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0022-538X/82/120772-10$02.00/0

Accurate Transcription of Simian Virus 40 Chromatin in

a

HeLa Cell Extract

JOHN N.BRADY, MICHAEL RADONOVICH,ANDNORMAN P. SALZMAN*

Laboratory of Biology of Viruses, National Institute of Allergy and InfectiousDiseases,Bethesda, Maryland

20205

Received 17 May1982/Accepted 23 August 1982

During simian virus 40 viral maturation,a series ofmodifications occurwhich

alter the composition of viral nucleoprotein complexes. As a consequence, the

chromatin that is extracted from extracellular simian virus 40 virions exhibits

properties thataresimilar tothose of transcriptionally active eucaryotic

chroma-tin.Theinfluence of this chromatin structureonspecificRNAinitiation by RNA polymerase IIwasexaminedby using the in vitro HeLa cellextractof Manleyet

al. (Proc. Natl. Acad. Sci. U.S.A. 77:3855-3859, 1980). The 5' ends of RNA

transcripts were positioned by the "run-off' assay, in which transcripts extend from the initiation sitetotermination sitescreated by restrictioncleavage and by

S1 nuclease analysis, using DNA probes labeled at their5' termini. Two major

earlyRNAtranscripts, which originated atmappositions 5,240 ± 10and5,145 ±

10, andtwomajor late RNA transcripts, which originatedatmappositions 325 ±

10 and 185 ± 10, were identified. Transcripts were initiated with comparable

relativeefficienciesatthesame5' site wheneither purifiedDNAorchromatinwas usedasthe template. Our resultssuggestthat extracellular simian virus40virion

chromatin modifications do notregulate simian virus 40promoter selection but

function to increase the accessibility of RNA promoter sequences to RNA

polymerase IIand allowefficient elongation of theRNAchain afterthe initiation

event.

The DNA of eucaryotic cells is associated with octomers of four histone proteins which condense the DNAinto the basicrepeatunit of chromatin, the nucleosome (29). The

nucleo-some structure effectively blocks the access of

enzymes to DNA and therefore inhibits RNA

chain initiationandelongation (7, 9, 46). Recent

studies have shown that changes in chromatin

structure occur asgenes areactivated for

tran-scription; thissuggeststhat chromatinstructure

isonedeterminant ofregulation oftranscription

ineucaryoticcells(6,8, 10, 12,16,25,32, 33,37,

44, 47,48). Analyses of severalgeneticlocihave

demonstrated that during periods of

transcrip-tionalactivity, chromatin DNAbecomes

hyper-sensitive to DNase I, apparently due to modifi-cations in the chromatin structure, including

histone acetylation, decrease in H1 stoichiom-etry,and interaction of nonhistoneproteinswith chromatin.

Two in vitro RNA transcription systems

which accurately transcribe purified DNA

tem-plateshave beendeveloped recently. Weiletal.

(43) useda combination ofpurified RNA

poly-merase II and a crude HeLa cell cytoplasmic

extract toprovide specifictranscriptionof DNA

templates, whereasManley etal. (26)promoted

specifictranscription byadding DNAtemplates

to aconcentrated HeLawhole-cellextract.

Us-ingavariety ofDNAtemplates, several investi-gators have demonstrated that both of these soluble systems recognize specificDNA

regula-tory sequences(e.g., theGoldberg-Hogness

se-quence TATA) and that RNA chain initiation

starts atsitesidenticaltothose thatoccurin vivo

(2, 18, 20, 21, 27, 38, 41, 42).

We have previously reported that the 110S

chromatin complex isolated from extracellular

simian virus 40 (SV40) virions is an efficient template forRNA transcription byEscherichia coli RNApolymerase(4, 5). We hadnot charac-terized the suitability of this complex as a

tem-plate forthehomologous eucaryoticRNA

poly-merase II or determined if the chromatin

structurealtered theabilityof RNApolymerase

II torecognize promoter sequences andinitiate

specific RNA synthesis. In this paper, we

de-scribe the transcription of extracellular virion chromatin by RNA polymerase II in the HeLa

extractofManleyet al. (26).

MATERIALSANDMETHODS

Viruses and cells. SV40 strain 776 was originally

obtained from K. Takemoto, National Institute of

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Allergy and Infectious Diseases. Theviruswasgrown

inBSC-1 cells.

Infection andpurification of SV40 virions.Confluent

monolayers of BSC-1 cells maintained in Eagle mini-malessential medium supplemented with 0.03% gluta-mine, 10% fetal calfserum, streptomycin, penicillin, ampicillin, andmycostatinwereinfected withSV40at amultiplicity of infection of 10PFU/cell. After2 h of

adsorption at 37°C, Eagle minimal essential medium

supplemented with 2% fetal calf serum was added. Whencelllysiswascomplete(approximately9days), theinfected cells and supernatantwerecollected,and

the viruswaspurifiedaspreviously described (5). Preparation of chromatin complexes and conditions for in vitro RNAsynthesis. Preparation of SV40

chro-matincomplexeswascarriedoutbyethylene glycol-bis(O-aminoethyl ether)-N,N'-tetraacetic acid

(EGTA)-dithiothreitol dissociation ofSV40 virionsas

described previously (4, 5), except that the NaCl

concentration was decreased to 100 mM. HeLa cell extractswereprepared bythemethodofManleyetal.

(26), using cells thatwereharvestedatacell concen-tration of5 x 105to6x 105 cellspermlinstead of 8 x 105cellspermlasoriginally described. The concentra-tion of template DNArequired for specific transcrip-tionwasdecreased whenthe cells wereharvestedat the lower cell density. Each standard transcription reaction mixture (60 ,ul) contained 10 mMHEPES

(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)

(pH 7.9), 8 mM Tris (pH 8.6), 50 mM KCl, 16 mM NaCl, 6.25 mM MgC92, 0.8 mM EGTA, 0.05 mM EDTA, 1.5 mMdithiothreitol, 0.16 mM phenylmethyl-sulfonyl fluoride, 0.008% Triton X-100, 8.5%glycerol, 0.4 mM ATP, 0.4 mM GTP, 0.4 mM CTP, 0.04 mM UTP,20,uCi of [c_-32P]UTP (500 Ci/mmol), and 0.5 to 2.5 ,ug of DNAas chromatin orpurified DNA. Tran-scriptionassaymixtureswereincubated for 60 minat 30°C. DNase I(RNase-free; Worthington Diagnostics) was then addedto afinal concentration of15 ,ug/ml, and the reaction mixturewas incubated for 5 min at 30°C. Sodium dodecyl sulfate (SDS)wasthen addedto afinalconcentrationof 0.2%,and thiswasfollowed by the addition of 25 ,ug of proteinase K per ml and incubation at 30°C for 15 min. The RNA was then purifiedasdescribed by Lavialleetal. (24).

Analysis of RNA transcripts by glyoxalgel

electropho-resis.SV40 DNA fragments whichwereusedas size markers were generated by restriction endonuclease cleavage ofsuperhelical SV40DNA with (i) BamHI,

(ii) HinFI, or (iii) BamHI and HpaII. Anequimolar mixture of the three digests was labeled at the 5' termini with phage T4 polynucleotide kinase and

[y-32P]ATPas described by Maxam and Gilbert (28).

Purified RNAsamples and DNA markerswere dena-turedbyglyoxalasdescribed by McMaster and Carmi-chael (30). Denatured samples wereelectrophoresed

on a 1.5% agarosegel (10 mM phosphate buffer, pH

7.0)at150V(50 mA) for 2.5 h. The gelwasfixed with

1% streptomycin for 30 min at room temperature, dried, and exposedtoX-rayfilm.

S1nucleaseanalysisof RNAtranscripts. SV40DNA

fragmentswerelabeledattheir 5'terminiasdescribed

above. RNAtranscripts and the labeled DNA probe weresuspended in 20 ,ul ofasolution containing0.4 M

NaCl, 0.04 M PIPES

[piperazine-N,N'-bis(2-ethane-sulfonic acid)] (pH 6.4), 1.25 mM EDTA, and 80% formamide and heatdenatured by incubationat85°C

for5min. Thesampleswerethentransferred

immedi-atelyto a50°Cwaterbath and incubated foratleast 3

h. Hybridization reactions were stopped by adding9

volumes of a solution containing 0.25 MNaCI,0.03 M

sodium acetate (pH 4.6), and 1 mM ZnSO4

(pre-equilibratedto4°C). SI nucleasewas addedtoafinal

concentration of 5,000 U/ml,and thereactionsample

was incubated for 45 min at 30°C. Protected DNA fragments were thenpurified and analyzed byglyoxal

gelelectrophoresis.

Analysis of the superhelical structure of chromatin

DNA.A 2.5-,ugsample of SV40 chromatinorpurified

SV40 F, DNAwas incubated with the soluble HeLa

cellextract asdescribedabovefortranscription

reac-tions.After0, 0.5, 5, 15, 30,and60 minofincubation,

portionswereremoved, andthereactionwasstopped

by adding an equal volume of20 mM EDTA-0.4%

SDS. Sampleswerethen treated withproteinaseK(50

,ug/ml) for 1 h at 37°C. The DNA was purified and

analyzed by gelelectrophoresisona1.5%agarosegel

at50Vfor16to20 h witha40 mMTris-acetate(pH

7.2)-20 mM sodium acetate-1 mM sodium EDTA

buffer. The gelwasstained withethidium bromide and

photographed underaUVlightsource. Todistinguish

betweennicked circularandcovalently closedcircular SV40 DNA molecules, DNA samples were

electro-phoresedin the presenceof6 ,ugof ethidium bromide

perml.

Isolation of RNAby using Southern blots.The sepa-rated strandsoftwo SV40 DNA fragmentsgenerated

by cleavagewithBamHIand HpaII restriction

endo-nucleases were electrophoresed and transferred to

nitrocellulosepaperbythemethod of Southern(36), as

previously described (3). Hybridization of

[c-32P]UTP-labeled RNAs was carried out in a buffer

containing 50% formamide, 2 mM EDTA, 0.75 M

NaCl, 0.5% SDS, and 200 p.goftRNA permlin 100

mMTris(pH7.4)for16hat37°C. Hybridization strips

werewashed with10successiverinse solutions (5 ml

each)containing0.2%SDS and2 mM EDTAin 10 mM

Tris (pH 8.0). Nitrocellulose strips were dried and

exposedtoX-rayfilm. Individual bandswereexcised,

and the RNA waselutedby incubation at 85°C for5 min inhybridization washbuffer.

RESULTS

Stability of SV40 chromatin in HeLa cell ex-tract. HeLacell extracts prepared by the

meth-od of Manley et al. (26) contain topoisomerase. This enzyme removes superhelical turns from covalently closed DNA molecules by introduc-ingasingle-strand nick inthe DNA andresealing

thenickafter the torsional tensionof the double

helix is relieved, allowing the release of one superhelical turn each time that the cycle is

repeated. By virtue of the torsional constraints that nucleosomes exert on the DNA duplex,

chromatin structure prevents the loss of super-helical turns (17, 29). Therefore, an analysis of DNAstructureafterincubationwith the extract

provides anassay for the stability ofthe SV40

chromatin complex. Control superhelical SV40 DNA

(F1)

and SV40 chromatin were added to

complete transcription assayreaction mixtures.

Sampleswereremovedatdifferenttimes,

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774

teinized, and analyzed by agarose gel

electro-phoresis. The action of topoisomerase on F1

SV40DNAwas apparent after 0.5 min of

incu-bation since most of the superhelical DNA had

been converted either to intermediates with a

lower superhelix density or to relaxed

covalent-ly closed DNA molecules and nicked circular DNA(SV40DNA

Fll)

molecules (Fig. 1,lanes A

and B). The latter two forms of SV40 DNA comigrated in this gel system. Further incuba-tion of purified DNA resulted in complete con-version ofF1 DNA to relaxed covalentlyclosed

DNA molecules and nicked circular molecules (Fig. 1, lanes C throughF). In contrast, incuba-tion of chromatin DNA under identical condi-tions revealed that the superhelical DNA was

stable in the presence of the enzyme (Fig. 1,

lanes G through L). After 0.5 and 5 min of

incubation, no significant change in superhelical

DNA structure was detected in the chromatin

samples. After10, 30,and 60 min ofincubation, there was a partial loss of the FI superhelical DNA band, with a concomitant increase in the amountofFII orrelaxed covalentlyclosed circu-lar DNA. To differentiate between F11 and

re-laxed covalently closed circular DNA

mole-cules, duplicate DNA samples were

electrophoresedin an agarosegel containing0.6 mg of ethidium bromide per ml. This analysis demonstratedthat inadditiontoDNAmolecules

with an unaltered superhelix density, the only DNA species present in the chromatin samples was DNA F1l(data not shown).

The stability of SV40 chromatin during incu-bation with HeLa cell extracts was also ana-lyzed by velocity sedimentation and isopycnic

banding in CsCl. [3H]thymidine-labeled SV40 virions were dissociated and incubated with the soluble HeLa cell extract as described above. After incubation, the samplewascompared with control chromatin on isokinetic 10 to 30%

su-crosegradients and CsCl density gradients. The

results of these experiments demonstrate that chromatin structure was preserved since the sedimentation coefficient (110S) and buoyant density (1.40 g/cm3)were notaltered after incu-bation with the extract (data not shown). The fact that no free DNAwas detected in eitherof

these analyses also demonstrates that the

F1l

circular DNA molecules noted above were in a chromatin structure before deproteinization of the DNA forgel analysis.

Transcription of SV40 chromatin in soluble HeLa cell extracts. SV40 virions were

dissociat-1*

II

FIG. 1. Superhelicalstructureof chromatinDNAafter incubation withHeLacellextract.A2.5-,ugsampleof

SV40 chromatinorSV40F,DNA wasincubatedinthe standardtranscriptionmixture,whichincluded allfour

nucleotide triphosphates. After 0, 0.5, 5, 15, 30, and 60 min ofincubation, portions were removed, and the

reactionwasstopped by addinganequalvolume of 20mmEDTA-0.4%SDS.DNAsampleswerethentreated

withproteinaseK(50,ug/ml)for1hat37°C,purified,andanalyzed by gel electrophoresison a1.5% agarosegel

at50Vfor16 to 20 hwitha40 mMTris-acetate (pH 7.2)-20mM sodiumacetate-1 mM EDTA(sodiumsalt)

buffer. Thegelwasstained withethidium bromide andphotographedunderaUVlightsource. LanesAthrough

F,zerotime and0.5-, 5-, 15-, 30-, and 60-minpurifiedDNAincubationsamples,respectively;lanesGthrough L,

zerotime,and0.5-, 5-,15-, 30-,and60-minchromatinDNAincubationsamples,respectively.IandIIindicate the migration positions of SV40 superhelical (F1) and nicked circular (F,,) DNAs, respectively. Relaxed

covalentlyclosed circularDNAmigratedatthesamepositionasF1, DNA.

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EcoRI* Bam HI

l I

I

AvaI1

Ta Ava 11 Hpa11Hind 11 HaeII* Eco RI

0 I I

1

1

lll1

I Il I

2.0 2.5 3.0 3.5 4.0 4.5 5.0 3.45

EARLY --I

.5 1.0 1.5

1.6

I 1.45

[image:4.491.97.397.76.170.2]

LATE

FIG. 2. TranscriptionalmapofSV40 DNA.ThediagramrepresentsanSV40 DNAtemplatewhich hasbeen

digested with endonuclease EcoRI (Fig. 3). The orientations andlengthsof RNAtranscriptsfromearlyandlate

SV40 promoters are shown below the map. The restriction endonucleasesites used fortranscription "run-of'

assays(single asterisks) (Fig. 3 and 4) andS1nucleaseanalysis (doubleasterisks)(Fig. 6)areindicated abovethe

map.The mapcoordinates andRNAtranscriptlengthsareindicated(inkilobases).

ed with EGTA and dithiothreitol as described

previously (4, 5), and the chromatin DNA was

restricted with endonuclease EcoRI (map posi-tion 1,783) (Fig. 2). The restricted chromatin

template was then added to the transcription reaction mixture at different concentrations

ranging from 10to50,ug/ml.Afterincubation at

30°C for 1 h,the RNAtranscriptswerepurified and analyzed by glyoxal gel electrophoresis

(Fig. 3). The majorRNA bandsdetectedin this

analysis were transcripts of approximately 3,450, 1,600, and 1,460 nucleotides. Assuming that transcription terminates at the

endonucle-ase restriction site, the 3,450-nucleotide

tran-script is consistentwithan RNAmolecule

origi-natingneartheearlySV40promoter and

extend-ingto the EcoRl restriction site. Similarly, the 1,600- and 1,450-nucleotide transcripts would

originatefrom late SV40 promoters. The radio-active band migrating at 1,800 nucleotides was not aSV40 transcript, but apparently was gener-ated by radiolabeling of the endogenous RNA present in the extract (26). The lengths of all RNAtranscriptshavebeencorrectedby5.6%to compensate for the different migration rates of RNAand DNA inglyoxal gels.

Thesurprisingaspectof this experimentwas

thataccurateinitiation oftranscription onSV40 chromatin occurred over awiderange of DNA concentrations. This is in directcontrast to the

strict DNA concentrationrequirementfor accu-rateinitiation oftranscriptionon nakedDNA,as

noted by Manley et al. (26) in the original description of the soluble transcription system.

We observed asimilar DNA concentration

de-pendence in our analysis of transcription of purified DNA templates. Specific transcription from SV40 early and late promoters occurred

only over averynarrowrange (20 to30,ug/ml)

when the template was purified DNA. Increas-ingpurified DNAconcentrations to 40 to 50,ug/

mlresultedina decrease inspecifictranscription

and aconcomitant increase innonspecific tran-scription(datanotshown).Transcription of both

DNAand chromatinatconcentrations lessthan

10 ,g/ml reduced transcription below the level of detection.

We havepreviously shown that extracellular SV40 virion chromatin is an efficient template

A B C D E F

5,243

3,036

2,190

- 1,087

768

uI 533

FIG. 3. Transcriptionof SV40 chromatin in HeLa

cell extracts. SV40 virions were dissociated by

treat-mentwith 5 mM EGTA-3 mM dithiothreitol-100 mM

NaCl in 50 mM Tris (pH 8.7) for 30 min at 32°C. After

dissociation, MgCl2was added to a concentration of 6 mM, and the chromatin template was restricted by

incubationat32°C for 1.5 h in the presence of 5 to 10 U

ofEcoRI per ,ug of DNA. [a-32]UTP-labeled RNAs

were synthesized in standard reaction mixtures con-taining 10 ,ug (lane A), 20 Fg (lane B), 30 ,ug (lane C),

40 ,ug (lane D), or 50 ,ug (lane E) of the

EcoRI-restricted chromatin template perml. Purified RNA

transcripts were purified and denatured with glyoxal

asdescribed by McMaster and Carmichael (30).

Dena-turedsamples wereelectrophoresed in a 1.5% agarose

gel in 10 mMphosphate buffer (pH 7.0) at 150 V (50

mA)for 2.5 h. Gels were fixed with 1%streptomycin

(30min,25'C),dried, and exposedtoX-ray film. Lane

F contained a 32P-end-labeled DNA marker. The

lengths of all RNA transcripts have been corrected by

5.6%tocompensatefor the differentmigrationratesof

RNAand DNAmolecules in glyoxalgels.

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776 BRADY,

for E. coli RNA polymerase (4, results were observed in a compan

transcriptional efficiencies of chro

SV40DNAin theextractof Manleye

quantitative analysis ofdensitometr [a-32P]UTP-labeled transcript auto

andhybridization data indicated thati, DNA concentrations, the chromatii

was approximately 75% as efficien

DNA(data notshown).

Termination of transcription occur!

tion endonuclease sites. To determir termination of transcriptionoccurrec

tion endonuclease sites, the chromat was restricted with either (i) EcoRI, and BamHI, or (iii) EcoRI and Hi restriction, the templates were ad( transcription reaction mixtureatacoi

of 30 ,xg/ml and incubated at 30°C f

The results of this experiment oriente tion oftranscription and demonstrate scription terminates at therestrictioi 4). Transcription of EcoRI-restricted (Fig. 4, laneB) produced dominantI

A B C D F

FIG. 4. Polarity and termination of ii

chromatin template transcripts. SV40 dissociated as described in thelegend t

restricted with either EcoRI (lane B),

BamHI (lane C), orEcoRI and HaeII

32P]UTP-labeled RNAsweresynthesizec

reaction mixtures, purified, andanalyze

gel electrophoresis. Lanes A and Ecor

markers.Thelengthsof all RNA transcril corrected by 5.6% to compensate for

migrationratesofRNA and DNA molecu

gels.

5). Similar of 3,450, 1,600, and 1,460 nucleotides.

Tran-ison of the scriptionof EcoRI-BamHI-restricted chromatin matin and (Fig. 4, lane C) produced RNA transcripts of tal. (26).A 2,700, 1,600, and 1,460 nucleotides. The specific

ic scans of decrease in the size of the 3,450-nucleotide

radiograms transcript to approximately 2,700 nucleotides atoptimum suggests not only that termination occurred at n template therestriction site but also that this RNA tran-t as SV40 scriptoriginatedat the earlySV40promoterand proceeded counterclockwise toward the EcoRI

satrestric- site. Transcription of the

EcoRI-HaeII-restrict-ne whether ed chromatin generated RNA transcripts of i atrestric- 3,450, 650,and515nucleotides (Fig. 4, laneD). :in complex Thespecificdecrease inlengthof the1,600- and (ii) EcoRI 1,460-nucleotide transcripts suggests that these

aeII. After transcripts originated at late SV40 promoter

ded to the sites andproceeded clockwise toward the EcoRI

ncentration site. An RNA transcript of approximately 850 for 60 min. nucleotides, which did not correspond to any of dthe direc- thetranscripts discussed above, was

reproduci-d that tran- bly observed in the EcoRI-HaeII transcription n site (Fig. samples. Although the origin of this transcript

chromatin was notstudied in detail, a preliminary analysis

RNA bands suggested that it is transcribed from the early SV40 strand and originates near map position

1,680.

Orientation ofRNA transcripts by hybridiza-tion,elution, andglyoxalgelelectrophoresis.

Re-striction of SV40DNAwithHpaII andBamHI,

5f243

followed by strand separation on agarose gels, allows for the separation of the main coding 3036 sequencesfor

early

andlate SV40 mRNAs

(3).

Hybridization of RNA transcripts synthesized

27

it90 from EcoRI-restricted SV40 chromatin to the 2 early/late Southern blot resulted in the pattern 1,829 shown inFig.5, lane A. RNAhybridizationwas

predominantly to the EA and LB DNA

frag-ments, which correspond to the major coding

0(J87 sequences of early and late mRNAs,

respective-ly. When the RNA that hybridized to the four _7 8. DNA fragments was quantitated, 70 to 80% of

768

the RNA hybridized to the EA and LB DNA

fragments. To determine which RNA species

533 hybridizedtothespecificDNAfragments,RNA was eluted from each fragment, purified, and

analyzedby glyoxal gelelectrophoresis.

Togeth-er with datapresented above, the distinct

hy-235 bridizationof the3,450-nucleotideRNA toDNA

fragment EA(Fig. 5, laneC)and the

hybridiza-n vitro SV40 tion of the 1,600- and 1,460-nucleotide RNAs to virions were the LB DNAfragment (Fig. 5, laneE) demon-.o Fig. 3 and stratedthat these RNAscorrespondtoinitiation

EcoRI and of RNAsynthesis at early and lateSV40

promo-(lane D). [a- ters, respectively. The heterogeneous smearof

din standard RNA in Fig. 5, lane C, apparently was due to

d byglyoxal degradation of the 3,450-nucleotide RNA

spe-ts

havebeen cies. The

light hybridization

of the

3,450-nucleo-the

different

tide RNAtoDNA

fragment

EB and the

hybrid-les inglyoxal ization of the 1,600- and1,460-nucleotide RNAs

to DNA fragment LA were due tooverlaps of

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ACCURATE SV40 777

AB C D F

EA

--LA

LB

EB

FIG. 5. Hybridization, elution, and

electrophoresis of RNAs synthesized froi

matin. RNAswere synthesized froman

matin reaction mixture and purified. [c

beledRNAswerehybridizedtothesepa

ofaBamHI-HpaIIdigest ofSV40(3)in

buffercontaining 50% formamide, 2 mM

MNaCl, 0.5% SDS, and 200 ,ug of tRNA

mMTris (pH 7.4) for 16 hat37°C. Hybric

werewashed with 10 successive rinsesc

each) containing 0.5% SDS and 2 mM ED

Tris (pH 8.0). The hybridization stripsw

exposedtoX-rayfilm. Radioactive band

ized andcutout,andthe radioactiveRN

by incubationat85°C for 5min in hybri(

buffer. RNA transcripts were then puril

lyzed by glyoxal gel electrophoresis. La

late hybridization (EA, early strand, A fi

latestrand, Afragment; LB, late strand

EB, early strand, B fragment); lanes (

[a-32P]UTP-labeled RNA transcripts h

EA,LA, LB, and EB DNA fragments,

lanes BandG, DNA markers.

RNAsequences atthe3' end ofthe

and the 5'end of thelate RNA. Si nudease analysis of RNA trai

determine thepositionof theinitiatio

RNA transcripts moreprecisely, we

Si nuclease mapping of unlabeled scripts with several DNA fragmentv their5' termini (Fig. 6). Hybridizati

matin RNAtoAvaIl fragmentD lab 5'termini(nucleotides 5,123and 561

the protection ofthree DNAfragm4 230, and 375(lower bandofdoublet)

(Fig. 6a, lanes D and E). The 43( DNAfragmentwasnotareproducib ase fragment and appeared to be tl

minor contamination in the probe.

tentative location of RNA initiatioi above), the 115-nucleotide bandwa:

with anearlyRNAtranscript thato

Gs map position 5,240 ± 10, whereas the 230- and 375-nucleotide bands correspondedtolate RNA

5,243 transcripts thatoriginated atmappositions 325

± 10 and 185 ± 10, respectively. These map 3,036 positionswere confirmed

by performing

a simi-lar S1 nuclease analysis with DNA fragments

2,190 labeledatthe

TaqI

site(Fig. 6a,lanes Gthrough

_

m.w.1829 L) (mapposition 4,741),theHpaIIsite(Fig. 6b,

lanes A through D) (map position 349), orthe Hindll site (Fig. 6b, lanes E through H) (map

#NO 1,087 position473). Briefly, the TaqI probe protected

two bands of 495 and 410 nucleotides (Fig. 6a,

~EM 768 lanesJ and K) the HpaII probe protectedaband _ 533 of 170 nucleotides (Fig. 6b, lanes C and D), and the HindIIprobe protected bands of 285 and 145 nucleotides (Fig. 6b, lanes G and H). Since the

structure of linearized chromatin might allow

ai 235 recognition of promoter sequences which are

notaccessibleinchromatinthat contains a cova-glyoxal gel lently closed circular DNA, each analysis

in-mSV40 chro- cluded an RNA samplesynthesized from uncut EcoRI chro- SV40chromatin. Our resultsdemonstrated that

X-32P]UTP-1a- specific initiation oftranscription occurred on

rated strands both intact and restricted SV40 chromatin and

hybridization thattheseinitiation sitescorrespondedto

initia-lEDTA,

0.75 tion sites used in vivo (2, 20, 27, 39). It is of

permlin100 interest that whereas the levels of RNA

synthe-)lutions (5ml sis fromthe late

SV40

promoter were

compara-)TA in 10 mM ble in intact and restricted SV40 chromatin ere dried and templates, RNA synthesis from the early SV40

Is were local- promoter appeared tobe enhanced in the intact

[A was eluted chromatin template. The significance of this

dlization

wash observation ispresently being investigated.

fied and

ana-me A, Early/ DISCUSSION

B

fragment;

Our results demonstrate that extracellular C

through

F SV40virion chromatin is

accurately

transcribed ybridized to in the in vitrotranscription systemofManleyet

respectively; al. (26). The lengths of RNA transcripts (Fig. 3 and 4), the hybridization patterns (Fig. 5), and

the S1 nuclease mapping data (Fig. 6) demon-early RNA strate that two major early RNA transcripts, which originate at map positions 5,240± 10 and nscripts. To 5,145 ± 10, and two major late transcripts,

nsite of the which originate at map positions 325 ± 10 and e performed 185 ± 10, are synthesized from the chromatin RNA tran- template. These map positions in turn

corre-s labeled at spond to RNA initiation sites which have been ion ofchro- mappedpreviously by either primer extension or yeledatboth Si nuclease analysis (2, 20, 27, 39). In contrast ) resulted in totranscription of purified DNA templates, ac-ents of115, curate transcription of SV40 chromatin is less nucleotides dependent upon the addition of specific DNA

)-nucleotide concentrations to the transcription assay

mix-leSi nucle- ture.

he result of Recent analyses of transcriptionally active From our and inactive eucaryotic genes suggest that one

n sites (see level oftranscriptional regulation isby

modifica-s consistent tion of chromatin structure. The nucleosome

riginated at structureof transcriptionally inactive eucaryotic

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83

FIG. 6. Sinucleaseanalysis of in vitro RNA transcripts. UnlabeledRNAtranscriptsweresynthesized from

EcoRI-restrictedorintact chromatin in standardreaction mixtures,exceptthat[a-32P]UTPwasomitted. Purified

RNAtranscripts andalabeledDNAprobewerelyophilized, suspended in20 ,ul of 0.4MNaCl-0.04 M PIPES

(pH6.4)-1.25mM EDTA-80% formamide,and heatdenatured byincubationat85°Cfor 5 min. Hybridization

samples were transferredimmediatelyto a50°Cwaterbath andincubated foratleast 3 h.Hybridization reactions were stopped by adding 9 volumes of 0.25 M NaCI-1 mM ZnSO4-0.03 M sodium acetate (pH 4.6)

pre-equilibratedto4°C. Sinucleasewasaddedto afinal concentration of 5,000U/ml, and the reaction mixturewas

incubated for 45 min at 30°C. Protected DNA fragments were then purified and analyzed by glyoxal

electrophoresis in a 2.0% agarose gel (10 mM phosphate buffer, pH 7.0) at 150 V (50 mA) for2 h. (a) Si

hybridizationwithAvallfragmentD(mappositions 5,123 and 561)orTaqI(mapposition 4,741)probe.LaneA,

AvaIl fragmentDprobe; lane C,AvaIl fragmentDprobehybridization, Si treated; lane D, AvallfragmentD

intact chromatinRNAhybridization,SItreated; lane E,AvaIl fragmentD-EcoRIchromatinRNAhybridization,

Sitreated; lane G, TaqIprobe;laneI, TaqIprobehybridization, SI treated;laneJ,TaqI-intactchromatinRNA

hybridization, S1treated;laneK, TaqI-EcoRIchromatinRNAhybridization,Si treated.LanesB, F,H, andL

containedDNAmarkers.(b)SihybridizationwithHpaII(mapposition 349)orHindll(mapposition473)probe.

LaneA, HpaII probe; lane C, HpaII-intact chromatin RNAhybridization, Si treated; lane D, HpaII-EcoRI

chromatin RNAhybridization, Si treated; lane E,HindIl probe, Si treated;laneG, Hindll-intact chromatin

RNAhybridization, Sitreated;laneH,HindII-EcoRIchromatin RNAhybridization,Si treated.Lanes BandF

containedDNAmarkers.

J. VIROL.

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genes consists of an octomer of four histones (H2A, H2B, H3, and H4) which are associated with 145 base pairs of DNA. Histone H1 inter-acts with this basic chromatin structure to con-dense the DNA into 25- to 30-nm higher-order chromatin fibers. (For a review of chromatin structure, see reference 29.) By virtue of the nucleosome structure, chromatin DNA is quite resistant to enzymatic activity and is an

ineffi-cient templateforeither the initiationor

elonga-tion reactions of RNA synthesis in vitro (7, 9, 46).Analyses oftranscriptionally active

eucary-otic genes, such as the genes for globin (44), ovalbumin (16), integrated viral genomes (6, 8, 12, 14, 15, 33), and the Drosophila GP-70 heat shock gene (47, 48), have demonstrated that thesegeneticloci reside within chromatinhaving an altered structure, as indicated by DNase I

hypersensitivity.Wuetal.(47,48) demonstrated thisclearly in theircomparisonof the transcrip-tionallyactive and inactive chromatinstructures

of the major heat shock protein gene of

Dro-sophila. When the GP-70 gene was

transcrip-tionally activated by heat shock, disruption in nucleosomal organization was evidenced by a

loss orsmearing of discrete DNAfragments in micrococcal nuclease digests. When the cells recovered from the heat shock and the GP-70 gene returned to the inactive state (47, 48),

micrococcal nuclease DNA ladderpatternswere

restored. Further evidence thattranscriptionally active chromatinis modifiedcomesfromrecent

studies by Weisbrod and Weintraub (45), who demonstrated that "active" nucleosomes may be separated from "inactive" nucleosomes by affinity chromatography with HMG proteins

bound to a solid matrix (45). Although these

experiments demonstrated a cleardifference in

chromatin structure, the basis of nuclease hy-persensitivity and differential binding of HMG proteins to transcriptionally active chromatin remains to be established. The contribution of chromatin modifications which include histone acetylation, loss of histoneH1, and nonhistone protein interaction with chromatin all require furtheranalysistoclarifytheir role in chromatin

transcriptional activation. Chromatin

modifica-tionsmayaffectpromoter selection or function primarily to "open" chromatin toallowentry of thepolymerase at the promoter site and subse-quent elongation of the RNA chain through nucleosomes.

We have recently reported that extracellular SV40 viralchromatin, which is similar to

tran-scriptionallyactiveeucaryoticgenes, has a "re-laxed" chromatin structure which makes the viral DNA highly accessible toenzymatic activi-ties(4, 5). The conversion ofSV40 chromatin to

therelaxedconformation occurs asintracellular

75S chromatin sequentially matures into a 200S

previrion, a 240S nuclearvirion, and finally an extracellular virion (4, 5, 11, 23). These studies demonstrate that the maturation process is

ac-companied by significant changes in chromatin composition, including increased acetylation of histones H3 and H4, the lossof histone H1, and thespecific association of nonhistone viral pro-tein with the chromatin complex. After in vitro dissociation ofnuclear and extracellular SV40 virions, we have shown by comparative enzy-matic analysisthatextracellular virion chroma-tin is nucleasehypersensitive and 16- to 20-fold more efficient as a transcriptional template (4). To determine what effect chromatin

modifica-tions might have on promoter selection, we compared thetranscription of extracellular viri-on chromatin and purified DNA in three

inde-pendentsystemswhich included(i)E. coli RNA

polymerase, (ii)purified polymerase II,and (iii)

HeLa cell extracts (Manley extract). Although

the utilization of specific promoters differed

qualitativelyin eachsystem,wefoundno

signifi-cant difference in chromatin and purifiedDNA

transcription patterns, which suggests that extracellular SV40 virion chromatin structure

regulates neither strand selection nor promoter

recognition bythe RNApolymerase.Our results suggest that SV40 virion chromatin modifica-tions, rather thanregulatingpromoterselection, functionprimarilytoincreasetheaccessibilityof RNApromoter sequencestopolymeraseIIand allow efficientelongationof theRNAchain after theinitiation event.

In contrast, intracellular SV40 chromatin,

which has quite different structural properties

thanextracellularSV40 virionchromatin2 appar-ently has the potentialtoaffect SV40 promoter

selection. Jakobovitsetal. have shown that the initiation specificity of E. coli polymerase is altered when transcription is carried out on

intracellular SV40 chromatin rather than on pu-rified DNA (22). Initiation oftranscription on the viralchromatin occurredpreferentially from the late strandin theregion close to the in vivo latepromoter. Thisobservation isstriking since

no E.coli promoters, which initiatesynthesis of RNA onthe late strand, havebeen mapped in thisregion ofthe genome (24). Precisely within the later promoterregion, approximately 20%of

the intracellularchromatincomplexescontain a

nuclease-sensitivegapped stretch of DNA which is not contained in a typical nucleosome

struc-ture. These results may suggest that in vivo transcription initiates withinthisregion on such gapped molecules.Theobservationthatactively transcribed eucaryotic chromatin also displays nucleosome-deficient stretches ofDNA makes

thismodelanattractive mechanism for promoter selection forsomegenes in eucaryotic cells.

TheSV40genome is divided into two sets of

VOL.44, 1982

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(9)

780 BRADY, SALZMAN

geneswhichare temporally regulatedduringthe

infection cycle. Before DNA replication, stable SV40 RNAtranscriptsare synthesized

predomi-nantly from the early gene which codes for the

T-antigens. After expression of the early gene

products and subsequentDNAreplication,

tran-scription occurs predominantly from the late

SV40genes which code for viral structural

pro-teins. Transcriptional analyses basedon in vivo and in vitro studies demonstrate that regulation

oftheSV40earlygeneis under the control of the

Goldberg-Hogness-like sequence TATTTA which is located 25 to 30 nucleotides upstream

fromthe majorRNA cap site and a72-base

pair

tandem DNA repeat sequence locatedmorethan 100nucleotidesupstreamfrom the cap site(1,

2,

18-20, 27). Expression ofSV40early mRNA is

repressed by its own gene product,

large

T-antigen (31, 35, 40). The regulatory sequences

whichcontrol expression ofthe SV40late genes

and the mechanism by which accumulation of late RNA transcripts is suppressed early in the

infection cycle are not clear at present. The

observation that both late and early SV40

pro-moters were efficiently expressed in the invitro

transcription system of Manley et al. suggests

that promoterstrengthcouldnot accountfor the 20- to 100-fold-lower amount of late mRNA

found during the initial stages of infection

(34).

Sinceour transcriptional analyses of extracellu-larvirionchromatindemonstrate thatchromatin modifications do not regulate promoter

selec-tion, it also appears

unlikely

that chromatin

structure can accuratelyaccountfor the

unequal

expression ofearly and late SV40mRNAs

dur-ingtheearlystages ofSV40infection. It appears

more plausible, as

previously

hypothesized

by

Birkenmeieretal. (3),thatinitiation of lateSV40

RNA synthesis occurs in vivo

during

the

early

stages of infection but stable

transcripts

are not

producedduetoattenuation oflate RNA

synthe-sis by cellular proteins or

by specific

degrada-tion of late RNA after

synthesis.

LITERATURECITED

1. Benoist, C.,and P.Chambon.1980. Deletionscoveringthe putativepromoterregionofearlymRNAsof simianvirus 40 do not abolish expression. Proc. Natl. Acad. Sci. U.S.A. 77:3865-3869.

2. Benoist, C., and P. Chambon. 1981. In vivo sequence requirements oftheearlySV40 promoterregion. Nature (London)290:304-310.

3. Birkenmeier, E. H., E. May, and N. P. Salzman. 1977. Characterizationof simian virus 40 tsA58transcriptional intermediatesatrestrictivetemperatures:relationship

be-tween DNA replication and transcription. J. Virol. 22:702-710.

4. Brady,J.,M.Radonovich,C.Lavialle,and N. P.Salzman. 1981. Simian virus 40 maturation: chromatin modifica-tions increase theaccessibilityof viral DNAtonuclease and RNApolymerase.J.Virol. 39:603-611.

5. Brady, J. N., C. Lavialle, and N. P. Salzman. 1980. Efficient transcription ofa compact nucleoprotein

com-plex isolated from purified simian virus 40 virions. J. Virol. 35:371-381.

6. Breindl, M., and R. Jaenisch. 1979. Conformation of Moloney murine leukemiaproviralsequencesin chroma-tinfrom leukemic and non-leukemic cells. Nature

(Lon-don) 277:320-322.

7. Bustin,M. 1978.Binding ofE. coli RNApolymeraseto chromatin subunits. Nucleic Acids Res. 59:925-932. 8. Chae, C., T. Wong,andR.Gadski. 1978. Transcription

processing, and structure of chromatin in SV40-trans-formed cells. Biochem.Biophys.Res.Commun.

83:1518-1524.

9. Cremisi,C.,A.Chestier,C.Dauget,andM.Yaniv. 1977.

Transcription of SV40 nucleoprotein complexesin vitro. Biochem. Biophys. Res.Commun. 78:74-82.

10. Defer,N.,M.Crepin, C. Terrioux, J. Krun,and F.Gros. 1979.Comparisonof non-histoneproteinsassociated with nucleosomes withproteinsreleasedduringlimited DNase

digestions.Nucleic AcidsRes.6:953-966.

11. Fernandez-Munoz, R., M. Coca-Prados,and M.-T. Hsu. 1979.Intracellular formsof simian virus 40nucleoprotein complexes.I. Methods of isolation andcharacterization in CV-1 cells. J. Virol.29:612-623.

12. Flint, S. J., and H. M. Weintraub. 1977. An altered subunitconfigurationassociated withactively transcribed

DNAofintegratedadenovirus genes. Cell 12:783-794. 13. Frolova, E., and E. Zalmanzon. 1978. Transcription of

viralsequencesin cells transformedby adenovirus type5. Virology89:347-359.

14. Frolova, E.,E.Zalmanzon,E.Lukanidin,andG.Georgiv. 1978.Studies oftranscriptionof viral genome in adenovi-rus5transformed cells.Nucleic Acids Res. 5:1-11. 15. Garber, E. A., and M. M. Seidman, and A. J.Levine.

1978. The detection and characterization of multiple

forms of SV40 nucleoprotein complexes. Virology

90:305-316.

16. Garel, A., and R. Axel. 1976. Selective digestion of

transcriptionally active ovalbumin genes from oviduct nuclei. Proc.Natl. Acad.Sci. U.S.A. 73:3966-3970. 17. Germond, J.,B.Hirt,P.Oudet,M.Gross-BelHard,and P.

Chambon. 1975. Folding of the DNA double helix in chromatin-like structures from simian virus 40. Proc. Natl. Acad.Sci. U.S.A. 72:1843-1847.

18. Ghosh, P., P. Lebowitz, R. Frisque, and Y. Gluzman. 1981. Identification ofapromoter componentinvolved in

positioningthe5'terminiof simian virus 40earlymRNAs. Proc.NatI.Acad. Sci. U.S.A. 78:100-104.

19. Gruss, P.,R.Dhar,andG.Khoury. 1981. Simian virus 40 tandem repeated sequences as an element of the early

promoter. Proc.Natl. Acad.Sci. U.S.A. 78:943-947. 20. Handa, H., R. Kaufman, J. Manley,M. Gefter, andP.

Sharp. 1981. Transcriptionof simian virus40DNA ina

HeLawhole cell extract.J.Biol.Chem. 256:478-482. 21. Hu, S.,andJ. Manley. 1981. DNAsequencerequiredfor

initiation of transcription in vitro from the major late promoter of adenovirus2. Proc.Natl.Acad. Sci. U.S.A. 78:820-824.

22. Jakobovits, E.,S.Saragosti,M.Yaniv,and Y.Aloni.1980. EscherichiacoliRNApolymerasein vitromimics simian virus40 in vivotranscription whenthetemplateisviral nucleoprotein. Proc. Natl. Acad. Sci. U.S.A. 77:6556-6560.

23. LaBella, F., andC. Vesco. 1980. Late modifications of simian virus40chromatinduringthelyticcycleoccurin

animmature form of virion.J.Virol.33:1138-1150. 24. Lavialie,C.,Y.Reuveni,M.Thoren,and N. P.Salzman.

1982. Molecularinteractionbetweensimian virus40 DNA and Escherichia coli RNA polymerase. J. Biol. Chem. 257:1549-1557.

25. Levy,B., W.Connor, andG. Dixon. 1979. Asubset of

trout testis nucleosomes enriched in transcribed DNA sequencescontainshighmobilitygroupproteinsasmajor structuralcomponents.J. Biol.Chem. 254:608-620. 26. Manley, J.,A. Fire,A. Cano,P.Sharp,andM.Gefter.

1980.DNAdependenttranscriptionofadenovirusgenes

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ACCURATE TRANSCRIPTION OF SV40 CHROMATIN

in a soluble whole cell extract. Proc. Natl. Acad. Sci. U.S.A. 77:3855-3859.

27. Mathis, D., and P. Chambon. 1981. TheSV40 earlyregion TATA box isrequired for accurate in vitro initiation of transcription. Nature (London)290:310-315.

28. Maxam, A. M., and W.Gilbert. 1977.A newmethodfor sequencing DNA. Proc. Natl. Acad. Sci. U.S.A. 74:560-664.

29. McGhee,J., andG.Felsenfeld.1980.Nucleosome struc-ture.Annu.Rev.Biochem.49:1115-1156.

30. McMaster, G., and G. Carmchael. 1977. Analysis of

single-and double-stranded nucleic acids on

polyacryl-amide andagarose gels by using glyoxal and acridine orange. Proc.Natl.Acad.Sci.U.S.A. 74:4835-4838. 31. Myers,R., D. Rio, A. Robbins, and R.TUan.1981. SV40

geneexpression ismodulated by thecooperative binding

of Tantigen to DNA. Cell 25:373-384.

32. Nelson, D., M. Perry, and R.ChaLldey.1979. Acorrelation

between nucleosome spacer region susceptibility to DNAse I and histone acetylation. Nucleic Acids Res. 6:561-574.

33. Panet, A., and H. Cedar. 1977. Selectivedegration of

integratedmurine leukemiaproviralDNAby

deoxyribo-nucleases.Cell11:933-940.

34. Parker, B. A., and G. R. Stark. 1979.Regulation ofsimian virus 40 transcription: sensitive analysis of the RNA

speciespresentearly ininfections byvirusorviral DNA. J.Virol. 31:360-369.

35. Rio, D., A. Robbins, R. Myers, and R. Than. 1980.

Regulation of simian virus 40 early transcription bya purifiedTantigen.Proc. Nati. Acad. Sci. U.S.A. 77:5706-5710.

36. Southern,E.1975.Deletion ofspecificsequences among DNAfragmentsseparated by gelelectrophoresis.J. Mol.

Biol.98:503-517.

37. Stadler, J., T.Seebeck, and R. Braun. 1978.Degradation oftheribosomalgenesbyDNase I inPhysarum polyce-phalum.Eur. J.Biochem.90:391-395.

38. Talkington,C.,Y.Nishioka,and P. Leder.1980. In vitro

transcriptionof normal, mutant and truncated mouse

a-globingenes. Proc. Natl. Acad.Sci. U.S.A.77:7132-7136. 39. Thompson, J. A., M. F. Radonovich, and N. P.Salzman. 1979. Characterization of the 5'-terminal structure of simianvirus 40 early mRNA's. J. Virol. 31:437-446.

40. Tijan, R. 1978. The binding site onSV40DNAfor a T

antigen-related protein.Cell13:165-180.

41. Wasylyk, B., R.Derbyshire, A. Guy, D. Molko, A. Roget, R. Teoule, and P.Chambon. 1980. Specific in vitro tran-scription of conalbumin gene is drastically decreasedby

single-point mutation in T-A-T-A box homology se-quence. Proc.Natl. Acad. Sci. U.S.A. 77:7024-7028.

42. Wasylylk,B., C.Kedinger, J. Corden, B. Brison, and P.

Chambon. 1980.Specific in vitro initiation of transcription on conalbumin and ovalbumin genes and comparisonwith adenovirus-2 early and late genes. Nature (London) 285:367-373.

43. Weil,P., D.Luse, J. Segall, and R. Roeder. 1979. Selective and accurateinitiation of transcription at the Ad2 major late promoter in a soluble system dependent on purified RNA polymerase II and DNA. Cell 18:469-484. 44. Weintraub, H., and M. Groudine. 1976. Chromosomal

subunits in active genes have an altered conformation. Globin genes aredigested bydeoxyribonuclease I in red blood cell nuclei but not in fibroblast nuclei. Science 193:848-856.

45. Weisbrod, S., and H. Weintraub. 1981.Isolation of active-ly transcribed nucleosomes using immobilized HMG 14 and 17 andanalysis of a-6 chromatin. Cell 23:391-400. 46. WilHamson,P., and G. Felsenfeld. 1978.Transcriptionof

histone-coveredT7DNA byEscherichia coli RNA poly-merase.Biochemistry17:5695-5704.

47. Wu, C., P.Bingham,K.Livak, R.Holmgren,and S.Elgin.

1979. The chromatin structure ofspecific genes. I. Evi-dence forhigher order domains of defined DNA sequence. Cell16:797-806.

48. Wu, C., Y. Wong, and S. Elgin. 1979. The chromatin structure ofspecific genes. II. Disruption of chromatin structureduringgeneactivity.Cell 16:807-814.

1982

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Figure

FIG. 1.nucleotidereactionSV40atwithbuffer.thecovalentlyzeroF, 50 zero Superhelical structure of chromatin DNA after incubation with HeLa cell extract
FIG. 2.digestedassaysmap.SV40 Transcriptional map of SV40 DNA. The diagram represents an SV40 DNA template which has been with endonuclease EcoRI (Fig
FIG. Polaritynchromatin 4. and termination of ii template transcripts. SV40virions
FIG.electrophoresis
+2

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