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0022-538X/85/030822-05$02.00/0

CopyrightC 1985, American Society forMicrobiology

In Vitro Transcription of a Cloned Vaccinia

Virus Gene by a Soluble

Extract Prepared from Vaccinia

Virus-Infected HeLa Cells

P. DAVID FOGLESONGt

Department of Developmental Biology and Cancer, Albert Einstein College of Medicine, Bronx, New York 10461

Received 20 August 1984/Accepted 16 November1984

Faithful transcription of avaccinia virus gene was accomplished in vitro by using a soluble extract prepared

from vaccinia virus-infectedHeLa cells. Specific transcription of the cloned vaccinia virus gene was detected by

using template DNArestrictedwithin the transcribed region. The vacciniavirus gene was not transcribed by

extracts prepared from uninfected HeLa cells even with supplementation by purified vaccinia virus RNA

polymerase, nor was a clone of adenovirus 2 DNA bearing the

major

latepromotertranscribed by the extract

from vaccinia virus-infected HeLa cells. Thus, infection by vaccinia virus altered cellular transcriptional

specificity to favor expression of vaccinia virus genes. RNA synthesis by theinfected cell extract was resistant

toa-amanitinbut strongly inhibited by I,,y-imidoATP and novobiocin.

Vaccinia virus has been a very useful model systemfor analysis of eucaryotic transcription. Vaccinia virion cores

transcribe early viralgenesin vitro (13) and thus containall

ofthe enzymesrequired for early transcription.A number of

enzymes have been isolated from viral core particles and

have been characterized. Of these, the DNA-dependent

RNApolymerase (2, 32),guanylyltransferase and

7-methyl-transferase (18, 21), 2'-O-methyltransferase(1), and

polyad-enylate polymerase (6, 22)areclearlyrequired for transcrip-tion. Several other virion enzymes which may function in transcription have been described, including 5'-phosphate

polyribonucleotide kinase (31), protein kinase (14), two

distinct nucleic acid-dependent ribonucleoside triphosphate phosphohydrolases (25), asingle-strand-specific nuclease(s) (26, 28),endoribonuclease (24), twoabundantDNA-binding proteins (3, 11), and a type I DNA topoisomerase (3).

Purifiedpreparations of vaccinia virusRNApolymeraseare

similar to cellular RNA polymerase II isolates in their requirement forMn2+ and single-strandedDNAin vitro and theirinability to transcribe duplex DNA templates. Thus, otherproteins appear to berequired forproperinitiation of transcription from nativeDNAtemplates (19).Furthermore,

RNAsynthesis from intact vaccinia viruscore particlesbut notbypurified vaccinia virus RNA polymerase is sensitive

to

1,-y-imido

ATP(AMP-PNP) andalsotonovobiocin(7, 8,

30). Thus, vaccinia virus is an ideal model system for

analysis of the factors required for initiation oftranscription. Recently, the development of soluble cellular extracts

capable

of faithful initiation of

transcription

in vitro has provided a powerful method for analysis oftranscriptional initiation events (16, 35). Defined duplex DNA templates

have beentranscribed invitrotoyieldtruncated transcripts characteristic of initiation from a given promoter. Cloning

and RNA mapping ofthe vacciniavirus genome (10) have

made possible analysis of transcription ofdefined vaccinia

virus genes invitro.Inthispaper Idescribethedevelopment

and characterization of a soluble extract from vaccinia virus-infectedHeLacells which faithfully initiates

transcrip-tion of a cloned vaccinia virusgene invitro.

tPresent address: Division ofVirology and MolecularBiology, St.JudeChildren's ResearchHospital, Memphis,TN 38101.

MATERIALS AND METHODS

Materials. Unlabeled nucleotides and novobiocin were

obtained fromSigma Chemical Co.AMP-PNP waspurchased from Boehringer Mannheim Biochemicals. [c-32P]GTPwas

purchased from Amersham Corp.

Preparation of nucleic acids. Plasmid pSmaI-F bearing

adenovirustype 2(Ad2)SmaIfragmentF(11.6to 18.2 map

units) was provided by H. Furneaux. Plasmid pAG4 (34) bearinga960-basepair (bp) insertion of vaccinia virusDNA wasprovided byB. Moss. Supercoiled plasmid DNAswere

isolated as described previously (20). DNA products of restriction endonucleasedigestswereanalyzed on4% poly-acrylamide gels under conditions describedpreviously (3).

Virus.Vaccinia virus strainWR waspurified from infected

HeLa cells through two sucrose gradients, as described previously (9).

Preparation of extractsfrom vaccinia virus-infected HeLa

cells. HeLacells at a

density

of 5 x

105

cells per ml were

infected with 280 PFUof vaccinia virus percell.At 1and5 h afterinfectionextracts were preparedas described

previ-ously

(16).Extracts werestoredat

-70°C

in200-gil

portions.

Preparation of vaccinia virus DNA-dependent RNA

poly-merase. Vaccinia virus RNApolymerase was purified from

virions through phosphocellulose and was assayed as

de-scribedpreviously (32).

Protein determination. Protein concentrationswere

deter-minedby

using

the methodof Bradford (5).

Assay for in vitro transcription. The standard reaction

mixture (50

,ul)

contained 20 mM HEPES

(N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.9),

5 mM

MgCl2, 2 mMdithiothreitol,1 mMATP, 1 mM

CTP,

1 mM

UTP, 0.2 mM

[a-32P]GTP

(20,000 to40,000

cpm/pmol),

1.0

gig

ofDNA, and 20 gil ofextract. Theassay mixtureswere

incubated at30°Cfor 60min,andreactionswereterminated

byadding 1 gil of1 mMGTP and 150 gil of4 M urea-1.0% sodium dodecyl sulfate-20 mM EDTA. The RNAwas

ex-tracted with 200

gil

of

phenol-chloroform

(1:1), and the

organic phasewas extracted withasolution

containing

7M

urea, 0.35 M NaCl, 10 mM

Tris-hydrochloride

(pH 8.0), 2

mMEDTA,and0.5% sodiumdodecylsulfate. The aqueous

phases werepooled, extractedagain, and

precipitated

with

ethanol

by using

50

gig

of EscherichiacolitRNAas acarrier.

The pellets were suspended in 300 gil of 1 M ammonium

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E

0 I:

--Q) -0

>

450

FIG. 1. Restriction sites and truncated tran insertion region. A 960-bp SalI fragment of terminal repeatwasligated into the SalI site or The promoter of the 7,500-dalton gene is in( triangle.TranscriptionofpAG4 restrictedwith yielded transcripts of 450, 730, and 1,010 nucle

acetate andprecipitatedwith ethanol. The

suspended in300,ul of0.3 Msodiumacetal

with ethanol. Thefinal pellets were suspe

solutioncontaining50 mMboric

acid,

S rn

10 mM sodium sulfate, and 0.2 mM ED'

mented

with

2,ulof0.2 MmethylmercuryI

ofasolution

containing

50%sucrose, 0.0: and0.02% bromphenol blue. Sampleswei on 4%

polyacrylamide

gels in BBE c

methylmercury hydroxideat80Vfor3h.i

thedriedgelswere prepared byusingKo

RESULTS

Restricted DNAtemplatesfor in vitro trai

studiesof specific transcriptional events

by using DNA-dependent cell extracts ai

restricted within the coding sequence

transcripts of defined length are synthesi;

used in this study, plasmid pAG4, conta

fragment

isolated from the vaccinia virus

repeatand cloned into theunique SalI sil

Theinsertion contains thepromoterand7'

sequenceofageneencodingan earlypro

7,500, as well as about 230 bp of sequ

proximal to the site oftranscriptional inil

indicating

the relevant restriction sites in pAG4 insertion isshownin

Fig.

1. Restric SalI releases the

insertion, which,

if ti

result in a truncated 730-nucleotide transc

readily detected by denaturing polyacr trophoresis.

Therefore,

the Sall digest c

tinelyused as atemplate for in vitrotrans

deduced from the BamHI-DdeI double dif

not

shown)

that the orientation of the va

insertion within pAG4 was such that the

scription from the 7,500-dalton gene pr(

from the unique BamHI site ofthe vect(

Bgll site, asshowninFig. 1. Therefore,r(

with DdeI should have yielded a shortc

about 450

nucleotides,

and

similarly,

res

shouldhaveproduceda

transcript

of aboul

(Fig. 1). RNA products of these predicto

served when these DNAs were transcril

vacciniavirus-infectedHeLacells as desc notshown). These RNAs were notobser

in vitro transcription assays programme

sponding

restricted pBR322 DNAs. The, the independently reported conclusion (2 thesis initiates at the same site on this i

virus DNA in vivo and in vitro in exi

virus-infected HeLa cells. The SmaI digest of a

plasmid

bearing Ad2 SmaI fragment F containing the major late

o v promoter wasused as thetemplate for invitrotranscription

cT) byuninfected HeLa extracts toyield a560-nucleotide

tran-script (35).

Comparisonofuninfected and vacciniavirus-infected HeLa cellextractsfortranscriptionof adenovirusandvaccinia virus

- 730 genes. Extracts were prepared from uninfected HeLa cells

- 1010 and alsofromHeLa cells infected with 280 PFU of vaccinia

virus per cell at 1 and 5 hafterinfection, as described above.

scripts

of the pAG4 The protein concentrations of these extracts were

deter-f

pBR322cDNA (v34)s

minedto be about 8

mg/ml.

Thetranscription of adenovirus

dicated by the open SmaI fragment F and

Sall-digested

pAG4 was determined

DdeI, Sall, and BglI for all three extracts as described above (Fig. 2). Assay

otides, respectively, mixtures lacking an added DNA template (Fig. 2, lanes A)

showed no RNAsynthesis, demonstrating the DNA

depen-,n the pellets were dence oftranscription by these extracts. Other assay

mix-teandprecipitated tures (lanes B) were programmed with Ad2 SmaI-F DNA

nded in 15 ,ulofa and showed the expected RNA product of 560 nucleotides

iMsodium borate, for theuninfectedextract. This band wasgreatlyreduced in

TA (BBE) supple- the assay mixture containing the 1-h-infected cell extract

hydroxide and 4,ul (VH1 extract) and completely absent in the assay mixture

2% xylene cyanol, containing the 5-h-infected cell extract (VH5 extract),

indi-reelectrophoresed cating that vaccinia virus infection eliminated RNA

polymer-ontaining 10 mM ase II transcription of SmaI fragment F. In other assays

Autoradiograms of (lanesC) the extracts were programmed with the Salldigest

dak XAR-5 film. of pAG4. The 730-nucleotide RNA product was observed

clearly only with the VH5 extract,indicating that the

vacci-niavirus gene can be transcribed most efficiently by extracts

nscription. In vitro from infected cells prepared several hours after infection.

can be performed These results are consistent with those reported by other

nd template DNA workers(27). Thehigher-molecular-weightbands(especially

so that truncated the 960-nucleotide band seen clearly in HeLa cell lane C)

zed. The template evidentin all lanesprogrammed withexogenous DNA were

iins a 960-bp SalI determinedto besensitiveto DNase(data not shown). Other

inverted terminal investigators havealsoconsideredsuch bandsto be artifacts

teof pBR322 (34). of endlabelingby crude lysates (17). The 560- and

730-nu-30bp ofthecoding cleotide RNAs were resistantto DNasetreatment.

tein withan Mrof Optimal reaction conditions for vaccinia virus transcription

ences that are 5' in vitro. Optimal synthesis ofthe 730-nucleotide RNA

re-tiation. A diagram quired the addition of20 ,u1 ofextract (8mg ofprotein per

the region ofthe ml) and 1.5 p.g of DNA per 50-,ul assay (data not shown).

tionof pAG4with

ranscribed, would

criptthatcould be __________ H1 VH5

ylamide gel elec- A B C A B C A B C M

f

pAG4

was rou- U

scription

studies. I

_353

gest

of pAG4(data 960- 1078

iccinia virus DNA 730- 1 872

direction of tran-

X3

60'

omoter was away

560-or and toward the

estriction ofpAG4

ened transcript of X -

31n

;triction with BglI

t1,010nucleotides

led sizes were

ob-bed in extracts of

cribedabove

(data

vedasproducts of

,d with the

corre-se results support

'7)

that RNA

syn-region

of vaccinia tracts of vaccinia

FIG. 2. Invitrotranscription by uninfected andvaccinia virus-infectedHeLacellextracts.Products oftranscription by uninfected

(HeeLa), VH1, and VH5 cell extracts were programmed with no

DNA(lanes A),SmaI-digestedpSmal-F(lanes B),andSall-digested pAG4 (lanesC). Productsthatwere 560, 730,and 960nucleotides longareindicated.Lane McontainedHaeIII-digested4X174DNA thatwas5'-labeled with

[y-32P]ATP

andpolynucleotide kinase (used

assize markers).

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Higher concentrations of extract or of DNA resulted in synthesis of spurious products. Similar sharp optima for

extractand DNAconcentrations arecharacteristicof related

in vitrotranscription systems (16). Synthesis of the

730-nu-cleotide RNA required relatively high concentrations (1

mM) of ribonucleoside triphosphates, perhaps because of

the presence of vaccinia virus phosphohydrolases in the

infected cell extracts. Specific RNA synthesis occurred at

Mg2+concentrationsof5 to 10 mM, conditions similar to the

conditions required for transcription ofDNA within intact

vacciniaviruscores. However, addition of 5 to 10 mMMn2+

to assay mixturescontaining 5 mMMg2+ strongly inhibited

synthesisof the730-nucleotide transcript (data not shown),

whereas

Mn2+

was required for activity of the RNA

polymer-asepurified fromvaccinia virions. Thus, the soluble infected

cell extract retained an important characteristic of viral

RNA synthesis in vivo which was lost upon purification of thevaccinia virion RNApolymerase.

a-Amanitinresistance of in vitro transcription by vaccinia

virus-infected HeLa cell extracts.a-Amanitin, apotent

inhib-itor ofRNA polymerase II, has been shown tocompletely

inhibit in vitro transcription ofAd2 SmaI fragment F by uninfectedHeLacell extracts (35). The a-amanitin

sensitiv-ity of transcription by the VH5 extract was examined.

Transcription ofAd2 SmaI-F and transcription of

Sall-di-gested pAG4 were determined as described above for the

HeLacell and VH5 extracts, respectively, in the presence

andabsenceof1,ugofao-amanitin per ml (Fig. 3). Synthesis

of the 560-nucleotide RNA was completely abolished by

ot-amanitin (Fig. 3, HeLa cell extract, lane C). However,

synthesis of the 730-nucleotidetranscriptbytheVH5 extract

wascompletely resistant to 1 ,ug ofa-amanitinper ml (Fig. 3,

VH5 extract, lane C), indicating that RNA polymerase HI

doesnotcatalyzethesynthesis of thistranscript. This RNA

product is synthesized by thea-amanitin-resistant vaccinia

virus RNA polymerase since its synthesis by infected cell

extracts is inhibited by antibody directed against purified

vaccinia virus RNApolymerase (27).

Supplementation of uninfected HeLa cell extracts with

vaccinia virus RNA polymerase. Both vaccinia virus RNA

HeLa

VH5

A

B

C

A

B

C

-0

Om~~~~~~~~~~~~~~~~~~~~~

-W;4:....

730-

560-FIG. 3. a-Amanitinsensitivity of transcriptionby uninfected and vaccinia virus-infectedHeLaextracts.Uninfected(HeLa)and VH5 cellextractswereassayedfortranscriptionofpSmal-Fand

Sall-di-gested pAG4, respectively, in thepresenceandabsence of 1 ,ug of ox-amanitinperml.Lanes A,noDNA; lanesB,DNA; lanesC,DNA plus1 ,uofa-amanitinperml.

~730-

560-A

B

C

D

E

F

G

H

J

w

K

FIG. 4. Supplementation ofextracts with vaccinia virus RNA

polymerase. Uninfected and vaccinia virus-infected HeLa cell ex-tractswereassayedfor transcription of Sall-digested pAG4 with or withoutsupplementation with purified vaccinia virus RNA polymer-ase.Uninfectedextractassaymixtures containedno DNA(lane A), SmaI-F DNA (lane B), and Sall-digested pAG4 (lanes C,1D,andE). VH5extract assay mixtures contained no DNA (lane F) or Sall-di-gestedpAG4 (lanes G, H, and I).LanesDand Hcontained assay mixturessupplemented with 0.02 U ofvaccinia virus RNA polymer-ase, and lanesEandIcontainedassay mixturessupplemented with 0.1UofRNApolymerase.LanesJ andKcontained assay mixtures ofSall-digested pAG4 transcription by 0.02 and 0.1 U of vaccinia virus RNApolymerase, respectively.

polymerase and RNA polymerase II require additional

fac-tors toutilize duplexDNA as atemplate.Therefore, it was

of interest to determine whether those factors present in

extracts ofuninfected cells could substitute for the factors

present ininfectedcells or whetherthere werevirus-specific

proteins other than the RNA polymerase which were

re-quired forinvitro transcription. Assays were performed as

described above, using both HeLa cell and VH5 extracts

with Sall-digested pAG4 as the template with or without supplementationwith 0.02 or 0.1 Uof

purified

vacciniavirus RNA polymerase (Fig. 4). Supplementation with vaccinia

virus RNApolymerase didnotresultin detectable

synthesis

of the 730-nucleotide transcript by the HeLa cell extract

(Fig. 4, lanes D and E), nor did it significantly alter the

transcription of infected cellextracts(lanes H andI). RNA

polymerase alone did not synthesize any detectable 730-nucleotide RNA (lanes J and K). Therefore, in vitro

tran-scription of pAG4requires

virus-specific

factors other than

vacciniavirus RNA polymerase.

Sensitivity oftranscriptionby HeLa cellandVH5extracts to

AMP-PNP and novobiocin. Transcription by vaccinia virus

core

particles

requires hydrolysis

ofATPtoADPand

Pi

and

thereforeisinhibitedbyATPanalogs, suchasAMP-PNP, in

whichhydrolysis of the

P,y

phosphatebond isrestricted (8,

30). This analog inhibits the vaccinia virus ribonucleoside

triphosphate

phosphohydrolases

but notthe vacciniavirus RNA polymerase (30). Novobiocin, an inhibitor of the

vaccinia virus DNAtopoisomerase, also inhibits

transcrip-tionbyintact vaccinia viruscores (7).

Therefore,

the

sensi-tivity of in vitro transcription to these compounds was

determined. Assays ofthe VH5 extract with

Sail-digested

pAG4 were performed as described above in the presence

and absenceof 1 mMAMP-PNP or novobiocin. The assay

with the ATPanalogwasperformed intheabsenceofATP.

The results are shown in Fig. 5. Transcription ofthe

730-nucleotide RNA bythe VH5 extract was

strongly

inhibited

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A B

C

D

730-FIG. 5. Transcription ofSall-digested pAG4 by vaccinia virus-infected HeLaextracts in thepresence and absence of AMP-PNP

and novobiocin. Lane A containedanassay mixture programmed

withnoDNA, and lane B contained the 730-nucleotide transcript

synthesizedfromSall-digested pAG4. LaneC shows the results of

an assayperformedinthe absence ofATPandin thepresenceof 1 mMAMP-PNP. LaneDshowsthe results ofan assayperformedin

thepresenceof1mMnovobiocin.

by 1 mMAMP-PNP (Fig. 5, lane C). Thisresult is consistent

witharequirement for vaccinia virus phosphohydrolaseIor

IIactivity for in vitrotranscriptionin thissystetn. Complete

inhibition of transcription by 1 mM novobiocin was

ob-served(lane D), indicatingapossible requirement forDNA

topoisomerase Iactivity forinvitrotranscription. Synthesis

of the560-nucleotide RNA by uninfected HeLa cellextracts

programmed with Ad2 Smal fragment F DNA was also

completely inhibited by 1 mM novobiocin (datanot shown).

DISCUSSION

Significant insights into the molecular mechanisms of

eucaryotictranscription have been obtained from studies of

vaccinia virus transcription, notably the discovery of

polyad-enylation (12) and thecharacterization of themechanism of

RNA capping (29). Since vaccinia virus encapsidates the

enzymesrequired for earlygeneexpression, the virion core

particles have been used to analyze transcription in vitro.

However, understanding the precise mechanisms of

tran-scription required the development of a soluble system

capable of faithful transcription in vitro. Cloningand mRNA

mappingof thevaccinia virusgenomehaveprovided

numer-ous potential templates for transcriptional analyses (10).

Plasmid pAG4 was selected for these studies since it

con-tains anearly vaccinia virus gene which has been mapped

precisely (34). Thepromoterof thisgenehasbeen shownto

function in vivo (15), and the DNA sequence of the

5'-proximal region has been determined (33). Transcription of

SalI-digested pAG4 results in a 730-nucleotide transcript

whichcanbereadily detectedbydenaturinggel

electropho-resis.

Extracts for transcriptional assays were prepared from

vaccinia virus-infected HeLa cells by a procedure which

yields transcriptionally activeextractsfrom uninfectedHeLa

cells(16). The infected cell extract synthesizedtheexpected

730-nucleotide product which initiatesat the same site as the

in vivo transcript, as shown by other workers (27).

Interest-ingly, uninfected HeLa cell extracts fail to transcribe the

vaccinia virus gene, and conversely vaccinia virus-infected

HeLa cell extracts do not transcribe the adenovirus clone

bearing the RNA polymerase II major late promoter. Thus,

a virus-specific function(s) is required for vaccinia virus

transcription, and infection by vaccinia virus inactivates RNApolymerase II transcription. Greatly reduced levels of

cellular mRNA have been reported for vaccinia

virus-in-fected cells (4). SynthesisofRNA by infected cell extracts is

resistant to a-amanitin (27), as is RNA synthesized by

purified vaccinia virus RNA polymerase (2, 32). The

vacci-nia virus RNA polymerase is required for synthesis of the

730-nucleotide RNA since antibody directed against it

elim-inates pAG4 transcription (27).

Supplementationof uninfected extracts by purified

vacci-nia virus RNApolymerase did not result in any detectable

synthesisof the730-nucleotide transcript. Thus, a

virus-spe-cific factor(s) other than RNA polymerase is required for

specific transcription in vitro. Inhibition oftranscription by

the VHS extract by AMP-PNP indicates a requirement for

ATP hydrolysis to ADP and

Pi,

as has been observed for

transcription in vaccinia virus core particles (8, 30).

There-fore,phosphohydrolase I or II may berequiredfor

transcrip-tion by theinfected cell extract. AMP-PNPalsoinhibits the

vaccinia virustopoisomerase(7) but atconcentrationshigher

than 1 mM, which was used in thesestudies. Novobiocin, a

potent inhibitor of the topoisomerase, completely inhibits

transcriptionby bothinfected and uninfected HeLaextracts, suggesting a requirement for topoisomerase activity for in

vitrotranscription. It should be noted that novobiocin may

not bespecific for the topoisomerase, but it does notinhibit

vaccinia virus RNApolymeraseorphosphohydrolases I and

II (D. Foglesong and R. Guggenheimer, unpublished data).

The apparent requirement for topoisomerase activity is

surprising sincethetemplatesused werelinearDNAswhich

are not subject to any topological constraint. However,

replication of linear duplex adenovirus DNA in vitro also

requires a type I topoisomerase (23). Thus, topoisomerase

activity may facilitate the movement of transcription and

replication forks through duplexDNA.

Since vaccinia virus encapsidates all of the enzymes

required for earlytranscription,virion extracts were assayed

foractivity in transcribingpAG4 in vitro. Initial attempts in

which I used extracts prepared by conventional methods

(32) wereunsuccessful even when the extracts were

supple-mented with purified vaccinia virus RNApolymerase.

How-ever, virion extracts concentrated by ammonium sulfate

precipitation or negative pressure dialysis synthesized the

730-nucleotide

product (unpublished data). Thus, high

pro-teinconcentrations may berequiredfor this in vitrosystem,

asfor othertranscriptionalsystems (16). Virion extracts may

be moreuseful thaninfectedcellextractsforsome

transcrip-tion studies since the proteins required fortranscriptionare

ofmuch greater

specific

activityinvirion extractsthan those

of infected cellextracts. Otherinvestigators have also

tran-scribed cloned vaccinia virus genes in vitro by using virion

extracts (B.Moss,personal

communication;

F. Golini and J.

Kates, Int. Symp.Pox/Iridoviruses, Abstr. ThA6,presented

26 July 1984 atMadison, Wis.). Thedevelopmentofsoluble

in vitro systems for vaccinia virustranscription willgreatly

facilitate the analysis of the protein factors required for

accurate initiation of transcription and also the DNA

se-quences required forrecognition by

those

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ACKNOWLEDGMENTS

Ithank B. Mossfor communicatingunpublished results andH. Furneaux and J. Hurwitz for helpful discussions.

This work was supported by Public Health Service grant 5T32 CA09176 from the National Institutes of Health.

LITERATURE CITED

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2. Baroudy, B. M., and B. Moss. 1980.Purification and character-ization of aDNA-dependent RNA polymerase from vaccinia virions. J. Biol. Chem. 255:4372-4380.

3. Bauer, W. R., E. C.Ressner, J. R. Kates, and J. N. Patzke. 1977. A DNAnicking-closing enzyme encapsidated in vaccinia virus: partial purification and properties. Proc.Natl.Acad.Sci. U.S.A. 74:1841-1845.

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5. Bradford, M. M. 1976. Arapid arid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 6. Brakel, C., and J. Kates. 1974. Poly(A)polymerase from

vacci-nia virus-infected cells. J. Virol. 14:715-723.

7. Foglesong,P. D., and W. R. Bauer. 1984. Effects of ATP and inhibitory factors on the activity of vaccinia virus type I topoisomerase. J. Virol. 49:1-8.

8. Gershowitz, A., R. F. Boone,and B.Moss. 1980. Multiple roles for ATP in thesynthesis and processing of mRNA by vaccinia virus: specific inhibitory effects of adenosine (,B,y-imido) tri-phosphate. J. Virol. 27:399-408.

9. Hruby,D. E., L. A.Guarino, andJ. R. Kates. 1979. Vaccinia virus replication. I. Requirement forthehost cell nucleus. J. Virol. 29:705-715.

10. Isle,H. B.,L.Venkatesan, and B. Moss. 1981. Cell-free trans-lation ofearly and late mRNAs selected by hybridization to cloned DNAfragments derived from the left 14million to72 million daltons of the vaccinia virus genome. Virology 112:306-317.

11. Kao,S., E. Ressner, J. Kates, and W.Bauer. 1981. Purification and characterization ofasuperhelixbinding protein from vac-cinia virus. Virology111:500-508.

12. Kates,J. 1970.Transcription of the vaccinia virus genomeand the occurrence ofpolyriboadenylic acid sequences in messenger RNA. ColdSpring Harbor Symp. Quant. Biol. 35:743-752. 13. Kates, J. R., and B. R. McAuslan. 1967. Messenger RNA

synthesis bya "coated" viral genome. Proc. Natl. Acad. Sci. U.S.A.57:314-320.

14. Kleiman, J. H., andB.Moss. 1975.Characterization ofaprotein kinase and two phosphate acceptor proteins from vaccinia virions. J. Biol. Chem.250:2420-2429.

15. Mackett, M., G. L. Smith,and B. Moss. 1982.Vaccinia virus:a selectableeukaryoticcloning andexpressionvector.Proc.Natl. Acad. Sci. U.S.A. 79:7415-7417.

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Figure

FIG. off pBR322cDNA (v34)sdicated by the openDdeI, Sall, and BglIotides,scripts of thetriangle.Theinsertionterminal pAG4yielded1
FIG. 4.polymerase.withoutase.tractsgestedSmaI-FVH50.1ofmixturesase,virus Sall-digested Supplementation of extracts with vaccinia virus RNA Uninfected and vaccinia virus-infected HeLa cell ex- were assayed for transcription of Sall-digested pAG4 with or sup
FIG. 5.aninfectedandwiththemMsynthesized assay Transcription of Sall-digested pAG4 by vaccinia virus- HeLa extracts in the presence and absence of AMP-PNP novobiocin

References

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