The yeast GAL4 protein transactivates the polyomavirus origin of DNA replication in mouse cells.

0022-538X/91/073496-08$02.00/0

Copyright C 1991,AmericanSociety forMicrobiology

The

Yeast GAL4 Protein Transactivates

the

Polyomavirus Origin of

DNA

Replication

in

Mouse Cells

MOSHEBARU, MEIR SHLISSEL, AND HAIMMANOR*

Department of Biology, Technion-Israel Institute of Technology, Haifa32,000,Israel Received 19February 1991/Accepted5 April 1991

Wehavereplacedthepolyomavirus (Py)enhancer,which isanessentialcomponent of thePy originofDNA replication (ori), with five repeats ofa 17-bp oligonucleotide including the yeast GAL4 upstream activating sequence (5xGAL4sites). Plasmids containing this modified Py ori, designated test plasmids, and plasmids encoding either the GAL4 transcriptional activator protein or various derivatives of this protein were

cotransfected intomousecells whichconstitutively synthesizeatemperature-sensitive Py largetumorantigen (T-Ag). Replication ofthe testplasmids was monitored by Southern blotdeterminations of the amounts of plasmidDNA that becameresistanttocleavagebytheenzymeDpnI.Thesestudiesshowed that in thepresence ofafunctional T-Ag,the GAL4protein,andhybrid proteinsincludingthe GAL4DNA-bindingdomain andthe activating domainof theadenovirus Elaor herpesvirusVP16 proteintransactivated themodifiedPyori. A truncated proteinincluding just the GAL4DNA-bindingdomainwasinactive in these assays. The authentic GAL4proteinwasfoundtobea moreefficientreplicationtransactivator thanthehybrid proteins.Incontrast, chloramphenicol acetyltransferaseassaysshowed that thehybrid proteinsweremoreefficienttranscriptional activatorsthan theGAL4 protein.Theextentof theGAL4-dependent replicationofaplasmidinwhich thePy earlypromoterwasdeletedwas55 %lower than that ofaplasmidincludingthepromoter.However,the extents ofreplicationofplasmids includingtwotandem repeats of theremaining Pyorigincore and5xGAL4 sitesor twoorigincoresflankingasinglecluster of5xGAL4 siteswere4.8- and1.6-foldhigherthanthat of theplasmid includingasinglecopy ofeach element. The replication ofaplasmidincluding twoclustersof 5xGAL4 sites flankingasingle origincore wasbelow the limitof detection ofourassays.These resultsindicate that the GAL4 andhybridtransactivators donotactivate thePyoribyvirtue of their interactions withtranscriptionfactors thatbindpromoterelements.Rather,itappearsthat these activatorproteinsmayinteract with thereplication initiationcomplexes, therebyfacilitating orinhibitingtheinitiation ofreplication.

The polyomavirus (Py) origin of DNA replication (ori)

consists oftwoelements: the origin core and the enhancer (7). The origin core was defined genetically as an essential and specific part ofthe Py ori (22, 26, 44). Itextends from nucleotides 5268 to 42 in the Py A2 strainnumberingsystem (11). The core contains three main sequence elements: (i) a central palindrome(nucleotides 5281to22), which includes two repeats of thelargeT-antigen (T-Ag) recognition

penta-nucleotide sequence motif [5'-(G)(A/G)GGC-3'] on each

strand; (ii)anA/T-rich sequence (nucleotides 5268 to5283) that maps on the late side of the palindrome; and (iii) a

polypurine-polypyrimidine sequence (nucleotides 27 to 42) that maps onthe early side of the palindrome (7). The sites of initiation of bidirectional DNA replication have been mapped ontheearly sideof the core (15).

The Py replication enhancer overlaps with the

transcrip-tionalenhancerforthe early transcription unit (nucleotides 5021 to

5265)

and with the late promoter (4, 9, 14, 43, 45). Deletionof the entire enhancer inactivated the origin (9, 45).

However, a functional origin could be reconstituted by

reinsertion ofvarious parts of the enhancer (3, 33, 39, 45). Moreover,functionalorigins were also obtained in which the Py enhancer was replaced with sequences from other viral andcellular enhancers (la, 9).

Previous work on eukaryotic enhancers focused on their role in transcription. It was found that enhancer sequences bind specific protein factors which act as transcriptional modulators (18, 20, 30, 38). Similar proteins bind to some

* Correspondingauthor.

upstream promoter elements, which are also found in en-hancers (28). A typical transcriptional modulator protein consists of a DNA-binding domain that specifically recog-nizes a short DNA sequence and a relatively nonspecific activating domain that interacts with general transcription factors and with other enhancer-binding proteins, thereby

enhancingorsilencingtheinitiation oftranscription (18,38). Anumber of mammalianand yeasttranscriptionalmodulator proteins were also found to regulate DNA replication, but

detailedknowledgeof the molecularinteractionsthat under-lie this regulationislacking (5, 19,24, 33, 34, 35).

Wereport here that the yeast GAL4transcriptional acti-vator protein and hybrid derivatives of this protein, which have been extensively used for studies of transcriptional

activation (38), also induce initiation of replication at a modified Py oriin which thePyenhancer hasbeenreplaced with aclusterof GAL4 upstreamactivatingsequences. We have begun a systematic study of this system and present here the dataobtained sofar, which indicate that transacti-vation of the Py ori by the GAL4 and hybrid proteins cannot be accounted for by their interactions with transcription

factors that bind promoter elements.

MATERIALS ANDMETHODS

Plasmids. Partial maps of the various plasmids used are shown inFig.1 to4.Plasmid

pPyAcat26.2'

(45) isapBR322 derivative which containsa PyDNAfragment (nucleotides 4632 to 152), in which the enhancer (nucleotides 5021 to 5262) has been replaced with two repeats ofa 26-bp

oligo-nucleotideincludingthePy oxenhancerelement(nucleotides 3496

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5108 to 5130). Italsocontainsachloramphenicol

acetyltrans-ferase (CAT) gene attached to the Py early promoter. Plasmid pPyLT1 (49) contains a Py genome from which the large T-Ag intron has been deleted, which was cloned into

plasmid pAT153. Plasmid pGSE472cat (25) contains adeno-virus type 5 E4 promoter sequences (-240 to +32 from the transcription initiation site) inserted upstream of the CAT gene in apSP72 vector (Promega Biotec). In addition, this plasmid contains five repeats of a 17-mer oligonucleotide includingabindingsite forthe yeastGAL4 protein(SxGAL4 sites) insertedupstreamofthe E4 promoter. PlasmidspAG4

and pAG147 (21) include genes encoding the whole GAL4 protein and the truncated N-terminal GAL4 protein [GAL4

(1-147)protein], respectively, whichareexpressed under the control of the adenovirus major late promoter. Plasmid

pSGVP(40) includesageneencodingahybrid proteinwhich

consists of the N-terminal 147 amino acids of the GAL4

protein fused to thecarboxyl-terminal78amino acids ofthe herpesvirus protein VP16 (GAL4-VP16 protein). Plasmid pGAL4-Ela (25) includes a geneencoding a hybrid

protein

which consists of the N-terminal 147 amino acids of the GAL4 protein fused to amino acids 121 to 223 of the adenovirus Ela protein (GAL4-Ela protein). Plasmid

pSG147(25)includesthe geneencodingthetruncatedGAL4

(1-147) protein.The genesmentioned above in the last three

plasmidsareexpressed under thecontrol ofthesimianvirus 40 (SV40) earlypromoter.

Additional plasmids used were as follows. Plasmid pM96

was constructed by insertion of a fragment of Py DNA

extendingbetween the BamHI and EcoRI sites

(nucleotides

4632to1560) intothecorresponding restrictionenzymesites

in plasmid pUC119 (47). In this Py DNA fragment, the enhancer (nucleotides 5021 to 5264) was replaced with a

XhoI linker. PlasmidpMlOlwasconstructedby replacement ofthePy DNAfragment extending between the BamHIand

XhoI sites in plasmid pM96 with a fragment

extending

between the same two enzyme sitesin

plasmid pG5E472cat

(see above). The latter fragment includes the cluster of

5xGAL4 sites. Plasmid pM107 contains the cluster of

5xGAL4 sites fused to the late side of a Py DNA segment

(nucleotides5265to 90). Toconstruct this

plasmid,

plasmid

pMlOl

was digested withthe enzymes BamHI andBglI. A

fragmentextendingbetween these twoenzyme

sites,

which includestheabove-mentioned sequences, was

purified

from

this digest. This fragment was exposed to the enzyme T4 DNApolymerase firstat 12°C and thenat

37°C

to

digest

the 3' extension formed

by

the

BglI cleavage

andtofillinthe 5'

extension formed by the BamHI

cleavage (41)

and was inserted into the SmaI site of

plasmid

pUC119.

Plasmid

pM108 contains two repeats of the above-mentioned BamHI-BglI fragmentinserted in tandem into theSmaI site of plasmid

pUC119.

Plasmid pMlll was constructed

by

insertion ofaPy DNA segment

extending

between nucleo-tides 5265 and 90, which was obtained

by cleavage

of

plasmid

pM108 withBamHI and

XhoI,

into

plasmid pM107

between the BamHI and Sall sites in the multiclonal seg-ment. To construct

plasmid

pM112, a

fragment

containing

the cluster of5xGAL4 sites was

prepared by

cleavage

of plasmid pM108 with the enzymes EcoRI and XhoI. This

fragment waspurified and inserted between the

recognition

sites of the enzymes EcoRI and

KpnI

in the multiclonal segmentof

plasmid

pM107.Theinsertionwas

performed by

first ligating the

matching

EcoRI

sites;

then T4 DNA

poly-merase was used to

digest

the 3' extension

produced

by

KpnI

and to fill in the 5' extension

produced by

XhoI as

described

above;

finally,

the two blunt ends

generated by

this

procedure

were

ligated.

Transfection-replication

assays. Mouse WOP cells

(23)

were

propagated

at

37°C

in Dulbecco modified

Eagle's

medium

supplemented

with 5% fetal calf serum in a

CO2

incubator as described

previously (29).

Twenty-four

hours

before the

transfection,

3 x

105

cellswereseeded in60-mm

plates

in 5 ml ofthesamemedium

containing

20% fetalcalf

serum. Each

plate

was transfected with

plasmids by

the

calcium

phosphate

technique (10)

as follows. A

240-pAl

vol-ume of a solution

containing

0.25 M

CaCl2,

0.05 M

N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic

acid

(HEPES;

pH 7.05),

and 10 ,ugof

plasmid

DNAwasmixed with240

IlI

of anothersolution

containing

0.05 M

HEPES,

0.28 M

NaCl,

0.75 mM

Na2HPO4,

and 0.75 mM

NaH2PO4 (pH 7.05).

The mixturewasincubatedfor20minat

25°C

andthenaddedto the

plate.

The cultures were incubated 6 h at

37°C,

after which time the medium was removed and the cells were

extensively

washed with fresh medium. Then 5 ml of me-dium

containing

5%fetalcalfserum wasaddedtoeach

plate,

and thecellswereincubatedfor 48 hateither33or

39°C,

as

specified

in the

figures.

The cells werethen

harvested,

and

small-molecular-weight

DNA was

selectively

extracted as

previously

described

(16,

31).

The DNA was

digested

with the enzyme

DpnI

andwithother enzymesas

specified

in the

figure legends.

The

digests

were

analyzed by

electrophoresis

in 1% agarose

gels

and then

subjected

to Southern

blotting

and

hybridization

witha

32P-labeled

plasmid probe

as

previ-ously

described

(31).

The

probe

used for these assays was

pUC119

plasmid

DNA

(47)

prepared by

oligonucleotide

labeling (17).

For

quantitative

analysis,

autoradiograms

of

the blots were scanned with a Cliniscan 2 densitometer

(Helena

Laboratories).

Therelativeextentsof

replication

of

the test

plasmids

were determined

by

estimations of the

following

ratio:

intensity

ofaband

containing

atest

plasmid/

intensity

ofa band

containing

a reference

plasmid.

These

ratioswere normalized relativetothe smallest

ratio,

which was

given

the value 1.0.

CAT assays. WOP cellswere transfected

by

the calcium

phosphate

technique

as described above. After 48 h of incubation at

33°C,

the medium was removed and each 60-mm

plate

was washed twice with 5 ml of a solution

containing

0.13 M

NaCl,

0.026 M

KCl,

0.008 M

Na2HPO4,

and 0.001 M

KH2PO4.

Protein extracts were

prepared

at

23°C

asdescribed

by

Seed and Sheen

(42).

Briefly,

thecells

were

scraped

with arubber

policeman,

suspended

in 1.5 ml

of the solution described

above,

and transferred to an

Eppendorf

tube. Thetubewas spunfor 10sat

11,000

xgin aSorvall

microfuge,

and the

supernatant

wasremoved.The

precipitated

cells were

resuspended

in 1 ml ofa solution

containing

20mMTris-HCl

(pH

7.5)

and 2 mM

MgCI2

and were incubated for 5 min at

23°C.

Then the cells were

precipitated

again by

centrifugation

asdescribed above. The

precipitated

cells were

resuspended

in 100

,ul

ofa solution

containing

20 mM

Tris-HCl

(pH

7.5),

2 mM

MgCl2,

and0.1%

Triton andincubated

again

for5minat

23°C.

Thistreatment

separated

the nuclei from the

cytoplasm.

The nuclei were

precipitated by

spinning

thetube for 5 minat

11,000

x g;the

supernatant

containing

the

cytoplasmic

extract was heated for5 min at

70°C

and used for the CAT assaysas follows.

Fifty

microliters of theextractwas mixedin a

plastic

vialat

4°C

with 200

[lA

ofa solution

containing

1.25 mM

chloram-phenicol

and 100 mM Tris-HCl

(pH

7.8).

To this mixture were added S

,ul

of

'4C-labeled

acetyl

coenzyme A

(0.01

,uCi/,A;

specific

activity,

4

Ci/mol)

and 5 mlofthe

scintilla-tion fluid Econofluor

(Dupont).

Themixture wasincubated

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at37°C and counted inaPackard scintillationcounterafter6 h. Theacetylatedchloramphenicol diffused into theorganic phase; hence, the recorded radioactivity represented the extentof the reactionandgavea measure of CATactivity. It should be noted that the radioactivity increased linearly in ourassaysforatleast 10 h.

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RESULTS

Experimental strategy. Two typesof shuttleplasmidswere used for intracellular replication assays. Plasmids of one type, designated test plasmids, contained modified Py

ori-gins, in which the yeast GAL4 upstream activating se-quences orPyenhancer elements were insertednext to the

origin cores. Plasmids of the second type, designated acti-vators, encoded the yeast GAL4proteinor various deriva-tives of this protein but did not contain active origins of

replication. These plasmids were transfected into Py-trans-formed mouse cells ofa line designated WOP (23), which continuously synthesize a temperature-sensitive Py large T-Ag, the viralproteinrequired forinitiation ofreplicationat the Py ori (7). All transfection-replication assays were

per-formedwith aconstant input ofplasmid DNA; i.e., plasmid pUC19 or pUC119 was added to the transfection mixtures such that the cells in each 60-mmplate were transfected with 10 ,Ig of plasmid DNA. Following transfection, the cells wereincubatedfor 48 h at either 33 or 39°C, the permissive

or nonpermissive temperature, respectively, for the large

T-Ag. Small-molecular-weight DNA was selectively ex-tracted from the cells at the end of these periods and was

digested withtherestrictionenzymeDpnI and with another enzymethat cutsthe testplasmidused for each experiment once. Thedigestswere analyzed by agarosegel electropho-resis, Southern blotting, and hybridization with a plasmid

probe. The amount of DpnI-resistant plasmid DNA, as

determined by blot hybridization, was a measure of the extentof replication of theseplasmids (36). In someassays,

referenceplasmids that had been propagated in Escherichia

coli dam were also cotransfected into the cells. These

plasmids were DpnI resistant even though they had not

replicatedin the mouse cells andwereused forquantitative

assessmentsofthe extentsofreplicationof thetestplasmids.

The yeastGAL4 proteincaninduce replication of aplasmid containing a Py origin core and GAL4 upstream activating sequences. The test plasmids designated pM1Ol and pM96 wereusedfor the firstseries of experiments shown in Fig. 1. AsillustratedinFig. 1C,plasmidpM1Olcontainsafragment

of Py DNAincluding the Pyorigincore,theearly promoter, andapartof the earlyregion encoding the small and middle Py T-Ags and a truncated large T-Ag. The Py enhancer was replaced in this plasmid with five repeats of a 17-bp DNA

oligomer containing a yeast GAL4 upstream activating se-quence (5xGAL4 sites; 21). Plasmid pM96 contains a frag-mentofPy DNAincluding the part of the early region which is also included in plasmid pM101 and a part of the late region. In this plasmid, the Py enhancer has been deleted. One of theactivatorplasmids used for these series of assays, pAG4, encodes the GAL4 protein, which includes a

DNA-binding domain and activating domains (21). A second activator plasmid, pAG147, encodes a polypeptide desig-nated GAL4 (1-147), which consists of the N-terminal 147 amino acids of the GAL4 protein; this polypeptide includes theDNA-bindingdomain of the protein (21).

The transfection-replication assays were performed in mouse WOP cells as described above. Autoradiograms of

theblots obtained inthese assays are presentedFig. 1A and

WSl-al

test _

396 35'

C

P1-t)SMIDES

i:( .4S I1-S

pUC(r

I

Pcv

5205

IIV

p'(.1t19 4f.1(2

.6 5e2 52(A1

ACTIIVATORS

AN IVA I 1KI

A1 (A. .1

FIG. 1. TransactivationofthePyoriby theyeastGAL4protein.

The plasmids whose structures are illustrated in panel C were

transfected into WOP cells as indicated above each lane. The transfection mixtures contained 10jigofDNA,including 1 ,ugofa testplasmid and/or2 ,ugofeachactivatorplasmid, the rest being pUC119 plasmid DNA, except for lane

pMlOl+GAL4+GAL4(1-147), in which themixture contained7 ,ugofplasmidpAG147, 1 ,ug

ofthe testplasmid, and2 ,ugof theactivatorplasmid. The cultures

wereincubated for48 h attheindicatedtemperatures.Then small-molecular-weightDNAwasselectively extracted and digested with DpnI and BamHI exceptforlaneGAL4, forwhichXhoIwasused instead of BamHI. The digests were analyzed by agarose gel electrophoresis, Southernblotting,andhybridizationwithapUC119 probe as described in Materials and Methods. Arrows indicate positions of the linear forms of thetestplasmids.

B.Thearrowspoint to the expectedpositionsofpMlOl (Fig.

1A)andpM96 (Fig. 1B) plasmidmolecules that have under-goneatleastoneround ofreplicationin themousecells,that

is, theDpnI-resistantlinear forms of thesetestplasmids. In theexperiments performed at33°C, the permissive temper-ature for the Py large T-Ag synthesized in WOP cells, no detectable replication ofplasmidpMlOl occurred whenthe cellsweretransfected with thisplasmid alone (Fig. 1A). This

plasmid did replicate in cells that had also been cotrans-fected with the nonreplicating plasmid pAG4, encoding the whole yeast GAL4 transactivator protein. In contrast, no detectable replication ofplasmidpM96 wasobserved when this plasmid was transfected into the cells either alone or with the plasmid encoding the GAL4 protein. Weconclude

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B

pPyAcat26.2+

2xaENHANCER

ori CAT

4632

A5021-5264

Py

pPyl.T1

4632

A409-795

152

pAT153 4631

FIG. 2. Demonstrationthatawild-type large T-Agcanactivatea

Py ori in WOP cells incubated at

39°C.

Transfection-replication

assaysoftheindicatedplasmidswere carriedoutasdescribedin the

legendtoFig. 1exceptthat in lane pPyLT1 theDNAwascut with DpnI and XbaI. Each transfection mixture contained 1 p.gofatest plasmid, 0.5

,ug

of plasmidpUC19propagatedin E. colidam(ref.), and 8.5

,ug

of plasmid pUC19propagatedin E.colidam'.TheDpnI cleavageproductsare not shown.

that the GAL4 protein can transactivate the Py ori in a

plasmid that contains GAL4 upstreamactivating sequences

instead of the Pyenhancer.

In another assay shown in Fig. 1A, the cells had been

cotransfected with plasmid pMlOl and plasmid pAG147, encoding the truncated protein GAL4(1-147). It canbe seen

that this protein was unable to activate the Py ori. In

addition, when both the plasmid encoding the whole GAL4 protein andthe plasmid encoding the truncated GAL4

pro-tein were cotransfected into the cells with plasmid pMlOl,

replication induced by the whole GAL4 protein was

inhib-ited. This result is compatible with the notion that both proteinscompetefor bindingthe clusterof5xGAL4sitesand indicated that theinability of the truncated GAL4 proteinto

activate the Py ori is not due to a rapid turnover of this

protein. It appears, therefore, that the GAL4DNA-binding

domain alone cannotactivate the Py ori and that additional GAL4 protein sequences are required for this function.

Requirement for the Py large T-Ag. The experiments

described so far were carried out at

33°C,

the permissive

temperaturefor thePylargeT-Agsynthesized in WOPcells. As shown in Fig.1A, at

39°C,

the nonpermissive tempera-tureforthe large T-Ag, plasmidpMlOl didnotreplicateeven

in the presence of the whole GAL4 protein transactivator.

This result, however,wasnotnecessarily duetoinactivation

of the T-Ag at

39°C.

It was possible, for example, that

replication of this plasmid was inhibited at

39°C

because

cell-encoded protein components of the replication

appara-tus were damaged at the higher temperature. To examine

this question, we performed transfection-replication assays

of two plasmids including functional Py origins, whose structuresareillustratedin Fig. 2B. Inoneoftheseplasmids,

designated pPyAcat26.2', the complete Py enhancer was

replaced with two tandem repeats of a 26-bp sequence

including a segment of the Py enhancer designated a, which

provide the enhancer function needed for Py DNA

replica-tion (45). This plasmid does not encode any Py-specific proteins. The second plasmid, pPyLT1, contains a whole Py genome in which the large T-Ag intron has been deleted. Hence,this plasmid encodes the large T-Ag and the late viral proteins but not the middle and the small T-Ags. Each of these plasmids was transfected into WOP cells with a nonreplicatingDpnI-resistantreference plasmid (seeabove). The results of the transfection-replication assays per-formed with these plasmids arepresented in Fig. 2A, which showsjust the DpnI-resistant bands found in the gels. It can be seen that plasmid pPyAcat26.2', whichdoes notencode a Py large T-Ag, replicated at

33°C

but not at 39°C. In contrast, plasmid pPyLT1, whichencodes awild-type large

T-Ag,replicated atbothtemperatures. Theseresults showed that the cell-encoded proteins required for DNAreplication could functionat

39°C.

Hence, the inability of the WOPcells

to support the GAL4-induced replication of plasmid pMlOl at

39°C

wasduetoinactivation of the endogenous tempera-ture-sensitivelarge T-Ag.

Transactivation ofthe Py ori by hybrid proteins including the yeast GAL4 DNA-binding domain and viral activating domains. Previous studies have indicated that hybrid pro-teins consisting of the GAL4 DNA-binding domain and activating domains derived from the viral transactivator protein adenovirus Ela (GAL4-Ela) or herpesvirus VP16 (GAL4-VP16) can enhance transcription from promoters

containing GAL4 upstream activating sequences in mamma-lian cells (25, 40). It was therefore of interest to determine whether the hybrid proteins can also transactivate the Py ori. For this purpose, plasmids pGAL4-Ela and pSGVP encod-ing these proteins were cotransfected into WOP cells with testplasmid pMlOl, and the extent of replication of the test plasmid was determined. Replication of plasmid pMlOl occurred at 33 but not

39°C

in the presence of either of the twohybrid transactivators (Fig. 3A). As shown previously, no replication was observed when plasmid pMlOl was transfected into the cells alone or with plasmid pSG147 encoding the truncated GAL4 (1-147) protein. Furthermore, in three plasmid cotransfection assays shown in Fig. 3A,the GAL4 (1-147) protein apparently competed with the hybrid transactivators for binding the 5xGAL4 sites and thereby reduced the extent of replication of plasmid pMlOl to an undetectable level.

Relative efficiencies of transactivation of replication and transcription by the yeast GAL4 activating domains and the viral activating domains. A comparison ofthe intensities of the bands of test plasmid pMlOlin Fig.1A and 3A indicated that the authentic yeast GAL4 protein transactivated the Py ori more efficiently than did the hybrid proteinsincludingthe viral activating domains. Figure 3B shows an experiment that further substantiated this conclusion and allowed a quantitative assessment of the relative potencies of these proteins as replication transactivators. In this experiment, similar assays of the GAL4 and GAL4-Ela proteins were performed in the presenceof anonreplicating DpnI-resistant reference plasmid. In parallel, the extent of replication of plasmid pPyAcat26.2'was assayed again. Scanner readings of the intensities of the bands revealed that the whole yeast GAL4 protein was 10 times more efficient in inducing the replication of plasmid pM1Ol than was the hybrid protein GAL4-Ela. The scanner readings also revealed that the extent ofreplication of the GAL4-activated plasmidpMlOl was 1.7 times lower than that of plasmid

pPyAcat26.2',

which was activated by endogenous Py enhancer-binding

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A

test-_

test-_ *

ref._- g

390 330

TEST PLASMIDS

5.GAL4SITES

pM101 PUC 5265

10 1 17 330

Py

2.0iENHANCER

oni pPyAcat26.27

ACTIVATORS

CAT

4632

A5021-5264

152

,A414ALA(1 881) GiAIA(1-147)L| GAIA(I- 147) i (GA1.4 -f.1a GALA (1-147)

GAIA VF'l6 GALA (1-147)

EIa (121-223)

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0iNT6(41041})

FIG. 3. Transactivation of the Py ori by hybrid proteins

includ-ing the yeast GAL4 DNA-binding domain and viral activating

domains. Transfection-replicationassaysofthe indicated plasmids

werecarriedoutasdescribed in the legendtoFig. 1. Thereference

plasmid (ref.)used intheassaysshowninpanelBwasthesame as

that used inFig.2. Numbersatthe bottom ofpanel Bindicatethe relative extents ofreplication of the test plasmids determinedas

described inMaterials and Methods andnormalizedwithrespect to theextentofreplication obtained inthe assayof plasmids pMlOl

plus pGAL4-Ela, whichwasgiven thevalue 1.0.

proteins. Similar estimations basedonthe data shown inFig.

2andonadditionalassaysof thesametyperevealed that the extentofreplication of plasmid

pPyAcat26.2'

was2.5to3.0 times lower than that of plasmid pPyLT1, which contains a

wholePyenhancer. Theseestimationswerecompatible with previous data of Veldman et al. (45), who compared the replication of plasmid

pPyAcat26.2'

with that of another plasmid containing awhole Py enhancer. Hence, under the

conditions of our assays, the extent of GAL4-activated replication ofplasmid pMl0lwas4.0to5.0 times lower than thatof plasmids containingacomplete Pyori.

In view of the data described above on the relative

efficiencies of the GAL4 and hybrid proteins as replication

transactivators, it wasof interest todetermine the relative efficiencies of theseproteins astranscriptional activatorsin WOP cells under the conditions in which the replication

assays were performed. For these transcription activation assays, weused a reporterplasmid (pG5E472cat) in which the gene encoding the enzyme CAT was linked to an

adenovirus E4promoterincludingaclusterof 5xGAL4 sites of the type shown in Fig. 1 and 3 (25). This plasmid was cotransfected into WOP cells with theplasmids encodingthe activator proteins. CAT activities were determined in ex-tracts prepared from the transfected cells after 48 h of incubationat33°C. In contrast totheirrelative activities in transactivation of the Py ori, the hybrid proteins GAL4-VP16 and GAL4-Ela transactivated the modified E4 pro-motermuchmoreefficiently than did theyeastGAL4protein (Table 1). Also, the truncated GAL4(1-147) protein didnot activate thepromoter.These resultsarecompatible with the results obtained inother celltypes(25, 40).

Activation ofthePyori occursintheabsenceofpromoter elements. Since plasmid pMlOl contains the Py early pro-moter,itwaspossible that the activation of the Py ori by the GAL4 and hybridproteinswascausedby their interactions

withtranscriptional elements. To examine this question,we

took advantage of the observation that in Py, unlike the related papovavirus SV40, thepromoterelementsmap out-side ori (7), and we deleted fromplasmid pM101 the early viral promoter elements, that is, the RNA start sites and about 60 bp upstream of these sites, including the TATA box.Replication of thenewplasmid, designated pM107,was then assayedas described above. As Fig. 4 shows, replica-tion of plasmid pM107 was induced by the intact GAL4 transactivator but not by the truncated protein GAL4 (1-147). Hence, the early promoter elementsare not required

TABLE 1. Transactivation of the adenovirus E4promoter by the yeast GAL4 protein and the hybrid proteins GAL4-Ela andGAL4-VP16 inmouseWOP cells

Radioactivity CAT

Transfected DNA' Transactivator

_protein6

extractedinto activity RelativeCAT

organic activity'

solvent(cpm)c (cpm)d

HerringDNA 1,436±43

pG5E472cat 1,809± 103 373

pG5E472cat+ pAG147 GAL4 (1-147) 1,557 ± 55 121

pG5E472cat+ pAG4 GALA 2,970 ± 390 1,534 1.0

pG5E472cat+ pSGVP GAL4-VP16 4,890± 790 3,454 2.25

pG5E472cat+ pGAL4-Ela GAL4-Ela 13,040± 580 11,604 7.6

a WOpcellsweretransfected withherringDNA or plasmid DNAs asdescribedinMaterialsandMethods.

bProteins encoded by the transfected activatorplasmids.

cCATassays wereperformed by thephaseextractionassay (42) as described in Materials and Methods. Each assay was carried out in duplicate. Numbers represent averagevalues andstandard deviations.

dValues in the firstrowofthe previous column weresubtractedfrom values in the other rows.

'TheCAT activitiesrecordedin thetwo rows atthebottomwereexpressedrelativeto theCATactivityinthe fourth row, whichwasgiven the value 1.0.

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n1 ax~~~4. 1.6 TFST PL.ASMID)S

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ilt'S 11I2

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FIG. 4. GAL4-dependent replication of a plasmid

promoterelementsandofplasmidscontainingduplications

origincoreand the 5xGAL4sites.Transfection-replication

the indicated plasmids werecarriedoutat33°C

legendstotheFig. 1and2exceptthat1.4instead pMlOl

plasmid DNAwas used. The reference plasmid

that had beenpropagatedinE. colidam. Numbers

indicatethe relativeextents of replicationofthe

for these assays, normalized relative to the extent

obtained in the assay ofplasmids pM107 plus GAL4,

giventhevalue 1.0.

forPy origin activation by the GAL4 transactivator.

ever, thedeletion didaffecttheefficiency oftransactivation

by GAL4, sincetheextent ofthe GAL4-induced of plasmid pMl07 was apparently less than one-half

plasmid pMlOl (Fig. 4). Thus, some involvement

promoterelements intheactivationofreplication

beruledout. Onthe otherhand, the reduction

of replication ofplasmid pMlO7couldbe due to

stronglarge T-Agbinding sites whichwere also

the deleted segment (6, 37).

Activationof replicationofplasmidscontainingduplications

of the origin core and of the cluster of 5xGAL4

determine whether deletion of the segment containing

early promoter elements and the T-Ag binding

plasmidpMlOl couldbe compensated byduplication

elements of the origin of replication, we constructed

assayed the replication of plasmid pM108, which contains two tandem repeats ofthe Py origin core and the cluster of

5xGAL4 sites but no promoter elements. As Fig. 4 shows, the extentof replication of plasmidpM108in thepresenceof the GAL4 transactivator was 4.8 times higher than that of

plasmid pM107. Apparently, the presence of two origin cores and two clusters of 5xGAL4 sites increased the probability of initiation of replication in a synergistic way

(see Discussion). Figure4 also shows that the extent of the GAL4-induced replication of plasmid pM108 was 2.3 times

higher than the extent of replication ofplasmid pMlOl and that replication of plasmid pM108 could not be induced by the truncated protein GAL4 (1-147).

To assess the contribution ofeach ofthe two duplicated elements to the synergistic effect on replication of plasmid

pM108,

we constructed and assayed the replication of

plas-mids pMll1 and pM112. Figure 4 shows the structures of these plasmids and the results of these replication assays. PlasmidpM1ll includes oneclusterof5xGAL4 sitesflanked by two origin cores. Theextentofreplicationof thisplasmid in the presence of the GAL4 transactivator was 1.6 times

higher than that of plasmid pM107. It should also be noted that some replication ofplasmid pMlllwas induced bythe

truncated protein GAL4 (1-147). Plasmid pM112 includes oneorigin core flanked bytwoclusters of 5xGAL4 sites. No detectable replication of this plasmid was observed even when it was cotransfected with the plasmid encoding the whole GAL4 protein or the plasmid encoding the GAL4 (1-147) protein. The origin core was not mutated in this plasmid, as revealed by DNA sequencing and by the ability of the same coretocausereplication of another plasmid into which it has beeninserted (1). Apparently, thepresence ofa second cluster of 5xGAL4 sitesnext tothe early boundaryof the core inhibited the initiation of replication of plasmid

pM112.

It should be noted that replication of another

plas-mid includingjustonecluster of 5xGAL4 sites inserted next

to the early boundaryof thecore wasbarely detectableinthe

presence of the GAL4 protein (not shown). DISCUSSION

We have shown in this report that the yeast GAL4 transcriptional activator protein, which also enhances

tran-scription in mammalian cells (21,48), caninducereplication

of plasmids containing aPy ori in mouse cells. Replication

transactivation by the GAL4 protein wasfound to be abso-lutely dependentonthepresenceof GAL4upstream activat-ing DNA sequences next to the late boundary of the Py origin core and ofa functional Py large T-Ag. These data indicated that GAL4 protein molecules bound next to the origin core induced initiation of DNAreplication atthecore. It appears unlikely that the GAL4 protein also affected the replication indirectly by enhancement oftranscription from genes whose products are required for replication, because GAL4 recognition sequences are not found in mammalian promoters.

Since plasmid pMlOl, which was initially used in these

studies, contained the Py early promoter, we thought that

the GAL4 protein and its hybrid derivatives might activate the Py ori by virtueof their ability tostimulatetranscription from the promoter, or through interactions with general transcription factors, without actually causing transcription. Such interactions could, for example, affect the chromatin structure at the adjacent Py origin core and thus facilitate

assembly of thereplication initiation complex. The observa-tion that the truncated protein GAL4 (14147) could not

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activate transcription and replication indicates that these two activities might be causally related. However, other results didnotsupport this hypothesis. First, wefound that theefficiencies ofactivation of the Py ori in plasmid pMlOl by the hybrid proteins GAL4-Ela and GAL4-VP16 were considerably lower than the efficiency of activation by the authentic yeast GAL4 protein. If activation of the Py ori were a consequence of transcriptional activation, then the relative efficiencies of these proteins as activators of

tran-scriptionand replication should have been correlated. How-ever, contrary tothis expectation, the hybrid proteins were foundtobemoreefficient transcriptional activators than the GAL4protein in thesame cells. Second, the GAL4 protein wasfoundtoefficiently induce replication of plasmidpM107, which contains thePyorigin coreand thecluster of 5xGAL4 sites but no Py promoter elements. Furthermore, even though the extentof theGAL4-dependent replication of the plasmid pM107wasless than half that ofplasmid pMlOl, the extentofreplication of plasmid pM108, which contains two repeats of the origin elements found in plasmid pM107 and also lackspromoterelements, was2.3 times that ofplasmid pMlOl (Fig. 4).

Other studies on the role that transcriptional activators playinreplication also supported the notion that replication transactivation by these factors may not result from the interactions that lead to transcriptional activation. In one study, it has been found that the CTF/NF-I transcriptional activators also stimulate initiation ofadenovirus DNA rep-lication (19). However, thereplication enhancement activity in this system required just the N-terminal portion of the CTF/NF-I proteins, whereas transcriptional activation re-quired, inaddition, aC-terminal domain (32). Similarly, the DNA-binding domain (POU domain) of the transcription factor Oct 1 (NFIII) suffices for stimulation ofadenovirus DNAreplication but doesnotactivate transcription(34, 46). Also, whereas the c-Jun/c-Fos heterodimer was found to activateboth transcription and replication, the cyclic AMP-responsive element-binding protein, CREB, stimulated

tran-scription but did not transactivate a Py ori containing a cyclic AMP-responsive element site (33). Thus, not all transcriptional activators alsostimulate replication. Finally, whereastransactivator proteins that bind enhancer elements stimulatetranscriptionevenwhenthese elementsareplaced atrelatively long distances from thepromoters oneither side of the transcription units, the same proteins could activate the Py ori only when the DNA sequences binding these proteins were placed at relatively short distances fromthe lateboundary of the core (14, 33).

It isinterestingtoconsiderourdataonthe GAL4-induced

replication of plasmids pM107, pM108, pMlll, and pM112 (Fig. 4)in relationtoother models that have been proposed

to account for the activation of SV40 and polyomavirus origins by transcriptional activators(8). One modelassumes

thatbinding of suchproteinstoaDNA-binding site inserted

next tothe origin core prevents chromatin assembly atthe originandthusfacilitates formation of the replication initia-tioncomplex. Suchamechanism appearsto accountforthe

invitro stimulation of SV40 DNAreplication byCTF/NF-I proteins (5). This model is compatible with the positive synergistic effectof the origin duplications onthe replication

ofplasmid pM108, for the repeat arrangement in this plas-mid, 5xGAL4 sites-core-5xGAL4 sites-core, could inhibit nucleosome assembly in a synergistic way and thus cause

theobserved4.8-fold increasein theextentof replication of this plasmid compared with thatof plasmid pM107, which contains the single-copy 5xGAL4 sites-core. However, to

accountfor the observation that the truncatedprotein GAL4 (1-147)did not activate the Pyorigins in these plasmids, one would have to assume that not only the DNA-binding domain but also the activating domains of the GAL4 or hybrid proteins were required for inhibition of chromatin assembly.Furthermore, it is difficult to reconcile this model with thecompleteinhibition of replicationof plasmid pM112

containing the repeat arrangement 5xGAL4 sites-core-5xGAL4 sites, because the presence of two clusters of 5xGAL4 sites in this plasmid should presumably prevent

chromatin assembly at the core at least as well as does a single cluster.

A second model assumes that, in analogy to the current models oftranscriptionalactivation, the DNA-binding

trans-activating proteins interact with proteins of the replication

initiationcomplex, e.g., the Py large T-Ag, thereby stabiliz-ing the complex and increasing the rate of initiation of

replication. Suchan interaction might also lead to enhance-mentofthelarge-T-Ag-dependentunwinding of the Py origin core, as suggested in studies of SV40 DNAreplication (12, 13). Thepositivesynergistic effects oftheduplicationsof the clusterof5xGAL4 sites and theorigincore onreplication of

plasmids pM108 and pM1ll could result from interactions between theproteinsthatbind theduplicatedelements. The inhibitory effect of the second cluster of 5xGAL4 sites inserted next to the early boundary of the origin core in plasmid pM112 could be accounted for if GAL4 protein

molecules bound to this element were to interact with the

replication initiation complex such that the initiation of

replication would be inhibited rather than stimulated.

Clearly, definitive experiments designed for testing these

models,whicharenotmutuallyexclusive,canbeperformed onlyin anin vitroreplication system.

ACKNOWLEDGMENTS

Wethank P. Clertant, M. R. Green,R. Kamen, D. Last, J. W.

Lillie,M. Ptashne,andG. M. Veldman for sendingusmanyofthe

plasmids used for this study. Wealso thank C. Basilico andF. G.

Kern for sending us the WOP mouse cells and A. Razin for providing theE. coli dam bacteria.

This workwassupportedbyagrantfrom theCouncil for Tobacco

Research (USA), bygrant 87-00267 from the UnitedStates-Israel Binational Science Foundation, and by a grant from the Israel Cancer ResearchFund.

REFERENCES 1. Baru,M. Unpublished data.

la.Bennet, E. R., M. Naujokas, and J. A. Hassel. 1989.

Require-ments for species-specific papovavirus DNA replication. J.

Virol.63:5371-5385.

2. Brand, A. H., G. Micklem, and K. Nasmyth. 1988. A yeast

silencer containssequencesthatcanpromote autonomous

plas-midreplication andtranscriptional activation. Cell51:709-719. 3. Campbell,B.A., and L. P. Villarreal. 1988.Functionalanalysis ofthe individual enhancer core sequences of polyomavirus: cell-specific uncoupling ofDNAreplication from transcription.

Mol. Cell. Biol. 8:1993-2004.

4. Cereghini, S., P. Herbomel, J. Jouanneau, M. Saragosti, B. Katinka, B. Bourachot, B. de Crombrugghe, and M. Yaniv. 1983.

Structure and function of the promoter-enhancer region of

polyomaand SV40. Cold Spring Harbor Symp. Quant. Biol. 47:935-944.

5. Cheng, L., and T. J. Kelly. 1989. Transcriptional activator

nuclear factorIstimulatesthereplication of SV40 minichromo-somes in vivoand in vitro. Cell59:541-551.

6. Cowie, A., and R. Kamen. 1984. Multiple binding sites for

polyomavirus large T antigen within regulatory sequences of

polyomavirusDNA.J. Virol.52:750-760.

on November 10, 2019 by guest

http://jvi.asm.org/

7. DePamphilis, M. L. 1987. Replication ofSV40and polyomavirus chromosomes, p. 1-40. In Y. Aloni (ed.), Molecular aspects of papovaviruses. Martinus Nijhoff, Boston.

8. DePamphilis, M. L. 1988. Transcriptional elements as compo-nents of eukaryotic origins of DNA replication. Cell52:635-638.

9. de Villier, J., W. Schaffner, C. Tyndall, S. Lupton, and R. Kamen. 1984. Polyomavirus DNA replication requires an en-hancer. Nature (London) 312:242-246.

10. Graham, F. L., and A. J. van der Eb. 1973. Transformation of rat cells by DNA of human adenovirus 5. Virology52:456-467.

11.

Griffin,

B. E., E. Soeda, B. G. Barrell, and R. Staden. 1981.

Sequence and analysis of polyoma virus DNA, p. 843-910. InJ. Tooze (ed.), DNA tumor viruses, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

12. Guo, Z.-S., C. Gutierrez, U. Heine, J. M. Sogo, and M. L.

DePamphilis. 1989. Origin auxiliary sequences can facilitate initiation of simian virus 40 DNA replication in vitro as they do in vivo. Mol. Cell. Biol. 9:3593-3602.

13. Gutierrez, C., Z.-S. Guo, J. Roberts, and M. L. DePamphilis. 1990. Simian virus 40 origin auxiliary sequences weakly facili-tate T-antigen binding but strongly facilifacili-tate DNA unwinding. Mol. Cell. Biol. 10:1719-1728.

14. Hassel, J. A., W. J. Muller, and C. R. Mueller. 1986. The dual role of the polyomavirus enhancer in transcription and DNA replication. Cancer Cells 4:561-569.

15. Hendrickson, E. A., C. E. Fritze, W. R. Folk, and M. L. DePamphilis. 1987. The origin of bidirectional DNAreplication in polyoma virus. EMBO J. 6:2011-2018.

16. Hirt, B.

1967.

Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26:365-369. 17. Hodgson, C. P., and R. Z. Fisk. 1987. Hybridization probe size

control: optimized "oligolabelling." Nucleic Acids Res. 15: 6295.

18. Johnson, P. F., and S. L. McKnight. 1989. Eukaryotic transcrip-tional regulatory proteins. Annu. Rev. Biophem. 58:799-839. 19. Jones, K. A., J. T. Kadonaga, P. J.Rosenfeld,T. J.Kelly,and R.

Tjian. 1987. A cellular DNA-binding protein that activates eukaryotic transcription and DNA replication. Cell 48:79-89. 20. Jones, N. C., P. W. J. Rigby, and E. B. Ziff. 1988. Trans-acting

protein factors and the regulation of eukaryotic transcription: lessons from studies on DNA tumor viruses. Genes Dev. 2:267-281.

21. Kakidani, H., and M. Ptashne. 1988. GAL4 activates gene expression in mammalian cells. Cell 52:161-167.

22. Katinka, M., and M. Yaniv. 1983. DNA replication origin of polyomavirus early proximal boundary. J. Virol. 47:244-248. 23. Kern, F. G., and C. Basilico. 1986. An inducible eukaryotic

host-vector expression system: amplification of genes underthe control of the polyoma late promoter in a cell line producing a thermolabile large T antigen. Gene 43:237-245.

24. Kimmerly, W., A. Buchman, R. Kornberg, and J. Rine. 1988. Roles of two DNA-binding factors in replication, segregation and transcriptional repression mediated by a yeast silencer.

EMBO J. 7:2241-2253.

25.

Lillie,

J. W., and M. R. Green. 1989. Transcription activation by

the adenovirus Ela protein. Nature (London) 338:39-44. 26. Luthman, H., M. G.Nillson,and G. Magnusson. 1982.

Noncon-tiguous segments of the polyoma gene required in cis for DNA replication. J. Mol. Biol. 161:533-550.

27. Ma, J., and M. Ptashne. 1987. Deletion analysis of GAL4 defines two transcriptional activating segments. Cell 48:847-853.

28. Maniatis, T., S. Goodbourn, and A. J. Fischer. 1987. Regulation of inducible and tissue-specific gene expression. Science 236: 1237-1245.

29. Manor, H., and A. Neer. 1975. Effects of cycloheximide onvirus

DNAreplication inaninducible line of polyoma-transformed rat

cells. Cell 5:311-318.

30. McKnight, S.,and R.Tjian. 1986. Transcriptional selectivityof

viral genes in mammalian cells. Cell 46:795-805.

31. Mendelsohn, E., N. Baran, A. Neer, and H. Manor. 1982.

Integrationsiteofpolyoma virus DNA in the inducible LPTline ofpolyoma-transformed cells. J. Virol. 41:192-209.

32. Mermod, N., E. A. O'Neill, T. J.Kelly, and R. Tjian. 1989.The proline-rich transcriptional activator of CTF/NF-I is distinct fromthereplication and DNA binding domain. Cell 58:741-753. 33. Murakami, Y., M. Satake, Y. Yamaguchi-Iwai, M. Sakai, M. Muramatsu, and Y. Ito. Proc. Natl. Acad. Sci. USA, in press. 34. O'Neill, E. A., C. Fletcher, C. R. Burrow, N. Heinz, R. G. Roeder, and T. J. Kelly. 1988. Transcription factor OTF-1 is functionally identical to the DNA replication factor NF-III.

Science 241:1210-1213.

35. Passmore, S., G. T. Maine, R.Elble,C. Christ, and B.-K. Tye.

1988. Saccharomyces cerevisiae protein involved in plasmid maintenance is necessary for mating ofMATa cells. J. Mol.

Biol. 204:593-606.

36. Peden, K. W. C., J. M. Pipas, S. Pearson-White, and D. Nathans.

1980.Isolation ofmutantsofananimal virus inbacteria. Science

209:1392-1396.

37. Pomerantz, B. J., C. R. Mueller, and J. A. Hassel. 1983.

Polyomavirus large Tantigen binds independently tomultiple, unique regions onthe viral genome. J. Virol. 47:600-610.

38. Ptashne, M. 1988. How eukaryotic transcriptional activators work. Nature(London)335:683-689.

39. Rochford, R., C. T. Davis, K. K. Yoshimoto, and L. P. Villar-real. 1990. Minimal subenhancer requirements for high-level polyomavirusDNAreplication: acell-specificsynergyofPEA3

andPEAl sites. Mol. Cell. Biol. 10:4996-5001.

40. Sadowski, I., J. Ma, S. Triezenberg, and M. Ptashne. 1988.

GAL4-VP16 is an unusually potent transcriptional activator.

Nature(London) 335:563-564.

41. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular

cloning: a laboratory manual. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.

42. Seed, B., and J.-Y. Sheen. 1988. A simple phase-extraction

assay forchloramphenicol acetyltransferase activity. Gene 67: 271-277.

43. Tang, W. J., S. L. Berger, S. J. Triezenberg, and W. R. Folk. 1987. Nucleotides in the polyomavirus enhancer that control viral transcription and DNA replication. Mol. Cell. Biol.

7:1681-1690.

44. Triezenberg, S. J., and W. R. Folk. 1984. Essential nucleotides

in the polyomavirus origin region. J. Virol. 51:437-444. 45. Veldman,G.M.,S. Lupton, and R. Kamen. 1985.Polyomavirus

enhancer contains multiple redundant sequence elements that activate both DNAreplication and geneexpression. Mol. Cell. Biol. 5:649-658.

46. Verrijzer, C. P., A. J. Kal,and P. C. vander Vliet. 1990. The

DNA binding domain (POU domain) of transcription factor oct-1 suffices for stimulation of DNA replication. EMBO J.

9:1883-1888.

47. Vieira, J., and J. Messing. 1987. Production ofsingle-stranded plasmid DNA. MethodsEnzymol. 153:3-11.

48. Webster, N., J. R. Jin, S. Green, M. Hollis, and P. Chambon.

1988. The yeast UASG is atranscriptional enhancerin human

HeLa cells in the presence of the GAL4 trans-activator. Cell

52:169-178.

49. Zhu, Z., G. M. Veldman, A. Cowie, A. Carr, B. Schaffhausen,

and R. Kamen. 1984. Construction and functional

characteriza-tion ofpolyomavirus genomes thatseparatelyencode thethree

earlyproteins. J. Virol. 51:170-180.

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