Structure and Function of the Polypeptides in Simian Virus 40 II. Transcription of Subviral Deoxynucleoprotein Complexes In Vitro

JOUIRNAL OFVIROLOGY, June 1972, p. 930-937 Copyright @,1972 AmcricanSociety for Microbiology

Vol. 9, No. 6 PrinitedinU.S.A.

Structure

aiid Fuiiction

of

the

Polypeptides

in

Simian

Virus

40

II.

Transcription

of

Subviral

Deoxynucleoprotein

Complexes

In

Vitro

ENG-SHANG HUANG, MEIHAN NONOYAMA, AND JOSEPH S. PAGANO

Department oJ Bacteriology aticl Immnunology andtc Departnlent ofMelicine, School oJ Medicine, Uniiversiti of North Cciroliia, CliapelHill, North Carolina 27514

Receivedforpublication 21 January 1972

A deoxynucleoprotein complex (DNP-1) isolated from simian virus 40 (SV40)

after disruption of the virus in an alkaline buffer contains the viral

deoxyribonu-cleic acid (DNA) and four minor structural polypeptides. Dissociation of DNP-I

by equilibrium centrifugation in CsCl yields a complex (DNP-II) that contains a

smallamount ofpolypeptide tightly bound to the viral DNA. Studies ofthe

tem-plate activity ofthese deoxynucleoprotein complexes in vitro with Escherichia coli transcriptase show that therate oftranscription of DNP-I and DNP-IL is 30 and

80%,,

respectively, compared with that of deproteinized SV40 DNA component I.

Indimethylsulfoxide gradients, thecomplementary ribonucleic acid (cRNA)

syn-thesized from DNP-I is one-third to one-half the size ofthe cRNA species from DNA-I and DNP-IL. Competition hybridization experiments show that with the E. coli transcriptase only a portion (about one-half) ofthe SV40 genome is

tran-scribed with DNP-I astemplate, whereas mostorallof the genomeis transcribed

with DNP-II as template. The template activity of the deoxynucleoprotein

com-plexeswithahighlyactiveform lI ribonucleicacid polymerase prepared from SV40-infected permissivecells follows similartranscription kinetics. The results indicate

that structural nucleoproteins of SV40 bind nonrandomly to the viral DNA and

effect thetranscriptionofsomesubsetof itssequencesinvitro. The biological significance of

deoxyribonu-cleic acid (DNA)-bound proteins of mamma-lian chromation (10, 19, 23), plant chromatin

(3, 6, 10), and Escherichia coli colicinogenic factor (4, 9) has been extensively investigated. Several lines of evidencethat the

binding

of chro-mosomal proteinsto DNA inactivates transcrip-tionsuggest that these proteins act as regulatory

molecules in gene expression (3, 10, 23). In the

colicinogenic factor system, the protein of the

DNA-protein relaxation complex may act as an endonuclease and open a single-strand nick to

initiateDNA replication (9).

These observations led us toinvestigatethe pos-sible role in the regulation of simian virus 40

(SV40) geneexpressionoftheviralstructural pro-teinsasociatedinthevirion with the viral DNA.

There is increasing evidence for the existence of

nucleoproteinsin SV40andpolyomavirus (2, 11, 15;P. M.FrearsonandL. V.Crawford, inpress).

We show here the template activity of SV40

deoxynucleoprotein complexes in vitro with DNA-dependent ribonucleic acid (RNA) poly-merases ofbacterial and mammalian origin. The

data sLIggest that SV40 may carry with it

struc-tural components thatexert anonrandom limita-tion on transcription so that a particular subset rather than the full complement of viral DNA sequencescanbetranscribed.

NIATERIALS AND METHODS

Cellsandvirus.Themethods forcultivation,

purifi-cation, isotopic labeling, andalkaline degradation of

virus areinprecedingreports (11, 15, 22).

SV40DNA and DNAcomponentI.SV40 DNAwas

extracted from purified virions bythephenol-sodium

dodecyl sulfate (SDS) method (21).SupercoiledSV40

DNA (conponent 1) was separated from SV40open

circles (component II) with ethidium bromide (100-200,ug/ml) andequilibrium densitycentrifugation in

CsCl with an initial density of 1.56 g,'cm3 in the

Spinco T-65 rotor at 43,000rev,'min for 48 hr (22).

The band corresponding to DNA component I was

collected; the ethidium bromide was removed by

repeated extraction with isoamyl alcohol until there

was no color in thewater phase. The DNA solution

was dialyzedagainst

tris(hydroxymethyl)aminometh-ane (Tris)-buffered isotonic saline solution, pH 7.4

(TBS).

Preparation of subviral deoxynucleoprotein cot' 930

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SV40 DEOXYNUCLEOPROTEIN COMPLEXES. IX plexes. Two types ofdeoxynucleoprotein complexes,

DNP-I and DNP-II,consistingof the viral DNA and structural virion polypeptides were isolated from degradedvirus (15).

Purification of DNA-dependent RNA polymerase fromE. coli. DNA-dependent RNApolymerase

con-tainingo-factor wasisolated from E.coli, strainQ13,

by the methodof Burgess(7).Enzyme ofhigh specific

activity (400 units/mgprotein,200units/ml) from low salt followed by high salt-glycerol gradientswas used for thepolymerization assay.

Transcription assay with E. coli DNA-dependent

RNA polymerase (transcriptase). The stock reaction mixture (2.5 X concentration) for the enzyme

con-tained 0.1 M Tris-hydrochloride, pH 7.9, 0.025 M

MgCl2, 0.25 mM

ethylenediaminetetraacetic

acid (EDTA), 0.25 mMdithiothreitol(DTT),and 0.375 mM uridine triphosphate (UTP), guanosine triphosphate

(GTP), cytidine triphosphate (CTP), and adenosine triphosphate (ATP) (7). For the assay,0.1 ml of the stockmixture,0.01 mlofE.coli RNApolymerase,0.1 ml of templatecontaining 1.0 ,ug of DNA, 0.01 ml of 3H-UTP

(100,4Ci/ml,

25Ci/mmole), and 0.03 mlof distilledwater tomake thevolume0.25mlwere incu-bated ina37C waterbathfor 15, 30,45, or60 min. The mixtures were precipitated with 0.05 ml

100%/-0

trichloroacetic acidcontaining0.2 Msodium

pyrophos-phate, collected on a membrane filter (Bac-T-flex,

Schleicher and Schuell), and washed eight times by filtrationwith5% trichloroacetic acidcontaining0.02 Msodium pyrophosphate.After drying, the filterswere

counted intoluenescintillationfluid.

Synthesis of virus complementaryRNAinvitro. The synthesis of 3H-UTP-labeled viral complementary

RNA (cRNA) wascarried out byslightly modifying

themethod ofWestphaland Dulbecco (31). To 0.25 mCi of3H-UTP (25Ci/mmole) lyophilizedina3-ml

ampoule wereadded0.1 mlof the stockreaction

mix-turewithout coldUTP, 0.03ml of E. coli RNA

poly-merase, 0.1 ml of SV40 DNA component I (100 ,ug/ml), or the equivalent quantity ofnucleoprotein complex, and 20,ulitersofdistilledwater. After incu-bation for 100 min in a 37 C water bath, 20 Ag of

deoxyribonuclease (Worthington)wasaddedtodigest

the DNA (1 hrat 37 C) and stop the reaction. The mixtureswerethentreated with SDSat afinal concen-tration of

1%<7;o

and chromatographed on Sephadex G-50(mediumgrade;bedvolume, 5ml;diameter,0.5

cm; flow rate, 0.5 ml/min) in O.1X SSC containing 0.01% SDS. Thefirst peak thatelutedwas collected, extracted three times with chloroform, and dialyzed

against thiee changes of0.1X SSCcontaining 0.01%

SDSfor24hrat4C.Cold viral cRNAwassynthesized

as above except that cold nucleotide triphosphates

wereusedin the reaction mixture.

RNA-DNAhybridizationandcompetitive hybridiza-tion. SV40 DNAcomponentI wastreated with deoxy-ribonuclease (31) and denatured by heating in boiling

water for 15 min and then quicklychilled inanice bath. The denatured DNA(diluted to 0.01,g in 3 ml

of6X SSC) was immobilized on membrane filters

that hadbeen soaked in 6X SSC and thenwashed in 40 ml of 6X SSC.Thefilters weredried inavacuum

at roomtemperature overnight and at 80 C for 2 hr.

The hybridization ofRNA to immobilized

dena-tured DNA was by the method of Gillespie and Spiegelman (13). The DNA filters wereimmersed in scintillation vials in 1.0 ml of 6X SSCcontaining0.05 gg of 3H-cRNAtranscribed from SV40DNA compo-nent I (4X 105 counts/min;specific activity, 8 X 106

countspermin perMg) or, in thecompetition experi-ments, together with graded amounts of cold cRNA fromDNP-I, DNP-II, or DNA-I.

Two variations of the competitive-hybridization technique were employed. (i) After the first hybridiza-tion with 0.05 Mug of 3H-cRNA of DNA-I and increas-ing amounts of cold competing cRNA, the filters containing denatured viral DNAwererinsed with four changes of 2X SSC and washed by suction filtration

oneachside with50ml of 2X SSC. The filters were thentreatedat 37Cfor1 hrwith pancreatic ribonu-clease(20ug/ml) thathad beenpreheatedat80 C for

15mintoinactivate contaminating deoxyribonuclease (13, 31). Thefilterswereagain rinsed four times with 2X SSC and washed byfiltration (17). The 3H-cRNA from DNA-I (0.05 Mg) was then added for a second hybridization in the presence of 1.0 mg of yeast RNA which wasreprecipitated with alcohol and0.1%OSDS. The filters were then subjected to the same washing andribonucleasedigestion procedure. Finally the

fil-terswererewashedoneachsidewith 2X SSC by

suc-tion filtration and were dried, and the radioactivity

wascounted.

(ii) After hybridization with amounts of hot and cold cRNA and denatured DNA comparable to those in(i),thefilterswerewashed with fourchanges of 2X SSC and heated ina70 Cwaterbath for 10 minwith 6X SSCto removeremainingnonspecifically

hybrid-izing materials. The filters were rinsed with four

changesof2X SSC andsubjectedtothesame ribonu-cleasetreatmentand finalwashing proceduresas in (i) withoutasecondhybridization.

Preparation of mammalian DNA-dependent RNA

polymerasefrom SV40-infected monkey kidney cells.

Confluentmonkeykidneycells (MA-134) growing in 30 roller bottles (surface area, 1,000 cm2) were in-fected with SV40 (50 plaque-forming units/cell).

Twenty-four hours later, the cells were rinsed twice with ice-cold Hanks solution (BSS), removed from thebottles with glassbeadsorrubberpolicemen, col-lected bycentrifugation, and washed twice with cold BSS.

The purification of DNA-dependent

RNA-poly-meraseessentiallyfollowed the method ofB. Sugden andW.Keller(personalcommu/iicatioti), inwhich the enzymes were eluted from the cells and purified

through ammonium sulfate fractionation,

diethyl-aminoethyl (DEAE)-cellulose chromatography, and

centrifugation in a low salt-glycerol gradient. RNA

polymeraseform II (RNAP-II) (25, 26) was purified byfurthercentrifugationina5to

20%,-

sucrose

gradi-entinahigh-saltbuffer.

Invitro mammalianDNA-dependent RNA polymer-asetranscription assay. The reaction mixture is modi-fied from the formula of Sugden and Sambrook (28). The stock reactionmixture (2.5X concentration) for the assaycontained0.2MTris-hydrochloride, pH 7.9, 0.01 M MgCl2, 0.005MMnCI2,0.25 mM DTT, 0.08 M

KCl, 1.25 mgofbovine serumalbumin per ml, 0.25

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HUANG, NONOYAMA, AND PAGANO

mM EDTA, 0.125 M (NH4)2SO4, 10 ,uCi of 3H-UTP perml, and 0.6 mmATP,GTP,and CTP.

The standard assay system consisted of 0.1 ml of stockreaction mixture, 0.1 ml of template containing 4,g of DNA, 20 ,uliters ofRNAP-1L from SV40-infected MA-134 cells, and 2 mm adenosine-3 ',5'-monophosphoric acid (cAMP). The rest of the proce-dure followed the E. coli system as described above.

RESULTS

Subviral deoxynucleoprotein complexes. Purified SV40virionscanbedissociatedin alkalinebuffers

atpH 10.5 into asoluble proteinand a

deoxynu-cleoprotein complex containing the viral DNA and the fourminor ofthesixstructural viral

poly-peptides (15), followedbyvelocitysedimentation

insucrosegradients. DNP-I canin turn be

disso-ciated into solubleprotein and another

deoxynu-cleoprotein complex (DNP-II) by equilibrium density centrifugation in CsCl. DNP-IL contains mainly the viral DNA and a small remnant of

viral protein, apparently chiefly VP3, tightly boundtotheDNA anddifficult todetect (15).

Transcription in vitro of deoxynucleoprotein complexes with E. coli DNA-dependent RNA polymerase. We examined the kinetics of

tran-scriptionofDNP-I, DNP-II, and SV40 DNA com-ponent I (supercoiled DNA). The concentration of DNA was assayed by the diphenylamine

method (8) and verifiedby extinctioncoefficients and theratio of absorbanciesat260 and 280nm

(30).

DNP-I,

DNP-II, and SV40DNA-I, diluted withTBS tocontain equalamounts of DNA (1.0

,g) wereusedastemplates.Asindicated inFig.1, the rateof

incorporation

of soluble 3H-UTP into acid-precipitable materials with DNP-I (Fig. la,

fractions

10-12)

as

template

is only about

30%0

of the rate obtained with DNA component I.

With DNP-IItherate is about

80%,o.

Theresults

showclearly thatDNA componentI andDNP-I

have a different template activity. Removal of

protein from DNP-I to form DNP-II increased thetemplate

activity,

and the difference in

activity

between DNA-Iand DNP-IIis not so obvious.

Themorerapidlysedimenting material (Fig.

la,

fractions 15-17) in the gradient used to isolate

DNP-I contained on electrophoresis much more viral coat protein (VP1 and 2) than did

DNP-I;

in otherwords, the degradation of the virus was

less complete and the template activity of this material waslower.

Sedimentation properties of cRNA species syn-thesized in vitro. After 30min of

synthesis,

SDS was addedto thereaction mixtures to a concen-tration of0.1%tostop thereaction. The mixtures

immediately were sedimented in a

sucrose-dimethylsulfoxide

gradient

to avoid

degradation

of the RNA.

Figure

2 shows the sedimentation

.4 yuulieL,.

DNP-I DNP-IL

DNA-I

15 10 5tOp0 5 1

DP0to

-.0

X4

I Fc10 12 3

12

o

P~~~~~~~~~c

16 -17

15

30

~

45

603

[image:3.495.268.456.79.318.2]

TIME VINUTES

FIG. 1. Kinieticsof ribonlcleoside triphosphate

inicor-porationt

withSV40 DNA-I, DNP-I, or DNP-II asthe

templates. DNP-I was

obtainied

by

degradationz

ofthe virusinanalkalinebuffer,pH 10.5,for 15hr,followed

by

sedimenitationi

in a sucrose

gradienzt.

DNP-Il was

producedby dissociation of DNP-I

durinig

equilibrium

dentsity cenitrifugationl

in CsCl (see

referenice

16 for

details.) The stock

reactionz

mixture(0.1 ml),0.1mlof

the different templates niormalized to 1.0

Mg

of DNA, 0.01 mlofE. coli

trantscriptase,

0.01mlof3H-UTP (100

yuCi/ml),

and 0.03mlof distilledwaterwere mixed

anld

incubated at 37 C. After different time intervals, the incorporated material was precipitated with ice-cold trichloroacetic acid

anid

collectedon membranefilters.

Tlhe

amounlt

of3H-UTP incorporated with DNA-Ias

template, (0 *);DNP-IIas template

(0

---0),

DNP-I

(fractioni

10-12) as

temnplate

(O--O), frac-tioII 15 (A -A); and 16 anid 17 (A A) as

templates.

profiles ofthe cRNAspeciessynthesizedinvitro.

The RNA transcribed from DNP-I sediments

more slowly than the cRNA from DNA-I and

DNP-II.

The cRNA transcribed from DNP-II

shows no significant difference from that of

DNA-I.

The peaks of cRNA from DNP-I are

locatedatapproximatelythe6and 10S

positions;

the cRNA species of DNP-I and DNA-I are at 18S.

Other preparations were analyzed by

velocity

sedimentation in 0.1 % SDS-sucrose

gradients

after chromatography of the cRNA

species

through Sephadex G-50; thecRNA fromDNP-I sedimented in a broad peak of about 6S,

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SV40 DEOXYNUCLEOPROTEIN COMPLEXES. II

FRACTION N1O. TOP30

FIG. 2. Sedimentation analysis in a dimethyl sul-foxide (DMSO) gradienit ofthe cRNA species

synthe-sizedin vitro withDNP-I, DNP-II, and DNA compo-inent I astemplates. The3H-cRNA speciessynthesized for 30 minfrom DNA-I, DNP-I, andDNP-IH were

sedimentedin a2.5 to10% sucrose gradientpreparedin

100% DMSOmodifiedfromTonegawaetal.(29). After

24 hrof centrifugation at 45,000 rev/minintheSpinco SW 50.1 rotor, 20

piliters

ofsample fromeachfraction

of thegradient were driedonglass filtersandwashed byfiltrationwith5%trichloroacetic acid with 0.02M 50-diumpyrophosphate, dried, andcoutnted. The

sedimen-tation isfrom righttoleft: 23, 16, and4S RNAfrom

E.coliwereusedasmarkersin aseparate tube.

Distri-bution ofDNA-I cRNA (AL); DNP-I cRNA (@);

DNP-11 cRNA (0).

compared with 16 to 18S for cRNAspeciesfrom

DNP-II andDNA I.

Competition study between the cRNA

species

of DNA component I,DNP-I,andDNP-II. To

exam-ine whether the binding of the DNA-associated viralstructural

proteins (VP3,

4, 5,

6)

totheviral

DNAresults in specific or randomlimitation of

transcription,

competitive hybridization

studies

weredone.

We first determined the amount of 3H-cRNA

ofDNA-IneededtosaturatetheamountofDNA usedinthecompetitive

hybridization experiments.

Theresults, shown in Fig. 3, indicated that0.05

,g

of 3H-cRNA

produced

close to maximal

hybridization; application ofthreetimesasmuch cRNA(0.15Mg) caused hybridization ofonly 24%

more counts.

WecalculatedtheamountofDNAimmobilized

in membrane filtersbylabelingit with

'4C-thymi-dine.Thespecificactivityof'4C-SV40DNA-I was

7,333 counts per min per

Mg.

Theaverage counts of DNA usedforhybridizationwere72countsper

minperfilter. Thepercentage of DNA remaining immobilized on thefilters was 82.6%

(±

3.3%) ofthe DNA applied; after22 hrof competition hybridization there was no significant loss of DNAfrom thefilters.

Figure4 shows theresults of two competition studies. Cold eRNA synthesized in vitro from

DNP-I, DNP-II, or DNA-I was used in compe-tition against3H-cRNAof DNA componentI. In the presenceof 0.5

,ug

of cold cRNA from

DNP-I,

when theratio of cold cRNA to 3H-cRNA was 10, about half (51.2%) of the control counts remained on the filters (Fig. 4A). With cold cRNA from DNP-II or DNA-I, the counts

re-maining bound to thefilters were about

12%o

of

the control counts. The results obtained with

method (ii) (Fig. 4B) with a single simultaneous hybridization confirmed the results showninFig.

4A. These dataindicate that with DNP-Ias

tem-plate only half of the SV40 genome sequences were transcribed with E. coli transcriptase; in

contrast with DNP-II as template, most of the DNA was transcribed. These data support the

hypothesis that theproteins ofDNP-Iarebound

nonrandomly to theviral DNA and that bound areas orblockedinitiation sites (E. coli

transcript-ase) allow the transcription ofonly about half of the SV40 genome.

In the converse experiment, cold cRNA from DNA-I in competition with labeled cRNA from

DNP-I prevented thehybridizationof morethan 95%of thecountshybridizing in the control filters

toDNA-I intheabsenceofcold cRNA.

Purffication

andtemplate specificityofRNAP-II

from SV40-infected cells. RNA polymerase form

I (nucleolar enzyme) and formII (nucleoplasmic enzyme) (25, 26) obtained after DEAE-cellulose column elutionwerepooled and centrifugedin a

2

x

O

0I

0.05

3.10 0.15

H3 c-RNA

(1ig)

FIG. 3. Determination of thesaturation levelof

3H-cRNA synthesizedfrom SV40DNA-IforSV40DNA. Aconstanit

amoun1t

ofdenaturedSV40 DNA (0.01

plg)

wasimmobilizedonntitrocellulosemembranefilters. Thle

filters wereimmersedin6X SSCwithgradedamounts

of3H-cRNA (8 X 106 countsperminper,ug)

syntthe-sizedfrom SV40 DNA-I, 1.0 mg ofyeast RNA, and 0.1% SDS. Hybridizationwascarriedoutfor22hras

in method ii (see text). With 0.05

pAg

of3H-cRNA in

the reaction, thenumberofcounts perminute that

hy-bridizedisclosetotheplatealuofthe curve.

10---7

0

v

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HUANG, NONOYAMA, AND PAGANO

100

A

60-er)

40

-20

<=:

o

--g

°

0.1

0.2

0.3

0.4

C 5

z

620-0

0

0.1

0.2

0.3

0.4

0.5

0

0.1

0.2

0.3

0.4

0.5

cold

c-RNA

(Aug)

FIG. 4. Competitive hybridizationt ofcRNA species

synthesizedin vitrofromDNA-I,DNP-I, andDNP-II.

Thefilters withconistantamountsofimmobilized DNA wereimmersedin1mlof 6X SSC containing 3H-cRNA (0.05jig; specific activity, 8 X 106 countsperminper I jig) synithesized from SV40 DNA-I, graded amounlts

ofcold cRNAfrom DNP-I, DNP-II, orDNA-I, Img of yeast RNA, and0.1% SDS. The hiybridizationi ex-periments were performed as described in Materials and Methods (i) and (ii) (see text). A, After thefirst

competitive hybridization, thefilters weresubjectedto

washing andribonucleasedigestiont;0.05 jAgof3H-cRNA ofDNA-Iwasthentaddedforthe secondhybridizationi.

B, After a single competitive hybridization thefilters werewashed and heatedat70Cfor10mm inplaceof

the seconidhybridization. The counits of3H-cRNA

hy-bridizedto immobilized SV40 DNA in the absenceof

cold competing cRNA wereusedas the 100%

hybrid-ization control; inexperimenits A anidB, the control countswere13,516anld13,402 counts/minl,respectively.

Thecounts onfilters withoutSV40 DNA were58 and 56counts/min in A and B, respectively. ColdcRNA synthesizedfrom DNP-J (0);coldcRNAsynzthesized

fromDNP-II(A);coldcRNAsynithesizedfromDNA-I

(0i)

low-salt-glycerol

gradient.

Twocomponents were

resolved (Fig.

5A);

RNAP-I

migrates

asa

dimer,

and RNAP-IIasa monomer (J. Sambrook, per-sonal communication). With denatured calf

thy-mus DNA as the

template,

the

activity

of

RNAP-IIfromSV40-infectedcellsisatleast four timesasgreatasthatof RNAP-I.

After the fractionswere sedimented ina

high-salt gradient,onedistinct RNAP-II peak appeared

(Fig. SB). Theenzymeobtained from this gradient was free from detectable host cell DNA or

con-tamination with viral DNA. Table 1 shows the template specificity of RNAP-II. The results indi-cate that in vitro RNAP-II prefers denatured DNA as a template. IfcAMP was added tothe reaction mixture, there was a slight increase in 3H-UTP incorporation when native SV40 DNA componentIwasthetemplate.

Transcription of deoxynucleoprotein complexes in vitro with mammalian RNA polymerase. RNAP-II (16, 25, 26) from permissive cells

infectedwith SV40wasusedforin vitro

transcrip-tion assays.DNA-I, DNP-I, or DNP-II

contain-ing equal amounts of DNA (4

,ug/0.1

ml) were

used as the templates. The rates of transcription with DNP-I and DNP-IIare28 and82%,

respec-tively, compared withthatof DNA componentI

(Fig. 6). The results with mammalian enzyme

from infected cellsarereproducible andare

com-parable to the results with the E. coli enzyme.

Withboth types ofenzymes, there was a

signifi-cantreduction in transcription when DNP-Iwas

the template and a slight reduction when

DNP-IIwas thetemplate.

DISCUSSION

Deoxynucleoprotein complex I isolated from alkali-degraded SV40isactive inDNA-dependent RNA synthesis with the transcriptase fromE.coli

we

I

I2

-1I

I

/ 1

.'I

I

I

.-

I...

_ r)

J . '\

e)OffO><>t)d°

X- * *sx

1VS 1 5 .- , 15

T

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F T. T?-L

...

FIG. 5. Purificationandsedimentationofmammalian RNApolymerases. Polymerase forms I andIIeluted

from a DEAE-cellulose column were pooled,

precipi-tated, anld dissolved in buffer. A, The enzymes were

centrifugedinalow-salt20to50%glycerol gradientat

26,000rev/mimlinaSpinco SW27rotorfor24 hrat4 C. B, RNAP-II was isolatedfrom fractionis 9, 10,and 11

ofa low-salt-glycerol gradient, precipitated, dialyzed,

dissolvedinahigh-salt buffer,andthencentrifugedina

high-salt5to20%Osucrosegradienttinthe SW 27rotor at26,000 rev/minfor30 hr.

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SV40 DEOXYNUCLEOPROTEIN COMPLEXES. IX TABLE 1. Templatespecificity of mammalian

DNA-dependent RNA polymerase (RNAP-II) iso-lated from S V40-infected MA-134 monkey

kidney cellsa

Templateb

Amt of _

DNA NativeSV40

used as Denatured Native DNAtiven I

tem- calfthymus calfthymus mponent I

plate DNA DNA

(pg) (without (without (with (without

cAMP) cAMP) CAMp)d cAMP)d

30 15,689c 1,630

10

11),733

2,949

4 16,402 2,054 3,954 3,396

1 13,664 975 1,388 716 a Method of B. Sugden andW. Keller (personal communication). Abbreviations: DNA,

deoxy-ribonucleic acid; RNA, deoxy-ribonucleic acid; SV40,

simian virus 40; cAMP, adenosine-3',

5'-mono-phosphoric acid. Enzyme control, 143 counts/

min; 30 gg of denatured calfthymus DNA con-trol, 143 counts/min; 30jugofnativecalfthymus

DNA, 72 counts/min; 4Ag of SV40 DNA 1, 132 counts/min.

IThe amount of RNAP-I in all assays is 20

,lAiters.

c3H-Uridine triphosphate incorporation,

ex-pressed in counts per minute.

d Finalconcentration of2.0mmoles/ml.

andanRNA

polymerase

from mammalian cells.

However, the

template

activity

of DNP-Iismuch

lessthanthatofan

equal

amountof

deproteinized

SV40 DNA component I. The decrease appears tobe due toviral

proteins

boundtothe

DNA;

the

template

activity

increases after the removal of

most ofthe

protein

from DNP-I

by

CsCl

centrif-ugation. DNA-cRNA

competition hybridization

experiments

show that the cRNA

synthesized

in

vitro with DNP-I as

template hybridized

with

only

a fraction of the viral DNA transcribable with the E. coli enzyme. This indicates that the viral

protein

prevents

transcription

ofasubset of theviral DNA sequences either by

binding

with itorblockingE.coli

transcription

initiation sites

orboth. WithDNP-IIasthe

template,

transcrip-tionwas not asreduced, andwecouldnot assess the small differences in the transcribed RNA

species in the

competition

hybridization

experi-ments.

Thereare technicalpoints worthconsideration.

Lucas andGinsberg (17)forhybridization

inhibi-tion testsappliedtoimmobilized viral DNA cold messenger RNA (mRNA) from infected cells, treated withribonuclease and washedextensively,

and thenapplied3H-cRNA of viral DNA. We first assessed the amount of 3H-cRNA synthesized

fromDNA-I needed to saturate theimmobilized

viral DNA and then used two different methods toascertainboth the existence of differentcRNA

species astranscribed fromDNP-I, DNP-II,and DNA-I and also the percentage of the genome

transcribed in eachcRNA.

Inthe first method weapplied cold cRNA from DNP-I, DNP-II, or DNA-I together with 3H-cRNA fromDNA-I, treated it with ribonuclease, washeditextensively,andthenapplied additional

3H-cRNA from DNA-I so as toinsuresaturation of any remaining unbound sites. In the second method, we simply applied a mixture of cold cRNA and 3H-cRNA from the appropriate

sources and in varying ratios simultaneously. In both cases, up to a10-foldexcessofthe cold

com-peting cRNAwasused;wepresumedthat thiswas a saturating amount as aplateauwasreachedin the competitive effect (Fig. 4). The results were

virtually identical. For analysis of these in vitro products,where theprecise quantities of the reac-tants are known, we suggest that simultaneous

application of both kinds of cRNA allows for a

quantitative competitive effect, whereas

applica-tion of the cRNA species sequentially does not

allow competition for binding to sites on the

DNA, but onlyindicatesthe final percentages of

the genome to which the different RNA species

bind. With the sequential

technique,

there is a

3

*DNA-I

b

2-~N-T_

U

~~~~~DNP-1

TIME

MINUTES

FIG. 6. Kinetics oftranscription in vitro ofSV40

DNA-I,DNP-I, andDNP-IIwith a mammalian RNA

polymerase. The mammalianRNApolymerase II assay isdescribedinthetext. Theamountof3H-UTP

incor-porated withDNA-I (-),DNP-I (A),orDNP-IIas

template (0).

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[image:6.495.250.443.376.609.2]

HUANG, NONOYAMA, AND PAGANO

dangerthateven aminor contaminationwith, for

example,

"late" RNAsequences in the

predomi-nately "early" cold RNA appliedfirst will mask

real differencesbased on the relativepopulations oftwokinds ofRNAspecies.

When chromatin from animal or plant tissues

is used as a template for synthesizing cRNA in

vitro, only a part of theDNAis transcribed (6,

10, 19). Chromosomal proteins persisting in a

complexwiththe DNA make specificportions of

it unavailable for transcription and account for

this effect. SV40DNA-bound protein may play a

similar role. However, the existence of viral

deoxynucleoprotein complexes and their

func-tional significance in vivoarestillopenquestions.

Tovalidate this concept, it would be necessary to find in permissive infected cells virus-specific

mRNA corresponding to thein vitrotranscripts ofDNP-I and DNP-II. If in lytic infection the

early viralgenes areexpressed beforeviralDNA

replication and the other late genes after DNA

replication (20,24, 27,29), thentheblockto

tran-scription

of lategenes islifted so that the entire

genomeis ultimately expressed in lytic systems. In contrast, in transformed systems such as SV 3T3cells, theentiregenomeispresent but the late

functions arenever expressed. Sauer andKidwai (27) haveshownthat thesections of viralgenome

transcribed in SV40-transformedGMK (18) cells

are twice as large as in SV40-transformed 3T3

cells. Therefore, Oda and Dulbecco (20) and Sauerand Kidwai(27) invoketwotranscriptional control

mechanisms:

theblocktotranscription of lategenesequences thatoccursin

permissive

cells beforeviralDNA

replication

isduetosome prop-erty of the virus, whereas in thetwo systems of transformed cells cited the

persistent

block to

transcription ofmostlate genes is based on

cel-lularproperties.

A deoxynucleoprotein complex similar to

DNP-I might be released in vivo after

un-coating ofthe virus. Then, after removal ofthe

capsid,

the

nucleoprotein

byitselforin

coopera-tion with some other factor

might

repress late

genefunctions and

permit

onlythe

expression

of

early genes. After this, the internal viralprotein is removed from the viral DNA, perhaps by an

early gene product. Viral DNA

replication

and

late gene expression are then favored. There is another

possibility. Newly synthesized

viral

pro-teinmaybindtonewly

synthesized

viralDNA at

theearlygenesitessothat thelate genes involved withvirus maturation continuetobefavored. This

possibilityisunlikely, however,

if,

as Alonietal. (1) showed, early mRNA is present throughout

theinfectiouscycle.

The mammalianRNA polymeraseusedin this

work

(RNAP-II)

was highly active,

especially

when denaturedDNA was used as template, its

specific activity being as high or higher than the E. coli polymerase. A template preference for

native DNA may depend on recovery of some

factor lost during DEAE-cellulose chromatog-raphy(B.Sugden and W.Keller,personal commu-nication). Since the binding sites ofE.coli enzyme

and mammalian enzyme may not be identical,

with three to nine sites for the bacterial enzyme butperhapsonly one for themammalian enzyme

(14),

the useofRNA polymeraseofmammalian

origin may be crucial. Viral nucleoprotein may

onlyblock a specific initiationsite for

transcrip-tion, which could

still

allow theE.colipolymerase

to initiate nonorthodox transcription and yield cRNAthat mightapproximatebut not match the

corresponding mRNA ininfected

cells.

The high

activity of the newly available mammalian polymerases

will

allow synthesis of adequate

3H-cRNA

from

DNP-I

for competitive

hybridi-zation experiments with "early" mRNA from

SV40-infected cells.

ACKNOWLEDGMENTS

We thank Norman S.-T. Chen for assistance and Joseph

SambrookandRobertWeinberg for helpful criticism. B. Sugden

andW.Keller generously gave details for the preparation of the

mammalian RNA polymerases.

This workwassupportedby a grant to theUniversityof North

Carolinafrom theAmericanCancerSociety(IN15-LInstitutional

Grant),by Public HealthServicegrant 5SO0 FR05406from the

Division of Research Facilities and Resources, and by grant

VC-48 from the American Cancer Society. Joseph S. Pagano holds Public Health Service Research Career Development Award 5 K04 Al 13516from theNational Institute of Allergy

andInfectious Diseases.

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