transformed with the gene 3-containingrecombinant plasmid pKC3OA1 (19) as a source of nativeprotein p3.The extracts were passed through a DEAE-cellulose col-

umn (Fig. 3A). Most of the protein was eluted at 0.3 M NaCl(see also Fig. 4A, lane b), whereas the major peak of proteinp3 was eluted at 0.7 M NaCl, together with nucleic acid (Fig.3A; Table 1; and Fig. 4A, lane c). Nucleic acids were elimi-nated from the latter fractions by precipitation with polyeth-yleneimine. About 97% of the nucleic acid was removed inthe precipitate, whereas about 80% of protein p3 remained inthe supernatant. This fraction was passed through a phos-phocellulose column to further purify protein p3 and to re-move the remaining nucleic acid and the polyethyleneimine.Protein p3 was eluted at 0.6 M NaCl (Fig. 3B) and finally wasconcentrated by passage through a small phosphocellulosecolumn and elution with buffer B containing 1 M NaCl (Ta-ble 1). The protein, stored in this buffer at -70°C, was stablefor at least 6 months. After phosphocellulose chromatogra-phy, protein p3 was highly purified as judged by NaDodSO4/PAGE (Fig. 4A, lane d). Densitometric analysis of the gelindicated that the protein was more than 96% pure (Fig. 4B).Amino acid analysis of the purified protein p3 gave valuesvery similar to those deduced from the DNA sequence (34,35) (Table 2).The purified protein p3 at the concentrations used for the

in vitro initiation reaction was free of nuclease activity whenassayed with M13 DNA, either single- or double-stranded(RFI). No DNA polymerase activity was detected with poly-(dA) (dT)1218 as template. ATPase activity was also assayedbecause the formation of the initiation complex required thepresence of ATP, and the low stimulation obtained with non-hydrolyzable analogs ofATP suggested that ATP hydrolysismight be required (18). No ATPase activity was foundwhether in the absence or presence of native or denatured429 DNA-protein p3.Formation of p3-dAMP Initiation Complex with Purified

Protein p3. The protein p3 present at various purification

x

E

WU

Volume, ml

FIG. 3. Purification of protein p3 by DEAE-cellulose and phos-phocellulose chromatography. E. coli N99XCI857 cells transformedwith the recombinant plasmid pKC30A1 were ground with aluminaand extracted with buffer A (20 mM Tris HCl, pH 7.5/7 mM 2-mer-captoethanol/1 mM EDTA/5% glycerol) containing 25 mM NaCI.(A) After centrifugation at 27,000 x g for 45 min, the extract waspassed through a DEAE-cellulose column equilibrated in buffer Acontaining 0.3 M NaCl. Retained material was eluted with buffer Acontaining 0.7 M NaCl, dialyzed against 1 mM Tris HCI (pH 7.5),and lyophilized. The sample was resuspended in 10 mM Tris HCl(pH 7.5) at a concentration of 1.5 mg of DNA/ml and dialyzedagainst 20 mM Tris HCl (pH 7.5). Nucleic acids were precipitatedwith 0.2% polyethyleneimine at 1 M NaCl. (B) The supernatant,containing protein p3, was diluted with 3 vol of buffer B (20 mMTris'HCl, pH 7.5/0.1% Nonidet P-40) and passed through a phos-phocellulose column. Protein p3 was eluted at 0.6M NaCl. The loca-tion of protein p3 (o) was determined by radioimmunoassay. o, A260.

steps was active in formation of the p3-dAMP initiation com-plex when supplemented with extracts of B. subtilis infectedwith a sus3 mutant of )29 (Fig. 5A). Quantitation of proteinp3 showed a recovery of p3 activity of 12% with a 200-foldpurification (see Table 1). No activity was obtained wheneither the sus3 extract, the purified protein p3 or the 429DNA-protein p3 template were omitted or when proteinaseK-treated 429 DNA was used instead of 429 DNA-proteinp3 (Fig. SB).The in vitro formation of the initiation complex was as-

sayed at various dATP concentrations, and a Km value for

Table 1. Summary of the purification of protein p3Total Total Total Specific

protein, DNA, activity,* activity,Fraction mg mg pmol pmol/mg

Extract 1470 129.5 97.6 0.07DEAE-cellulose/

0.7 M NaCl 2.7 30.1 21.7 8.1Phosphocellulose 0.8 <0.001 11.7 14.1*These values were obtained by excising the 32P-labeled p3-dAMPband from the gel and measuring the Cerenkov radiation. Similarvalues were obtained by densitometry of the autoradiographs.

Biochemistry: Prieto et aL

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(1 Table 2. Amino acid composition of purified protein p3

'70(

48

-40_'. -

P-T1

Residue

Asp + AsnThrSerGlu + GlnProGlyAlaValMetIleLeuTyrPheLysHisArg

Predicted no.*

311218378

1119126

1816131527219

*Amino acid composition predicted from the nucleotide sequence(35, 36). The amino-terminal Met was presumably removed byprocessing.tAmino acid composition of the purified protein. Each value repre-sents the average after 24-, 48-, and 72-hr hydrolyses except forMet, which was after 48-hr hydrolysis, and Val, Ile, and Phe, whichwere after 72-hr hydrolysis. Values represent residues per mole-cule. Trp and Cys were not determined.

B120

80

40-

Migration -

FIG. 4. NaDodSO4/PAGE of purified protein p3. (A) Proteins atvarious purification steps were subjected to NaDodSO4 electropho-resis in slab gels containing a 10-20%o acrylamide gradient. Afterelectrophoresis, the proteins were stained as described (31). Lanes:a, extract, 78 jig; b, DEAE-cellulose, 0.3 M NaCl eluate (78 jig); c,DEAE-cellulose, 0.7 M NaCl eluate (3.2 Mg); d, phosphocellulose,0.6 M NaCl eluate (1.4 jAg); e, structural proteins of phage 429 12-particles (25 j.g). Sizes are shown in kDa. (B) Densitometric analysisof purified protein p3 (phosphocellulose fraction).

that can detect as little as 100 pg of this protein. With use ofRIA to quantitate the protein, we have purified biologicallyactive protein p3 from extracts of E. coli transformed withthe gene 3-containing recombinant plasmid pKC3OA1 (19).The purified protein p3 by itself, in the presence of 429DNA-protein p3 as template, is not active in formation of theinitiation complex p3-dAMP, but it can be complementedwith extracts of B. subtilis infected with a 429 sus3 mutant.This indicates that the initiation reaction requires other pro-tein(s) in addition to p3, in agreement with previous workshowing that the gene 2 product is also needed for initiation(18). The initiation of adenovirus DNA replication also re-quires, in addition to the precursor of the adenovirus termi-

A

8l h c d

- -84- 17()

dATP of about 3 x 10-6 M was obtained (data not shown).Thus, since the data of Table 1 were obtained using a con-

centration of dATP of 0.25 gM, the amount ofdAMP linkedto protein p3 can be increased by a factor of about 10 byraising the dATP concentration in the assay. The initiationreaction was not inhibited by NaCl up to 0.15 M or by 0.03%Nonidet P-40. Pretreatment of protein p3 for 30 min with 1%Nonidet P-40 at 40C or with 0.1% NaDodSO4 at room tem-

perature had no effect on its activity if the detergents were

removed by gel filtration in Sephadex G-25 prior to assay.Preincubation of purified protein p3 for 30 min at 40C at pHvalues as extreme as 2 or 12 had essentially no effect on its

activity in formation of the initiation complex. Heating for 5

min at 650C did not affect protein p3 activity, and at least50% remained after 5 min at 80'C. The activity was essential-ly lost after 5 min at 950C (data not shown).

DISCUSSION

We describe in this paper a simple method for isolation of

denatured protein p3 from phage 429 particles and an RIA

w-

-48

4_ I7_~~~~m_- /.

-\8tiX~

-48

-40

-7

p 1-t-d A M11)

FIG. 5. Formation of the p3-dAMP initiation complex with puri-fied protein p3. (A) Samples of protein p3 at various stages of purifi-cation were incubated with extracts of sus3-infected B. subtilis sup-plemented with 429 DNA-protein p3 complex and assayed as de-scribed. Lanes: a, extract (3.7 pig); b, DEAE-cellulose, 0.7 M NaCleluate (0.016 ,ig); c, phosphocellulose, 0.6 M NaCl eluate (0.009 Pg);d, )29 structural proteins labeled with [35S]sulfate. (B) Incubationwas as indicated in A with 0.018 jig of purified protein p3 (phospho-cellulose fraction). The following components were omitted fromthe reaction mixtures in lanes: a, none; b, sus3-infected B. subtilisextract; c, purified protein p3; d, 429 DNA-protein p3 complex; e,proteinase K-treated 429 DNA instead of 429 DNA-protein p3 com-

plex. Sizes are shown in kDa.

A I-

m_

:-

Observed no.t30.011.315.036.98.9

12.818.712.35.0

16.816.312.614.426.72.3

19.2

cl h c d e

-84-iI-1

1642 Biochemistry: Prieto et al.

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nal protein, a virus-encoded protein that was shown to be aDNA polymerase (36, 37). Partially purified protein p2 alsohas DNA polymerase activity (unpublished results). Proteinp2 might catalyze the formation of the covalent linkage be-tween the terminal protein p3 and the 5' terminal nucleotide.Although it remains to be determined whether the terminalprotein has any catalytic activity, its main role may be astructural one, by providing the serine residue throughwhich covalent linkage to the terminal dNMP takes place(38). The quick arrest of DNA elongation when extracts ofcells infected with temperature-sensitive mutant ts3(132) areincubated at 420C suggests that protein p3 may also be re-quired for elongation (39).The two protein p3 molecules linked at the ends of 429

DNA can interact, giving rise to circular structures and con-catemers (2, 40). The parental protein p3 has been shown tobe required for transfection (20, 41) and for initiation of 429DNA replication in vitro (10, 11, 42). In contrast, there is notan absolute requirement for the parental terminal protein inthe formation of the adenovirus initiation complex (17, 43).This difference could be due to the fact that the invertedterminal repetition of 029 DNA is only six nucleotides long(44, 45), whereas that of adenovirus is about 100 nucleotidesin length (46). Thus, protein-protein interaction might play amore important role in the initiation of 429 DNA than in thatof adenovirus replication.At present we cannot rule out the possibility that protein

p3, in addition to its interaction with the parental protein,also specifically recognizes regions at the ends of q629 DNA.

This investigation has been aided by Grant 2 R01 GM27242-04from the National Institutes of Health and by grants from the Co-misi6n Asesora para el Desarrollo de la Investigaci6n Cientifica yTdcnica and the Fondo de Investigaciones Sanitarias. We are grate-ful to E. Mendez for the amino acid analysis, to L. Enjuanes for hisadvice with the immunological techniques, and to L. Blanco for hishelp in the initiation assay. I.P. and J.A.G. were Fellows of theSpanish Research Council.

1. Sogo, J. M., Inciarte, M. R., Corral, J., Vifiuela, E. & Salas,M. (1979) J. Mol. Biol. 127, 411-436.

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18. Blanco, L., Garcia, J. A., Pefialva, M. A. & Salas, M. (1983)Nucleic Acids Res. 11, 1309-1323.

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20. Salas, M., Garcia, J. A., Pefialva, M. A., Blanco, L., Prieto,I., Mellado, R. P., Lazaro, J. M., Pastrana, R., Escarmis, C.& Hermoso, J. M. (1983) UCLA Symposia on Molecular andCellular Biology, New Series, ed. Cozzarelli, N. R. (Liss, NewYork), Vol. 10, in press.

21. Camacho, A., Jimenez, F., Vifiuela, E. & Salas, M. (1979) J.Virol. 29, 540-545.

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23. Moreno, F., Camacho, A., Vifiuela, E. & Salas, M. (1974) Vi-rology 62, 1-16.

24. Jimdnez, F., Camacho, A., de la Torre, J., Vifiuela, E. & Sa-las, M. (1977) Eur. J. Biochem. 73, 57-72.

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Mellado, R. P., Vifiuela, E. & Salas, M. (1976) Eur. J. Bio-chem. 66, 229-241.

29. Fairbanks, G., Steck, T. L. & Wallach, D. F. H. (1971) Bio-chemistry 10, 2606-2617.

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