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Restriction Endonuclease Mapping of the Proviral DNA of the Exogenous RIII Murine Mammary Tumor Virus

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0022-538X/82/030855-13$02.0O/0

Restriction

Endonuclease

Mapping of

the

Proviral

DNA

of

the

Exogenous RIII

Murine

Mammary

Tumor

Virus

POLLY R. ETKIND,* PAUL SZABO, ANDNURULH. SARKAR

Laboratory ofMolecular Virology,Memorial Sloan-Kettering Cancer Center, and DepartmentofGenetics andMolecular Biology,Sloan-KetteringDivision, GraduateSchool ofMedical Sciences, Cornell University,

New York, New York 10021

Received 17February 1981/Accepted12 March1981

Cellular DNA containing integrated murine mammary tumorvirus (MuMTV)

was isolated fromFeI/C6 feline kidney cells and CCL64 mink lung cells infected

with miLkborne RIII MuMTV. By using restriction enzyme HpaI, intact RIII

MuMTVprovirus (length, 8.7 kilobases [kb])wasexcisedfrom the cellular DNA.

Subsequent restriction endonuclease analysis of this HpaIfragmentwith KpnI,

HindIII,EcoRI,BamHI,BglII, PstI,SstI,Sall,and XhoIenabledusto construct

amapofthe RIII virus genome. Acomparisonof this map with the maps of the

GR and C3H MuMTV's revealed that there are greater sequence differences

between theRIIT virus and the GR and C3H MuMTV proviruses than there are

between the GR and C3H proviruses. The following are featuresof the restriction

map unique to the RIII provirus: the presence of three BamHI and two EcoRI

cleavage sites, a HpaI cleavage site in the terminal 3'-5' repeat unit of the

provirus,andthe absenceofan XhoIcleavagesite.Anotherdistinguishingfeature

of the RIIIprovirusis that the sizes of some of the restrictionfragments produced

by cleavage ofthe RIII provirus with PstI are different from the sizes ofthe

fragments obtained byPstIcleavageof the GR and C3Hproviruses. Like the GR

proviral DNA, the RIII proviral DNA has three Sstl (SacI) cleavage sites,

whereas the C3Hprovirus has only two Sstl sites. Hpal digestion of

MuMTV-infected mink lung cell DNA revealed only one class of provirus (an 8.7-kb

fragment); however, we observed severalminor classes of RIII proviral DNA in

addition to the major class ofprovirus DNA in infected cat kidney cells. PstI

digestion ofthe Hpal8.7-kbfragmentsfrom both feline and mink cells generated a

3.7-kb DNA fragment identical in size to a PstI-generated fragment that has been

found in GR andC3Hmilkborne virus-infected cells.Althoughafragmentsimilar

insize to the milkborne3.7-kb PstIfragment has been found as an endogenous

component in many C3H and GR mouse tissues, we did not observe such an

endogenous fragment in the RIII mouse strain. Therefore, the 3.7-kb fragment

maybeuseful as a marker for the milkbome RIII MuMTV provirus in RIII mice.

Murine mammary tumor virus (MuMTV) is

the etiological agent of spontaneous murine

mammaryadenocarcinomas in several strains of

mice. A comparison of the oncogenicities and

host rangesof MuMTV's and of the influence of

hormones on the developmentofmammary

tu-mors inGR, C3H, and RIII mice has revealed

interesting similarities and differences (5, 34).

Forexample, these three strainsof mice contain

high titers of MuMTV in theirmilk,andbetween

7and 10 months of age mammary tumors occur

at ahigh frequency (80 to 90%) (3, 7, 36). Foster

nursing of RIII andC3Hmice on

low-mammary-cancer-incidence strain mothers whose milk

does not contain MuMTV results in a low

inci-dence(5 to30%)and late development (15 to 24

months) of mammary tumors (1, 3). These late

tumors are thought to result from vertically

transmittedMuMTVsequenceswhicharefound

endogenously in the DNAs of RIII and C3H

mice (12). However, foster nursing of GR

fe-males onlow-mammary-cancer-incidence strain

mothersdoes not delay the onset or decrease the

incidence of early tumor development in this

strain (4, 24, 36, 40). An important biological

property of the RIII and GR viruses that

distin-guishes them from theC3Hvirus is that the RIII

and GRviruses infect the

low-mammary-cancer-incidence strain C57BL mice more efficiently

thandoestheC3Hvirus(33, 35, 49).Inaddition,

theGR and RIII viruses induce

hormone-depen-dent tumors, whereas the C3H virus produces

hormone-independent tumors (3). Type-specific

differences have been detected amongthe

glyco-proteins gp52 and gp36 and the viral core

pro-teinsp28andplO of these three MuMTV strains

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(30, 46, 47). These findings are in agreement

with the results obtained by a tryptic peptide

analysis of isolated viral proteinsgp52, p28,and

plO (20). Although hybridization experiments

haverevealed only minordifferencesamongthe

RNAs of these MuMTV's (32, 37), RNase T1

fingerprinting (19) has revealedsome

character-istic sequence differencesbetween the GR and

C3H strains. Therefore, it would be interesting

tostudy thestructureofRIIIMuMTV RNAby

RNase T1 fingerprinting to determine to what

extent, if any,the RNA of this virus differs from

the RNAs of the GRandC3Hviruses.

The biochemical and immunological

proper-ties of the GR, RIII, and C3H viruses have been

studied inorder toestablish the similarities and

differences among these viruses. A powerful

method of analyzing variation among MuMTV

strains is restriction enzyme analysis of

virus-specific DNA isolated from MuMTV-infected

cells. Using 12 restrictionenzymes,Shanketal. (42) analyzed unintegrated GR and C3H viral DNAs. Ten of these restriction enzymes yielded identical DNA fragments from both C3H

MuMTVDNAand GR MuMTV DNA.

Howev-er, two of the enzymes yielded different DNA

fragments; these were XhoI, which cleaved at

onesite in GR MuMTV DNA andattwosites in

C3H MuMTV DNA, and SstI. (SacI), which

cleaved atthree sites in GRMuMTVDNA and

at two sites in C3H MuMTV DNA (18, 42).

However, no studies of the genome structure of

the RIII MuMTV provirus have been reported.

Therefore, we used variousrestriction

endonu-cleases to map the genome of the RIII virus.

Using restriction enzyme HpaI, weexcised

in-tact MuMTV RIII provirus from the DNAs of

infected catkidney cells and infected mink lung

cells. Subsequent restriction analyses of this

HpaI fragment revealed that the RIII virus

genome was distinguishable from the genomes

oftheGR and C3Hviruses. Byusingrestriction

enzyme PstI, we isolated distinct DNA

frag-ments as markers for distinguishing between

exogenousandendogenous RIII proviralDNAs.

MATERIALSAND METHODS

Cells. Asingle-cellclone(FeI/C6)of theCrFKfeline

embryokidney cell line which had been infected with RIIImilkborneMuMTV(14)waskindly provided by

Jeffrey Schlom and David Howard, National Cancer Institute. The growth conditions and characteristics of thisinfected cellline have beendescribedpreviously.

As a result of filter disk and in situ hybridization studies and from a restriction analysis (Szabo and

Sarkar,unpublished data), itwasdetermined that the cloned infected FeI/C6 cell line contained

approxi-mately 10 to 11 copies of MuMTV proviral DNA. Mink lung cells (CCL64) infected with RIIImilkborne

MuMTVwerekindly provided by Etienne Lasfargues

(48). Bycomparingdensitometertracingsof restriction

fragments generated byBamHI or PstI digestion of

equal amounts ofFeI/C6 cell DNA and CCL64 DNA, we determined that each CCL64 cell contained ap-proximately 100 to 110 copies of MuMTV proviral DNA.

DNA extraction. Totalcellular DNA for restriction analysis was prepared from FeI/C6 cells by the method of Hirt (25). Cells were removedfrom culture vessels

by incubating them in 0.6% sodium dodecyl sulfate

(SDS)-0.01M EDTA(pH7.5) for20 to30 min at25°C,

followed by scraping with a rubber policeman. NaCl wasadded to the cell lysate at a concentration of 1 M, andthe mixture was allowed to stand for 12 h at 4°C to precipitate large DNA. The precipitate was collected by centrifugation at 17,000 x g for 30 min, and the resulting DNA pellet was dissolvedin 0.01 M EDTA (pH 7.5). The solution was adjusted to 50 mM

Tris-hydrochloride(pH8.1), 0.005 M EDTA, and 200 ,ug of proteinase K per ml and incubated at37°C for 6 h. The DNA wasthenextracted twice with an equal volume ofphenol-chloroform and once with an equal volume of chloroform and finally precipitated with ethanol. The ethanolprecipitate was collected, dissolved in 10 mMTris-hydrochloride (pH7.5)-0.1mMEDTA,and dialyzed extensively against the solvent buffer.

The mink cell DNA was released from cells by

incubatingconfluent cultures inaminimum volume of a solutioncontaining50 mMTris-hydrochloride (pH 8.1), 5 mM EDTA, 200 ,ug of proteinase K perml,and

1.0% SDS at 370C for 12 h. The viscous

DNA-containing solution was adjusted to 0.5 M sodium

perchlorateandincubated foranadditional 10minat

37°Cwithgentleagitation.The DNAsolutionwasthen

deproteinized,and the DNA waspurifiedasdescribed above.

Mouse spleens weredispersedin DNA extraction buffer (0.02 M Tris-hydrochloride, pH 8.1, 0.01 M

EDTA, 0.1 M NaCl) with a Sorvall motor-driven

homogenizer.Proteinase K(final concentration,1mg/ ml)andSDS (finalconcentration, 1%)wereaddedto thedispersedspleen cell preparation, which was then

incubated for at least 12 h at 37°C. The DNA was extracted twice with an equal volume of

phenol-chloroform and then dialyzed extensively against 5 mMTris-hydrochloride (pH7.4)-0.1mM EDTA.

Digestion of DNA with restriction endonucleases.

Restriction endonucleases EcoRI,HindIlI, KpnI,and

SstI (Sacl)were purchased from BethesdaResearch Laboratories, Rockville, Md.; HpaI andBglII were purchased from New England BioLabs, Beverly, Mass.; and PstI and BamHI were obtained from Boehringer Mannheim, St. Laurent,Quebec,Canada. Thedigestion mixtures used for these enzymeswere thosesuggested by thesuppliers. Toensurecomplete digestionof theDNA,a5-to10-foldexcessof enzyme wasused (8). For HpaIdigestions 30 to 50 ,ug of DNA per 100,ul of reaction mixturewasusuallyused. For subsequent digestions of the HpaI fragments with otherrestrictionendonucleases,5to10,gof DNA per 25,ul of reaction mixturewasused.

GelelectrophoresisandDNAtransfer.DNAsamples digestedwith restriction endonucleaseswere electro-phoresed in either 0.8or1.0%/ agarose(Seakem)gels containing0.05 MTris-acetate (pH 8.0), 0.02 M sodi-umacetate, 0.018MNaCl, and 0.002 M EDTA (23). Lambda DNA cleaved with HindIII was used as a molecularweight reference andwasvisualized under UV light after ethidium bromide staining. Gels were

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prepared for transfertonitrocellulose sheetsbyusing

a minor modification of the methods described by

Southern(44). The gelswereshaken inamixtureof 0.5 MNaOHand 1.0 M NaClatroomtemperaturefortwo

consecutive 12-min periodsand thenrinsed with dou-ble-distilled waterandneutralizedbyshakingatroom

temperaturefortwoconsecutive 22-min periods in0.5 MTris-hydrochloride (pH 7.0)-3 MNaCl. The gels

werenextplaced upside down onWhatmann 3 MM paperwicks previously soakedwithand keptin

con-tactwithareservoir of20 x SSC(1 x SSCis0.15 M NaCl plus 0.15M sodium citrate) buffer. Nitrocellu-lose filterpaper was placed ontop ofeachgel, and

papertowelswere placedontopofthenitrocellulose filters. After the DNAwastransferred, the filterswere

driedat80°C ina vacuum ovenfor3.0h.

Preparation of DNA probes for hybridization. (i) cDNA probes. A 32P-labeled cDNA representative probe (cDNA,.p) wassynthesized in areaction cata-lyzedby avianmyeloblastosisvirusDNApolymerase (supplied by J. Beard and the Office of Program Resources andLogistics, NationalCancerInstitute), using oligomers of calfthymus DNAasprimers (21). TheMuMTVRNAusedin thereactionswaspurified from MuMTV C3H virions supplied by the National Cancer Institute. The reaction mixture forpreparing

the cDNA,p probe contained 0.05 M Tris (pH 8.1),

0.01%bovine serumalbumin, 0.002 Mdithiothreitol,

100 ,uMdATP, 100 ,uM dTTP,100 p.MdGTP,0.5mCi of[32P]dCTP, 2 pLg of MuMTV RNA, 60,ug ofcalf

thymus DNA, 100 ,ug of actinomycin D, 0.005 M MgCl2, and40 U of avian myeloblastosis virus DNA polymerase inavolume of100 ,ul (11). A32P-labeled cDNA specific for the 3' end of MuMTV RNA (cDNA3)wassynthesized ina200-pul reaction mixture containing 5.0 p.g of oligodeoxythymidylic acid1218,

5.0,ugof MuMTV 70SRNA,0.05M Tris-hydrochlo-ride (pH 8.1), 0.1% bovine serum albumin, 20 mM

dithiothreitol, 100 p.MdATP, 100,uM dTTP, 100 ,uM dGTP,8 mMMgCl2,4mMsodiumpyrophosphate, 50 mMNaCl, 28Uof avianmyeloblastosis virusreverse

transcriptase, and 500 puM [32P]dCTP (16). After incu-bationat37°C for1.0h,0.02 MEDTA and 0.5% SDS

wereadded,andthe cDNAwasextracted with phenol-chloroform. Thepreparationwasthenpassed through a Bio-Gel A-0.5 (Bio-Rad Laboratories, Richmond, Calif.) column equilibrated with TNE buffer (0.1 M NaCl, 1 mMTris, pH 7.4,1 mMEDTA) to remove

unincorporated radioactivity. The cDNA in the ex-cludedvolumewasthen treatedwithalkaliat60°Cfor 30min, neutralizedwith HCl, andethanol precipitat-ed. ThemeansizeofthecDNA3 probe,asdetermined byagarosegelelectrophoresis,was400to500 bases, although the probe ranged in size from 50 to 1,000 bases.

(ii) Nick-translated cloned probe. 32P-labeled

nick-translated pBR322 containing the 1.1 kilobase (kb) PstIfragment ofGR MuMTV DNAwaskindly

pre-pared byElenaBuetti, Swiss Institutefor Experimen-tal Cancer Research, Lausanne, Switzerland. This clonedPstIfragmentrepresents sequencesfromonly

the 5'halfoftheMuMTVgenome(9).

Hybridizationconditions.Nitrocellulose filterswere treatedforaminimum of 6 hat37°Cinhybridization buffercontaining50%formamide, 5x Denhardt solu-tion(15),0.1%SDS, 200 ,ugofsingle-strandedsheared calfthymus DNAorsalmonspermDNAperml, 3x

SSC, and mouse or rabbit liver RNA. The

hybridiza-tion buffer was removed, and freshhybridization buff-er which contained approximately 5 x 106 cpm of [32P]dCTP incorporated into either cDNArep or cDNA3 was added. Hybridization with a nick-trans-lated probe was done by E. Buetti with 3.5 x106 cpm (30ng/ml).Hybridizationwasdoneat41°Cfor 24to48 h. Filters were washedextensivelyat68°C in 2xSSPE containing 1% SDS (1x SSPE is 0.15MNaClplus0.01 MNa2PO4, plus0.001 M Na3EDTA, pH 7.0) and then at55°C in 0.2x SSPE containing0.1%SDStoremove nonspecifically absorbed radioactivity. The filters werewrapped in Saran Wrap andexposed toKodak RP-Royal X-Omat filmat -70°C in the presence ofa DuPont Cronex Lightning Plus intensifying screen (45). Thefilters usedfor comparisons ofhybridization tovarious probes were washed inhybridizationbuffer at68°C betweenhybridizationstoremovetheprevious

probe.

RESULTS

MuMTVsequencesintheFeI/C6cell line.The

FeI/C6 cell line which we used as a source of

MuMTV proviral DNA for restriction analysis

wasinfectedat ahighmultiplicity with MuMTV

isolated from the milk ofRIII mice (26). As a

result of filter disk and in situ hybridization

studies and from restriction enzyme analysis

(SzaboandSarkar,unpublisheddata), weknow

that the cloned infectedFeI/C6cell linecontains

approximately 10 to 11 copies of MuMTVDNA

percell. These cells also contain smallamounts

of unintegrated MuMTV-specific DNA.

There-fore,ourstrategy was to usetheintegratedform

of theMuMTVproviralDNAfor mapping since

it was present in sufficient quantities in these

cells. Wefirst determinedthe sensitivity ofthe

RIIIMuMTVprovirusin theFeI/C6 cell line to

digestion with a number ofrestriction

endonu-cleases(Table1).Acomparisonof these results

with those obtained for the othertwo MuMTV

strains which have been mapped (C3H and GR)

showed that thereweremanysimilarities in both

the number of restriction enzyme recognition

sites and the sizes offragments generated by

enzymes which cleave more than once in the

MuMTV genome. The GR and C3H strains

differ in that the C3H virus has an additional

XhoIsitenotfoundinGR,whereas theGRvirus

has an additional SstI site not found in C3H.

However, there were several differences

be-tween the cleavage sites ofthe RIII strain of

MuMTV and the cleavage sites ofthe C3Hand

GRstrains;forexample,there was noXhoI site

but there were additional BamHI and EcoRI

sites in the RIII virusgenomecomparedwith the

GRand C3H genomes. Inaddition,there was a

HpaI sitein theterminal3'-5' repeat unitofthe

RIIIprovirus butnosuch site in the GR or C3H

virus genome. The sizes of some of the PstI

fragments obtained fromtheRIII, GR,andC3H

viruseswere also differentdespite thefact that

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858 AND

TABLE 1. Comparisonof the cleavage sites for restriction endonucleasesand the sizes of theinternal fragments obtained by restrictionendonucleasedigestion

No.of cleavage sites Size(s) ofinternalfragment(s) (kb)

Enzyme RIIIa GRb C3Hb RIIIa GRb C3Hb

KpnI 1 1 1

HindIII 1 1 1

EcoRI 2 1 1 1.6

BamHI 3 2 2 2.5, 1.3 1.3 1.3

HpaI 2 0 0 8.7

BgIII 2 2 2 3.6 3.6 3.6

PstI 5 5 5 3.7, 1.8, 3.7, 1.6, 3.7, 1.6,

1.4,1.4 1.3,0.9 1.3,0.9

SstI(Sacl) 3 3 2 6.4, 2.3 6.4,2.4 7.7

Sall 0 0 0

XhoI 0 1 2 0.45

a DNA was isolated from RIII MuMTV-infected

FeI/C6

cells and digestedto completion with aspecific

restriction enzyme. Digests were electrophoresed in 0.8% agarose gels, transferred to nitrocellulose filters,

annealedwith MuMTVcDNArep,andautoradiographed asdescribed in thetext.

bDatafor GR and C3H proviral DNAsarefrom references11, 18, and 42.

all three virus genomes had five PstI cleavage

sites.

HpaI sites. Digestion of FeI/C6 cellular DNA

with restriction enzyme HpaI yielded several

fragments detectable by Southern blotting and

hybridization with MuMTV cDNArep (Fig. 1,

lane A). These fragments were also labeled

whenhybridization was with the cDNA3 probe

(Fig. 1, lane B). In addition, other fragments

werelabeled more prominently by the cDNA3

probe than by the cDNArep probe. The major

fragment labeledbyboth cDNAprobeswas8.7

kb long (Fig. 1). This fragmentconstituted

ap-proximately 50% ofthe MuMTV-specific DNA

presentin these cells andwas similar in sizeto

the fragment ofthe linear unintegrated formof

RIIIDNA, whichweobservedin theseinfected

catcells (datanot shown), and the fragmentof

the MuMTV genomes of GR and C3H viruses

(42).Therefore,itappearedthat theHpaI

cleav-agesites in themajorform of theRIIIprovirusin

FeI/C6cellswerelocated in the 3'-5'long

termi-nal repeat (LTR) at each end of the provirus.

Inaddition to the 8.7-kbHpaIfragmentwhich

we considered to be the predominant form of

MuMTV provirus in FeI/C6 cells, other minor

proviral DNA sequences were detected. We

found at least three restriction endonuclease

fragments which were larger than the 8.7-kb

HpaI fragment and contained MuMTV

se-quences (Fig. 1). These fragments were not

partial digestion products since they were

ob-tained in the same yield in four independent

HpaI digests, including digestsfrom digestions

inwhich a10-fold excess ofHpaIenzyme was

used. This class of minor proviruses may not

havecontained HpaI sites inatleast oneofthe

LTRs, and thus HpaI cleavage generated

frag-mentslargerthan8.7 kb.HpaI digestion ofthe

FeI/C6DNAalso resulted inanumberof bands

smaller than themajorHpaIfragment; the sizes

ofthese fragments ranged from 1.25 to 5.3 kb

(Fig. 1, lanes A and B). Most of these fragments

probably represented virus-host cell junctions,

Durnior

notMigration

A.

8.7kb 5.3 3.7 1.7 1.25

FIG. 1. HpaI restriction pattem of DNA isolated from milkborne MuMTV-infected FeI/C6 cells. The

cellularDNAwas digested to completion with restric-tion enzyme HpaI, subjected to electrophoresis in a

0.8% agarose gel, transferred to nitrocellulose filters, annealed with 32P-labeled MuMTV

cDNA,r,p

(lane A) andcDNA3 (laneB),andautoradiographed as described inthetext.Thedensitometertracings of hybridizationto MuMTVcDNArep(solidline) and cDNA3' (dashed line) arealso shown. Themolecular weights were determined for all of the gels shown in this figure and the other figures from known molecular weights of marker DNA

fragmentsobtained byHinduIIdigestion of lambda DNA

electrophoresedinparllelgels and detected by ethidium bromidestaining. For the gels shown here and those in otherfigures in whichcomparisonswere made between different probes, the same filter was used for both

hybridizations, asdescribed in the text.

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whereas others may have beengeneratedfrom

other RIII proviruses consisting of truncated

viralgenomes with deletions of varying lengths,

eventhough they had terminal HpaI sites.

KpnI and Hindu sites. As Table 1 shows,

restriction endonucleases KpnI and HindIII

each cleaved only once in the RIII genome. To determine the location of the KpnI site relative

to thatof the HpaI sites in the major form of the

integrated viral DNA,FeI/C6 DNA was digested

first with HpaI and then with KpnI. We found

that there was a single KpnI cleavage site that

yielded two major fragments (6.1 and 2.6 kb) from the HpaI 8.7-kb fragment (Fig. 2, lane A).

Figure 2, lane B, shows the result ofhybridizing

a cDNA3' probe to a Southern blot of

HpaI-KpnI-digested Fel/C6 DNA. Since 6.1- and 2.6-kb

fragments

were labeled at about the same intensity with the 3' probe, each fragment must

have contained aportion of the 3'-terminal

se-quences, and therefore, the HpaI sites must

havebeen located in the 3' portion of the LTR.

Several minor DNAfragments were detected by

the cDNA3 probe but not by the cDNArep

probe. This finding suggested that these minor

fragments were composedof 3' portions of the

LTRand host DNA. Digestion ofHpaI-cleaved

FeI/C6 DNA with HindIII yielded two major

restriction fragments,which were 5.6 and 3.2 kb

long (Fig. 2, lane C). Hybridization of

HpaI-HindIII-digested FeI/C6 DNA with a cDNA3

A B

C

D

6.1kb-s

5.6-* "

~3.2-

W

2.6-L;

FIG. 2. Digestion of DNA obtained from HpaI-cleaved RIII MuMTV-infected FeI/C6 cells with either restriction endonuclease KpnIorrestriction endonu-cleaseHindIII. Thecompletedsequential digestswere

subjectedto electrophoresis asdescribedin thetext. Lanes A and C, Results ofhybridizations of

HpaI-KpnI and HpaI-HindIII digests, respectively, to

cDNA,,p;lanesB andD, results ofhybridizationsof

HpaI-KpnIandHpaI-HindIII digests, respectively,to

cDNA3,.

A.B

C

D

-6.1kb

5.6-w

.'l.I%

3.-.

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..6

FIG. 3. Digestion of DNA obtained from

HpaI-cleaved RIIIMuMTV-infectedFeI/C6cellswith either

restrictionendonuclease KpnI orrestriction endonu-cleaseHindlIl. Thecompletedsequentialdigestswere

subjected toelectrophoresis asdescribedin thetext. Lanes A and B, Results ofhybridizations of HpaI-HindIlI and HpaI-KpnI digests, respectively, to

cDNAr,p;lanesC and D, results ofhybridizations of

HpaI-HindIIIandHpaI-KpnI digests, respectively,to nick-translated pBR322 containing the 1.1-kb PstI

fragmentofGR MuMTV.

probe resulted in the labeling of bothfragments

with equal intensity, again indicating that the

HpaI sites were in the 3' portion ofthe LTR.

Toorient the cleavageproducts of both

HpaI-KpnI-digestedDNAandHpaI-HindIII-digested

FeI/C6DNArelative to the 5' and 3' ends of the

viralgenome,bothdigests werehybridizedwith

32P-labeled

nick-translated pBR322 containing

the 1.1-kb PstI fragment of GR MuMTV (9).

This cloned 1.1-kb PstI DNAfragment is part of

the gagregion andis found on the 5' sideofthe

genome immediately nextto the LTR(9). Both

the2.6-kb fragment and the 6.1-kbfragment of

HpaI-KpnIcleavage ofFeI/C6DNAhybridized

to cDNArep (Fig. 3, lane B), whereas only the

2.6-kb fragment hybridized to the

nick-translat-ed probe specific to the 5' end of the viral

genome (Fig. 3, lane D). Also, both fragments

(3.2 and 5.6 kb) ofthe HpaI-HindIII digest of

FeI/C6 DNA hybridized to

cDNA,,p

(Fig. 3,

laneA), but only the5.6-kbfragmenthybridized

to the nick-translated probe (Fig. 3, lane C).

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ETKIND, SZABO,

These results indicated that both the 2.6-kb

HpaI-KpnI fragment and the 5.6-kb HpaI-Hin-dIII fragment were derived from the 5' side of

the viralgenome.

Having identified the positions of the two

enzymes(KpnI and HindIII) known to cut once

in the RIII genome, we then chose the KpnI site

as ourpoint of orientation and began cleavages

withEcoRI, BamHI, and PstI, which cut more

than once in the RIII genome (Table 1).

EcoRI sites. Figure 4 shows the results

ob-tained by digestingFeI/C6DNAwith HpaI plus

EcoRIand HpaI plusKpnI plus EcoRI, followed

by hybridization with

cDNA,,p

(Fig. 4, lanes A

andC), as well as the results obtained with the

samedigests annealed with cDNA3 (lanesBand

D). The HpaI-EcoRI digestion generated three

major 5.3-, 1.8-, and 1.6-kb DNA fragments. The

5.3-kb fragment must have contained the 5'

portion (Fig.4, laneC)of the viral genome since

this was the only fragment cleavedby KpnI to

yieldtwo otherfragments (2.7 and 2.5kb). The

2.7-kb fragment hybridized efficiently to the

cDNA3' probe, indicatingthatitwasthe

HpaI-KpnI fragmentatthe 5' endofthegenome. The

2.5-kb fragmentappeared to contain theportion

oftheviral genome from the KpnI sitetothe

5.3-kb EcoRI site since it did not hybridize to

cDNA3. The major 1.6-kb fragment resulting

fromHpaI-EcoRIdigestionappearedtobe

inter-nal in the viral genome since it was also

ob-served in EcoRI digests ofproviral RIII DNA

(Table 1).

BamHI sites.Figure4alsoshows the results of

BamHI digestion ofHpaI- and

HpaI-KpnI-di-gested FeI/C6DNA(Fig. 4,lanes E toH). The

A 3 - Jr

locations of the fragments of the viral genome obtained by digestion at the HpaI sites and the

BamHI sitesclosest to each end of the genome

were determined by comparing the abilities of

the fragments to hybridize with cDNA3 and

cDNA,,p

(Fig. 4, lanes E and F). Of the

frag-ments produced byBamHI digestion and

detect-ed with the cDNArep probe, only the 3.0- and

1.8-kb fragments hybridized to the cDNA3

probe, indicating that these two fragments

com-prised the end portions of the viral genome.

Further cleavage of theHpaI-BamHI fragments

withKpnI slightly reduced the size of the 3.0-kb

fragment (Fig. 4, lanes G and H), showing that

this 3.0-kbfragmentcamefrom the 5' portion of

the genome. It should be noted that BamHI

digestionresulted in twointernal fragments 2.5

and 1.3 kb long in addition to the 3.0- and 1.8-kb

externalfragments (Fig. 4, lane E). The

orienta-tions of the 2.5- and 1.3-kb BamHI fragments

were established (see below) by utilizing the

knowledgeof the PstI cleavage sites of the viral

genome.

Pstl sites. Hybridization of thePstI digest of

HpaI-cleaved cellular FeI/C6 DNA with

cDNA,,p

revealed an unexpectedfinding, name-ly, that the sum of the total lengths of the major

DNA fragments (5.7, 3.7, 1.8, and 1.4 kb) was

greater than the length (8.7 kb) of a single viral

genome(Fig. 5, lane A). These results suggested

that at least two different MuMTV proviruses

were present inthecellularDNA. The

predomi-nantsizesof theHpaI-PstI-generatedfragments

were 3.7, 1.8, and 1.4 kb. The lesspredominant

5.7-kb fragment was observed consistently in

HpaI-PstI digests and, therefore,did not appear

E F G H

5.3kb-g _

.6 4:

,3.d- ". h" -3 215-Oil

$0. a4

1.3-d_e -_) 7

1

8--

*_-"

[image:6.491.104.392.461.612.2]

--3_

FIG. 4. Digestion of DNA obtained fromHpaI-cleaved RIIIMuMTVFeI/C6cells with eitherrestriction enzyme EcoRI orBamHIalone or restriction enzyme KpnIfollowed by EcoRIorBamHI.Thecompleted sequentialdigests weresubjected to electrophoresis, transferred to nitrocellulosefilters,andannealedtoMuMTVcDNA,porcDNA3

asdescribed in the text. Lanes A, C, E, and G, Results ofhybridizationsofHpaI-EcoRI,HpaI-KpnI-EcoRI, HpaI-BamHI,andHpaI-KpnI-BamHI digests,respectively,tocDNAr,p;lanesB,D,F, andH,resultsofhybridizationsof

HpaI-EcoRI, HpaI-KpnI-EcoRI, HpaI-BamHI,andHpaI-KpnI-BamHI digests, respectively, tocDNA3.

J. VIROL.

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A 8 C D

5.7kb-m_,

3.7-_052-a-3.2-q

1.8- * 1.8-_i

1.4- 14-Om*

0.8-

0.8-

0.5-FIG. 5. Digestion of DNA obtained from

HpaI-cleaved RIII MuMTV-infectedFeI/C6 cellswith the

restriction enzyme PstI or KpnI, followed by PstI digestion. The digests were electrophoresed, trans-ferred to nitrocellulose filters, and annealed to MuMTV cDNA,p and cDNA3. as described in the text. Lanes A and C, Results of hybridizations of

HpaI-PstI andHpaI-KpnI-PstI digests,respectively,

tocDNArp;lanes B andD, resultsofhybridizationsof

HpaI-PstI andHpaI-KpnI-PstI digests, respectively,

tocDNA3.

to be a partial digestion product. Digestion of

cellular DNA with PstI alone (Fig. 6, lane A)

generated fragmentswith sizes(5.7, 3.7, 1.8,and

1.4kb)similar to the sizes of fragments obtained

by HpaI-PstI digestion, suggesting that these

A B

5.

C D

fragmentswerederivedbyPstIcleavageswithin

the provirus. A small 0.8-kb fragment, which

was notobservedafter digestion with PstIalone

(Fig. 6, lane A), was present in the HpaI-PstI

digest andwasheavily labeled when

hybridiza-tion was to the cDNA3 probe (Fig. 5, lane B).

This 0.8-kb fragmentwasprobablyderivedfrom

sequencesbetweenaPstIsite and theHpaIsite

presentin theLTRunitsateach endof theviral

genome. When HpaI-KpnI-cleaved FeI/C6

DNA was digested with PstI, both the 5.7-kb

fragment and the 3.7-kb fragmentwerereduced

in sizeby 0.5 kb; in addition, a0.5-kbfragment

wasproduced (Fig.5, laneC). Thisobservation

suggested thattherewas aPstIsite 0.5 kbtothe

5' side of the KpnI cleavage site on the viral

genomeand that this sitewasthe origin of both

the 5.7-kb fragment and the 3.7-kb fragment.

These results also implied that the 5.7- and

3.7-kb fragments were derived from two different

formsofproviruses. Since the sumof the sizes

of the 3.7- and 1.8-kb fragments approximately

equaled 5.7 kb, it waspossible that the 5.7-kb

fragmentarosefromamutationatthe PstI site

whichseparatedthe 3.7- and 1.8-kb fragmentsof

theprovirus. This hypothesis was confirmed by

subsequent sequential digestion of the cellular

DNAwith PstIfollowed by HindlIl, EcoRI,or

BamHI. Ifthe PstI siteatthe 5' end of both the

3.7-kb fragment and the 5.7-kb fragment was 0.5

kbfrom the KpnIsite, itwaspossibletopredict

thenumber and sizes of the cleavage products

which would be generatedbydigestion withPstI

and anyof the other restriction endonucleases

described above (HindIII, EcoRI, and BamHI)

E F G H j

.7kb- - 5.2

3.7-t

~3.2-"

31

<- 2

.5-1.8- "_ .5-1.8-^ 12.4i 16- 5

1.4--b t 1.4-. 1W 1.4- 1.4-

I4-1.4- 1.-

1tw3-0.8

0.8-0.6-

05-FIG. 6. Results ofdigestion of the DNA isolated from RIII MuMTV-infectedFeI/C6 cells with restriction

enzymePstI,aswellasPstIdigestionfollowedby further digestion with eitherKpnI,HindIII, EcoRI, or BamHl.

Thedigests were electrophoresed and annealedto eitheranMuMTVcDNA,pprobe or acDNA3' probe, as described in thetext.LaneA, PstIdigest hybridizedtocDNA,p;lane B,PstIdigest hybridizedtocDNA3.;lane

C,PstI-KpnIdigest hybridizedtocDNA,p;lane D, PstI-KpnIdigest hybridizedtocDNA3.;laneE,PstI-HindIII

digest hybridized tocDNA,p; lane F,PstI-HindIII digest hybridized tocDNA3.; lane G, PstI-EcoRI digest

hybridizedtocDNA,,p;lane H,PstI-EcoRIdigest hybridizedtocDNA3.;laneI,PstI-BamHIdigesthybridized

to

cDNA,.p;

lane J,PstI-BamHIdigest hybridizedtocDNA3.

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(Table 2). For example, HindIlI should have

cleaved the proviruses containing the 3.7-kb

PstI fragment, yielding 3.1- and 0.6-kb

frag-ments,aswellas anuncleaved 1.8-kb fragment.

Similarly, the provirus containing the 5.7-kb

fragment should haveproduced 3.1-and 2.6-kb

fragments. Our results (Fig. 6, lane E) confirmed these predictions. The predicted sizes of the

restriction fragments that were generated by

PstI-EcoRldoubledigestion (Table2)werealso

observed (Figure 6, laneG).

For PstI-BamHI digestion itwas possibleto

predict different results depending onthe

rela-tive positions of the 2.5- and 1.3-kb internal

fragments(Fig.4,laneE).If itwasassumed that

theorder of the fragmentswas5'-2.5kb,1.3

kb-3', then BamHI cleavage of the3.7-and 1.8-kb

PstIfragments would have resulted in fragment

sizes of approximately0.9,2.5, 0.4, 1.0,and0.8

kb, whereas cleavage of the 5.7-kb fragment

would have generated fragments of0.9,2.5, 1.3,

and 1.0 kb (Table 2). On the other hand, if the

order of thefragments was 5'-1.3 kb, 2.5 kb-3',

no0.4-kb fragments could have been produced

by BamHI cleavage (Table 2). Since we

consis-tently observeda0.5-kbfragment inourdigests

(Fig. 6, lane I),webelieve that thecorrectorder

of theinternalBamHIfragments is 5'-2.5 kb, 1.3 kb-3'.

Restriction map of the major form of RI

provirus found in RII-infected FeI/C6 cells. As

discussedabove, FeI/C6 DNA mustcontain at

leasttwodifferent MuMTV-specific proviral se-quences,the predominant proviralsequence

be-ing that which generates the 3.7- and 1.8-kb

internal fragments, as well as a 1.4-kb

frag-ment(s), byPstIdigestion. Therefore, the 1.4-kb

fragment which hybridized with cDNA3' (Fig.6,

lane B) must have been a terminal fragment.

When the HpaI-cleaved provirus was cleaved

withPstI (Fig. 5, lanes A and B), theextentof

hybridization between the cDNArcp probe and

the 1.4-kb fragmentwasreducedbutnot

elimi-nated, and the HpaI-PstI 0.8-kb fragment

hy-bridized strongly with cDNA3, suggesting two

possible PstI mapsof the provirus. Inonemap

(Fig. 7A), the location of thePstIsitesresults in

two 1.4-kb fragments located in tandem with

each other at the left side of the proviral

genome. One fragment extends from a PstI

cleavage siteattheextremeleftofthe3'portion

of the LTRtoasecond PstI cleavage site 1.4 kb

totheright. The other fragment extends 1.4 kb

from this second PstI cleavage site to another

PstI site located at the leftside of the internal

3.7-kb fragment. It should be noted that

approxi-mately 0.8 kbtotheright of thePstIsite foundat

the extreme left of the 3' portion ofthe LTR

there anHpaI site. Since the 3' portion of the

LTR is present also on the right side of the

proviral DNA, aPstI cleavage site should also

existattheextremeleft side of the 3'portion of

theright LTR.Cleavageatthis siteshould result

inheterogenously sized fragmentsderived from

thesequences between the PstI site locatedat

the 3' end of the 1.8-kbfragment (whichisatthe

extreme left end of the 3' portion ofthe right LTR) and the host DNA. When these

heterogen-ously sized fragments arecleavedby HpaI, an

additionalcopyof the 0.8-kbfragmentis

gener-ated. From the alternative map (Fig. 7B), it is

possible that the PstI site in the LTR may be

locatedatthebeginning of the 5'portion ofthe

LTR, thereby generating two1.4-kb fragments

(oneateach end of theprovirus).Onefragment

isderivedfromthePstIsiteattheorigin ofthe 5'

region of theleft LTRtoaPstI site 1.4 kbtothe

right, whereas the other fragment is derived

from thePstI sitelocatedattherightend of the

internal 1.8-kb fragment to the PstI site atthe

origin ofthe 5'sequencesof therightLTR. PstI

cleavageattheoriginof the 5'sequencesof the

left LTR should result in the generation of

[image:8.491.52.449.534.618.2]

heterogenously sizedfragments extendingfrom

TABLE 2. Predicted and observed MuMTV DNAfragments generated byKpnI,HindIII, EcoRI,and BamHI digestions of the 5.7-kb (provirus A) and 3.7-and1.8-kb(provirus B)PstIfragments

Predicted fragment sizes (kb)

Enzyme Observedfragmentsizes(kb)l

Provirus A(5.7kb) Provirus B(3.7and 1.8kb)

KpnI 5'-0.5,3.2, 1.8-3' 5'-5.2,0.5-3' 5.2, 3.2,1.8,1.4,0.5b

HindIII 5'-3.1,0.6, 1.8-3' 5'-3.1,2.4-3' 3.1,2.4, 1.8, 1.4,0.6c EcoRI 5'-2.8, 0.9, 1.0, 0.8-3' 5'-2.8,1.6, 1.0-3' 2.8, 1.6, 1.4, 1.0, 0.9,0.8d BamHI

5'-1.3,2.5-3' 5'-0.9,1.3, 1.5, 1.0, 0.8-3' 5'-0.9,1.3,2.5, 1.0-3'

5'-2.5, 1.3-3' 5'-0.9,2.5, 0.4, 1.0, 0.8-3' 5'-0.9,2.5, 1.3, 1.0-3' 2.5, 1.4, 1.3, 1.0,0.8,0.5e aThe 1.4-kbfragmentwasobservedconsistentlyin alldigestsandwas notincluded in thepredictionssince it was aterminalfragmentandwas notcleavedby any of the enzymes tested.

bSeeFig.6,lane C.

cSeeFig.6, laneE.

dSeeFig.6, lane G.

eSeeFig. 6, lane

I.

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A.

B.

C.

LLTRJ

1LTRJ

14

3.7

1.8 'j1.4

__

-i~~I

I

1LTRJ

LLTRJ

I I I I I II I I I II

0 1 2 3 4 5 6 7 8 9 10 11

Kb

FIG. 7. (A and B) Alternative physical maps of restriction endonuclease PstI sites (0) in RIII MuMTV proviral DNAisolated fromFeI/C6cells and CCL64mink lungcells infected with milkborne RIII MuMTV. The distances between cleavage sites are

expressedinkilobases. Thewavylines indicate cellu-larsequences. (C)Physicalmapofrestriction

endonu-clease sites for HpaI (O), KpnI (A), BamHI (A), EcoRI(0),andHindIII (U)inRIIIMuMTVproviral DNAisolated from FeI/C6feline cells infected with milkborneRIII MuMTV.

thissite into the hostDNA; HpaI-PstI digestion results in the generation of 0.8-kb fragments. These 0.8-kb fragments in this alternative map

are apparently derived from cleavage at the HpaI site which is located approximately 0.8 kb

totheleft of the PstI sitepresent attheorigin of the 5'partof the left LTR. Wewerenotableto

determine whetherminor proviruses containing the 5.7-kbPstlfragment had thesamerestriction

map at the 5' and 3' ends as the predominant

provirus.

Figure 7C shows the KpnI-BamHI-EcoRI-HindIII restriction map of the 8.7-kp HpaI-generatedfragment which represents the entire

RIII virus genome. However, since HpaIcutin the3'portion of theLTR, this fragmentwasnot

identical in size to unintegrated proviral DNA.

Therefore,the sizesof the noninternalfragments

produced by restrictionenzymes inour

experi-mentswererelatedtothe 8.7-kbHpaI fragments rather than to the entire unintegrated proviral DNA.

Inmany respectsthemap showninFig.7Cis

similar tothepreviously published maps ofthe

C3Hand GR MuMTVgenomes(Table 1)(9-11,

21, 39, 42). However, we found the following

threerestriction endonuclease sites in the RIII

genome that are notpresentin the C3H or GR

provirus genome: the HpaI site in the 3' region

of theterminal3'-5' LTR,anadditionalBamHI

site, and anadditionalEcoRI site.Since the size

of theinternalviral restrictionfragmentobtained

by digestionof FeI/C6 DNA withBglII (Table1)

was identical to the sizes of the fragments

ob-tained fromC3H and GRDNAs, itwaspossible

that the BglII cleavage sites are identical in all

three genomes. The number of DNAfragments

generated by SstI digestion of RIII proviral

DNA was the same asthe number generatedin

GR proviral DNAbut differed byone cleavage

site from the number in C3H proviral DNA

(Table1).

Restriction analysis of RUT MuMTV-infected

minkceilDNA. Since the FeI/C6 cellsappeared

to contain more than one MuMTV provirus

sequence, we attempted to determine whether

other heterologous cells infected in vitro with

MuMTV contained more than one type of

MuMTV provirus. Therefore, using restriction

endonucleasesweanalyzedaminklung

epitheli-al cell line which was infected with milkborne

RIII MuMTV andcontained 100to110copiesof

RIIIproviral DNA per cell. The results obtained

afterHpaI and PstI digestions of cellular mink

DNA are shown in Fig. 8. The only

provirus-containing fragment after HpaI digestion was

the 8.7-kb fragment that was also observed in

MuMTV-infectedcatcell DNA(Fig. 8, lane B).

After PstI digestion both cat and mink cell

DNAs produced 3.7-, 1.8-, and 1.4-kb

frag-ments;however, unlike PstI digestion of FeI/C6

DNA, no 5.7-kb fragment was observed in the

minkDNAdigests. Digestion of mink cellDNA

with BamHI, EcoRI, and SstI produced the

same size internal fragments that were found

with FeI/C6 DNA (data not shown). These

re-sults confirmed that the 8.7-kb HpaI fragment

found inthe MuMTV-infectedcatcellswas the

majorMuMTV provirus and that thisfragment

represented the exogenous(milkborne) RIII

vi-rus.

Restriction analysis of RIlIf mouse spleen

DNA.Todeterminewhether the 3.7-kb fragment

observed after digestion of RIII virus-infected

catandminkcell DNAs with HpaI-PstIorwith

PstIalonewas specific forRIIIexogenous

pro-viral DNA, the cellularDNAfromRIlIf mouse

spleens wasisolated and subjected to digestion

withHpaIandPstI(Fig. 9). When digested with

HpaI, the RIIIfmouse spleen DNA contained

twofragments (8.0and 8.7 kb) (Fig. 9, lane B),

whereas HpaI digestion ofthe DNA from the

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A B C D

AE3

CDo15i

8.7kb- .

-57

MWQ1

-3:7

*^_ -1.8

*_

-1.4

FIG. 8. Cleavage of DNA isolated from RIII MuMTV-infected FeI/C6 cells and mink lung cells

withrestriction enzymesHpaI andPstI. The digests

were electrophoresed and annealed to an MuMTV

cDNAr,,p probe. Lane A, HpaI digestof FeI/C6cat

DNA;lane B, HpaIdigestof mink lung DNA; laneC,

PstI digestof cat DNA; lane D, PstI digestof mink lung DNA.

RIII virus-infected mink lung cells resulted in

onlyan8.7-kbfragment (Fig.9, lane A).

Howev-er,PstIdigestion of the spleen cellDNA

gener-ateda5.7-kbfragment; the 3.7-kb fragment that

weobserved (Fig. 9, laneD)after PstI digestion

of RIII MuMTV-infected mink lung cell DNA

(lane C)was notobserved. These results

indicat-ed that the 3.7-kb PstIfragmentwas

characteris-tic of theexogenous RIIIprovirus, whereas the

5.7-kb PstI fragment represented endogenous

RIIIprovirus.

DISCUSSION

MuMTVcaninfectheterologouscells

by

inte-grating into the host cellgenome.

Although

the

efficiency of infection is low (26,

27, 29, 38),

mostinfected cellsproducevirus.Using

restric-tion endonucleases, we

investigated

MuMTV

proviralsequencesintwoheterologouscell lines

infected with the milkbome MuMTV derived

frommouse strain RIII. Both ofthesecelllines

wereoriginally infectedathighmultiplicitiesand

containedmultiplecopiesoftheMuMTV

provi-ruses.

About50% ofthe MuMTV-related sequences

in the FeI/C6 cell line were full length and

containedan HpaI site in the 3' end of the LTR

of the provirus. Cleavage at this HpaI site

produced an 8.7-kb fragment, representing the

complete genome(Fig. 1). The restriction map

of this fragmentwassimilar, butnotidentical,to

the mapsof the othertwoexogenousmilkbome

strains ofMuMTV (C3H and GR). The

differ-ences which we observed between the map of

MuMTV strain RIII and the maps ofthe other

two strains were greater than the differences

previously reported between the maps of GR

and C3H MuMTV's (18, 42). This finding is

compatible with previously reported data ob-tained by serological and tryptic peptide

analy-sis, which showedthat thestructural proteinsof

the C3H and GR MuMTV's are more closely

relatedtoeach otherthan they are tothe

struc-tural proteins of the strain RIII virus(2, 41).

Inadditiontothe major8.7-kbHpaIfragment,

we found that FeI/C6 DNA produced other

MuMTV DNA fragments (larger as well as

smaller than 8.7 kb) as aresult of HpaI

diges-tion. Theminor proviruses thatwerelarger than

the 8.7-kbfragmentmay notcontainone orboth

of the terminal HpaI cleavage sites. The absence

of the HpaI site in this class ofMuMTV

provi-ruses may be due to deletion since deletions

have been reported in the LTRs of integrated

endogenous avian proviruses (31; J. M. Coffin,

personal communication) and in the LTRs of

unintegratedforms of both aviansarcomavirus

DNA (43) and MuMTV DNA (42). A simple

A B C D

kLit - v8

ei-5.7kb

3,7-E

.18_ .owe

i.4-4IS-FIG. 9. HpaIandPstIcleavageproductsof exoge-nous(lanes A and C) and endogenous(lanesB andD)

MuMTVproviralDNAs.Exogenous MuMTVproviral

DNA was obtained from milkborne RIII MuMTV-infected mink cells. Endogenous MuMTV proviral

DNA wasobtained fromRIIIfmouse spleens. Each DNApreparationwasdigested with eitherHpaI(lanes

AandB) orPstI(lanes C and D) andanalyzed by the

Southernblotting procedure,asdescribed in thetext.

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base substitution may also accountfor the loss

ofHpaI sites. Digestion of FeI/C6 DNA with

EcoRI alone generates a 1.6-kb internal

frag-ment, whereas digestion withBamHI alone

gen-erates 2.5- and 1.3-kbinternal fragments. These

internal fragments (for example, 2.5and 1.3 kb

with BamHI) are more abundant than the

termi-nalfragments (i.e., 3.0 and 1.8 kb with BamHI)

derived from cleavage from the HpaI sites to the

BamHI sites (Fig. 4, lane E). Ourfindings

sug-gest that although many, if notall, of the minor

proviruses may have lost HpaI cleavage sites,

they have the same EcoRI and BamHI sites as

thepredominant form of the RIIIprovirus. Our

results did not permit us to prepare complete

restriction maps of the minor proviruses, and we

could not determine whether these minor

provi-rusesgive rise to the 5.7-kb PstI fragment.

HpaI digestion of FeI/C6 DNA results in a

numberofbands smaller than themajor 8.7-kb

HpaI fragment (Fig. 1, lanes A and B). Mostof

these fragments probably represent virus-host

cell functions, whereas others may have been

generated from RIII proviruses consisting of

truncated viral genomes with deletions of

vari-ous lengths, even though theseproviruseshave

terminal HpaI sites. Deletion mutants ofboth

avianand murine sarcoma viruses and leukemia

retroviruses occur naturally (6).Itiswell known

that the propagation of avian sarcoma virus in

permissivecellsfrequently leadstothe

accumu-lation of transformation-defective deletion

mu-tants(17,50). Ananalysis by Hughesetal. (28)

ofthe DNAfrom cloned avian sarcoma

virus-transformed rat cells revealed the presence of

proviruses containing ahigh frequency of

vari-ousdeletions.

UsingPstIdigestionofFeI/C6 DNA,we were

able todistinguishtwoclasses of MuMTV

pro-viruses, one which contains a 5.7-kb fragment

and one which contains 3.7- and 1.8-kb

frag-ments. In contrast to the different types of proviruses produced by RIII viral infection of

catFeI/C6cells,infection of minklung cells with

RIIImilkborne virus ledtointegrationofasingle

type of MuMTV proviral DNA sequence.

HpaI

digestion ofmink DNA yielded

only

the 8.7-kb

proviral DNA fragment

containing

the PstI

cleavage fragmentsof 3.7, 1.8, and1.4kb.Since

infection of these two cell lines was doneintwo

differentlaboratories, it is possible that the mink

and cat cells wereinfected with variants of RIII

MuMTVor that in the case of the catcellsdual infection occurred with more than one RIII

MuMTV variant. It is also possible that the

cellular factors necessary for retroviral

integra-tion can restrict viralintegration to certain types

ofagiven virus. Alternatively, there may have

beencellularfactors presentinthe cat cells but

not intheminkcells which increased variability

in the viral DNA copies made after infection.

Like the FeI/C6 cat cells, the infected CCL64

mink lung cells had been in culturefor 3 years.

Therefore, the types of virus that originally

infected thesecells were unknown to us, and it is

possible that the other classes of provirus were

lost during cell culture. Since the infected mink

lung cells have not increased the variability of

theproviral DNA during continued cell passage,

it seems unlikely that the increased variability

observed in cat cells was due simply to their

time in tissue culture.

Thedifference in the number of PstI cleavage

sites in the RIII proviral DNA may be more than just the loss of a single PstI cleavage site. A 3.7-kb PstI-generated DNA fragment is thought to

be a fragment specific for milkborne C3H and

GR MuMTV's (11). The presence of a 3.7-kb

DNAfragment in thepredominant class of RIII

milkborne provirus ofinfected FeI/C6 cat

kid-ney cells andinCCL64 mink lung cells indicates

that this fragment is also a marker for RIII

milkborne MuMTV. Thus, all three exogenous

proviral DNAs of the three MuMTV strains

(GR, C3H,and RIII) possess thisPstI-generated

DNA fragment. This 3.7-kb fragment is also

found endogenously in many C3H and GR

mouse strains (12, 13), but it does not appear

endogenously in the RIII mouse strain. We

foundthat only the5.7-kbfragmentispresentin

the endogenous MuMTVsequences of RIII(Fig.

9) mouse spleencells, suggesting that this

frag-ment may bespecific for endogenous RIII virus.

The presence of the 5.7-, 3.7-, and 1.8-kb

frag-ments in infected FeI/C6 cells suggests that

these cells may have been infected with RIII

milkcontaining bothendogenous and exogenous

MuMTV's, whereas the mink cells may have

been infected with milkcontaining only

exoge-nous MuMTV. Our ability to identify specific

markers for both exogenous and endogenous

RIIIproviral DNAs may enable us to study RIII

virusinfection and tumorigenesis in the natural

host, RIII mice.

ACKNOWLEDGMENTIS

We thank S. L. Marcus for discussion and A. J.McClelland for editorialassistance.Weespecially thank Elena Buetti and HeidiDiggelman for allowing us to use their cloned 1.1-kb PstI DNAfragment of GRMuMTV.

This workwas supported byPublic Health Service grants CA-16599, CA-17129,andCA-08748 from the National Insti-tutesof Health.

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Figure

TABLE 1. Comparison of the cleavage sites for restriction endonucleases and the sizes of the internalfragments obtained by restriction endonuclease digestion
FIG. 3.cleavedfragmentrestrictionHindIlIcleasecDNAr,p;HpaI-HindIIIsubjectednick-translatedLanes Digestion of DNA obtained from HpaI- RIII MuMTV-infected FeI/C6 cells with either endonuclease KpnI or restriction endonu- HindlIl
FIG. 4.EcoRIBamHI,wereHpaI-EcoRI,as described Digestion ofDNA obtained from HpaI-cleaved RIII MuMTV FeI/C6 cells with either restriction enzyme or BamHI alone or restriction enzyme KpnI followed by EcoRI or BamHI
FIG. 6.ThehybridizedtodigestC,describedenzyme cDNA,.p; PstI-KpnI Results of digestion of the DNA isolated from RIII MuMTV-infected FeI/C6 cells with restriction PstI, as well as PstI digestion followed by further digestion with either KpnI, HindIII, EcoRI,
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