DNAs of simian virus 40 and polyoma direct the synthesis of viral tumor antigens and capsid proteins in <i>Xenopus</i> oocytes

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Reference

DNAs of simian virus 40 and polyoma direct the synthesis of viral tumor antigens and capsid proteins in Xenopus oocytes

RUNGGER, Duri, TURLER, Hans

Abstract

Purified simian virus 40 and polyoma DNAs injected into nuclei of Xenopus oocytes were transcribed and subsequently translated into virus-specific tumor antigens and capsid proteins. Simian virus 40 large and small tumor antigens synthesized in the oocytes were indistinguishable, by gel electrophoresis and [35S]methionine-labeled tryptic peptide mapping, from the corresponding polypeptides synthesized in CV-1 African green monkey cells. The synthesis of large simian virus 40 tumor antigen implies the correct splicing of its mRNA, which is complementary to nonadjacent nucleotide sequences in the early region of the viral genome. Polyoma DNA directed synthesis of two polyoma tumor antigen polypeptides, 57,000 Mr and small tumor antigen, and of the main capsid protein.

RUNGGER, Duri, TURLER, Hans. DNAs of simian virus 40 and polyoma direct the synthesis of viral tumor antigens and capsid proteins in Xenopus oocytes. Proceedings of the National Academy of Sciences, 1978, vol. 75, no. 12, p. 6073-6077

DOI : 10.1073/pnas.75.12.6073

Available at:

http://archive-ouverte.unige.ch/unige:127555

Disclaimer: layout of this document may differ from the published version.

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Vol.75, No. 12, pp.6073-6077, December1978 CellBiology

DNAs of simian virus 40 and polyoma direct the synthesis of viral tumor antigens and capsid proteins in Xenopus oocytes

(microinjection/coupledtranscriptionandtranslation/immunoprecipitation/mRNAsplicing) DURI RUNGGER* ANDHANSTORLERt

*Departmentof AnimalBiology,UniversityofGeneva, CH1224Chene-Bougeries, Geneva;andtDepartmentof MolecularBiology,UniversityofGeneva, CH1211Geneva,Switzerland

Communicated byV.Prelog, September18,1978

ABSTRACT Purified simian virus 40andpolyomaDNAs injected intonuclei ofXenopus oocytesweretranscribedand subsequently translatedintovirus-specifictumorantigens and capsi proteins. Simianvirus 40largeandsmalltumorantigens synthesizedintheoocyteswereindistinguishable,by gelelec- trophoresisand[35Smethionine-labeled tryptic pepti emap- ping, from the correspondingpolypeptides synthesizedinCV-1 Africangreenmonkey cells.Thesynthesis of largesimian virus 40. tumor antigen implies thecorrect splicing of its mRNA, whichiscomplementarytononadjacent nucleotidesequences inthe earlyregionofthe viralgenome.PolyomaDNAdirected synthesisof twopolyomatumorantigenpolypeptides,57,000 Mr andsmalltumorantigen, andofthemaincapsidprotein.

Nuclei from variouseukaryoticcellsinjectedintofrogoocyte nuclei (germinalvesicles)continuetodirectsynthesisof ori- gin-specific proteins(1).Inaddition,genesthatwere not ex- pressed in thedonorcell may be activatedafterinjection (2).

Purifiedviral,phage,orplasmidDNAsinjectedintothe nucleus of Xenopus oocytes serve astemplatesfor the transcription of specificRNAsequences(3).Thesynthesisof 5S RNA(4)and tRNA (5) is greatly stimulated upon injection ofthe corresponding templates.Ithasbeenclaimedthat, afterinjectionof simian virus 40(SV40)DNA I(6), muchof thevirus-specific RNAsynthesized inthe Xenopus oocyte has the same size and istranscribed fromthe same region asthelateviralmRNAs present inlytically infected monkeycells (7, 8), and proteins that migrate ontwo-dimensional gelsinthesamemanner as theSV40capsid proteins VP 1 and VP 3 weredetected(8).

Inthis articlewereportthatXenopus oocytes transcribe and translatemostorall ofthe genetic information containedinthe SV40genome. Inparticular,weobserved the synthesisof large (T)andsmall(t)SV40tumor antigenshavingthe samechar- acteristics asthose synthesizedinmonkeycellsundergoing lytic infection.Injectionof polyoma DNAI(6)resulted in the synthesis of three polyoma-specific polypeptides comigrating, duringgelelectrophoresis, withtwo minorpolypeptides related topolyomatumorantigen and with the major capsid protein, respectively.

MATERIALS AND METHODS

SV40 DNA I (covalently closed circular) was isolated from SV40-infectedCV-1Africangreenmonkeycells (Flow Labo- ratories, Irvine, Scotland)40-48 hr afterinfection by selective extraction, deproteinized with phenol, and purified by CsCl/ethidium bromide density gradient equilibrium cen- trifugation (9). Polyoma DNA I was isolated from polyoma- infected mouse kidney cell cultures 30-35 hr after infection by the same procedure. For injection, the viral DNA was dissolved in88mMNaCl/1 mMKCI/15 mMTris-HCl,pH7.6 at con- centrationsbetween 300 and 500

Ag/ml.

Theprocedureused forinjectingviralDNA into thegerminal vesicle ofXenopusoocyteswas amodification ofalreadyes- tablishedmicroinjectiontechniquesforDNA(4, 10)andmRNA (11, 12). Piecesofovarieswereexcised fromXenopuslaevis females anesthetized with tricaine (MS 222; Sandoz, Basel).

Stage5and6oocytes(13)weredefolliculatedbycollagenase

treatment(14), extensivelywashed with modifiedBarth'sso- lution(15),and maintainedinthis bufferat200Cduringall further manipulations and incubations. The oocytes were

placedonto a700-Sm meshgrid(Zfiricher BeuteltuchFabrik, Ruschlikon)fixedtothebottom ofa6-cm petridish andcen-

trifuged (10)in aMSE2-literswing-out rotorat400Xgfor 10 min. Duringcentrifugation the germinal vesiclesrise to the surface of theoocytesandtheirposition isindicatedbyaclear area inthe dark animalpole.Theoocytes weregently removed from thegrid,transferredto adepression slide, andpositioned for injection by means of watchmaker's forceps. The glass capillariesusedfor injections hadatipdiameter of about 15 ,4m andwerecalibratedtodeliver about 15nl,containing5-7ng of viral DNA. Amicromanipulator (Singer InstrumentsCo., Somerset,England) anda micrometersyringe(Agla Wellcome ResearchLaboratories, Beckenham, England) allowedrapid injection.The injectedoocytes(about50-100perseries)were incubated for about24hrinBarth's solutioncontaining50 units per ml each of streptomycin, penicillin, and kanamycin.

Thereafter 1 ml of Barth's solution containing 300 ,uCi of [35S]methionine (500-1000 Ci/mmol, the Radiochemical Centre, Amersham, England)wasadded and incubationwas continued for another24hr(1Ci=3.7 X1010becquerels). The surviving oocytes(60-90%)werewashed first withBarth'sso- lution, then with distilled water, and stored at -80'Cafter removalof thewater.

For extractionof viral proteins, the oocytes weredisrupted in asmallDouncehomogenizerin 2mlof abuffercontaining detergent (0.1 MTris-HCl, pH9/0.1MNaCl/5mMKCI/1 mMCaCl2/0.5mMMgCl2/0.7mMNa2HPO4/0.5%Nonidet P-40). The homogenate was sonicated in a MSE ultrasonic disintegrator (Mk 1) for1minwith anamplitude of 10

Atm

^and

kept on ice for 20 min. Aftercentrifugationat 15,000 X gfor 30 min at 40Cthe supernatant wasdivided into aliquotsand 1Oulof antiserum wasadded to each.Theimmune complexes wereisolatedby means ofstaphylococcalprotein A-Sepharose (16)(Pharmacia,Uppsala, Sweden) and theproteins wereanalyzed byelectrophoresis on a 12.5% sodium dodecyl sulfate (NaDodSO4)/polyacrylamidegel (17).

For trypticpeptideanalysis, the 88,000 Mr and 18,000Mr polypeptides shown in Fig. 1, slot 1, were eluted from a preparative 12.5% NaDodSO4/polyacrylamidegel with 0.25M ammoniumcarbonatecontaining 0.1%NaDodSO4 and 50

Ag

Abbreviations: SV40,simian virus40;tantigen,smalltumorantigen;

T antigen, large tumorantigen; NaDodSO4, sodium dodecyl sulfate.

6073 Thepublicationcosts of this article were defrayed in part by page chargepayment. This article musttherefore be hereby marked "ad- vertisement"inaccordancewith 18U. S. C. §1734 solely toindicate this fact.

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6074 Cell Biology: RunggerandTurler

of bovineserumalbuminper ml as carrier. Large (88,000 Mr) and small (18,000 Mr) tumor antigens synthesized in SV40- infected CV-1 cells were isolated by the method of Schwyzer (16)from 10 cultures that had each been labeled with 100

,gCi

of[a5S]methionine from 27 to 31 hr after infectionandwere elutedafter preparative gel electrophoresis as described above.

The proteins were precipitated with trichloroacetic acid, oxi- dizedwith performic acid, and digested with trypsin (18, 19).

The digests were dissolved in 10,l^{of water}and spotted on 20 X 20 cm Polygram Cell 300 precoated plastic sheets (Ma- cherey-Nagel,Duren, Germany). The solvent for the first dimension was pyridine (175ml)/isoamylalcohol (175 ml)/water (150 ml) and for the second dimension n-butanol (150 ml)/

pyridine (120ml)/aceticacid (30 ml)/water(120 ml) (20). For fluorography the sheets were run in acetone containing 20%

2,5-diphenyloxazole (PPO). Preexposed (hypersensitizing light flash)(21) Kodak RP Royal X-Omat films were exposed to the chromatograms for 2-3 weeks at -700C.

RESULTS

Identification of SV40-specific polypeptides synthesized inthe oocyte

Virus-specific polypeptides were isolated from the oocyte lysate by using specific antisera, characterized by NaDodSO4/polyacrylamidegel electrophoresis(17),and comparedtoknown virus-specific proteins extracted by the same methods from infected cells. Intheexperimentshown in Fig. 1,SV40-specific proteins wereisolated fromoocytesinjectedwith SV40 DNA

1 2 3

T88,000-

_O.._

_

78,000 UOh

VP146,000-a - m

VP239,000-.- VP332,000_

ei4.

Marker -116,000

- 94,000

- 77,000 - 69,000

- 53,000

- 41,000

- 29,000

I with two different antisera. Anti-SV40 T serum from tumor-bearing hamsters (22) strongly complexed two polypeptides with apparent Mr values of 88,000 and 18,000 (Fig.

1, slot 1). A commercial SV40 hyperimmune rabbit serum (Microbiological Associates, Bethesda, MD) also complexed the 88,000 and 18,000 Mr polypeptides, but in addition precipitated apolypeptidewith an apparent Mr of about 46,000 (Fig. 1, slot 2). Fig. 1,slot 3, shows the pattern of all radioactive proteins present in the oocyteextract. Radioactive bands that might correspond to 18,000, 46,000, and 88,000 Mr SV40-specific polypeptidescanbedetected in this autoradiogram. As deter- mined by trichloroacetic acid precipitation, the immune complexes representedinthese experiments 0.25-1% of total radioactivity incorporated in the oocytes.

Inorder to testthe specificity of the antisera used, a number ofcontrol experiments were done, using preimmune serum as well as anti-polyoma serum to precipitate extracts of SV40 DNA-injectedoocytes. The results are shown in Fig. 2. The oocytelysate(slot 1)wasdivided intoaliquotsthat weretreated withnormal hamster serum (slot 2), hamster serum directed againstpolyomaTantigens(23)(slot 3),anti-SV40 Tserum(22) (slot4), and rabbit antiserum directed againstpurifiedSV40 capsids (slot 5) (a gift from E. May, Institut deRecherches Scientifiquessurle Cancer,Villejuif, France). Slot6shows the patternof[35S]methionine-labeledproteinsfrompurifiedSV40.

Although in this experiment the isolated immunecomplexes werecontaminatedwith oocyte proteins, Fig.2shows that the 46,000 Mrpolypeptide wasspecificallyboundbyantiserum against SV40capsid (slot 5) and that it comigratedwith the maincapsidprotein(VP 1) of SV40(slot 6). The88,000and 18,000Mrpolypeptideswerespecifically boundbyanti-SV40 Tserum (slot 4) but notby preimmune serum (slot 2), anti-

1 2 3 4 5 6

Marker

116,000-__

96,000- 3 -T88,000

77,000 -14

69,000- M

53,000-_53,000-_~ _~ _~~~~* 11

-~VP 1I46,000

41,000-_41,000- ₅

:JVP

²39,000

-VP 332,000 29,000-r°

t 18,000- M

- 17,000

17,000-

m

FIG. 1. Polypeptidessynthesizedin oocytesinjectedwithSV4o DNA I.Theproteinsof 50 oocytes,injectedwithSV40DNAand incubated from26 to 52hrafterinjectionwith[35S]methionineat300

gCi/mlwereextractedwith2mlof extraction buffer. Theextract was divided intotwoaliquotstowhich10,uofeitheranti-SV40Tserum (22)(slot 1)or serumdirectedagainst SV40virus(Microbiological Associates) (slot2)wasadded.The immunecomplexeswereisolated bymeansofstaphylococcalproteinA-Sepharose(16)and theproteins

wereanalyzedby electrophoresisona12.5%NaDodSO4/polyacryl-

amidegel (17).Slot3: 2Alofthe oocyteextractin 10,lofloading bufferwasdirectlyputonthegel.Thearrows tothe leftindicate the expected positionofSV40proteins(T,t,VP)and thefigures givetheir nominal Mr. The figurestotherightindicate thepositionofmarker proteins: 116,000Mrf3-galactosidase, 96,000Mrphosphorylasea, 77,000Mrtransferrin, 69,000Mrbovineserumalbumin, 53,000Mr glutamatedehydrogenase,41,000 Mralcoholdehydrogenase,29,000 Mr carbonicanhydrase,17,000Mrmyoglobin.

a ^-t ^18.000

FIG. 2. Characterization ofSV40-specificpolypeptides synthe- sizedinoocytes.From47oocytes,injectedwithSV40DNAI and labeled with[35S]methioninefrom20to48hr afterinjection,proteins

wereextracted with2ml ofextraction buffer. A smallaliquotof the extract wasdiluted inloadingbuffer forgelelectrophoresis (slot 1).

To0.5-mlaliquotsof theextract,5,lofserum wasadded. Slot2:

normal hamster serum; slot3:anti-polyomaT^serum(hamster)(23);

slot4:anti-SV40Tserum(hamster) (22);slot5:anti-SV40capsid

serum(rabbit).Theisolated immunecomplexesweresubjected^to NaDodSO4/polyacrylamide gel electrophoresis (12.5%gel)together

withasampleof[35S]methionine-labeledSV40(slot6)purifiedfrom alysateofSV40-infectedCV-1cells. Thepositionsof the marker proteins(seelegendtoFig. 1)areindicated^totheleft;^totheright,

the apparentMrvalues ofSV40-specificproteinssynthesizedinthe oocytesorpresent inSV40virus.

Proc.Natl.Acad.Sci. USA 75

(1978)

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polyoma T serum (slot 3), or anti-SV40 capsid serum (slot 5).

In this experiment a faint band representing most likely a contaminating oocyte proteinthat comigrated with the strong 18,000 MrSV40-specificpolypeptide can be seen in slots 2, 3, and 5. Furthermore, neither the 88,000 nor the 18,000 Mr polypeptide bands were observed when extracts of oocytes that had been injected with polyoma DNA I were treated with the sameanti-SV40 T serum (see Fig. 5, slot 2). The specific reaction with antisera and the electrophoretic mobilities observed suggest that the strong bands ofSV40-specific polypeptides synthesized inthe injectedoocytescorrespond to large SV40 Tantigen (88,000MT) (16, 22, 24), small t antigen (18,000 Mr (25, 26), and the main capsid protein VP 1 (46,000 Mr) (24, 27).The electrophoretic mobilities of the SV40 T antigens and of thecapsidproteinssynthesizedinthe oocytes werecompared tothose of the corresponding proteins synthesized in monkey CV-1cellsundergoing lytic infection with SV40 and run on the samegel. The anti-T and anti-capsid sera used were the same asinFig. 2,slots4and5,respectively.As isshown in Fig. 3,the putativelargeTand smalltantigensisolatedfromoocytes(slot 3)comigratedwith large T and small t antigens isolated from SV40-injectedCV-1cells(slot4).Themaincapsidprotein(VP 1) wasresolved into two or three bands, which differed in in- tensity in VP 1isolated from cells or from oocytes (slot 2); this might reflect differences in the extent of modification of the polypeptides.Inthis experiment contamination of the immune complexeswith oocyte proteins was very low and no contami-

1 2 3 4

Marker 116,000-

96,000^-

- _ -T88,000 69,000-

53,000-

41,000-

--I

^{VP 1} 44,000-48,000 -VP 239,000

nating 18,000Mroocyte polypeptide could be observed in immunecomplexes isolated with anti-capsid serum (slot 2).In all extracts of oocytesinjectedwith SV40 DNA and treatedwith commercial anti-SV40serum(Fig. 1)oranti-SV40capsidserum (Figs. 2 and 3), a faint discrete band with an electrophoretic mobility corresponding to VP 3 (32,000. Mr) (24, 28) and a rather diffuse zoneinstead of a discrete band of VP 2 (39,000 Mr) (24, 28) wereobserved (see Fig. 1, slot 2, and Fig. 3, slot 2).For reasonsthat will be discussed below, the formation of largeSV40 T antigen ininjected oocytes is of major interest.

Therefore, the putative large and small SV40 tumor antigens werefurtheridentifiedbytwo-dimensionalchromatographic analysisofthe

[a5S]methionine-labeled

trypticpeptides. The patternsobtained from the 88,000 and 18,000 Mrbandsisolated inthe experimentshown in Fig. 1, slot 1, are representedinFig.

4 a andc,respectively, and arecomparedtothose obtained from largeand small SV40 tumor antigenssynthesizedin infected CV-1 cells(Fig.4b andd, respectively). Chromatog- raphy of the tumor antigens from the oocytes and from infected cells was done in different tanks and at different times, which might account for the minor differencesinmigration ofsome spots.Nevertheless,the overall pictureclearlyshows that the fingerprintsrepresentedinFig. 4 a and c are very similar to thosegiven in Fig. 4b andd, respectively. Inbothcasesthe small t antigens(4candd)share spots with thelargeTantigens (4aandb),and the same spots that are presentinlargeTan- tigens(4 a andb)and indicatedbyarrows areabsentinsmall tantigens(4candd). Analysis of the 78,000 Mrband(Fig. 1, slot1) by the same method revealed that this band alsoisrelated tolargeSV40 T antigen(notshown).

Theseresults demonstratethatinjectionof SV40DNA I into Xenopusooc'ytesevokes the synthesis of SV40 largeTand small tantigens that areindistinguishable bytheanalyticalmethods used (immunoprecipitation, gel electrophoresis, and finger- printing) from theTandtantigenssynthesized inSV40-in- fectedmonkeycells.

a b

-VP 332,000 29,000^-

.e "I

I , 6

-t 18,000

17,000- C d

FIG. 3. ComparisonofSV40 proteinssynthesized in oocytes in- jectedwithSV40 DNA I and inSV40-infectedCV-1 cells. Fifty oocytes,injectedwithSV40DNA Iand labeledwith[35S]methionine

from 36 to 60 hrafterinjection, wereextracted as described for Fig.

1.One culture(9-cm diameter)ofSV40-infectedCV-1cells was labeledwith 50ACi^of

[35S]methionine

from41to 45hr after infection andextractedasdescribedearlier(16).The isolated immune complexes wereanalyzed by electrophoresis on a 12.5% NaDodSOJ polyacrylamide gel. Slot1:proteinsisolatedfromCV-1 cells with serumdirected againstpurified SV40 capsids.Slot 2: proteins isolated from oocytes withanti-SV40capsidserum.Slot3: proteinsisolated from oocytes withanti-SV40T serum(22).Slot 4:proteinsisolated fromCV-1 cells with anti-SV40 T serum (22).

..

9

.-..9

FIG. 4. Trypticpeptide analysisoflarge-and smallSV40tumor antigenssynthesizedinoocytesand in CV-1 cells.(a)The88,000Mr Tantigensynthesizedinoocytes;(b)88,000MrTantigensynthesized inCV-1cells; (c)18,000Mrtantigensynthesizedinoocytes;(d)18,000 MrtantigensynthesizedinCV-1cells. Thearrows indicate spots presentonlyin88,000MrTantigen.

Biology: Rungger

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6076 CellBiology: RunggerandTurler

Polyoma-specificpolypeptides synthesizedinthe oocytes

Usingthesameprocedures,wealsolookedforthe synthesis of polyoma-specific polypeptidesinoocytesthathadbeen injected withpolyomaDNA I.Theseexperimentsalsoserve as amock DNA injectioncontrol forthe resultsobtained withSV40DNA.

Fig. Sshowstheautoradiographsof proteinsisolatedby means of various sera. Aliquotsof the oocyte extract (slot 6) were precipitatedwithpreimmunehamsterserum(slot1),anti-SV40 T serum (slot 2), anti-polyoma Tserum (23) (slot3), anda commercial mouseantiserum against polyomavirus (slot4;

MicrobiologicalAssociates, Bethesda, MD). Slot5wasloaded withpolyoma-specificproteinsthatwereisolated frompoly- oma-infected mouse kidney cells by using anti-polyoma T serum,which also bindsthe viral capsidproteins(23).Inthis extractthe largepolyomaTantigen(98,000 Mr)small polyoma tantigen(23,000Mr),andVP 1ofpolyomavirus(47,000Mr) wereclearly visible. Twoadditional, minorbands related to polyomaTantigenand migratingasiftheirMrvalueswere about61,000and57,000 wererather weakinthisexperiment, buttheycanusually be detectedinextractsofmousekidney cells (23). Similar resultsonthecharacterizationofpolyoma tumorantigens inlytic infectiondifferingslightlyintheesti- matedMrhave beenpublishedbyothergroups(29, 30).The commercialpolyoma virusantiserum (slot 4)boundastrong

t

1 2 3 4 5

Marker 116,000-

96,000- X

69,000- 53,000

41,00-0

-T98,000

57,000 -VP 1 47,000

-VP235,000

29,000

-t23,000

17,000

FIG. 5. Characterization ofpolyoma-specific polypeptidessyn-

thesized inoocytes.From67 oocytes,injected with polyomaDNAI andlabeled with [35S]methionine from 20 to48 hr after injection, proteins^wereextracted with2ml ofextraction buffer.A smallaliquot oftheextractwasdiluted inloading buffer for gel electrophoresis (slot 6).To0.5-mlaliquots of theextract,5 Mlof^{serum was}added. Slot1:

normal hamster^serum;slot2:anti-SV40Tserum(hamster) (22);slot 3:anti-polyoma Tserum(hamster) (23);slot 4:anti-polyomavirus

serum(mouse); slot5:polyoma-specific proteins isolated, by using anti-polyoma Tserum (23), frommousekidneycells infected with polyoma virusat270Candlabeled with[35Slmethioninefrom40to 48hr afterinfection. On the leftareindicated thepositionsof marker proteins,and^onthe right,theapparentMr values of polyoma-specific proteinssynthesized in polyoma-infectedmousekidney cells. The arrowheads (slot 2) indicatethepositionsofSV40tumorantigens, whicharenotsynthesized after injectionofpolyomaDNA.

bandcomigratingwithVP 1present ininfected mousekidney cells (slot 5) and aslightlyfastermoving band. The same two bands were alsobound by the anti-polyoma T serum (slot 3), which in addition bound a rather strong band at about 57,000 Mr and aband at23,000Mr; the latter twocomigratedwith 57,000 Mr andsmallpolyoma t antigen from infectedcells(slot 5). None of these bands were present in oocyte extractstreated with preimmune serum oranti-SV40 T serum, nor werethey observedin an extractofSV40-injectedoocytestreated with thesameanti-polyoma T serum (see Fig. 2,slot3).Thespecific reaction with the antiserum andtheelectrophoreticmobilities observed suggestthatthe57,000and 23,000 Mrpolypeptides correspond to 57,000 Mrandsmall polyomatantigenandthat the 47,000 Mrband corresponds to VP 1 ofpolyoma virus.

While these three bands were observed in all experiments,the bandmovingslightlyfasterthanVP1wasobservedonlyinthis experiment; itmightrepresent unmodifiedor incompletely modifiedVP 1.Sofar none ofthepolyoma-specificpolypep- tidessynthesized intheoocyteshas beenidentified further.In theexperimentsdonesofarwe wereunabletodetect thesynthesisof anew proteincorrespondingtolarge polyomaTan- tigen. Itsdetection is,however,rendereddifficultbyastrong, Coomassieblue-staining, unlabeledoocyteprotein migrating at 100,000 Mrthat interferes withthe resolution of the radio- activebands in this region(seeFig. 5,slots1,2,and3).Although the total incorporation of

la5S]methionine

intoproteins was similar for oocytesinjected withSV40DNAandpolyomaDNA, synthesis ofpolyoma-specificpolypeptidesseemstobesome- what less efficientthanthat ofSV40-specificpolypeptides.

DISCUSSION

Ourresults show that Xenopus oocytes transcribe andtranslate SV40 DNA intosubstantial amounts ofSV40-specificproteins closely similar to thosesynthesizedinmonkey cellsundergoing lyticinfection. Proteinsynthesis directedby polyomaDNA I was less efficient; nevertheless, polyoma-specific proteins similar to thosefoundinlytic infectionswereformed.

The synthesis of full-size SV40Tantigeninthe oocyte is additionalproof that thisprotein isentirely codedby the viral genome. Furthermore, theformation of 88,000 Mr SV40T antigen isparticularlyinterestinginthelight ofrecentfindings thatsuggestthat itsmRNAhastobeprocessedbyasplicing mechanismsimilar tothatfound in mRNAsynthesis of ade- novirus 2(31). Fromstudies with SV40deletionmutants(26) and from DNA sequenceanalyses(32, 33)it seemslikelythat a certainnucleotidesequencelocatedwithin thecodingregion for 88,000 MrTantigenhastobe omittedinthemRNA.Our results wouldsuggest,then, that thisprocessingof SV40mRNA occurs inoocyteswith thesameaccuracyas in

monkey cells,

butperhapswithasomewhat decreasedefficiency,asindicated by theincreasedproportionof smalltantigenproducedinthe oocyte(seeFig.3).This wouldmeanthat thesplicingofmRNAs transcribedfromsplitgene sequencesisachieved

by

amech- anism commontodifferentcelltypesand species.

Ourresults donotallowus todrawconclusionsontherelative amountsofcorrect orabnormalvirus-specifictranscripts synthesized in the oocytes. Inexperimentsusing

purified

mRNAs (11, 12, 34, 35), synthesis of similar proportions of

specific

proteinscouldbeinducedbyinjectionof 1-10 ngofmRNA per oocyte,stronglydependingonthe type ofmRNAused.Because weinjected 5-7ngofDNAper oocyte (i.e.,about i0Pmole- cules), one or a few correct transcripts per

injected

genome might givetheobserved amount of protein

synthesis.

There were, however, no clearindications ofabnormaltranscription, becausethemajorityof the

SV40-specific

proteinsdetected

by

the immunoassaywereof thecorrect size. Inanother

coupled

Proc.Natl. Acad.Sci. USA75

(1978)

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transcription and translation system,derived fromEscherschia coli and using SV40DNA I astemplate,severalpolypeptides rangingfrom 12,000to59,000Mrwereimmunoprecipitated by the same anti-SV40Tserum(36).

Wedo not know yet whether SV40orpolyomaDNA isable toreplicate in the oocyte. Duringlytic infectionswith these viruses,viralcapsidsaresynthesizedinlargeamountsonlyafter the onset ofviral DNAreplication (6,37,38),whereas T antigens aresynthesized earlyininfection andarerequiredforthe inductionofhost and viral DNAreplication(39,40,41).Further experiments areneeded to determine whetherintheoocytes the control of late viral gene expression(i.e., synthesisof viral capsids)issimilar to or different from thatoperatinginmam- maliancellsundergoinglyticinfection.

Coupled transcription and translation of viral DNAs in Xenopus oocytesmight provide a tool forstudiesonthe control of geneexpressionand on processing of mRNAs.Inaddition, this systemmight be useful forcharacterizingthestructure and functionof proteinssynthesizedfrom DNA ofdefectiveviruses, inparticular ofnonviable deletion mutants.

We are verygratefultoConsuelo Salomon for her excellenttechnical assistanceandtoO. JenniandF.Bourquinforthephotographs.This workwassupported bySwissNationalScience FoundationGrants 3.707.76(to Prof.R.Weil)and3.716.76(to Prof.M.Crippa).

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