Characterization of the 5'-terminal capped structures of late simian virus 40-specific mRNA.

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0022-538X/78/0025-0824$02.00/0

Copyright01978 AmericanSocietyforMicrobiology Printed in U.S.A.

Characterization of the

5'-Terminal

Capped Structures of Late

Simian Virus 40-Specific

mRNA

G. HAEGEMAN AND W. FIERS*

LaboratoryofMolecular Biology, State University Ghent, 9000 Ghent, Belgium Received for publication25October 1977

32P-labeled,latesimian virus40-specificRNA was isolated frominfected CV1

cellsandcompletely degraded with RNaseT2 and bacterial alkaline phosphatase.

The RNase-resistant materialwas fractionated two dimensionally and further

characterized withPenicillium nuclease andnucleotide pyrophosphatase. Two

major 5' terminiwere identified in latesiirianvirus 40RNA, namely, 7-methyl

Gppp2',6-dimethyl ApUp and 7-methyl Gppp2',6-dimethylAp2'-methylUpUp.

Both 5' terminiarepresentinunfractionated viral RNA as well as in theseparated

16S and 19S species. Asboth caps differ onlyin secondary modification, it is

possiblethat theyarederived from the same site onthe DNA. The relatively

higher cap II content of the 16S mRNA may be related to its slowerrate of turnover.

Blocked5'terminiareageneralcharacteristic of eucaryotic cellular mRNA's (21, 24). Also,

mostof theeucaryoticviral mRNA's studied so

far are blocked attheir 5' end (24). The latter

contain exclusively a purine nucleotide in the

5'-penultimate position, whereas cellular mRNA'smay have eitherapurine or a

pyrimi-dine. This penultimate nucleotide can be

un-modified (cap 0structure), but in many cases

itappearstobemethylatedon theribose, and

in afew examplesanadditional base

methyla-tion alsooccursinthisposition (capIstructure).

Ribosemethylation of the second nucleotide is

oftenobserved in the mRNA's fromhigher

or-ganisms and the corresponding viruses, thus

leadingtothe formation of capII structures. It

is noteworthy that the methylation pattern of

the caps increases as one movesup the

evolu-tionaryladder.

Simian virus40 (SV40) isaDNA-containing

virus, whichproduces

virus-specific

RNA in the

cytoplasm oflyticallyinfected monkeykidney cells(3, 6, 17, 27, 31).Studies on themethylation

pattern of SV40-specific RNA revealed

5'-ter-minal ends of the type7mGpppNm(13). Because

of the general interest in SV40, we have

char-acterized in more detail the 5' termini of the

predominantcytoplmmicviralRNAs, and their

structuresarereportedhere.

MATERIALS AND METHODS

Preparationof32P-labeledlate SV40RNA.CV1

monkey cells (Bio-Cult, Paisley, Scotland) were cul-tured inplastic petridishes(Falcon Plastics,Oxnard,

Calif.) andinfected with60PFU ofSV40(strain776)

percell. At24hafterinfection,thegrowingmedium

waschangedto alow-phosphate medium (containing

10'- M phosphate, i.e., 1% of the normal concentra-tion),and3mCi of3Pradioactivity (The Radiochem-ical Centre Ltd., Amersham, England) was added per 14-cm petri dish for alabeling period of 24h. The mediumwasthenremoved,and the ceUs were washed afew times withphosphate-bufferedsaline and finally lysedwith1% Nonidet P40. The nuclei were removed by centrifugation, and subsequently the lysate was subjected toseveral extractions with

phenol-chloro-form-isoamylalcohol.The extracted RNA was

precip-itatedatleast twice with ethanoltominimize contam-ination withco-precipitating[tP]phosphate, originat-ing from thepoolpresent inthe cellularcytoplasm. Isolation of polyadenylic acid [poly(A)]-containing RNAonoligodeoxythymidylicacid-cellulose (P-L

Bio-chemicals, Inc., Milwaukee, Wis.) was followed by

selection of theSV40-specific sequencesby hybridi-zationonSV40DNAlinkedtoSepharose4B (Phar-macia FineChemicals, Inc.,Piscataway, N.J.) (9). The SV40mRNA thuspurified usuallyrepresented around 0.2%of the totalcytoplasmicRNAfraction.

Preparative gel fractionation of 32P-labeled

RNA. Insome casesthe RNApreparation was frac-tionatedbypreparative electrophoresison anagarose slabgel (160 by120by3mm). This stepwas carried outeither before (seeFig. 4)orafter the selection by hybridization. A solution of 1.4% agarose (Sigma Chemical Co.,St.Louis, Mo.) in running buffer (0.02 MTris-acetateplus 1 mM EDTA, pH 7.5) was used. The RNAsamplesweredissolvedinrunning buffer, which had beenmixed with 50% deionized formamide, heated for 1 minat 70°C, and loadedonto the gel. The electrophoresis wasstarted immediately inthe coldat50 to 100V forabout2 h.Next, thegelwas

dismantled, and the 3P-labeled RNAwas localized by autoradiography. The bands were cut out, and theirradioactivitywasestimatedbyCerenkov count-ing. The material was recovered from squeezed gel bandsby several extractions withsmall quantities (i.e., 824

on November 10, 2019 by guest

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100 to 200

pl

foragelvolume of 20by5by 3 mm) of 0.5MNaCl,containing5mMEDTA and 0.5% sodium dodecylsulfate. The addition ofafewdropsofphenol may facilitate the releaseof RNA from the agarose pieces.Finally, the 3P-labeled RNA was precipitated at-20°C togetherwith carrier yeastRNA,by adding two partsofethanol to the combined extracts. The recoveryof the elutionprocedureamounts to70%for RNA from the28Sregionandreachesmorethan 80% for material of smaller S values.

Isolation of 5'-terminal structures. For degra-dation with RNase T2, the pelleted RNA (i.e., 32P-labeled viral orcellular mRNA and 40 ug of carrier yeastrRNA)wasdissolved inavolatile buffer (0.1 M formicacid-pyridine, pH 4.5) anddigestedovernight at37°Cwith 5 U of T2 enzyme(SigmaChemicalCo.). Next, thehydrolysatewasevaporatedtodryness, and the residuewasdissolved in 0.1 M ammonium hydro-gencarbonate containing0.5U of bacterial alkaline phosphatase(WorthingtonBiochemicals Corp., Free-hold, N.J.) forafurther incubation ofafewhoursat 37°C.Thereaction mixturewasevaporatedagain and washedseveral times with distilledwater toremove remainingtracesof salt.

Theseparation procedureinvolvedaclassical elec-trophoresisoncelluloseacetateinthe firstdimension (23), followed by ion-exchange chromatography on thin-layer plates in the second dimension. The T2 hydrolysate,dissolved inaminimalvolume of water, was applied as a thin line on prewetted cellulose acetatestrips(Schleicherand SchiillGMBH, Dassel, WestGermany) andsubjected toelectrophoresis at pH3.5in the presence of5Mureauntil thepink dye hadmigrated 20 to 25cm. The material was trans-ferredonto athin-layer chromatographic plateof pol-yethyleneimine (PEI)-cellulose (Machery-Nagel and Co., Duren,WestGermany)bythe inversedblotting technique(26)anddevelopedinthe seconddimension byascending chromatographywith avolatile buffer solution of1 M (or1.3M) formic acid-pyridine (pH 4.3).Radioactive spotswerevisualizedby autoradiog-raphy at -70°C, using preflashed X-ray films (RX Fuji X-Ray Film) (12) together with intensifying * screens (CAWO, Schrobenhaussen, West Germany) (R.Laskey and A.Mills, FEBS Lett.,in press). The nucleotide materialcanbe recoveredeasily from thin-layerchromatographyplates bythemicroelution tech-niquedescribedpreviously(28).

Analysisofcappedtermini. RNase T2-resistant material was further degraded by Penicillium (P1) nuclease (P-L Biochemicals, Inc.). The dry residue, after elution from thePEIplate,wasredissolved ina volatile buffer(0.1Mformicacid-pyridine, pH6), and 10AgofPI enzymewasadded. The reaction mixture wasincubated for 30 minat37°Cand thenevaporated to dryness. The materialwasthoroughly washed by the addition ofasmall volume ofwaterfollowedby dryingandthenappliedontominiplates of PEI-cellu-lose (6.6 by9.9cm) fortwo-dimensional chromatog-raphy: the first dimensionwasdeveloped witha20% formicacidsolution;inthe seconddirection,abuffer system of1 Mformicacid-pyridine(pH 4.3)wasused (27a).

Cap structures,resistanttoP1hydrolysis,were an-alyzed by digestionwith nucleotidepyrophosphatase (SigmaChemicalCo.).Thedriedmaterialwaswashed

withwater to removeremaining traces of triethyla-mine carbonate. After it was redissolved in the appro-priateenzymesolution(10U ofnucleotide pyrophos-phatase per ml of 25 mM Tris-chloride-2 mM

Mg2e,

pH 7.5), the pH was checked andeventuallyadjusted. The mixturewasincubated for 30 min at 37°C, and thedigestwastransferreddirectly onto miniplates of PEI-cellulose(6.6 by9.9cm) fortwo-dimensional sep-aration. A less concentratedsolution (e.g., 0.5 M formic acid-pyridine, pH 4.3, in the second dimension) was usedtoobtain agoodresolution between singly and doubly methylated nucleotides. Methylated nucleo-tides used as references were purchased from P-L Biochemicals,Inc.

RESULTS

RNase T2 hydrolysis oftotal late SV40

RNA. 3P-labeled late SV40 mRNA was

pre-pared and completelydegradedwith RNase T2

andbacterial alkalinephosphatase,as described

above. A typical degradation pattern of SV40

RNA is shown in Fig. 1. Twospots are promi-nent, products 1 and 2, corresponding to two

different 5' termini in the late viral RNA.

Inor-ganic [32P]phosphate was run off the plate in

the firstdimension,andonlysometrailing

radio-activity from this giant spot isseen. The dark

spots at the topof the figure are cyclic

mono-nucleotides; these intermediates are due to

in-completeRNaseT2 hydrolysisandareresistant

tothephosphatase.

Theaforementionedproducts1and2contain

the7-methyl Gppp group, aswillbe shown

be-low;sugar(2') methylation ofthe following

res-idue(s) renders these resistant tothe action of

RNaseT2 and generatesthe longer 5'-terminal

oligonucleotides (1, 19, 32). That the caps are

derived from "late" SV40 mRNA follows froma

characterization of the cytoplasmic,

poly(A)-containing, SV40-specificRNAbyfingerprinting

[G. Haegeman and W. Fiers, Nature (London),

in press] and is in agreement with the

well-known abundance of late RNA relative to

"early" (3). Also, someminor spots maybe

ob-served in Fig. 1; they are probably not virus

specified, but they are derived from a small

contaminationwithcellularRNA.Indeed,in less

purified preparations ofSV40RNA, the entire

pattern of cellularcapstructures wasstillvisible,

but as the viral RNA was more rigorously

se-lectedby stringenthybridizationconditions,the

two majorspots becamemore andmore

promi-nent while the overall cellular contamination

disappeared. Thestrongestcontaminants

corre-spond tothe maincellular cap structures (e.g.,

products 3 and 4 in Fig. 5), but even in this

examplethetwoviralproducts1and2represent

65 to 70% of the total fraction of T2-resistant

material, whereas the latterspotsamount to at most 20 to 25%of the entire cap populationin

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Elec. pH

3.5

FIG. 1. Two-dimensional map on PEI-cellulose of a combined RNase T2 andphosphatase degradation of total late32P-labeledSV40 mRNA. The ion-exchangechromatographyin thesecond dimensionwascarried outwith1.3Mformic acid-pyridine (pH 4.3). Thedye marker, xylenecyanol FF,denotedby B, migrated10.5 cmstartingfromthefirst-dimensional originline.

thecaseofatotalcellularmRNAhydrolysate.

Furthermore, thesameRNAmaterial,inwhich

products 1 and2 are the majorcap spots, has

beenfingerprintedafter RNaseT1digestionand

correspondsindeedtothe viral RNA,asrevealed byanalysisof all of the T1oligonucleotides

pres-ent and correlation with the known DNA

se-quence.

Furthermore, combined P1 hydrolysis and

phosphatase treatment of total cellular RNA

yieldstwodifferentcap structures, anA-cap and

a G-cap, both of which are present in nearly

equimolaramounts(unpublished data). On the

otherhand, similaranalysisofthehighly

puri-fied SV40 RNA showed almost exclusively an

A-capstructure.

Themolar yield of the 5'-terminalgroupwas

calculated from an analogous experiment, in

which theradioactivitypresentinthe

Pa-resist-antcap structureandrepresenting exactly

three

phosphateresidueswascomparedwiththe total

radioactivity of the viral RNA. Assuming an

average chainlengthofapproximately1,600

nu-cleotides for the mRNA molecules, the

5'-ter-minalstructuresappear in 0.5 molar ratio only.

Atpresent, we do notknow whether this

phe-nomenonis due to aninefficientcapping mech-anism or is the result ofpreferentiallosses of the 5'terminusduringtheisolationprocedure; how-ever, T1 oligonucleotides originating from the spliced 5'-terminal leader sequence appear in molarquantity (Haegeman and Fiers, in press).

Analysis of T2-resistant cap structures.

The T2-resistantproducts 1 and 2 were eluted

from the PEI plate and further characterized

by digestion with P1 nuclease (Fig. 2). Both

patternsshow a major componentcloseto the

bluedye andalesseramountofradioactivity in

the mononucleotide region; some undegraded

product remainsneartheapplication point. The

majorcompound,identical in both T2-generated

"caps," contains a

Pi-resistant

structure with the general formula 7mG(5')ppp(5')X. As

men-tionedabove,complete

P1

hydrolysis of cellular

RNA, followed by dephosphorylation, yields

mainlytwoproductsderived from the 5' termini,

namely,7-methyl GpppA and 7-methyl GpppG

(our unpublished data). The position of these

on the miniplates in the neighborhood of the

bluedyewasverifiedbyseveral control

experi-mentswith

32P-labeled

material and withoptical density (OD) references. In thecase ofthe two

SV40products (Fig. 2),the 5'terminus behaves

as anA-cap (G-caps are more delayed inboth

dimensions),buta morerefinedanalysis,aswell

asthe determination of the degree of

methyla-tion,isdescribed below.

Furthermore, enzymatic digestion of product

1 releasesa mononucleotide, which behaves in

this systemaspU. Indeed, carrier

mononucleo-tide pU, added to the reaction mixture as an

internalODmarker, chromatographsinexactly

thesameposition.Inthecaseofproduct2,two spots are visible in the mononucleotide region:

the slowestoneisagain pU,whereas the

faster-migratingspotmightbe the methylated analog

(it chromatographs like pU inthe first

dimen-sion, which separates mainly according to

charge) (27a). It actually coincides with

2'-O-methyluridine-5'-phosphate, added as an OD

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marker, but further confirmation was needed

since one cannotunambiguously determine from

*the mobility pattern in thissystemwhether the

methylationoccurs onthe base or onthe sugar

moiety ofthe nucleotide. Therefore, the

com-pound was rechromatographed on cellulose

platesin amixture ofisopropanol-anmonia-0.1

Mboric acid (7:1:2). Urnodifiedor

base-meth-ylatednucleoside-5'-phosphatesarestrongly

de-layedbecause of thecomplexing activityofboric

acid with thecis-diolgroups. The radioactivity

recovered was not retained at the origin, but

moved together with 2'-O-ribose-methylated

pU,thusproviding furtherproof for the

identi-fication(datanotshown). This analysis leadsto

thepartialstructure 7mGpppXmpUforproduct

1 and 7mGpppXmpUmpU for product 2, where

Xm indicates a ribose-methylated nucleotide,

since the residue to the 3' end of Xm is not releasedby RNaseT2 digestion.

The identical Pi-resistant cap structures of

theT2 products 1 and2 (Fig. 2) were reeluted

from the PEIminiplate and independently

ana-lyzed bydigestion with nucleotide

pyrophospha-tase (Fig. 3). The experimentswere performed

in duplicate for each product, applying in one

2'-O-methyladenosine-5'-phosphate (pAm)as an

internal OD reference andinthe other

N6,2'-O-dimethyladenosmie-5'-phosphate (6mpAm).Both

sets offractionations showed an identical

pat-tern: inorganicphosphate (above the blue dye,

butmoving differentlyfrom undegraded P1-re-sistant caps), 7mpG (the fastest spot in both dimensions, also checked by an internal OD

reference), and the pA residue, in both cases

coincidingperfectlywith theinternal OD

refer-ence

'mpAm

andmovingprecisely oneposition

a

b

fasterthan pAm (or6mpA),which inturnmoves

faster than pA. Although most of the capped

termini studied so far contain a

2'-O-ribose-methylated purinenucleotide, someviralRNAs

other than SV40 display a doubly methylated

A-residue in this position (11, 15, 16, 20, 25);

doubly methylated pG, however, has notbeen

observedsofar.

Thestructureof thetwoT2-resistant products (1and 2) isfully determinedby these analyses. They are 7mG(5')ppp(5')6mAmpU and

7mG-(5')ppp(5')6mAmpUmpU, respectively, and will

befurther indicatedby thetermscap Iandcap

II.Itisquite possible thatcapIIisderived from

cap Ibya secondmethylation step, a

phenom-enonthatalready hasbeenobserved in' thecase

of viral RNA (16, 20, 25) and appears rather

general even in cellular mRNA populations (4,

5, 7, 19, 29,32). However,asthelate viral SV40

RNA contains at least two RNA species (27,

31), we cannotexcludeapriorithat onetype of

cap structure maybe specifically derived from

one RNA species. This was checkedby

identi-fication of thecapsinfractionated SV40mRNA.

RNaseT2hydrolysisof fractionatedSV40

RNAspecies.Cytoplasmic, poly(A)-containing RNA wasisolatedfromSV40-infected cells and

fractionatedon a1.4%agarosegel. The

fraction-ation pattern was divided into a set of

banids,

from which the RNA material was eluted and

hybridized toSV40DNA. A distributionprofile

of the SV40-specific sequencesisshown inFig.

4a. Themajoritywaspresent in the 17Sto 18S

region andcorrespondstothemajor, late RNA

species,whichsedimentsin sucrosegradientsat

16S (30, 31). The 19S species is less prominent

and represents about25% of the 16S material.

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c

d

FIG. 2. Two-dimensionalchromatographyonminiplatesofPEI-celluloseofaP1 digest ofT2products 1

(a) and 2(c) and the corresponding diagrams, respectively, (b)and (d), showing the position of relevant referencemononucleotides(revealedunder UVlight).Theposition of theblue dye is indicatedbyadashed

circle.

so

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QpUm

|BpU

a ~~~~* . 20 B'

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828 HAEGEMAN

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1 ~ ~ ~

b

c

FIG. 3. Nucleotidepyrophosphatase analysis of Pi-resistantcapstructures,derived from products1and 2

(spotsnearthedyemarkerB inFig. 2). (a)and(c)showtherespective autoradiographs ofatwo-dimensional

separationonPEI-cellulose. (b)denotesthepositionofmethylatedreference nucleotides, whichwereadded asinternal markers indifferentcombinations and whichwerevisualized under UV light.Thetworadioactive spots, in (a)as well as in (c), correspond preciselytothe 7'pG and "'pAm internalreference compounds, respectively. Themonomethylated 6'pA and(2')pAm moveexactlythe samein thissystem. Bdenotes the position ofthe bluedye. ThespotthatmovesjustaheadofB in bothdimensionsisinorganic phosphate.

All of theSV40-specificfractionsweresubjected

tohydrolysis by RNase T2, followed by dephos-phorylation, and subsequently fractionated in two dimensions on PEI-cellulose plates. Both

cap I and cap II were present in all of the

fractionsanalyzed, and theyrepresented about 70%of thetotalamount ofT2-resistant products. As an example, the digestion patterns of the

18S and 19S SV40-specific material are

com-pared in Fig. 5. There seems to be relatively morecap IIin thematerial, whichruns at18S orfaster,ascompared with the 19SRNA. This is illustrated in Fig. 4b, in which the relative amountofcapIIisplotted for the different size classes; these results, however, are based on

countingverylowamountsofradioactivities and mustbe considered tentative.

DISCUSSION

The 5' termini of late poly(A)-containing

SV40-specificRNAhavebeenisolatedand

char-acterized in detail. It hasbeenclaimedthatPi RNasehydrolysisofSV40 mRNAreleases both 7mG(5')ppp 2'-O-methyladenosine and 7mG-(5')ppp2'-O-methylguanosine (13). Accordingto

ourdata, however,nosubstantialamountof

2'-O-methylguanosine containing capwaspresent in SV40 mRNA; furthermore, we have shown

thatthe modified adenosine residueis actually

NV,2'-O-dimethyladenosine.Infact,weconclude

thatonlyonemajorPI RNasecapispresentin late SV40mRNA, namely,7mG(5')ppp(5')SmAm.

Analysis with RNase T2 reveals two major types of cap structure, cap I and cap II, in cytoplasmic, late SV40 mRNA. Cap Iwas

iden-tifiedas7mG(5')ppp(5')6mAmpU,andcapIIwas

identified as7mG(5')ppp(5')(6mAmpUmpU. Both

types of 5' termini are also found among the wide variety of monkey cellular mRNA cap

structures (our unpublished data). It is quite possible that the 5' ends of both 16S and 19S

late mRNA'sarederived fromthesamesiteon

the genome, but differ only in the degree of secondarymodification. Themolecularbasis for suchahypothesiswasnotreadily evident until

veryrecently,asthe 16SRNA isbelievedtobe a processing product of the 19S molecule and

sharescommon sequences with the3'-terminal half of the 19Sspecies (2, 10, 30). Recent results, however, indicate a common5'-terminalleader

sequenceforthe two lateSV40mRNA's,which becomelinkedtotranscriptsfromanonadjacent

region on the genome (14, 22; Haegeman and

Fiers,inpress;S.Lavi andY.Groner,inpress). SimilartranslocationofRNAsegmentshasbeen showninadenovirusmRNA,and in thissystem thesharingofa commonleadersequenceiseven moreobvious (3a, 8,10a). Moreover,the locali-zationof theSV40capstructureintheHind C fragmentatposition0.72orcloser to the origin

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[

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[

0.1 1

50

25

FIG. 4. (a) Separation of the total cytoplasmic poly(A)-containingRNAbyelectrophoresison a1.4% agarosegel,followedby selectionoftheSV40-specific sequencesby hybridization.Theposition ofthe rRNA markers is indicated. (b)The percent amountofcap II structure present in the totalpopulation (cap I plus capII)isplottedagainstthecorresponding gel fraction.

a

%

1I

B

I1

4

I 0 0

3

Elec. pH 3.5

1

do

2

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of replication (Haegeman and Fiers, in press;

Lavi and Groner, in press) suggests that the

putatively common 5'-terminal structure is

di-rectly derived from the 5' end of the primary

nucleartranscript. Indeed, aflow of the

meth-ylated cap structures from the heterogeneous

nuclear RNA tothe cytoplasmicmRNA

popu-lation has been observed inL-cells(18).

The relatively higher percentage of cap II

structure in the 16S mRNA mayberelated to

a difference inhalf-life,as aslower turnover of

the latter mRNA as compared with the 19S

species has been reported (2). In the case of

cellular mRNA's,it has in fact beenfound that

theformation ofcap IIstructures iscorrelated

with thelongevityof the mRNAmolecules (18).

Moreover, in the current view of "spliced"

mRNAmolecules, as is the caseforlate SV40 mRNA (see above), the relationship between the methylation of the cap structure and the

turnover of the RNA may perhaps be more complex. Indeed,ahigherrepresentation ofcap

IIin the 16S RNApopulationascompared with

the 19S may be either directly related to the

higherstability of the mRNA itselfor formed

byalongerexposuretomethylatingactivity of

the conserved 5'-terminal leader fragment,

which functions in the 19S mRNA and, after

cytoplasmic conversion (2), again in the 16S

mRNA. Alternatively, the 5'-leader sequence

containing thecapII structurecould be

prefer-entially used for transposition and linkage to

the 16Scoding portion.

V

b

p %

t A

11_%% B _J.;

1-'C,

0. 0

0i

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FIG. 5. Two-dimensionalmap onPEI-celluloseofacombined RNaseT2andphosphatase degradation of 18S (a)and19S (b) SV40-specificRNA. The second dimensionwasdevelopedwith 1 Mformic acid-pyridine (pH 4.3). The bluemarker moved 7.5cmin the second dimension.

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ACKNOWLEDGMENT1S

We thank J. Van derHeydenforculturingcells and virus

stocksand A. Van de Voorde for advice. Weareindebted to

M.Revel,in whoselaboratorypart of this workwasoriginally initiated.

G. H. holdsafellowshipfrom theNationaal Fondsvoor

Wetenschappelijk Onderzoek of Belgium. This project was

supportedbygrant 20.298 from theFondsvoorGeneeskundig

Wetenschappelijk Onderzoek.

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