Nucleotide sequence deletions within the coding region for small-t antigen of simian virus 40.

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0022-538X/79/06-0674/09$02.00/0

Nucleotide Sequence

Deletions Within the Coding Region for

Small-t Antigen

of

Simian

Virus 40

GUIDO VOLCKAERT,'JEANFEUNTEUN,2 LIONEL V.CRAWFORD,3 PAULBERG,4 AND

WALTERFIERSI*

Laboratoryof Molecular Biology, StateUniversity of Ghent, B-9000 Ghent,Belgium';Institut deRecherches

ScientifiquessurkCancer,94800Villejuif,France2;Department of Molecular Virology, Imperial Cancer

Research Fund, London WC2A 3PX,England";andDepartment of Biochemistry, Stanford University

Medical Center, Stanford, California 94305w

Received for publication 28 November 1978

Simian virus40earlymutants withdeletions mapping in the 0.53-0.60 region

havebeen sequenced by the Maxam and Gilbert approach. All these deletions

affect the small-t gene. The size of the shortened small-t-related polypeptides

produced by several of themutantshasbeen compared with the molecular weight

as deduced from the nucleotide sequence. There was good agreement for the

mutants dl890, d1891, and d12102. For d12121 and dl2122 the small-t-related

proteinwasconsiderably larger thanexpected. It ispossible to explain this result

onthe basis of thenucleotidesequence:thenormalsplicingeventofthesmall-t

mRNA stilloccurs,butasthedeletion shifts the reading frame, translationofthe

small-t-relatedpolypeptidecontinues beyond thesmall-tsplice,butinadifferent

reading frame thanlarge-T. Mutants d1883, d1884, and d12112have lost one of

the small-t splicing boundaries, and no (or minute amounts of) small-t-related

protein has been observed in mutant-infected cells. The possible relationship

between splicingandtransportofpolyadenylic acid-containing mRNAfromthe

nucleustothecytoplasm in vertebratecellsisdiscussed.

Theearly regionof the simianvirus 40(SV40)

genome extends counter-clockwise from about

position 0.66 to 0.17 on the standard

physical

mapofSV40DNA(13,23).It codes foratleast

twoproteins,small-t and

large-T

antigens (4,

18,

21), whicharetranslatedfromtwo

different,

but

overlapping,mRNA's(1).

The early region has been characterized by only one complementation group of

tempera-ture-sensitive mutants: tsA (14).

However,

Shenketal. (24)described another class of

early

regionmutants, whichwereconstructed by

in-troducing deletions in the 0.60-0.53 segment of

theviral DNA. Thesemutantsresemble inmany

aspects thepolyomahr-tmutantsisolatedbyT. Benjamin and co-workers(7,9,28).Themutants

still producenormallarge-T,butthesizeofthe

small-tprotein is affectedbythedeletion(4).In

permissive cellstheygrowonlyslightlylesswell

thanwildtype, but whentestedunder

appropri-ate conditions their transforming ability is

greatly reduced (2, 3, 6,25).

Examination of thenucleotidesequence of the

beginning of theearlyregion and theN-terminal

amino acid oflarge-Tandsmall-t showed that both proteins initiate at the same AUG start

codon,correspondingtomapposition0.649

(nu-cleotidesE80-82,numberingsystemasinref.8),

and have a common amino-terminal sequence

(19, 30).An openreadingframe extendingtothe

information for a UAA termination signal at

0.547(E602-604) allows the deduction ofa

174-amino-acids-long polypeptide. Considering the universality of the genetic code and the

alloca-tion ofthe firstboundary of the splice for

small-tmRNAtoE 606 (22),wehave concluded that

this deduced polypeptide sequence witha

mo-lecularweight of 20,503 does indeed represent

the small-tantigen (30).

The isolation of the aforementioned viable

deletionmutants andthe occurrenceoftriplets

correspondingtoterminationcodons in all three

reading frames in the region 0.53-0.55 of the

DNA message strand suggested that the seg-ment 0.60-0.53doesnotencode information for

large-T. Since neither of theuninterrupted

cod-ing regionsflankingthissegmentwould suffice

to specify a94,000-dalton large-T protein, and

considering the existenceofpeptidescommon to

large-T and small-tantigen, anintragenic

splic-ingevent washypothesizedfor thelarge-Tgene

(4).Direct characterization oftwoearlymRNA

species (1) in addition to nucleotide sequence

information in theearly region (30)clarified the

splicingoflarge-TmRNA andestablishedmore

precise boundariesfor theinterveningsequence.

674

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NUCLEOTIDE SEQUENCE OF SV40 SMALL-t MUTANTS 675

Large-T antigenis coded forbytwononadjacent

DNA segments: onefrom 0.65 (E 80) to about

0.60 andasecond from about 0.53 to the

infor-mation foraterminationcodon at 0.17(E 2550).

Analysis of the small-t mRNA showed that it

also has aspliced structure, lacking onlya

rela-tively short sequencearoundmap position0.54

(1). Considering the characterization of the

ge-nomesof small-t deletionmutantsby digestion

with restrictionenzymes(6, 24),andconsidering

also the terminationsignalsin the different

po-tential reading frames (8,30),it can be concluded

that the intervening sequence in the large-T

genecan start no further than nucleotide E 380

and endno sooner than E 670 (Fig. 3). As

dis-cussed before (30), the real boundaries cannot

be much removed from these limits. Indeed,

according to more recent results, the large-T

spliceextends from E 326 to E 671(22).

SV40DNA contains aunique TaqI cleavage siteat 0.565mapunits.Taking advantageofthis

feature,several laboratories have isolated

addi-tional mutants with deletions in the 0.60-0.53

region by selection forTaqI-resistantderivatives

(6, 25). These mutants were characterized by

restrictionenzymemappingandbytheir biolog-ical properties. We have sequenced the DNA

region spanning the deletion in mutants from

the800series(24)andfrom the 2100 series (6),

which were derived from SV40 wild-type 830

and fromlarge-plaque (LP)strain SV-1 ofSV40,

respectively. Thimmappaya and Shenk (29)

have obtained identical nucleotide sequence

re-sults for the series d1800mutants.

MATERIALS AND METHODS

Preparation of viral DNA. DNA from the 800

series of dlmutants waspreparedasdescribed

previ-ously (31). Extraction of viral DNA from the 2100

seriesof dlmutantsis describedby Feunteunet al.

(6).

Degradationof DNA withrestrictionenzymes.

Hinfl and TaqIrestriction endonucleaseswere

puri-fied in the laboratory accordingto standard

proce-dures. HaeIII and MboII endonucleases were

pur-chased from NewEngland Biolabs (Beverly,Mass.).

Digestionof DNA andseparationof restriction

frag-ments was asdescribedpreviously (31).

Labelingof DNAfragments.The restriction

di-gestof viral DNAwastreated with 1yg ofbacterial

alkaline phosphatase for45min at37°Cin the

pres-enceof 0.1% sodiumdodecylsulfate, extracted twice

withphenol, andprecipitated with ethanol. A

three-fold molarexcessof[y-YP]ATP, prepared according

tothe method ofGlynn andChappell(12),wasadded,

andtheprecipitate wasdried down. The residuewas

redissolved in20

Ad

of0.02MTris-hydrochloride (pH

7.6)-0.01 M MgCl2-0.025 M 8-mercaptoethanol, and

0.5,l-(1.5 U) of T4polynucleotide kinase (P-L

Bio-chemicals,Milwaukee,Wis.)wasadded.Thereaction

mixture was incubated for 25 min at 37°Cand was

then loadedonto a 5% polyacrylamide slab gel

con-taining 0.04 M Tris-hydrochloride, 0.02 M sodium

acetate, and 0.002 M EDTA (pH 7.8). After

electro-phoresis, the bands were located by autoradiography

and eluted. Thelabeled DNA fragments could then be

cleaved with another restriction enzyme.

DNA sequencing. The sequencing technique of

Maxam andGilbert (16) was used with thefollowing

modifications: only 1

iLg

of carrier DNA was used

during thechemicaldegradation reactions, and 4 ug of

tRNAwasadded after the chemical degradation. The

digestswerefractionated on 10%polyacrylamidegels

with dimensions of 90by30by0.2 cmand

autoradi-ographed at -70°C with intensifying screens from

CAWO(Schrobenhausen, F.R.G.) (15).

RESULTS

Strategy for sequence analysis. The Hinfl fragment D encompasses the 0.53-0.60 region (Fig. 1), and the following approach was used to

sequence the segmentof this fragment relevant

to eachmutant DNA. The wild-type or mutant

Hinfl fragment D was labeled at both 5' ends

with T4polynucleotide kinase and

[y-32P]ATP

and cleaved with a suitable endonuclease to

generate a singly labeled subfragment which

contained the deletion and which could be

se-quenced by the Maxam and Gilbert approach.

The particular enzyme used for this second

cleavagewasHaeIII for d1884 and d12102, MboII

for dl2112, dl2121, and dl2122, and TaqI for dl883, d1890, and wild-type large plaque. For analysis of d1891 the viral DNA was cleaved

with TaqI, 32P-labeled, and subsequently

di-gested with Hinfl. The resulting subfragments

weresequenced by the procedure of Maxam and

Gilbert (16).

Mutantsof the800series. Figure 2a shows

the chemical degradation pattem of the DNA

region encompassing the deletion in mutants

d1883, dl884, dlI90, and dl891. The number of base pairs deleted from each mutant DNA is compiledinTable 1. The deletion indl890 and

in dl891 (27 and 25 residues, respectively), is

located closer to theorigin or5'-proximal part

of the early mRNA, whereas the deletions in

dl883 and dl884 are more counterclockwise and

include the termination triplet of the small-t

gene. dl884 hasby far the largest deletion: 247

base pairs. The deletion ofd1883 comprises 57

basepairs. Because the original d1883 stock was

suspected of being contaminated by wild-type

virus(see Discussion),the sequence analysis was

repeated on a

dl83

stock which was again

plaque purified and which no longer gave any

small-t-relatedpolypeptide; the results were the

same. Adl883 stock obtained by rescue from a

transformedcelllinealso gave an identical

pat-tern.

Mutants ofthe2100series.Figure 2b shows

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676

A

Hinf

I

D

bl

1 I

G

Hinf Hinf

11

100 20 300 T00 6 700

89 mS

Uw

883

2112~21

FIG. 1. Schematic representation of the location of deletions in the 0.53-0.60 region. The heavy horizontal

line representswild-type SV40 DNA and is calibrated in units of 100 base pairs. Restriction cleavage sites

thatwereused in the sequence determinations (see Results) are indicated by vertical arrows above the DNA

line;Hinfrestriction fragments are noted above this line by capital letters. Deletions are represented by light

horizontal lines.

thegel pattern of wild-typeSV-1 (LP) and

mu-tantfragmentsofd12102,-2112, -2121, and -2122.

Thewild-typeLPpattern showsonlyone

differ-ence in the sequence of thisregion when

com-pared with wild-type strain776: atnucleotideE

601 anA.T basepairoccurs, which isaC.Gin

the776sequence. Thismutation,whichdoes not

change the primary amino acid sequence of

small-t antigen,mayonly representadifference

between virus stocks. d12102 ismissing 15base

pairs, from E 488 to E 502. Remarkably, this

deletion is asymmetric: all residues removed

duringconstruction of thismutant werelocated

atthe left of the TaqI site (alternatively, it is

notexcluded that themutant was notgenerated

by theexonuclease treatment, but wasalready

present in thestock).

Bothd12121 and d12122 have lost thesegment

from E 361 to E 594. However, restriction

en-zyme analysis showed that d12122 gave a less

shortened Hinfl fragmentD. A closerinspection

of the DNAsequence revealedthatdl2122 has

an additional

point

mutation (G C -* T-A) in theHinflrecognition site between fragmentsD

andI,resultingin alarger(fused) fragment.The

possibility that Hinfl fragment I is duplicated

canbe ruledout,because theAluI fragment C,

whichincludes theHinflfragment I,isidentical

inboth mutants (data notshown). Another

in-terestingpointisthattheligationof the mutant

DNAhasnotbeenstraightforward: there is an

inserted A-A-A-Csequence intheDNA message

strand. It isunclear how thissequencearose. The mutantd12112 has lost the segment be-tween E 339and E 607 butcontains an insertion of two A-residues. Hence a series of three A-residues hasbeenelongatedto five. The addition couldpossiblybecaused bysomeslippageof the

polymeraseduring therecircularizationevent. It

should be noted that thesephenomenawere not observed in the deletion mutants of the 800 series.

DISCUSSION

Thedeletions in the 800 series and 2100 series

mutantDNAs aresummarizedinFig. 3.

Knowl-edge of theexactposition andextent of the lost

segment allows us to compare the size of the

small-tfragmentsproducedin vivoand the

trun-catedproteins predicted from themutant DNA

sequences;the differencesaretabulatedinTable

1.The sizeof these small-t-related polypeptides

hasbeen determinedbyelectrophoresisin

poly-acrylamide gels after immunoprecipitationwith

serum from animals bearing SV40-induced tu-mors (5, 6, 17, 20, 21, 25). Since about the first

half of thesmall-tproteinisalsopresent in

large-T antigen, both proteins may share one (or

more) antigenic determinants. Paucha and

Smith (20) have also shown that analysis via

immunoprecipitation does notleadto selective

loss of small-t-related polypeptides. Based on

theresultsinTable 1,itispossibletoassign the

mutants to different classes. Sequence data

about thecorresponding regioninpolyoma virus

DNAandamutantwith adeletion thereinhave

also beenreported recently (10, 11,26).

Mutantswithanin-phasedeletion in the

small-t gene. The deletionindl890andd12102

comprises 27 and 15 base pairs, respectively,

within the small-t coding sequence; hence the

respectivemutantsmall-tproteinswilllacknine

andfiveamino acids.This is in excellent

agree-ment with the decrease in size of the

small-t-related fragment actually observed (4-6, 25)

(Table 1). It is noteworthy that a deletion of

only five amino acids reduces drastically the

transforming ability; in bothcases the deletion includes one of thecysteine-rich clusters which occurtwice inSV40 and also inpolyoma

small-tantigen (10). Sleighet al. (25) presumed that

all the deletions in mutants they had isolated were"in phase", becausetheyobserved agood

correlation of deletion size and decrease in

mo-lecular weight of the resulting small-t-related

polypeptide. However, the fact that we found

onlytwoin-phasedeletionsoutofeightmutants

sequencedindicates that suchin-phasedeletions

offer noselectivebiological advantage.

Mutants with "out-of-phase" deletions

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producing a small-t protein of a size pre-dictable onthe basisof the location of the deletionin DNA. The deletion of d1891 starts

at E 352 and comprses 25 base pairs. Hence,

after thedeletion thecoding sequence proceeds

inanotherreadingframe until it terminates with

aTGA tripletatE432-434.Theprotein specified

by this coding sequence would be 109residues

long and have amolecular massof 12,443, i.e.,

8,060 daltonsshorter than wild-type small-t. A

small-t-related polypeptide is present in cells

infected withd1891; its decrease in sizerelative

towild-typesmall-thas been estimatedas6,500

to7,000daltons(6, 17). The agreement between

thepredictedand observed decrease in size may beregardedasreasonably good, especiallysince

the loss of most of thecysteineresidues from the

mutant small-t may greatly alter its mobility

behavior on sodiumdodecylsulfategels.

Mutants without-of-phase deletions pro-ducing atruncated small-t with a greater thanexpectedchainlength.Thesmall-t

pro-teinsynthesized by d12121 and d12122 is

consid-erably larger than the expected size based on

the extent of the deletion. Both mutants have

an identical deletion, and nucleotide E 594

marks the end of themissingDNAsegment; this

is only seven nucleotides before the wild-type

small-t termination codon.Sincefouradditional

nucleotides have beeninserted, the deletion is

not amultipleofthree,andtherefore thereading

frame changesphase and the stop signal at E

602-604 no longerfunctions. However, another

TAAtripletoccursverysooninthe newreading frame, viz., at E 607-609. Based on the DNA sequence, the mutant small-t would have a

mo-lecular weight of 11,367, which is considerably

lower than the molecularweightestimate of the

small-t-related polypeptide actually observed

(6). However, the segment spliced out of the

small-t mRNA is 66 nucleotides long, from E

606 to E 671 (22); it starts just after thenormal

small-t TAA termination signal andbefore the

second TAA codon (at E 607), which would

terminate the small-t-related polypeptide.

Hence the splicing event eliminates a series of

terminationcodons from the mRNA andpermits

translationtoproceed into the regioncoding for

the second part of large-T antigen, but in a

differentreading frame, longenough to code for

about 5,900 daltons of protein. As aresult, the

small-t-related protein terminates with a TAG

codonatE821-823. It seems very likely,

there-fore, that the same splice which in wild type

occursafter thecoding region of the small-t gene

is actuallylocated inside the coding region for

the d12121-2122-directed, small-t-related

poly-peptide. On this basis, and considering the

boundaries of the deletion in the DNA and of

the splice in the RNA, one would predict a

fragment that is 3,395 daltons shorter than

wild-type small-t, in reasonable agreement with the

2,000 to 2,500 daltonsobserved directly (6, 17).

The explanation is further strengthened by the observation of May et al. (17), who found that

translation in vitro of the mutant cytoplasmic

early mRNA results in the synthesis of a 17K

protein (asobserved invivo), whereas a nuclear

mRNA extractgave rise to both a 17K andan

11K derivative. According to the model

pro-posed above, the latter would constitute the

translation product of the prespliced mutant

mRNA, and its size corresponds indeed with what one would expect from the nucleotide se-quence.

Mutantsproducing no small-t fragments

invivo. No small-t-related fragment has been

detected in vivo after infection with deletion

mutantsdl883 and d12112 (5, 6, 17). The

small-t that was previously found in d1883-infected

cells has now been shown toresult from

contam-ination with wild-type virus (5). The situation

withd1884 islessclear.Sleigh etal. (25) could

detect (a small amountof)asmall-t-related

poly-peptide that was 3,500 daltons shorter than

wild-type small-t. AlsoKhoury (personal

communi-cationcited in ref.29) observeda12,000-dalton

polypeptide in cells infected with d1884.

Craw-ford and O'Farrell (5) could detect a

small-t-relatedpolypeptideof 15,000daltonsonlyafter

alkylation. On the other hand, Paucha and

Smith (20) did notfind putative fragments

re-latedtosmall-t ind1884-infected cells,

irrespec-tive oflabeling conditions. The reason for the

different results isnotknown,butnevertheless,

wheneverasmall-t-relatedprotein ofa

molecu-larweightin agreement with thatpredicted from

the d1884 DNA sequence was detected, its

amount wasconsiderablyreduced.

Acommonfeature of this class of mutants is

that the deletion extends beyond the small-t

terminationtriplet. Assumingthat thesplice of

thesmall-t mRNA extends fromE 606toE671,

all threedeletion mutantseliminate one of the

boundaries of the spliced sequence. Mayet al.

(17) and Paucha and Smith (20) showed that

the absence of a truncated small-t protein in

cells infected with thesemutants is due tothe

lack of corresponding cytoplasmic mRNA.

Moreover, translation in vitro of a nuclear

ex-tractdidyield a small-t fragmentwhose

reduc-tion inmolecularweightwasingood agreement

with the decrease predictedfromthe DNA

se-quence (17; E. May, personal communication).

It therefore appears that mutant premessage

RNAispresentinthe nucleusbut is notspliced

and not transported to the cytoplasm. Hence

splicingcould be an obligatoryevent for

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FIG. 2. Chemicaldegradationpatternscoveringthe relevantarea of each deletionmutantDNA. (More extensivegelpatternswhich substantiatethesequencedeductionswerealsoobtained [datanotshownJ.)(a)

mutantsfrom the 800series;(b) wild-typeLPandmutantsfrom the 2100 series. TheDNA restriction fragment bearing the deletionwaslabeledatonlyone5' endanddegraded accordingtothe procedure ofMaxamand Gilbert(16). The base specificity of each reaction is indicated atthe top of each lane (note that the lane referredtoasTalsoshows Cbands; theAlanealsoweakly reveals the C residues). The nucleotidesequence

isshownalongside eachautoradiogram. Nucleotides that denote the boundaries of the deletion, the additional

nucleotides ind12112, d12121, andd12122, and thesingledifference between wild-type LP and wild-type 776 areallunderlined.

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TABLE 1. Deletionmutantsin the0.53-0.60region

Decrease in mol. wt. based on: Ammno acids No. of

nucleotides

Additional

Virus beforestart deleted amino acids Total Observed

of deletion DNA sequence small-t

frag-ment

WT - - - 174 -

-883 164 57 3 167 842

-884 111 247 3 114 7,356

890 111 27 1 +53b 165 1,055 1,000

891 91 25 18 109 8,060 6,500

2102 136 15 33" 169 631 500C

2112 86 269(-2) 0 86 10,471

-2121 94 234(-4)d 5 99 9,136' 2,000

2122 94 234(-4)d 5 99 9,136' 2,500

aThe occurrence ofasmall-t-related polypeptide in

dl884-infected cells

isunclear, as discussed in the text.

bUnderlined numbers refertoaminoacids read in thesameframeaswild type.

'The decrease in sizewasoriginally reported as 1,500 daltons (7), but this estimate has been reevaluated (J.

Feunteun, unpublished data;seealsoref. 17).

d Numbersin parentheses refer to additional nucleotides present in the mutant DNA (as a result of the

ligationevent?),butnotinthewild-typeDNA.

'Adecreaseof 3,395 daltons is calculated when the small-t mRNA splicing event (which occurs here in the

coding sequence of this mutant) is taken into account (see the text).

A CT.GAG.GTA.TTT.GCT.TCT.TCC.TTA.A AT.CC T.GG T.G T T.G A T.GC A.ATGITAC.T G C.A A A.CAA.T GG.CC T.

38 .

- 2121-2122

GAG.TGT.GCA,AAG.A A A.ATG.T CT.G CT.AAC.T GC.A TA.TGC.TTG.CT G.T GC.TTA.CTG.AGG.ATG.AAG.CAT.

445

84

G AA.AAT.AGA.A AA.TTA.T AC.AGG.AAA.GAT.C C A.CT T.G TG.TG G.G TT.GAT.T GC.TAC.TGC.TTC.GAT.T G C.

508 2102 _

T T T.A GA.AT G.T G G.T T T.G GA.CTT.G AT.CT T.TGT.G AA.GG A.A CC.T TA.CTT.CTG.T G G.T G T.G AC.AT A.A T T.

t,71

GGA.CAA.ACT.ACC.TAC.AGA.GAT.TTA.AAAG.CTC GC TAAAATATAAAATATTTTAAGTGTATAATG

6,34 2121- 22 A AA C

884_

1611TAAACTACTGATTCTAAIIT611161TGTATTTTG:IA6TITCCCA.AC C.TAT.GGA.ACT.GAT.GAA.

FIG. 3. SV40mutantswith deletions inthe 0.53-0.60region ofthe DNA. The nucleotidesequenceofthe

DNA message strand(samepolarityasearly mRNA)is shown(8).Theextentofthe deletionsofthe800series

mutantsisrepresented by horizontalarrowsabove thesequence, andarrowsbelow the sequence indicate that ofthe 2100seriesmutants.Nucleotide additions observed in the2100seriesmutants(see text)arenoted below

theirrespectivedeletionrepresentation.The boxed TAA tripletisthe termination codonofthe small-t gene.

The codon boxed in broken lines is thetheoreticallyearliestpositionwherelarge-Tcanpickupagain after

thesplice. TheA-G sequences thatarecovered withabracketdenote thesplicingsitesofthelarge-T (E326

andE671) and small-t (E606andE671)genesasreported byReddyetal.(22).

680 VOLCKAERT

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port of polyadenylic acid-containing mRNA

from the nucleustothecytoplasm in vertebrate

cells. If so, the presence ofa small amount of

small-t-related polypeptide in d1884-infected

cells couldoriginatebysomeleakage of the

pre-mRNAthrough the nuclear membraneor,

alter-natively, by the occurrence ofanother splicing

event onthesamemRNA.

Deletion mutants and splicing. All

mu-tantsused in thisstudy produceapproximately normalamountsoflarge-T antigen. Theregion

spliced outof the large-T mRNAhas been

re-portedtoextendfrom E326 toE671 (22).Since

thedeletions in thesemutantsfall withinthese

boundaries, we conclude thatmost of the

nu-cleotide sequence in the intervening region of the large-Tgene isunnecessary for thecorrect splicing of the large-T mRNA. Some of the

deletions go rather close to these crossover

points (e.g., d12112 at 13 nucleotides from the

first crossoverandd1884at 12nucleotides from

thesecond). These resultssuggestthat the

sig-nals forsplicingarerestricted to alimited area

around thecrossoversite.

Thespliceof thesmall-t mRNA extends from

E606 to E 671 (22) and only the mutantsd1883,

d1884, and d12112 eliminate part of thisregion.

These deletions interfere with thesplicingof the

small-t mRNA, and so far no small-t-related

polypeptide (orcorresponding mRNA)hasbeen

detected in the cytoplasm, except for d1884,

wheresmallamountsof therelevantpolypeptide

have been occasionally detected, as discussed

above. This suggests thatsplicingcould play a

role in thetransportofapolyadenylic

acid-con-taining mRNA from the nucleus to the

cyto-plasmorinitsstabilityinthecytoplasm.All the

other mutantsproduce a detectable

small-t-re-lated polypeptide whose size corresponds to

whatonewouldexpect onthebasis of the DNA

deletion. It is of interest that, for the mutant

d12121-2122,thereadinggoesintoanother frame and, as aresult, the normal small-tsplicenow

occursinside thecoding region for the

small-t-relatedmutantpolypeptide. Itfollows from this

that theway anmRNAissplicedisindependent

oftheway itisbeing used for translation. The

fact that about normal amounts of large-T

mRNA arepresent in all these mutants shows

that theshortsplicepresent insmall-t mRNA is

not anobligatoryprecursor for the normallarge

splice in large-TmRNA. An analogous

conclu-sion may also be relevant for the relationship

betweenSV40 19Sand16SlatemRNA's.

All SV40 mRNA's are spliced, either before,

beyond,orwithin thecodingregion(8,22). We

know of noexample of avertebrate, cellular or

viral, polyadenylic acid-containing mRNA that

reaches the cytoplasm without undergoing at

leastonesplicingevent (onepossibleexception

is tumor virus 35S RNA, which apparently

reaches thecytoplasm partly intactafter

injec-tion intonuclei; 27). Nevertheless, the link

be-tweensplicing andtransport from the nucleus to

the cytoplasm could be more complex and/or

indirect, since Paucha and Smith (20) found that

inseveral mutants (d1885, d1890) the amount of

cytoplasmic mRNA coding for the

small-t-re-lated polypeptidewasmarkedlyreduced relative

towildtype.

ACKNOWLEDGMENTS

We thank A. Van de Voorde for helpful discussions, Jose

Vander Heyden for expert technical assistance, and T. Shenk for communicating hisresults to us prior to publication.

This research wassupported by grants from the Kanker-fonds of the AlgemeneSpaar- en Lijfrentekas (ASLK) and the Geconcerteerde Akties of the Belgian Ministry of Science.

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