Mapping of the three species of polyoma mRNA

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Reference

Mapping of the three species of polyoma mRNA

TURLER, Hans, et al.

Abstract

The polyoma mRNAs present in the cytoplasm of primary cultures of mouse kidney cells during lytic infection were characterized by sedimentation velocity analysis and by hybridization to polyoma DNA fragments generated by a specific endonuclease of Hemophilus parainfluenzae (Hpa II). Three distinct species of polyoma mRNAs were found and mapped on the viral genome: (a) early 19S polyoma mRNA, which is complementary to about 45% of the early strand and maps from approximately 78 (5' end) to 23 (3' end) map units; and (b) two related late mRNAs, a larger species (19 S), which is complementary to about 44% of the ate strand and extends from approximately 70 (5' end) to 26 (3' end) map units, and a smaller species (16 S), which is complementary to about 31% and maps between 61 (5' end) and 30 (3' end) map units. Thus, the entire sequence of the late 16S mRNA is comprised within the late 19S mRNA, a finding which was also supported by two-dimensional chromatographic analysis of the RNase T1 oligonucleotides of the two late mRNAs.

TURLER, Hans, et al. Mapping of the three species of polyoma mRNA. Proceedings of the National Academy of Sciences, 1976, vol. 73, no. 5, p. 1480-1484

DOI : 10.1073/pnas.73.5.1480

Available at:

http://archive-ouverte.unige.ch/unige:127869

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

Vol. 73, No. 5, pp. 1480-1484,May 1976 Biochemistry

Mapping of the three species of polyoma mRNA*

(restrictionenzymes/hybridization)

HANS TURLER, CONSUELO SALOMON,BERNARD ALLET, AND ROGERWEIL

Departmentof Molecular Biology,University of Geneva, 1211 Geneva, Switzerland

Communicated byErwinChargaff,February 11, 1976

ABSTRACT Thepolyoma mRNAspresent in the cytoplasm ofprimary cultures of mouse kidneycells during lytic infection werecharacterized bysedimentation velocity analysis and by hybridization to polyoma DNA fragments generated byaspe- cific endonuclease of Hemophilusparainfluenzae (Hpa II).

Threedistinct species of polyoma mRNAs were found and

mapped

on theviral genome: (a) early 19S polyoma mRNA, whichiscomplementary to about 45% of the early strand and mapsfromapproximately 78 (5' end) to23(3'end) map units;and (b)two related late mRNAs,a larger species (19S), which is complementary toabout44% ofthe ate strand and extends fromapproximately70(5' end) to 26 (3' end) map units, and a smallerspecies (16 S), which iscomplementary to about 31%

andmaps between61(5' end) and 30 (3' end) map units. Thus, theentiresequenceofthelate16SmRNA iscomprised within the late 19SmRNA, a finding which was also supported by two-dimensional chromatographic analysis of the RNase T1 oligonucleotidesof thetwolatemRNAs.

Polyoma mRNA synthesized early in lytic infection (i.e., earlier than12hrafter infection) sedimentsinsucrose densitygradients as ahomogeneous band withasedimentation coefficient of19 Sandisdesignated early 19S polyomamRNA(1). Polyoma mRNAsynthesized late,when therateof viralDNAsynthesis hasreachedamaximum(i.e., laterthan25hr afterinfection), comprisestwoclasses ofRNAwithsedimentationcoefficients of16Sand19S, whicharedesignated late16Sand 19Spolyoma mRNAs (2). As determined by exhaustive hybridization to polyomaDNA,early19SmRNA(labeledfrom8to 11hrafter infection)accountsforabout0.03%and latemRNAs(labeled from27 to 30hrafterinfection)accountforabout 2% ofthe totalradioactivityincytoplasmicRNA.Thesedimentationand hybridizationpatternswerecloselysimilarforpolyomamRNAs isolatedfrompolysomes (2) bychromatographyonoligo(dT)- cellulose (3)orfrom totalcytoplasm.

By usingseparated strands ofpolyomaDNAand of DNA fragments generated by an endonuclease of Hemophilus parainfluenzae (Hpa II),Kamen et al. (4)concludedthatcy- toplasmic

polyoma-specific

RNAs

synthesized

earlyandlate inlyticinfectionaretranscribed from oppositestrands, with both transcripts startingnearthe origin ofDNAreplicationin

fragment5(5)and terminatinginfragment6[seethe

physical

map(6)ⁱⁿFig.3].

Toidentify the different species ofpolyomamRNAssyn- thesized duringlyticinfectioninprimarycultures ofmouse

kidney cells andtomapthemon the viral genomewehave combined sedimentation

analysis

of

cytoplasmic

RNA with hybridizationtoHpaIIfragmentsofpolyomaDNA.Here,we Abbreviations: Hpa II, endonuclease from Hemophilus parain-

fluenzae;SSC, standard salinecitrate(0.15Msodiumchloride-0.015 Msodiumcitrate,pH7);^{x X}SSC,concentrationofthesolutionisx

timesthat of SSC.

*Partof these results have beenpublishedinpreliminaryform[(1975) Experientia31,746Abstr.]and^werepresentedatthe9thMeeting of theEuropeanTumor VirusGroupinMariehamn(Finland)inMay 1975.

presentevidence for the existence of three distinct species of polyoma mRNAs, i.e., early 19S, late 16S, and late19SmRNA, and show that the complete sequence of late 16SmRNA is comprised within late 19S mRNA.

MATERIALS AND METHODS

Cultures of mouse kidney cells (1, 7) were infected 2 days after confluence with 0.4 ml of a suspension of wild type, plaque- purified polyoma virus containing5X 108plaque-forming units (PFU)/ml.

Purified polyoma DNA1(8) wasdigested with HpaII,and the fragmentswereseparated bypolyacrylamidegel electro- phoresis (9). They were eluted from the gelelectrophoretically intodialysisbagsand concentrated byethanolprecipitation.

PolyomaDNA Iwasdenatured and fixed oncellulose filters (30 mm) asdescribed (10). HpaIIfragments weredenatured in0.2MNaOHfor10min at roomtemperature,neutralized withaceticacid,and fixedonfiltersbythesamemethod. The 30-mmfilters werecut into 25squarefiltersof3.5mm,each carrying0.2,ugofpolyomaDNAor0.05-0.1 ugofanHpaII fragment. These quantities assuredDNA excessfor hybrid- ization.Usingradioactive HpaIIfragments,wefound that90%

of the inputDNAoffragments 1, 2,and4 werefixedonthe filters,butonly65%of fragment6.

EarlyRNAwaslabeledfrom8to 11hr after infectionwith 500

MCi

of

[5-3H]uridine

(25 Ci/mmol, NEN Chemicals, Langen,Germany)in1mlof culture medium.Late RNA was

labeled from 27 to 30 hr after infection with 250 MCi of

[3H]uridine

in 1ml ofmedium,orwith100,uCiof

[32P]ortho-

phosphate (91

Ci/mg

ofP.RadiochemicalCentre,

Amersham,

England)in1ml of

phosphate-free

medium.

Cytoplasmic RNA wasextractedwith

phenol-choroform-

isoamylalcohol (2).Approximately500,gofRNAwere

layered

onto a10-25%

(wt/vol)

linearsucrose

gradient

in0.05 M

NaCl,

0.01 M

Tris1HCl,

¹mMEDTA,pH7.0, and

centrifuged

in a

SpincoSW40 rotor at

29,000

rpm for16hrat7°.Fractions werecollectedfrom the bottom of the tube.

Hybridization ofRNA to DNA filterswasdoneeitherin 4 XSSC(SSCis0.15MNaCl,0.015MNa-citrate,pH

7)

for48 hrat65°(10)followed

by digestion

withpancreaticRNase

(EC

3.1.4.22;20

Mg/ml; Worthington

Biochemical

Corp.)

^orin50%

(vol/vol)formamide,2 XSSC, for60hrat370

(11).

After

hy-

bridization in

formamide,

filters were washed once in SSC containing0.5%sodium

dodecyl sulfate,

then incubatedinthe hybridization buffer for S0 min at

370,

followed

by

two

washingsin0.1XSSC.These filterswereeitherused forelution of the

hybridized

RNA or

they

^were

assayed

for

radioactivity

andthentreated with RNase. Assay ofthe filters intoluene scintillation

liquid

followed

by

two

washings

withtoluene and ether didnotreduce the

efficiency

of the

subsequent

RNase treatment. Polyoma RNA is

partially degraded during hy-

bridization in 4 X SSC at

650; although hybridization

ⁱⁿ formamideis somewhat less efficient

(11), polyoma

mRNAs 1480

Proc.Natl.Acad. Sci. USA 73(1976) 1481

q- ^? T1 I x .- L

21 23 25 27 29 31 21 23 25 27 29 21 23 25 27 29 FRACTION NUMBER

FIG. 1. Hybridizationofearly 19S mRNA containing fractions toindividualHpa ^IIfragmentsofpolyomaDNA. EarlyRNAwas

labeled, extracted, and sedimented through^sucrosegradients; aliquots ofthe fractions^werehybridizedtototal polyoma DNAtodetect early 19SmRNA.(A) The bulk of the oddfractions containing19S mRNA

washybridizedinformamidetofragment1(late) (o). Radioactivity of the filterswasmeasured before(solid line) and after (dashed line) RNasedigestion. The bulk of the^evenfractions^washybridized to

fragment2(early); radioactivitywasdeterminedwithout RNase di- gestion (0).

Afterthefirsthybridization,everyfractionwashybridizedtoafilter with fragment5andtoafilter withfragment 6. (B)Hybridizationto

fragment5without (solidline)andwith(dashedline) RNase diges- tion.(C) Hybridizationtofragment6without (solidline) and with (dashed line)RNasedigestion.

remainedintact,^asjudged bysedimentationinsucrosegradi-

ents.

Polyoma RNA ^was eluted from the filters that had been washedasdescribed aboveby incubatingtwice inwarmed 2 mM EDTA, pH 7.2, for 3min at85° (12). The first eluate contained60-70%of the hybridized polyoma RNAand the second about 10%, whereas 10-20% of theradioactivity re-

mained^onthe filter.

RESULTS

Early19SmRNA.EarlyRNA^waslabeled with[3H]uridine from8to11 hrafter infection, extracted,andsedimented^as described in Materials and Methods. To localize polyoma mRNA, ^we hybridized ^an aliquot of each fraction to total polyoma DNA. The bulk of the odd fractions containing polyomaRNAwashybridizedinformamide^tofilters with Hpa IIfragment1(late)and thatof theevenfractionstofilters with fragment2(early). Morethan80%of polyomaRNAhybridized

tofragment 2,forming^asharpbandaround 19S(Fig. IA).

However,^somehybridization^wasalso observed with fragment 1; ^wedo^notknow whether this^wasduetosynthesis ofvery

smallamountsoflate 19SmRNAortosome"anti-late"RNA hybridizingtotheearly strand of fragment1. In^asecond hy- bridization every fraction ^was hybridized with ^a filter of fragment5and^afilteroffragment6. Fragment 5(Fig. 1B) bound lessRNA thanfragment6(Fig. IC), both beforeand after RNase treatment. The expected differencein hybrid-

izationbetween the oddfractions (which hadnotbeen hybri- dizedtofragment 2)andthe^evenfractions (which hadbeen hybridized to fragment 2) was also more pronounced for fragment6than for fragment5.Similar resultswereobtained

Table 1. Hybridizationofearly19SmRNA (labeledwith [3H]uridine from 8to11 hr afterinfection)

cpm hybridized*

Hpa IIfragment Before After %RNase-

onfilter RNase RNase resistant

2 206t 43t 20.8

5 75 7 9.3

6 242 14 5.8

NoDNA 66t 6f

*Filters of the different fragmentswere hybridizedin50% form- amide in separatetubes, eachcontaining800,000cpmof RNA.

tSum of cpm of^twofiltersfromwhich twicethevalue of the blank filterinthesametubewassubtracted.

I Mean value of all blank filters.

with anotherpreparationofearly19S mRNA that washybri- dized inequalparts to filters withfragments 2, 5,and 6. The results(Table 1) show thatbefore RNase treatmentfragments 2and6bound similaramountsof RNA,whereasfragment5 bound only one-third of thatamount.AfterRNase treatment fragment 6 again retainedmore RNAthan didfragment5.The RNase-resistant radioactivityonfragment2mayseemquite low. Itshould be pointed out, however, that with lateRNA hybridized to total polyomaDNAonly50% of theRNAre- tained beforeRNase treatment was RNase-resistant. This seems tobe due tothe fact that the fixedDNAis notfreelyavailable for idealhybrid formation (unpublished observation).

To testwhether early 19SmRNAisthe contiguous transcript oftheearly region,wehybridized early 19S mRNA in form- amidetoeitherfragment2or 4. HybridizedRNA waseluted andaliquots of theeluateswererehybridizedtofragments2 and4.The results(Table 2) show that both eluates rehybridized inthe same proportion to the two fragments (thetheoretical ratioof fragment 2 to fragment 4 is 1.6). This observation in- dicates that thereisonlyonespeciesofearly 19SmRNA.The low proportionof rehybridization seems to be due to detach- mentof DNA fromthe filters during elution of RNA and to hybrid formationinsolution (unpublishedobservation)

Bysedimentation in dimethylsulfoxide-sucrose gradients, the molecular weight of early 19S mRNA was estimated to be around 700,000(1), corresponding to about 2,000 nucleotides.

Fromlength measurements by electron microscopy, polyoma DNA I has been estimated to contain about 4,600 base pairs (Hirt, personal communication; and refs. 13 and 14). Based on this value, early 19S mRNAis the transcript of 44% of one strand.Ourhybridization results suggest that early 19S mRNA istranscribed from fragments 4, 8, 7, and 2 and partially (about

Table 2. Rehybridization of early19SmRNA eluted from fragments 2 and 4*

RNA % cpm hybridized

eluted to fragment Ratio of

from fragment 2

fragment cpm/tube 2 4 to fragment 4

2 325 5.6 6.4 0.9

4 350 4.6 5.0 0.9

*Aliquots of early 19S mRNA were hybridized in formamide to filters with fragments2and 4.Hybridized RNA was eluted from the filters and each eluate was rehybridized in two equal parts to filters with fragments2and 4, respectively, in 4 x SSC followed byRNasedigestion.

Biochemistry:

Tfirleretal.

Proc.Natl. Acad.Sci. USA 73(1976)

27 29 31 33 35 27 29 31 33 35 27 29 31 33 35 FRACTION NUMBER

FIG. 2. Hybridizationoflate polyomamRNA-containingfrac-

tionsto individualHpa II fragments.Late RNAwaslabeled, ^ex- tracted, and sedimented throughsucrosegradients; late polyoma mRNAs^weredetected by hybridizingaliquotsof eachfractiontototal

polyoma DNA. Other aliquots of the polyoma mRNA-containing fractionswerethenhybridizedseparatelytoHpa II fragments1,2,

3, and4in 4XSSCfollowed by RNase digestion.(A) Hybridization tothe late fragments1and³(-)andtotheearly fragments2and4

(0). (B) Hybridizationtofragment 1 (-)and fragment3 (0). (C) Hybridizationtofragment2(0) and fragment4(0).

3mapunits)fromfragment6,whichcomprisealtogether45%

ofthegenome.Bythe methods useditwasnotpossibletolo- calizemorepreciselythebeginningandend oftranscription ofearly19S mRNA.

Late mRNAs.Late RNAwaslabeled with[3H]uridinefrom 27to30hrafterinfection, extracted,and sedimented^asde- scribedabove. Analiquotof each fractionwasfirsthybridized

to totalpolyoma DNA. Otheraliquots of thefractions^con- tainingpolyomaRNA^werethenhybridized separatelytoHpa IIfragments 1, 2, 3, and4 (in4 X SSC followedby RNase treatment).Fig.2Ashows thatpolyomaRNAhybridized mostly

tofragments ¹and3; this resultsuggeststhat thetwoclassesof late mRNA (2) are both transcribed from the late region.

Fragment 1bound^moreRNAthan didfragment3; theratio of RNA boundtofragment1overRNAboundtofragment3 increased from 19Sto 16SRNA(Fig. 2B). Small amountsof RNA hybridizingtofragments2and 4 (early mRNA) were

foundinaratherbroadpeak (Fig. 2A)andhybridizedinsimilar proportionstofragment2and4(Fig. 2G). Hybridizationof polyomaRNAinthe16S region^tofragments2and 4maybe duetoincreasedturnoverofearlyRNAlateininfection(1).

To characterize the late mRNAs further, ^we pooled and analyzedthe fractionsof thesucrosegradientcontaining 19S and16SRNAasfollows.

Late16SmRNA.Thepooledfractionscontaining 16SRNA

werehybridizedinformamidetofilters withfragments 1, 3, 5, and6.The filters^werewashed andradioactivity^wasdeter- mined.Theyweretreated with RNase andassayedforradio- activityagain(Table 3). Before RNase treatment,fragments 1 and³ bound similaramountsof RNA; fragments5and6 bound 10-20% of thatamount. After RNasetreatment,frag-

ment1retained3.8 timesmoreRNA than didfragment3.More

than90% of the RNA^wasremovedfromfragment5or6by^the RNasetreatment. Ifonecomparesthe RNase-resistantradio- activityonfragment5or6tothat^onfragment1,itcorresponds

toaspecific hybridizationofabout 0.5 mapunitor25 nucleo-

Table 3. Hybridization of late168mRNA (labeled with(3H]uridine from 27 to 30 hr after infection)

cpmhybridized*

Hpa II % Ratio of

fragment Before After RNase- fragment1 to onfilter RNase RNase resistant fragment 3

1 1575 672 42.7 3.8

3 1436 159 11.2

5 171 12

6 238 13

NoDNA 2 0

*Eachtube contained 40,000 cpm of RNA. For details, see foot- notes ofTable 1.

tides for each of the two fragments. In three independent ex- perimentsthe ratio ofRNase-resistant radioactivity bound to fragment 1 overthat bound to fragment 3 was determined seven times.The averagevalue found was 3.3⁴ 0.3.

To testwhether there is only one species of late 16S mRNA, wedidthesametypeof experiment as described for early 19S mRNA.The results (Table 4) indicate thatinthe firsthybrid- izationboth fragments1and3bound the same speciesof RNA, which, afterelution, rehybridized to the two fragments in similarratios as inthe firsthybridization.

Fromsedimentationindimethylsulfoxide-sucrose gradients, the molecular weight of late 16S mRNA was estimated to be about 500,000 (2). This corresponds to approximately 1,400 nucleotides or to a transcript of 31% of the late strand. Ac- cordingtothe hybridization results, this has to be divided be- tweenfragments1and3 with a ratio of 3.3 to 1, yielding 24 mapunitsonfragment 1and 7 units on fragment 3. On the physical map showninFig. 3, late 16S mRNA istranscribed counterclockwise(4), the5'end beinginfragment3near 61 unitsand the3'endinfragment1 near 30 units.Taking into accountthesizeestimateandthehybridizationtofragments 1and3oflate16SmRNA,itappearsunlikely that thisRNAis transcribedpartiallyfromfragment5or 6, as wassuggested by the small proportion ofhybridizationobserved withthesetwo fragments(Table 3).

Late19S mRNA. Hybridizationinformamide ofthe pooled fractions containing 19SRNAtothe HpaIIfragments 1, 2, 3, 5,and6showed that late 19SmRNAalso

hybridized

predom- inantlyto

fragments

1and3(Table 5). BeforeRNasedigestion

Table 4. Rehybridization of late16SmRNA eluted from fragment1 or3*

%cpm

RNA hybridized

eluted tofragment Ratio of

from fragment1

fragment Exp. cpm/tube 1 3 tofragment3

1 (i) 800 26.1 9.2 2.8

(ii) 500 29.4 12.6 2.3

(iii) 2100 26.7 6.5 4.1

3 (i) 2150 30.3 9.7 3.1

(ii) 600 22.0 19.2 1.1

(iii) 1400 14.0 9.8 1.4

*Independentpreparationsof late16SmRNA[(i)^and(ii)labeled with [3H]uridine, (iii) with [32P]phosphate] ^were hybridized in formamide to fragments ¹ and 3, eluted, and

rehybridized

in equalpartstofragments1 and3in4x SSC followedbyRNase digestion.

Biochemistry:

Proc.Natl.Acad. Sci. USA 73

(1976)

1483 Table 5. Hybridizationof late 19SmRNA

(labeled with[3H]uridinefrom 27to30hr afterinfection) cpmhybridized*

Hpa II % Ratio of

fragment Before After RNase- fragment1 to onfilter RNase RNase resistant fragment 3

1 1859 524 28.7 1.9

3 1650 250 15.2

5 103 9

6 328 24

2 195 23

No DNA 3 1

*Each tube contained 49,000 cpm of RNA. For details, seefoot- notesofTable1.

the twofragmentsbound similar amountsof RNA.Fragments 6and 5 bound5and 15 times less RNA,respectively,than did fragment1or 3.AfterRNasetreatment,fragment1retained 1.9 times more RNAthan didfragment3.RNase-resistantra- dioactivityboundtofragments6and5, whencomparedtothat onfragment 1, wouldcorrespond to a transcript ofabout1.5 map units(about70nucleotides)onfragment6and about0.5 map unit onfragment5, assumingthatall 19SmRNAmolecules areidentical. Wecannot exclude,however, that this low degree ofhybridizationisthe result of some sizeheterogeneity ofa fraction of 19SmRNAmolecules (2). Asjudged from the hy- bridization tofragment 2, bothbefore and after RNasetreat- ment, less than 10% ofthe19S RNAwasearly19S rnRNA.The ratioof RNase-resistant RNA bound tofragment 1 over that bound tofragment3wasdetermined four times in two inde- pendentexperimentsandgave anaverage value of 1.9I0.1.

Therehybridization experiment showninTable 6 suggests that thereisonlyonespecies oflate 19S mRNA.The molecular weight of 19S mRNA was estimated to be approximately

Table6. Rehybridizationof late 19S mRNA eluted from fragments1 and 3*

%cpm

RNA hybridized

eluted to fragment Ratioof

from fragment1

fragment cpm/tube 1 3 tofragment3

1 2940 15.0 7.5 2.0

3 2470 9.8 10.0 1.0

*Late 19S mRNA labeled with [32P]phosphate. For details see footnote of Table4.

700,000(2),i.e.,about2,000nucleotidesor atranscriptof44%

of thelate strand. Fragments 1 and3 accountfor44%ofthe genomeandgive aslightlysmallerratio(1.7)than the experi- mental result. Takingintoaccountthesize estimateandthe hybridization results (Tables5and6),weassumethat late 19S mRNA iscomplementarytothe late strand between approxi- mately70(5'end) and26(3' end)map units(see Fig. 3).

Chromatographic Analysis of Late 16S and 19S mRNA.

The hybridization results obtainedwiththetwolatemRNAs suggested thattheentirenucleotidesequenceof late16SmRNA is comprised within the late 19S mRNA. This has beencon- firmedby two-dimensional chromatographic analysis (15) of RNaseT1 (EC 3.1.4.8) digests of[32P]phosphate-labeled late 19Sand16S mRNAs.The autoradiographsinFig. 4showvery similarpatterns. About75spots canbe detectedinthedigest ofthe late 16SmRNA;all but one arealso foundinthedigest oflate19S mRNA. Onthe otherhand, thepatternobtainedwith late19S mRNAshows ninespotsthat are either absent or

mqch

LATE 16SmRNA LATE 19SmRNA

N

-

..

~ ~ w ^*

4 _

FIG. 3. Regionsoftranscriptionofthe threepolyoma mRNAs.

ThephysicalmapofpolyomaDNA(6)shows theHpa^IIfragments numbered 1 to8ontheoutside andis divided into 100mapunits (numbersinside) startingatthesinglesite cleavedbythe endonu- cleaseEcoRI.OR^istheoriginofbidirectionalDNAreplication (5).

The direction oftranscriptionforearlyandlate RNAshasbeen de- terminedbyKamenetal. (4).

CELLULOSEACETATE ELECTROPHORESISpH 3.5 FIG. 4. Two-dimensional chromatograms of the RNase T, oligonucleotides of late 16S and19S polyoma mRNAs.RNAwasla- beledfrom 27 to30hrafterinfection with 100 MCiof[32P]ortho- phosphatein1ml ofphosphate-freemedium, extracted, andsedi- mentedthrough^sucrosegradients; polyoma mRNAswerelocalized byhybridizing aliquots of the fractionstototalpolyoma DNA and subdividedinto16Sand 19S mRNAs.Each of thesewashybridized informamidetototalpolyoma DNA(5,Mg^ona30-mmfilter). The filterswerewashed, treatedwithRNase T1 (WorthingtonBiochem.

Corp.)at10units/ml for30minatroomtemperature,and washed again.RNAwaseluted by incubatingthefilterstwicewith0.5ml of warmed2mMEDTA, pH7.2,at850 for3mineachandprecipitated afteraddition of40,gofpurifiedyeastRNA. The drypelletwasdi- gested with 5AlofanRNaseT1solution (Sankyo Co.,Tokyo;0.5 mg/ml) for 30 minat37°.Chromatogramswereobtainedby cellulose acetateelectrophoresis (pyridineacetate,7Murea,pH 3.5) in the first dimension andhomochromatography^onDEAE-cellulose thin layers withhomomixture bat600 in the second dimension (15).

I_0L (90

0Ir C-)0 0 I1

Biochemistry:

Tfirler^etal.

Dow

A^ioT,O..

I ,I

: if

-94M

weaker in the 16S mRNA digest. It is unlikely that the addi- tional spotsareduetothepresence of early19SmRNA because it amounts toless than 10% in the late19S RNA preparations (Table5).

DISCUSSION

In thispaper we present evidence that three distinct species of polyoma mRNAs are synthesized during lytic infection in mousekidney cells: an early19S mRNA, a late 19S mRNA, and a late 16S mRNA. Their positions on the physical map of polyoma DNA are shown in Fig. 3.

Ourresults are based on hybridization of the two size classes (19S and 16S) of polyoma mRNAs to individual Hpa II frag- mentsofpolyoma DNA. To locate the 5' and3'termini of the mRNAs we used the published estimates of the molecular weights of the mRNAs [700,000 for 19S RNA (1, 2) and 500,000 for 16S RNA (2)] and of polyoma DNA [3 X 106 molecular weight (13) or 4,600 base pairs (14)]. Assuming that the true molecular weights of the polyoma mRNAs are within +10%

of thepublished estimates, the indicated positions of the 5' and 3'ends have to be regardedasapproximationswith anaccuracy ofabout+3map units.Therefore,ourresults donotexclude that the 3' ends of thetwolatemRNAscoincidenear 28map units. LatepolyomamRNAs aresynthesizedinthe nucleus as molecules equalto orlarger thanafulltranscriptof thelate strand (4, 10, 16), and thetwo late mRNAs aremostlikely formedbyexo- orendonucleolyticdigestionfollowedby po- lyadenylation (3). Detailed analysisof the end-terminal se- quencesof thetwolatemRNAsmightprovideinformationon the mechanism and accuracy of processingmRNAprecursors inmammalian cells.

Uptoabout12hrafter infectionearly19SmRNAisvirtually theonly virus-specific RNA foundin the cytoplasmofcells undergoing lytic infection. Thereafter increasingamountsof thetwolatemRNAsappearinadditiontoearly19SmRNA, whichissynthesized throughout lyticinfection. Results^tobe presentedelsewhereshow that synthesisof late 19Sand 16S mRNAbeginsbeforedetectable viral DNA

replication

andalso takesplaceunder conditions wheresynthesisof both cellular and viralDNA isinhibited by 5-fluorodeoxyuridine.

Early19S mRNA

(or

avery similarspecies)isalsosynthesized inabortiveinfection (1)andinpolyoma virus-transfor~medcells (4, 16).Itmostlikelydirectssynthesisofpolyoma-specifictumor

antigen, thehypothetical "pleiotropiceffector"(1), whereas thelatemRNAsdirectsynthesisof thecapsid protein(s) (17).

Inlyticinfection of thecloselyrelatedsimianvirus40,both the number ofmRNAsand their functionseem tobeanalogousto whatwefindwith

polyoma

virus

(18-20).

The

experimental

resultspresentlyavailablearecompatible with thehypothesisthat

polyoma

^{viruscontainsnotmore}than three genes.Twoof thesemost

likely specify

the viral

capsid

(21), whereas the other carries most of the information need for lytic infection and all the information for abortiveinfecti, initiation, and maintenance of the transformed phenotype a probably also for induction of tumors in animals. Therefo wepropose to designate the region of polyoma DNA fr(

which early 19S mRNA is transcribed as "(pleiotropic) effec gene" and the region of the viral genome that specifies the t latemRNAs as"structural gene(s)" (Fig.3).

Wethank Chantal Bonnet and Christine Berger for excellent te(

nicalassistance,MouradBensemanne for preparing the cell cultur andOttoJennifor drawing the illustrations. We are also grateful Daniel Kolakofsky and Gerald Smith for their helpand advice fort chromatographic analysis and to Gerald Selzer for reviewing I manuscript.This workwassupported by the Swiss NationalFoundati forScientific ResearchGrants3.097.073and3.287.074.

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