Long terminal repeat enhancement of v-mos transforming activity: identification of essential regions.

0022-538X/83/060726-11$02.00/0

Copyright©1983, AmericanSocietyforMicrobiology

Long Terminal Repeat Enhancement of

v-mos

Transforming

Activity: Identification of Essential Regions

T. G. WOOD,* M. L. McGEADY, D. G. BLAIR,ANDG. F. VANDE WOUDE Laboratory of Molecular Oncology, National Cancer Institute, Bethesda, Maryland20205

Received 29 November 1982/Accepted 9 March 1983

The transforming efficiency of recombinant DNA clones containing the

Mo-loney sarcomavirusv-mos sequence wasenhanced by introducing the Moloney sarcomaviruslong terminalrepeat(LTR) in either the 5' or3' position relativeto

v-mos. We analyzed the polyadenylated RNA expressed in cells transformed by

these recombinant DNA clones and examined the structural integrity of integrated copies of the DNA. In each case, we demonstrated the presence of v-mos

containing RNA transcripts in the polyadenylated RNA and showed that these RNAtranscripts are consistent with the structure of the transfected DNA. The analysis of DNA from these transformed cells showed that the relative positions of thev-mosand LTRsequenceswithin thetransfected DNAwereconservedin

the integrated DNA copies. These results demonstrate that a single LTR can

successfully enhance the transforming activity ofv-mos from eithera5' or a3'

relativeposition. The results from the transfection analysis of recombinant clones containing only portions of the LTR introduced 3' tov-mosdemonstrate that the

essentialregion of the LTR responsible for the enhancement of transformation isa

region within the unique 3' sequences of the LTR containing the 73-base-pair tandem repeat sequence.

The essential components of the Moloney murine sarcoma virus (MSV) proviral genome

responsible

for cell transformation are the

ac-quired v-mos sequence and the proviral long terminal repeat (LTR) (7, 18). The retroviral

LTRhas been shownto containtranscriptional control elements that function to ensure viral

RNAtranscription of the provirus (9, 15, 29, 30, 33). The results from transfection assays using

subgenomic

MSV

proviral

DNA clones have

shown that the LTR enhances the

transforming

activity ofv-moswithequivalent efficiencyfrom

eithera5' or a3'

position

relativeto v-mos

(7).

Although these results suggest that asingleLTR can enhance the

transforming activity

ofv-mos, these assays do not provide evidence that will exclude the

possibility

that tandem

integrations

or rearrangements ofthe transfected

recombi-nant DNA are responsible forthe enhancement

ofthe transforming

activity.

In this report, we

present the analysis of polyadenylated RNA

expressed in cells transformed by the transfec-tion ofrecombinant DNA clones containing

v-mosandasingleLTRand examine thestructure oftheintegrated formof the transfected DNA in these cells. Furthermore, to determine the

es-sential region of the LTR responsible for en-hancement, weconstructedaseriesof

recombi-nant DNAclonescontaining only portions ofthe

LTR introduced 3' to v-mos and tested these recombinant DNAs intransfection assays.

MATERIALS ANDMETHODS

DNA transfections and developmentof transformed cell lines. Maps of the restriction endonuclease sites present in recombinant DNAclones ofHT1 and ml strains of MSV proviral DNA have been published

elsewhere (19, 34). Descriptions of the subgenomic

proviral recombinant DNAclones used in this study have been reported elsewhere (7) or are described below. DNA transfection of NIH3T3 cells was per-formed by modifications of established procedures (11) as previously described (7). Morphologically transformed cellswereselected from individual fociby single-cell cloning either inagarorby serial dilutionin microwell tissue culture dishes. Thecellswere main-tained inDulbeccomodified minimal essential medium (GIBCO Laboratories) supplemented with 10% (vol/ vol)calfserumand antibiotics.

DNA analysis. The cells were lysed with abuffer containing 0.6% (wt/vol) sodium dodecyl sulfate, 10 mM EDTA,10 mM Tris-hydrochloride (pH 7.5),and 100,ugof pancreaticRNase Aper mlwhich had been incubatedat100°C for 5min.Thelysatewasincubated

at37°C for1 h.ProteinaseK(BoehringerMannheim) wasaddedto afinal concentration of250 ,u.g/ml, and the incubation was continued for 2 h at 37°C. The mixturewasthen extractedoncewithanequal volume

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ofphenol saturated with 1 MTris-hydrochloride (pH 8.0), twice with phenol-CHC13 (1:1), and once with CHCl3. DNA was precipitated with 2 volumes of ethanol anddissolved insterileH20.

Restriction endonuclease (New England Biolabs) digests were performed under the conditions recom-mendedby the manufacturer. DNA samples (12to15 ,ug)wereapplied to0.75% (wt/vol) agarose gels con-taining 0.5 ,ug ofethidium bromide per ml, and DNA

fragments were separated by electrophoresis as re-ported by McDonell et al. (20). HindIII digests of lambda DNAwereincluded in eachanalysisfor esti-mating DNA fragmentsize. The transfer of DNAto nitrocellulose membranes(Schleicher& Schuell Co.) was performed as described by Southern (31). The blots were dried for 2 hat80°Candpretreated witha solutioncontaining50%(vol/vol)formamide (Fluka),

5x SSC(1xSSC is0.15 M NaCl plus 0.015 M sodium

citrate), 0.2% (wt/vol) Ficoll (Pharmacia Fine Chemi-cals),0.2%(wt/vol)polyvinylpyrrolidone,50 mM sodi-umphosphate (pH6.5),1% (wt/vol) glycine,0.1% (wt/

vol) sodium dodecyl sulfate, and 250 ,ug of sheared salmon sperm DNA(SigmaChemicalCo.) per mlfor 16 h at 43°C. After hybridization, the blots were washed three times in 2x SSC at room temperature and three times in 0.1x SSC for 15 minat50°C.The blots were then air driedovernightat room tempera-ture.Autoradiography wasperformedwith Kodak SB-5film andanintensifying screen at -70°C.

RNA analysis. The cells (0.3 to 0.5 ml of packed cells) werelysed byhomogenizationinbuffer contain-ing 25 mMTris-hydrochloride (pH 7.5), 25 mM NaCl, 5mMMgCl2,1 mgofheparin (Sigma) per ml, and 2% Triton X-100(Bio-Rad Laboratories).After

centrifuga-tion of thelysateat15,000 x gfor 10 min at 4°C, the

supernatant wasmixed with an equal volume of the abovebuffercontaining200 mMMgCl2.Thismixture wasincubated for 2 h at0°Candthenlayered over a solution of 1 M sucrose, 25 mM Tris-hydrochloride

(pH 7.5), 25 mM NaCl, and 100 mM MgCl2.

Mg2+-complexed polysomes werepelletedby centrifugation at 15,000 x g for 20 min at 4°C. The pellet was suspended in 200

RlI

of 100 mM EDTAcontaining1mg ofproteinase K per ml and then mixed with 5 ml ofa buffercontaining25 mM Trisacetate(pH7.5),600 mM sodium acetate, 2mMEDTA, and0.5% sodium dode-cyl sulfate. The mixture washomogenizedand incu-bated at 45°C for 3 min. Polyadenylated RNA was selectedonoligodeoxythymidylate-cellulose(P-L

Bio-chemicals, Inc.) as previously described (2). RNA

samples were stored under ethanol at -20°C. RNA was collected by centrifugation at 12,000 x g for5 min, and the RNApellets weredried undervacuum. RNAsamples weredenatured in 15 mMmethyl mer-curyhydroxide (Alfa)for 10 minatroomtemperature and separated by electrophoresis on 1.2% (wt/vol) agarosegels containing5 mMmethyl mercury hydrox-ide (1, 3). The transfer ofRNA to diazobenzyloxy-methyl paper (Schleicher & Schuell) and the subse-quent treatment of the blots for hybridization and

autoradiography wereperformed asdescribedby Al-wineet al.(1).

DNA probes. The restriction endonuclease sites usedinisolating specificDNAfragmentsare shownin Fig. 1. p-mos, aplasmid containingtheentire c-mos sequenceand 1.9kilobases(kb) of normalmouseDNA 5'to c-mosclonedintopBRSc7 (see the legend to Fig.

5) at the Sacl and HindIll sties, was used as the source of the mos-specific DNAfragment. pmlsp, a plasmid containing one copy of the ml MSV proviral LTRplus the 5' and 3' cellular sequencesflankingthe ml MSV

pmos

mos

pmlsp

s I

I3

S ~~~~~~1~ 113

AHj3

P2

U3

R

3mlflank

P2 Pe

IL

pml5

989

PL J2

FIG. 1. DNA fragments used to prepare nick-trans-lated DNAprobes. Shown are restriction endonucle-asesites present inthree cloned DNA inserts (p-mos, pmlsp,pmlS) that have been cloned into pBR322 (7, 24). Theserestriction sites were used in isolating DNA fragments (see the text) that represent specific DNA sequences used in the preparation ofnick-translated probes. The size of the DNA fragments was estimated by comparison with the electrophoretic mobility of known DNA size markers in either agarose (20) or polyacrylamide gels (17). p-mos, a plasmid that con-tains theendogenous c-mos sequence (24), was used in isolating a 930-bp AvaI-HindIII mos-specific DNA fragment. pmlsp is a plasmid containing one copy of the ml MSV proviral LTR plus the 5' and 3' cellular sequencesflankingtheml MSVprovirus (19). A 215-bp DNAfragmentthat represents the U3region of the LTR was isolated from PvuII-SacI digests of pmlsp DNA. A 70-bpHhaI-Hinfl DNAfragment containing allof theRsequenceand aHinfl-AluIDNAfragment that contains the entireunique 5' regionof the LTR and 34bp of 3' cellular flanking sequence were also isolated from this plasmid. A700-bp DNA fragment representing the 3' cellular flanking sequences was isolated fromPvuII-PstI digests of pmlsp. pmi5is a

plasmidcontaining the 5' LTR and 2.0 kb of adjacent virus-specific gag sequences cloned from ml MSV proviral DNA (7). A 1.1-kbPvuI-PvuIIDNAfragment representing part of the gagcoding region of ml MSV wasisolated from thisplasmid. Restriction enzymes: A,AluI;Avl,AvaI;Bg2,BgII;H,Hinfl;H3,HindIII;

Hh,HhaI;P1,PvuI; P2,PvuII;Ps,PstI;RI,EcoRI; S, Sacl.

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Ri SB B S H3 H3 B RI

f lank LTP aag v-mos pBR322

45 kb

1 kb

t1 pBR U15 qa, I

(-r

t

F11

il.I:

RI.

b~i-, e.H1 -1 pBR u-)ia; t

7

ti-3

U _

TO-1

U Rn)v

F:i

Ri S .f

13-- a

i;8

4'-4

-..AT 3 4 5 6

FIG. 2. Analysis of RNA and DNA from pHT25 transfectants. A schematicdiagramof thestructureof the 12.2-kb pHT25plasmid DNA is shown, with various proviral, vector, and cellular flankingDNA sequences labeled. Restriction endonuclease sites utilized inanalyzingthestructureof theintegratedDNAareindicated.

pHT25 DNA was linearized by EcoRI digestion before transfection. The results from the analysis oftwo independentpHT25transfectantsareshown.(A andC)Results fromhybridizationanalysisofpolyadenylated

RNA(2.5,g/lane)isolated from thesetransfectants. Theprobesusedinanalyzingthe RNAblotsareindicatedat

the topof each lane, and theirpreparationis described in thetext.Theresultsfromhybridization analysiswitha mosprobeof restriction endonucleasedigestsof cellular DNAs isolated from nontransformed NIH3T3 cells(B,

lanes 4 and5)and thetwopHT25transfectants(B,lanes 1to4,andD)areshown. Therestriction endonucleases used in the variousdigestsareindicatedatthe top of each lane. Restriction enzymes:B,BamHI; H3,Hindlll;S, Sacl;RI,EcoRI.

provirus (19),wasusedas a sourceof DNAfragments

representing the unique 3' region (U3), the repeat sequence (R), and the unique 5' region (US) of the LTR,aswellasthat ofaDNAfragment representing

the 3' flanking cellular sequences. pmlS, a plasmid containing the 5' end of ml MSV proviral DNA,

including the LTR and 2.0 kb ofvirus-specific gag sequences(7),wasusedtoisolateagag-specificDNA

fragment and the US DNA fragment (HinfI-PvuI),

whichwasusedas aprobe in thehybridization analy-sis ofthe pml3 transfectants. A5.6-kbEcoRI DNA fragment that represents the mink cellularDNAtarget site ofHT1MSVprovirusintegration(34)wasusedas aprobe for theHT1 MSV cellularflankingsequences (datanotshown).

For restriction endonuclease digestion, plasmid DNA fragments were purified by electrophoresis on 5% (wt/vol) polyacrylamide gels (bis:acrylamide,1:25)

(17). The nick translation ofpurified DNAfragments

(1 x 108to2x108cpm/,ug)wasperformedasreported by Rigbyetal.(28),andallhybridizations ofRNA and DNAblots contained5 x 106to10 x 106cpmof the respective DNA probe. EachDNAprobewastested for sequence specificity before use in hybridization

assays.

RESULTS

Analysis ofcells transformedby DNA contain-ing an LTR 5'to v-mos. The resultsfrom DNA transfection assays using cloned recombinant

DNAscontainingv-mosand asingle LTR have previously been reported (7, 18). One of these clones, pHT25, was derived from HT1 MSV proviral DNA and contains the 5' LTR and

adjacent proviral sequences through v-mos

clonedinto pBR322(Fig. 2).

The analysis of RNA and DNA from two

independent pHT25

transfectants

is shown in

Fig. 2. If the LTRprovides transcriptional

con-trol elements that insure the expression of

v-mos, thentranscriptionshould beinitiatedatthe repeat(R)sequence within the 5' LTR, and the

resulting RNA transcripts would contain U5,

gag, and v-mos sequences. In onepHT25

trans-fectant, a single 10-kb RNA was detectedwith mos, US, and gag probes (Fig. 2A, lanes 1, 3,

and 4). The second pHT25 transfectant

con-tainedtwoRNA

transcripts

(8.9 and7.0kb)that

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hybridized with mos, gag, andU5 probes (Fig. 2C, lanes 1, 3, and 4). The predicted primary transcript for an RNA initiated at the R se-quencewithin the LTRandcontaining allofthe viral sequences encodedinpHT25is4.5 kb,but all of the mos-containing RNA transcripts ob-served in these cellswereinexcess of this size. Some of this additional information can be

at-tributed tothe exression of pBR322 vector se-quencessinceaprobetothisDNAdetectedthe same RNA transcriptsas the mos, gag, and U5

probes(Fig.2AandC, lane2).However, the

10-kb RNAtranscript (Fig. 2A) would still require additional sequencesderivedfrom hostor

carri-er DNA even if we assume that a complete

pBR322 vector sequence was expressed. A

proberepresenting the U3 region of theLTRdid

nothybridizeto anyof the mos-containingRNA

transcripts(Fig. 2A and C, lane 5). This

demon-strates that the discrete RNA transcripts ob-servedarenotderived from tandem integrations of the transfected DNA and suggests that the termination andpolyadenylation signals utilized by these RNA transcripts are acquired from either vector, host, or carrier DNA sequences. AU3 probe did detecta1.3-and a1.0-kbRNA

transcript (Fig. 2Aand C,lane 5)in the pHT25 transfectants analyzed; however, these

tran-scripts

did not

hybridize

with mos-specific

probes.

Todeterminethenumberofintegrated copies of pHT25 DNA and to assess the structural integrity of the

integrated

DNA sequencesin the

twopHT25transfectants,weanalyzed the cellu-lar DNA by digestion with restriction endonu-cleasesand

by

SoUthern

analysis.

EcoRI-digest-ed DNA from ndntransformed NIH3T3 cells containeda15-kbDNA

fragment

which

hybrid-ized with a mos

probe

(Fig. 2B,

lane

5).

This

DNA

fragment

corresponds

to the

endogenous

EcoRI c-mos DNA

fragment

found in normal

mouse cellular DNA

(14, 24).

The mos

probe

detected the 15-kb c-mos DNA

fragment

and a

new10-kb DNA

fragment (Fig. 2B,

lane

1).

The sizeof the latter EcoRI DNA

fragment

suggests

that EcoRI sites at the ends of the transfected pHT25 DNA have not been conserved in the integrated copy and thata

portion

of the trans-fected DNA was deleted. The extent of this deletion could be estimated from Sacl and

BamHI

DNA digests (Fig. 2B, lanes 2 and

4).

Sacl digests contained a 6.3-kb c-mos DNA

fragment and a 5.4-kb DNA fragment derived

from the transfected pHT25 DNA (Fig. 2B, lanes 2 and4). Asingle Saclsite was present in the LTR, whereas no SacI sites existed in the

adjacentviral orvectorsequences of the

trans-fectedpHT25DNA

(Fig.

2,schema).The 5.4-kb

mos-containing DNA fragment is, therefore, generatedfrom a secondSacl siteacquiredfrom

either host or carrier DNA sequences, and its

size implies that aminimum of 3 kb of pBR322 vector sequences has been deleted from the integrated DNA. pHT25 DNA also contained a

7.0-kb BamHI fragment (Fig. 2). This DNA

fragment was not conserved in the integrated

DNA,indicating that potentially all but 340 base

pairs (bp) of the pBR322 sequences have been deletedfrom the integratedcopy. A comparison

of the abundanceof the endogenousc-mos DNA

fragmentrelative to that of the new

mos-contain-ing DNA fragments observed in this pHT25

transfectant suggests that a single copy of the

pHT25 DNA is present in these transformed cells(Fig. 2B,

lanes

1 through 4),

which

would imply thatthe10-kb mos-containingRNA (Fig. 2A) detected in these cells results from

tran-scription from this singlecopy of pHT25 DNA

(Fig. 2B).

The 8.9-and 7.0-kbmos-containing RNA

tran-scripts(Fig. 2C) expressed in the second pHT25 transfectant alsoappear tobe transcribedfroma

single integratedcopyofpHT25 DNA(Fig. 2D,

lanes 1 through 4). EcoRI digests of cellular DNAfrom this transfectant contained the 15-kb

c-mos DNA fragment and a new 13-kb DNA

fragment that hybridized with the mos probe

(Fig. 2D, lane1).Probes representing pBR322or

the cellular flanking sequences presentin plas-mid pHT25DNAalso annealwitha13-kbEcoRI

DNAfragment (datanotshown). The mos probe

hybridized to a 7.0-kb BamHI DNA fragment (Fig. 2D,lane4)andan8.8-kb DNA fragment in EcoRI-SacI digests (Fig. 2D, lane 3). Both of theseDNAfragments correspondtothe

predict-ed sizeof theirrespective mos-containing DNA

fragments in pHT25 plasmid DNA. These

re-sultsimplythatessentiallyallofthetransfected pHT25 DNA has been conserved in this

trans-fectant. Sacldigestion of DNA from these cells produceda23-kbmos-containingDNAfragment (Fig. 2D, lane

2),

demonstratingthat the pHT25 DNA sequences are integrated in the cellular DNA. The results from the analysis of DNA

from both of these pHT25 transfectants show

that a single LTR introduced 5' to the v-mos sequence caneffect the expression of

mos-con-tainingRNAtranscripts.

Analysis of cellstransformedby DNA contain-ing an LTR 3' to v-mos. pHT21 is a recombinant DNA plasmid derived from HT1 MSV

proviral

DNA containing a single LTR 3' to the v-mos

sequence andcloned intopBR322(Fig. 3). Five mos-specific RNA transcripts ranging in size from 6.3 to 3.2 kb weredetected in thepHT21 transfectant(Fig. 3A,lane1). AnidenticalRNA pattern was obtained by

hybridizations

with a

U3probe (Fig. 3A, lane2),whereasnoneofthe

mos-containingRNAtranscriptsannealedtothe U5 probe (Fig. 3A, lane 4). This result shows

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730 AL.

V

pBR322

A

mos LJ3 pBR U5 gag flk

6.3-li

3.0

4.0

.;

X

HF3X

xi I x

~~IRI

v-mos LTR flank

8 .mos ...pBR . i

RI RI FI

Rl H3H13 Rl 131H3 RI Hf3 H3

15-9 .., _

9.o- - 0 -8.0

6.8- * Am - - 0-70

_ -4.8

4,8- S

4.0- -o

2 3 4 5 6 1 2 3 4 5 6 7 8 9

FIG. 3. Analysis of RNA and DNA from apHT21 transfectant. A schematicdiagram of the structureof pHT21 plasmid DNA is shown. The 9.9-kb plasmid DNAwaslinearizedby EcoRIdigestion before transfection. (A) Results from hybridization analysis of polyadenylated RNA (5 jig/lane) isolated from the pHT21 transfectant. (B) Results from hybridization analysis of DNA from these transformed cells digested with either EcoRI, HindIII, EcoRI-HindIII, orXbaI restriction endonucleases. The probes used in thehybridization assays are indicatedatthetopof the lanes andaredescribed in thetext.

that the enhancement of the transforming effi-ciency of v-mos by a 3' LTR does not result from the tandem integration of the transfected pHT21 DNA. The mos-containing RNA

tran-scripts expressed in the pHT21 transfectants

presumably terminate in the 3' LTR, utilizing

the LTRpolyadenylation signals. The promoter signals andtranscription initiation sites for these RNAtranscriptsarenotderived from the

trans-fected LTR andpresumablyareeitherpresent in

the Moloney leukemia virus sequences preced-ingv-mosor areacquired fromvector, host, or

carrier DNA sequences. The size of the

mos-containing RNA transcripts expressed in this

transfectant limit the use ofpossible promoter

signals encoded in the proviral DNA to the expression ofonly the 3.2-kb transcript.Two of

the five mos-containing RNA transcripts ex-pressed in the pHT21 transfectant contained

sequences that annealed to the pBR322 probe (Fig. 3A, lane 3).

The U5 probe annealed to a 6.5-kb RNA

transcript in the pHT21 transfectant (Fig. 3A, lane 4). However, the expression of this RNA transcript is apparently not directly related to

theexpression of pHT21-encoded sequences. A

probe made to the cellular flanking sequence downstream from the 3' pHT21 LTR did not

hybridizetothistranscript (Fig. 3A, lane 6), but a probe specific for the gag region of MSV, a region notpresent inpHT21 plasmid DNA, did anneal to the 6.5-kb RNA transcript (Fig. 3A, lane 5). These results are consistent with this transcript representing the expression ofan en-dogenous murine viral RNA. A similar RNA

transcript is sometimesobserved in polyadenyl-ated RNA from normal NIH3T3 cells, but the

level of theexpressionof thisRNA isincreased in this pHT21 transfectant and in several other MSV-transformed cells that have been exam-ined(T. Wood, unpublished data). However,all MSV transfectants do not express this RNA

transcript, asis shown forthetwopHT25

trans-fectantsanalyzed inFig. 2.

To determine the number ofintegrated copies of pHT21 DNA and to assess the structural

integrity oftheintegrated DNAsequences

pres-ent in thesetransformed cells, we analyzedthe

cellularDNA from thepHT21transfectant(Fig. 3B). Hybridization with a mos probe detected

onlya15-kbDNAfragmentinEcoRIdigests of cellularDNAfrom thepHT21 transfectant (Fig.

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mos x

2.8-i

2.4-10

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X XS

[image:6.488.50.442.76.315.2]

Rl Bg2 .i RI

...

pBR322 v-mos LTR flank

... 2.2kb

1kb

B mos pBR

Ri S X RIS X C

mos U3 pBR U5 gae filk

o mob pBP

HiS X Ri

I

6.1- _W

a

24-

2.1-1.6- *

!,23. 456

1 2 3 4 5 6 1 2 3 4 5 6

FIG. 4. Analysisof RNAandDNAfrompml3 transfectants. Aschematic diagram of thestructureof pml3 plasmid DNA is shown.Theresultsfromthehybridization analysis of polyadenylatedRNAisolatedfromtwo

pml3transfectantsareshown in(A) (4,ug/lane) and (C)(5.5Fg/lane). The results from thehybridization analysis ofcellular DNA isolatedfrom thesetransformed cellsareshown in(B andD).The restriction endonucleases used intheDNAdigestsareindicatedatthetopof eachlane.Restrictionenzymes:Rl,EcoRI;S, SacI;X,XbaI. Theprobes (seethetextusedinanalyzing the RNA andDNAblots)areindicatedatthetopof the lanesin each panel.

3B, lane 1). Aprobe representing pBR322orthe cellular flanking sequence in pHT21 plasmid DNAalso detecteda15-kb DNAfragment(Fig.

3B, lanes 4 and 6). Theseprobes didnot hybrid-ize to the endogenous c-mos 15-kb DNA

frag-ment (datanot shown), indicating that a DNA fragmentrepresentinganintegratedcopy ofthe pHT21 DNA comigrates with the c-mos 15-kb DNA fragment. This interpretation was con-firmedby analysis with other restriction endonu-cleases.HindIII digests contained thepredicted 9.0-kbc-mosDNAfragment andtwoadditional mos-containing DNA fragments of6.8 and 4.0 kb (Fig. 3B, lane 2). The lattertwoDNA

frag-ments were notaffectedby additionaldigestion withEcoRI, whereas the c-mosDNA fragment

was cleaved (Fig. 3B, lane 3). The XbaI site

immediately 5' to v-mos was 2.4 kb from the

single XbaI sitepresentin the3' LTR in pHT21 plasmid DNA (Fig. 3, schema). Hybridization

withamosprobe detectedtwoDNAfragments inXbaI digests of DNA from the pHT21

trans-fectant,a2.8-kbc-mosXbaIDNAfragmentand

a2.4-kb XbaI DNAfragment derived from the

integrated pHT21 DNA (Fig. 3B, lane 10). These results demonstratethattherearetwointegrated copies ofpHT21 DNAinthistransfectant,

nei-therof which has undergone rearrangement of the relative positions of the v-mos and LTR

sequences present inthe transfected DNA. Al-though the size of the EcoRI DNA fragment indicated the loss of EcoRI sites from both pHT21 DNA copiespresentinthistransfectant, pBR322 and cellular flanking sequences were retained in both of the integrated DNAs (Fig. 3B, lanes 4 through 9),offering further evidence that a major rearrangement of the transfected DNA has not occurred. Both the pBR322 and

mos probes detected the same DNA fragments in EcoRI, HindIII, and HindIll-EcoRI digests (Fig. 3B, lanes 1 through 6). However, the relativeintensityofpBR322 hybridizationtothe HindIll DNA fragments suggests that different

amounts ofpBR322 sequences are retained in

each of theintegrated copies. It should be noted thatthesetwointegrated copies of pHT21 DNA

areapparently responsible for theexpression of

fivemos-containing RNA transcripts.

Asecondexample of the enhancement of the transforming activity ofv-mos bya single LTR

introduced in a 3' relativeposition is presented

by the analysis ofpml3-transformedcells (Fig. 4). pml3 is derived from ml MSVproviral DNA and, likepHT21, this recombinant DNA plasmid

A

r75 IlJ F3EPBR U5 gag filk

C'- I

,~01i.t~- *

15_a

95

15-00

1$_

--

28-i ? : ,) 6

b.) >- so 4

2-28- i

IC *

1 2 3 4

63- 10

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containsasingle LTR 3' tothev-mos sequence

and cloned into pBR322. The circular 7.7-kb pml3 plasmid DNA was transfected without endonuclease digestion. The results from the

analysis ofRNA and DNA from two indepen-dent pml3 transfectants areshowninFig. 4.

In the first example, the mos probe detected

two RNA transcripts of3.6 and 3.0 kb which differed vastly in their relative abundance (Fig. 4A, lane 1). Theprolongedexposureof this blot revealedtwoadditional faint bands correspond-ingto 2.4and 2.1 kb (note dots inFig. 4A, lane

1). Each of these mos-containing RNA

tran-scripts also hybridizedtotheU3probe (Fig. 4A, lane 2), whereasnoneof these RNA transcripts encoded sequences that annealed to the U5 probe (Fig. 4A, lane 4). Only the 3.6-kb RNA

was detected with pBR322 probe, even after longexposures (Fig. 4A, lane 3). A probe made

tothe cellularflankingsequence presentinpml3 plasmid DNA didnotannealtoanyof the RNA transcripts expressed in this pml3 transfectant (Fig. 4A, lane 6).

The results from an identical analysis of the

RNA

transcripts

expressed inasecond

indepen-dent

pm13

transfectant are shown in

Fig.

4C. After prolonged exposure (10 days), the mos

probe detectedtwo RNA

transcripts

of 2.4 and

2.1 kb in these transformed cells

(Fig. 4C,

lane

1). Both of these RNA transcripts contained

sequences that

hybridized

to the U3

probe,

whereasneithertranscript annealed with the U5, flank, or pBR322

probes (Fig. 4C,

lanes 2

through 4 and 6). These results are consistent with the results from theprevious analysis of the pHT21

transfectant

but, in

addition,

show that the level

of

the

expression

of

mos-containing

RNA

transcripts

can vary

considerably

in cells

transformed with

subgenomic v-mos-containing

proviral DNAs. Note that the sizes of the

mos-containingRNAsshown in

Fig.

4Careidentical

tothoseof the smaller RNAtranscripts detected only after

prolonged

exposure in the

previous

example

(Fig.

4A).Weestimate the level ofmos RNA in the second example

pf

pml3-trans-formed cells tobe between 1 and 10copies per

cell (T. Wood, unpublished data). This

implies

that only afew

copies

of

mos-containing

RNA

arerequired toinduce celltransformation. Fur-thermore, these results suggest that the en-hancement of the transforming

efficiency

of

v-mosdoes not predispose the

expression

ofthe mosgene totranscribe

high

levels ofmosRNA.

However, we cannotexcludethe

possibility

that

highlevelsofmosRNAaretranscribed in

pm13-transformed cells butnotprocessedtothe

cyto-plasm.

A 6.1-kb RNA transcript was detected with

theU5andU3

probes

inbothexamples of

pml3-transformed cells

(Fig.

4Aand

C,

lanes 2 and4).

This transcript did not contain mos sequences

(Fig. 4A and C, lane 1) and appeared to be analogous to the 6.5-kb RNA transcript

ex-pressed in pHT21-transformed cells (Fig. 3A). The probe made to the gag

region

ofMSV, a sequence absent in pml3

plasmid

DNA,

an-nealedtothe6.1-kbRNAtranscript

(Fig.

4Aand

C,

lane 5). As was noted for the 6.5-kb RNA transcript detected in the pHT21

transfectant,

this 6.1-kb RNA transcript represents the expression of endogenous murine retroviral se-quences.

EcoRI

digestion

of DNA from the first pml3 transfectant

produced

a 15-kb DNA fragment

that

hybridized

to a mos

probe

(Fig.

4B, lane 1).

A15-kb EcoRI DNA

fragment

was

also

detected withapBR322probe,

suggesting comigration

of thec-mos15-kb DNA

fragment

andpml3 DNA

sequences

(Fig.

4B, cf. lanes 1 and 4). The

analysis

of SacI and XbaI

digests

of DNA from

thesecells withmosand

pBR322 probes

demon-stratesthepresence ofa

single integrated

copy

of pml3DNA

(Fig.

4B, lanes 2, 3, 5, and 6). The

mos probe annealed with a 1.6-kb XbaI DNA fragment from this pml3 transfectant

(Fig.

4B, lane3). This

corresponds

tothe

predicted size of

theXbaI DNA

fragment

inpml3

plasmid

DNA (Fig. 4, schema) and indicates that the v-mos

and LTR sequences in this integrated pml3

DNA have not been

rearranged.

The mos and

pBR322probes both detecteda10-kb SacI DNA fragment, suggesting that thevectorsequenceis oriented in a

5' position

relative tov-mos (Fig. 4B, lane 5). This result is consistent with the

expression of

pBR322 sequences in the 3.6-kb

RNA

transcript (Fig.

4A,lanes1 and 3).

Themos

probe

detecteda 15-kb c-mos DNA

fragment

and a new 11-kb DNA fragment in

EcoRI

digests of

cellular DNA

from

the second pml3 transfectant

(Fig.

4D, lane 1). The analysis of SacI

digests

of this DNA with themosprobe also demonstrates a

single integrated

copy

of

pml3

DNA

(Fig.

4D, lane 2).Asnotedwith the

previous pml3

transfectant (Fig. 4B, lane 3),a

1.6-kb XbaI DNA

fragment

was detected with

themosprobe, indicatingthat therelative posi-tions of the v-mos and LTR sequences in the

integrated copy of

pml3

DNA have been

con-served

(Fig.

4D, lane 3). The pBR322

probe

did

notanneal to DNAfrom this

pml3

transfectant, suggesting that thevector sequenceshave been deleted(Fig. 4D, lane 4).

The results from the analysis of RNA and

DNA isolated from cells transformed by the

transfection of cloned subgenomic proviral

DNA(Fig. 2through 4) haveprovided evidence

that a

single

LTR can

efficiently

enhance the

transforming activity ofv-mosfrom eithera 5' or a3'relative

position.

Thetransfection of recom-binantDNA

containing

anLTR 5'to v-mos

(i.e.,

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http://jvi.asm.org/

PIasmdW

DNA DNA

H3

pHT1O

C S

:

R

pHT22 L--!--- J 975

LTR

pHTS3 8 LIIILL1170

pHTX3 ] 1150

K

pHTR5

---I--I

-

<5

pmI

m

mosl

mF

4500

LTR

1kb

FIG. 5. Physical maps and transforming efficiency of recombinant DNA clones containingvarious LTR sequencesintroduced3'to v-mos.Descriptionsof pHT10,pHT21, pHT22,andpmlspplasmidDNAshavebeen previously published (7, 19). The recombinant DNA clones and the essential restriction endonuclease sites used inthe construction of the recombinant DNA clonesareshown. The DNAfragmentsused in these constructions werepurified by electrophoresis in agarose gels (20), and the DNAwasrecoveredby electroelution. The DNA fragments were extractedoncewithphenol-CHCl3(1:1) andprecipitatedwithethanol. The DNAfragmentswere

ligated(10), and thesamplewastransfected into competent LE392(32). Colonieswereselected and testedfor confirmation ofstructurebyrestriction endonucleasedigestion.pHTS3wasconstructed from the HindIII-SacI DNAfragmentofpHT22 byligationto aBgtII-HindIIIDNAfragmentfrompHT10andclonedinto the BamHI-Saclsites ofpBRSc7 (a plasmidvectorwithSacl linkers introducedatthe PvuIIsite inpBR322).pHTX3was

constructed inasimilarmannerwiththeHindIII-XbaI DNAfragmentofpHT22ligatedto aBglII-HindIIIDNA

fragment frompHT10and cloned into theBamHi-XbaI sites in pBRX19 (aplasmidvectorwith XbaI linkers introduced at thePvuIIsite inpBR322). pHTR5wasconstructed withaBgII-KpnIDNAfragmentpurifiedfrom apartialdigestofpHT21 DNA. This DNAfragmentwasligatedto aKpnI-EcoRIDNAfragmentderivedfrom pmlsp andcloned into theBamHI-EcoRI sites ofpBR322. Sequences representing v-mosandthe LTRare indicated. The dashed line represents cellularflankingsequences. The cloned DNAswerelinearizedby digestion

witheither EcoRI(pHTS3, pHTX3), SalI (pHT22, pHTR5),orBamHI(pHT10, pml)before DNAtransfection (see the text). Thespecificactivities represent the number of foci induced per 2.5 x I0 cells perpmolof DNA transfected and are expressedasfocus-forming units (ffu). They werecalculated fromatleastfourreplicate

determinations.

pHT25) requires the

acquisition

oftermination

polyadenylation

of RNA

transcripts

(9, 15, 29,

and

polyadenylation

signals

for RNA expres-

30,

33),

the enhancement of the

transforming

sion;

alternatively,

an LTR 3' to v-mos

(i.e.,

activity

ofv-mosdoesnot

apparently

result from pHT21, pml3)

imposes

the

requirement

of ob- these

transcriptional

elements,

and this

implies

taining signals

fortheinitiation of

transcription.

that the LTR contains other sequences that

Although the LTRdoes encode

transcriptional

influence the

expression

ofv-mos.If this istrue, control

signals

for

directing

the initiation and then

eliminating

the

polyadenylation signals

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[image:8.488.119.385.62.378.2]

ET AL.

from an LTR introduced 3' to v-mos should not

influence its transforming efficiency and could serve to identify the regions ofthe LTR

respon-siblefor enhancement.

Determination of the sequences responsible for enhancement by an LTR introduced3' to v-mos. To identify the essential sequences required for enhancementbyan LTR3' tov-mos, wetested thetransformingactivity ofaseriesof

recombi-nantDNAclones constructedtocontain various

sequences derived from the LTR (Fig. 5). As

previously shown, pHT10, aplasmid containing

v-mos and 900 bp of Moloney leukemia virus

sequences 5' to v-mos, butno LTRsequences,

exhibits a very lowtransforming efficiency (<5

focus-forming units per pmol of DNA) (7), whereasaplasmidcontaining the entire proviral DNA ofml MSV (pml) produced 4,500 focus-forming units per pmol ofDNA (Fig. 5). The

insertion ofa single LTR 3' to the v-mos se-quence (pHT22) resulted in a 200-fold stimula-tion of the

transforming

efficiency

observed with pHT10 (Fig. 5). An

equivalent

enhancement of thepHT10transformingefficiencywasobserved when DNA fragments containing only se-quencesderivedfromtheU3 region ofthe LTR wereintroduced 3'to v-mos

(Fig.

5, pHTS3 and pHTX3). However, when sequences derived from the R and U5 region of the LTR were present 3' to v-mos (pHTR5), thetransforming

efficiency

was

equivalent

to the level observed

with the transfection of pHT10 DNA (Fig. 5). These results demonstrate that theregion ofthe

LTR

responsible

for the enhancement of the

transforming

activity

of v-mos consists of

se-quences present in the U3

region

and that

se-quencesinthe RandU5

region (i.e.,

polyadeny-lation

signals)

do notenhance the

transforming

activity

ofv-mos.

DISCUSSION

The results from DNA transfection assays

have shown that thetransforming activity of v-mosis enhancedby introducingasingleLTRin

either

a5' or a3'

position

relativeto v-mos (7,

19). In this report, we have presented results

from the analysis of RNA and DNA isolated from cells transformed by the transfection of recombinantDNAclonescontaining v-mos and a single LTR. In each of the transfectants, we

demonstratedthe presence of additional copies ofv-moswithinthecellularDNAin contrast to a

single haploid DNA copy observed in normal

NIH3T3cells (14,24). Theanalysis ofrestriction

endonuclease digests of cellular DNA from these transfectants by hybridization with vari-ousprobes specific forsequences present in the

transfectedDNA confirms theobservation that asingleLTRis sufficientfor the enhancement of thetransforming activityof v-mos. Tandem

inte-grationsof the transfected DNA,recreating pro-virus-like DNAstructures, are not necessary for theexpression of v-mos RNAtranscriptsorfor

the induction of cellular transformation. It shouldbe notedthatin theseexperimentswedid

not distinguish between the introduction of transfectedDNAinto the hostchromosome and its insertion into high-molecular-weight carrier

DNA in a "pekelasome" structure (27).

The rearrangement of the v-mos and LTR sequencesis not required fortheexpression of

v-mos RNA transcripts, and the relative

posi-tions of the v-mos and LTR sequences within thetransfectedDNAs areconserved inthe inte-grated DNA copies. The fate ofthe vectorand cellularflankingsequences presentin the

trans-fected plasmid DNAs varied for each of the

transfectants that we examined. In one case

(i.e., pml3), theentire pBR322vector sequence present in the transfected plasmid DNA was

deleted from the integrated DNA copy. Our resultsdemonstrate that theLTRprovides

tran-scriptional control elements that insure the expression ofv-mos RNAtranscripts.

The cells transformed by the transfection of

DNA containing an LTR 5' to v-mos express RNAtranscripts thathybridize withmosandU5

probes, butnotwith probesrepresentingthe U3

region ofthe LTR. The termination and

polya-denylation signals utilized by the v-mos RNA

transcripts in these transfectants are not ob-tainedfrom the transfected LTR. Thesesignals

areacquired from eithervector, host,orcarrier DNA. Although downstream promotion

pro-vides one mechanism for the activation ofan oncogene, the

induction

of

transformation

by the insertion ofan LTRin a3' position relative

to v-mosisinconsistent withapromoter activa-tion model. Blairetal. first observed that these

constructs inducedtransformationasefficiently

as the 5' LTR v-mos constructs (7). We have

shown here that cells transformed by the

trans-fection ofDNA

containing

an LTR 3'to v-mos express RNA

transcripts

that

hybridize

with

mos and U3 probes. The U5

probe

does not annealto the v-mosRNAtranscriptsexpressed

inthesecells,demonstratingthattranscript initi-ation sites present in the transfected LTR are not utilized in the expression of these v-mos RNA transcripts. Again, these signals must be acquired from either vector, host, or carrier DNA sequences and,becauseofthehigh trans-formation frequency in their absence, must be

easily

acquired

during

transfection. Becauseof the size ofthe 3.2-kb

mos-containing

RNA tran-scriptin thepHT21 transfectant (Fig. 3)and the

size ofthe 2.1-kb

mos-containing

transcript in

pml3 transfectants (Fig. 4), we cannot exclude

thepossibleuseofproviralsequencespreceding

v-mos to provide the initiation sites. By

intro-J.VIROL.

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http://jvi.asm.org/

ducing an LTR 5' to v-mos, we provide

tran-scriptional control elements for the expression ofv-mos-containing RNA transcriptsanalogous

to the promoter insertion model suggested for avian leukosis virus(ALV)-inducedbursal lym-phomas (13, 22, 23, 25, 26). These studies show evidence for an increased expression of the chicken c-myc generesulting from the insertion ofan ALV LTR 5' toc-myc. Payneet al. have shown that in one example of ALV-induced bursal lymphomas, the ALV LTR can also in-creasetheexpression ofc-mycfroma3' position (26).

The results obtained witha numberof simian

virus40(SV40)deletionmutantshavesuggested thatimportant elements controlling the expres-sion ofSV40RNAtranscripts are contained in the72-bp tandem repeatslocatedneartheSV40 origin of replication (5,6, 12). Moreauetal. have shown that the 72-bp tandemrepeat fromSV40 can stimulate T-antigen expression in chimeric plasmids where theSV40 TATA box region has beensubstituted with conalbumin oradenovirus 2 major late promoter sequences (21). Further-more, the level of T-antigen expression is not

alteredby reversing the orientation of the 72-bp tandem repeat (21). The enhancement of gene expression forgenesother than thosepresentin

SV40genomic DNA has also been demonstrat-ed. Capecchi reported that the insertion ofan SV40 DNA fragment containing the 72-bp

tan-demrepeatintoaplasmidcontaining the thymi-dine kinase gene of herpes simplex virus in-creases the transformation frequency ofmouse LMTK- cells (8). The expression of the rabbit

P-globin gene is enhanced in HeLa cells when

this gene is inserted into a recombinant vector

that contains SV40 DNA sequences, including the72-bp repeatelement (4).

MSVproviral DNA has been shown to con-taina73-bp tandemrepeatwithin theU3 region of theLTR(9). Levinsonetal. have demonstrat-ed that the 73-bp repeat from ml MSV can replace the SV40 72-bprepeatas anactivator for theexpression of viralsequenceslocated

down-streamfrom the73-bprepeat(16). We tested the ability of various sequences, derived from the LTRandintroduced 3'tov-mos,toenhancethe transforming activity of v-mos (Fig. 5). The results from this analysis demonstrate that

se-quences within the first 300 bases of the LTR

canyield the samelevel ofenhancement of the

transforming activity ofv-mos ascan acomplete LTR.Sequencespresentinthe R andUS regions ofthe LTR(i.e., polyadenylation signals) donot

stimulate the transforming activity of v-mos.

These results show that the region of the LTR responsible for enhancement consists of

se-quences present in the U3 region, a sequence thatcontains the73-bp tandemrepeat.

ACKNOWLEDGMENTS

We aregrateful to A. Woodworth, M. Oskarsson, and T. Robins forcriticalreading of the manuscript and to K. Barry, L. Shaughnessy, and K. Cannon for assistance inpreparing thismanuscript.

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