Selective amplification of mouse mammary tumor virus in mammary tumors of GR mice.

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0022-538X/80/10-0109/06$02.00/0

Selective

Amplification of

Mouse

Mammary Tumor Virus in

Mammary

Tumors of GR Mice

THOMAS G. FANNING,JOHN P. PUMA,AND ROBERTD.CARDIFF*

DepartmentofPathology, SchoolofMedicine, UniversityofCalifornia,Davis,California 95616

DNAs extracted from the mammary tumors of GR mice were analyzed for mouse mammary tumor virus proviral sequences by the restriction enzyme-Southern blot procedure. The tumor DNAs contain more proviral copies of mouse mammary tumor virus than DNA from a

nonmalipant

tissue. The degree of proviral amplification issmall(ca. one to fiveadditionalcopies) andappears to be variable from tumor to tumor. The restriction patterns of the amplified proviral sequences suggestaclonalorigin forthe tumor mass. Inaddition, the restriction patterns observed after digestion with the enzymesBgl andSacI indicate that only one of theproviruses endogenous to GR mice is amplified. The amplified provirus found in GRmammar tumors is identical to the provirus thatis missing

inGR-Mtv-2- mice,acongenic line exhibiting a low mammary tumor incidence.

Mouse mammary tumor virus (MuMTV) is

transmitted fromone mouse generation to the

nextby twomechanisms:vertically through the

germ cells, and horizontally through the milk

(3). Although all laboratorystrainsof mice ap-pear to contain several endogenous, vertically

transmittedMuMTVproviruses(11, 26), notall

strainstransmitamilk-bornevirus. Forexample, BALB/c and C57BL mice containnodetectable MuMTV in the milk of lactating females, whereasGRandC3Hmice contain appreciable

quantities ofthe milk-borne virus(14).

Mouse strainsconaining noMuMTV in the milk of females have alow incidence of

mam-marytumorsand,conversely,strains

exhibiting

the milk-borne virus have a high incidence of mammary tumors. Foster-nursing the pups of low-incidence strains on females from high-in-cidencestrains increases thetumorincidence of the former (2). The DNAs oftumorsobtained from the foster-nursed animals contain addi-tionalMuMTVproviruses (MuMTV amplifica-tion) which are the result of infection by the milk-borne virus (6,15).

Foster-nursing the pups of mice of strains

containing the milk-borne virus on those of

strains that donotcontain the virus in the milk

has,ingeneral,theeffectofreducingthetumor

incidence of the forner (1). However, the GR

mouse is an exceptional case in which

foster-nursinghas noapparenteffect on themammary

tumorincidence.GRmicefoster-nursedonmice

of strains conaining no milk-borne virus (e.g.,

C57BL) have a97% tumor incidence,which is

identicaltothatof GRmice ingeneral(4).This

observation, together with the finding that no

increaseinMuMTV proviral sequences canbe

detectedin GR mammarytumorsby liquid

hy-bridization (13, 15), has led to the

speculation

thatneitherinfectionnorMuMTV

proviral

am-plification isrequired for mammarytumor

de-velopment inGRmice (3;P.Bentvelzenand J.

Hilgers, inG.Klein, ed., Virological

Oncology,

inpress).

We haveused restriction enzymes, in conjunc-tion with the blotting technique of Southern (22), to examine the question of MuMTV pro-viralamplificationinGRmammarytumors.Our

findingsindicate that

proviral

amplificationdoes

indeedoccur. In

addition,

the

proviral

integra-tion patternswe observe

imply

that the tumor

massis dominated bythe progenyofaunique

celloruniquesubsets ofcells,ashas been found

forBALB/cmiceinfected with the milk-borne

virus ofC3H animals (6). Further, our results indicate that only one of the endogenous MuMTV proviruses of GR mice is amplified.

Thisprovirusisnotpresent inGR-Mtv-2 mice,

a congenic line which lacks the genetic locus

responsibleforearlyGRmammarytumors(25).

MATERIALS AND METHODS

Mice, cells, and virus. GR mice were obtained

from the Cancer ResearchLaboratory,Berkeley,Calif. GR3A,acell line derived fromaGRmammary tumor (20), wasmaintainedinDulbecco-modifiedEagle

me-dium with 5%fetal calfserumand 5jLgof

dexameth-asoneperml.MuMTVvirions (from the Mm5mt/cl cellline) weresupplied by the Frederick Cancer

Re-searchCenter, Frederick, Md.,throughtheResearch Resources, Biological Carcinogenesis Branch,

Na-tional Cancer Institute.GR-Mtv-2 micewerekindly

provided by Jo Hilgers, TheNetherlands Cancer

In-stitute,Amsterdam, The Netherlands.

Extraction of nucleic acids. GRmammary

tu-109

on November 10, 2019 by guest

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110 FANNING, PUMA, AND CARDIFF

mors were minced and thendisruptedby

homogeniz-ing with a Douncehomogenizerin abuffercontaining

10mMTris-hydrochloride(pH 7.4)-10mMEDTA-10

mM NaCl(TEN buffer).The cellswerecollectedby centrifugation andresuspended in TEN buffer. GR 3A cellswerescraped fromtissuecultureplates, pelleted, andresuspendedinTENbuffer. Protease Kand

so-dium dodecyl sulfatewere added to thecell

suspen-sions to 100yg/ml and0.5%, respectively. The

mix-tures were incubated for 30 min at 37°C and then

extracted twice withphenol(9). The extracted nucleic acidsweredialyzedexhaustivelyat4°C against10mM

Tris-hydrochloride (pH8.0)-i mM EDTAtoremove

residualphenol and storedat4°C.

MuMTV virionsweredisruptedbytheaddition of

sodium dodecyl sulfate to 1%, and the RNAwas

ex-tracted twice with phenol. The MuMTV RNA was

heatedto90°C for3minand thencentrifuged ina 5 to 20% sucrosegradient (17). The materialsedimenting

in the 23-35S region ofthe gradient was collected,

ethanolprecipitated, anddissolvedinasmall volume of10 mM Tris-hydrochloride (pH 7.4)-50mMKC1-1

mMEDTA,andstoredat-20'C.

Preparation of MuMTV 32P-cDNA. Radiolabeled MuMTV-specific complementary DNA (32P-cDNA) wasprepared in a50-M1reaction mixturecontaining:

50 mM Tris-hydrochloride, pH 8.1; 75 mM KCI; 10

mMMgCl2;2mMdithiothreitol; 0.2mMeachdGTP, dCTP, and dTTP; 50 ILCi of[32P]dATP (2,000 Ci/

mmol; NewEngland Nuclear Corp., Boston, Mass.);

25,ugofactinomycinDperml;0.5ygofMuMTV

23-35S RNA;600ytgofcalfDNAprimers (24); and50U of avian myeloblastosis virus reverse transcriptase (providedbyJ.Beard, LifeSciences, Inc., St. Peters-burg,Fla.,through the Research Resources, Biological Carcinogenesis Branch, National Cancer Institute).

Themixturewasincubated for30min at37'C. EDTA, sodiumdodecyl sulfate, and NaOH werethen added

to final concentrations of0.05 M, 0.5%, and 0.3 M, respectively, and the mixturewas incubatedat65°C for 3 to4h tohydrolyzethe RNA.The32P-cDNAwas

separated from othercomponents ofthe mixtureby chromatography onSephadex G-50, ethanol precipi-tated, dissolved in 10 mM Tris-hydrochloride (pH

8.0)-imMEDTA,and stored at-40°C.

Enzyme digests and blotting. Restriction

en-zymes were purchased from New England Biolabs, Beverly, Mass. Digests were done in 100 mM Tris-hydrochloride (pH 7.4)-10 mM NaCl-5mM MgC12-100Mgoflysozymeper ml at 37°C. Approximately9

Mgofcellular DNAwasmixed with 1 Mgof bacterio-phage lambda DNA before enzyme digestion. The

completeness ofthe digestion reaction was verified, afterelectrophoresisin1%agarose gels, by the restric-tion pattern of the added lambda DNA. The transfer of DNAfragmentsfromtheagarose gels to

nitrocel-lulose filters was done essentially as described by

Southern(22). After transfer, the filters were treated

according to published procedures (6).

Autoradi-ograms were scanned with anSDC 300 density com-puter (Schoeffel Instruments, Westwood,N.J.).

RESULTS

We extracted thenucleic acids of four

mam-mary tumors and three livers obtained from

J. VIROL.

three female GR mice. Using the restriction

enzymes EcoRI, BamHI, PstI, BglI, BglHI and

Sac, we analyzed the three liver DNAs for

MuMTV proviral sequences by the Southern

blotting procedure (22).Eachenzymegenerated

a distinct restriction pattern, and for each

en-zyme the three liver DNAs had identical

pat-terns(not shown).Since the liver is not infected

byMuMTV (6, 15),these patterns were

consid-ered characteristic of the endogenous,

geneti-callytransmitted MuMTV proviruses (7).

We cleaved samples ofone of the GR

mam-marytumorDNAs with the restrictionenzymes

listed above and compared the digestion

prod-uctswithsamples of GR liver DNA cleaved with

the same enzymes. The autoradiograph of one

comparison, with EcoRI, BamHI, and PstI, is

presentedin Fig. 1. There were no major

differ-ences between the mammary tumor DNA and

liver DNA restriction patterns.

The restriction patterns of another GR tumor

DNA showed major differences in theform of

manyadditionalfragments when digested with

several of the enzymes (Fig. 2A, slot 2, andFig.

2B, slot 2). These additional restriction

frag-ments probably contained both MuMTV and

15o0+

*imui

6.4 -_

4.3 -_ -i

2.9

_4-1.44

0

1

2

3

4

5

6

FIG. 1. Comparison ofMuMTVproviralsequences in GR liver and GR mammary tumor DNAs. Both the

tumor and liverDNAs used in this example were

from animal 2 (see Fig. 2). Each DNA (9 MLg) was

digestedwith EcoRI(slots1 and 2),BamHI (slots 3 and4), orPstI(slots5and6) andanalyzed by the Southern blottingprocedure and autoradiography. The liver DNA wasrun inslots 1, 3, and 5; tumor DNAwas runin slots 2, 4, and6.Slot0is aHindIII

digestof32P-labeled bacteriophagelambda DNA (19). Themolecularweights(x106) ofthelambda markers

aregivenontheleft(16).

:. 10., k ddk--.Ak

.i -1:

am-l&

own mpmw

is'

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MuMTV AMPLIFICATION IN GR TUMORS 111

mousesequences, thus suggesting a clonal origin for the tumor mass (see below). Thus, amplifi-cation of MuMTV proviral sequences was easily detected insome GR mammary tumors, but was not readily identified in others. However, we could show that each tumor DNA contained unique MuMTV proviral fragments provided the appropriate restriction enzyme was used. Thus, tumors 2, 3, and 4 could be distinguished from oneanother by the appearance of unique

BglIfragments (Fig. 2B, slots 2, 3, and 4), and

tumor 1 could be distinguished from the other threeby a unique BamHI fragment (not shown). During the course of this investigation we observed that someautoradiographic bands in the GR mammary tumor DNAs were consis-tently darker (i.e., the fragments bound more cDNA) than comparable fragments from GR liver DNA; that is, specific MuMTV proviral sequences appeared to be amplified. The phe-nomenon was best demonstrated with theBglII orSaclrestriction enzyme (Fig. 3).

Of the eight major MuMTV DNA fragments found in BglII digests of GR mammary tumor DNA, one fragment of 2.6 x 106 molecular weight had bound more cDNA than the

com-parable liver fragment (Fig. 3A). Of the eight

major MuMTV-specific proviral fragments found inSacl digests oftu

bound more cDNA than t

A

'^F

*::.>...-v...:....3

*,. ,.2Af;."*

_ __ - i S :.

__ __ _ a a_

a = ;^s

s_ _= s_

_w.

*-lrB...f,.

.:::::B;:r

S

... ...:: :.

B

1

2

3

4

FIG. 2. EcoRI and BglI MIMTVproviralsequencesi

DNAs. Each tumor DNA (9

either EcoRI (A) orBglI (B,

Southern blottingprocedure

Slot 1, Tumor1fromanimal

animal1;slot3,tumorfromi from animal3.

A B

1212

FIG. 3. BglII and SadI restriction patterns of

MuMTVproviral sequences in OR liver and OR mammary tumor DNAs. Nine micrograms ofeach DNA (fr-omanimal2)wasdigestedwith eitherBglIII (A)orSadI(B)andanalyzedbytheSouthernblotting

procedureandautoradiography. Slot1, LiverDNA;

slot2,tumorDNA. Theamplified proviralsequences

in the tumor samples are indicated by the arrows. The numbersgive the molecularweights (x 106) of

thefragments.

nmorDNA, two had fragments (Fig. 3B). ThesetwoSacIfragments ;he comparable liver have molecular

weights

ofapproximately 4.0x

106

and 1.6 x

106,

the sum of which represents

virtuallythe entireMuMTV genome (6.0x 106)

(8). The otherGR mammarytumorDNAs also contained the amplified BgllI and SacI

frag-_

__

~~ments.

Densitometer tracings of the SacI digests shown in Fig. 3 are presented in Fig. 4. These confirm whatcanbe surmisedupon inspection.

Two SacI fragments from tumor DNA bound

more cDNA than the

comparable

liver

frag-ments. Integrationof the two curvessuggested

thattwo tothree additionalMuMTVproviruses

were present in this GR mammary tumor DNA.

Weinterpretthe results shown in Fig.3 and

4asfollows:oneof the GRMuMTVproviruses (or one class of provirus) contains two BglII restriction sites that give rise to a 2.6 x 106-- O *s_ daltonfragment and threeSacI restriction sites

generating

1.6x 106- and 4.0 x 106-dalton

frag-1

2~

3 4 ments. The other GR

proviruses

donotcontain

the same sites. The GRprovirus inquestion is

restrctio pattes selectively amplified in GRmammary tumors.

restriction patterns of The degree of amplification can range from a

,nOR

maimmary

tumor

ismgle

additionalcopytofourorfive

additional

pg was digested withsigead

)and

analyzed by the

copies.

In those cases where four or five

addi-and autoradiography. tionalcopiesarepresent,unique proviral

restric-1; slot 2, tumor 2from tion fragments can normally be detected with

animal2;slot4,tumor enzymesthatcleavethe

provirus

ata

single

site (Fig. 2A,slot2).Whenonlyoneadditional

pro-VOL. 36,1980

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virus is present, it may be difficult toidentify.

Unique proviral fragments may beobscured by

comigration with other high-molecular-weight

restrictionfragmentsorrelativelysmallamounts

of MuMTV sequences may be present in the

provirus-mouse fragments. However, the GR

mammarytumorsweexaminedcouldbeshown

to have selective MuMTV amplification using

BglII and SacI, which cleave at several sites

within theamplifiedprovirus.

The amplified provirus we detected in GR

mammary tumors was not present inthe tissue

DNAsofGR-Mtv-2- mice(Fig. 5).This linewas derivedthroughabreedingprogramdesignedto

eliminate thegeneticlocusresponsible for early

mammary tumors in GR mice (25). Thus, our resultssuggestthat thisgenetic locus is,atleast

inpart, one ofthe GRproviruses.We will

hence-forth refer to this provirus as the GR-MTV-2 provirus.

The low degree ofMuMTVDNA

amplifica-tion detected in GR mammary tumors wasin

FIG. 4. Densitometertracings of the SacI restric-tion patterns shown inFig. 3. (A) GR liver; (B) GR mammarytumor.The 1.6x106-and 4.0x106-dalton

proviralsequencesamplified in thetumorsampleare

indicatedby thearrows.

A '.-...;A

~~~~~~~~~~~~~~~~4.~~~~~~~g

1.6-+ .. 4.

[image:4.510.275.465.73.210.2]

2 34 3 4

FIG. 5. SacI and BglII restriction patterns of MuMTVproviralsequencesin GRtissue,

GR-Mtv-2-tissue, andGR 3A cell DNAs. (A) Each tissue DNA

(9 ug)andGR 3A cell DNA (2 ,ug)wasdigested with

either SacI (A) or BglII (B) and analyzed by the

Southern blotting procedure and autoradiography. Slot 1, GR mammarytumorDNA; slot 2, GR liver DNA; slot 3, GR-Mtv-2- liverDNA; slot 4, GR 3A cell DNA. The 1.6x 106- and 4.0x 106-dalton SacI and 2.6x106-dalton BglII fragmentsareindicatedby the arrows.

contrast to the large degree of amplification

observedin mammary tumors fromothermouse

strains (13, 15). Thissuggestedthat the degree

of amplification of theGR-MTV-2 provirusmay

be limited. Comparison ofSacl digests of GR

liver and GR 3A tumorcell line DNA,however,

demonstrated extensiveamplificationofthe 1.6

x 106-and 4.0 x 106-dalton SacIfragments(Fig.

5A). The 2.6 x 106-dalton BglII fragment was

also (and exclusively) amplified in GR 3A cell

DNA (Fig. 5B). Since theGR 3A cell linewas

derived from a GR mammary tumor, we

con-clude thatamplification of theGR-MTV-2

pro-virus can be extensive under some

circum-stances.

DISCUSSION

Amplification ofproviral geneshas been

ob-served in severaltumorsystems (5, 10).

Ampli-fication ofMuMTVproviralsequences in mouse

mammarytumorshas beendocumented in

sev-eral mouse strains whichtransmitamilk-borne

virus (e.g., BALB/cfC3H and RIII) (13, 15).

However,noamplificationofMuMTVcould be

detected in the mammary tumors of GR mice

(13, 15). This, togetherwith thefact that

milk-borne viral infection is notnecessary for tumor

development (4),hasledtothespeculationthat mammarytumorigenesis in GR micemay

rep-resent aspecialcase(BentvelzenandHilgers,in

press).

Our results indicate that MuMTV proviral

amplification does occur in the mammary

tu-J. VIROL.

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mors of GR mice. Thus, mammary tumors of GR mice resemblethose of otherstrains exhibit-ingamilk-borne virus. The relatively small de-greeofamplificationweobserved in GR

mam-mary tumorsundoubtedly accounts for its

fail-ure to be detected byliquid hybridization (13,

15).

Theproviralamplificationweobservedin GR mammary tumors appears to involve only one of the endogenousGR proviruses. Thisprovirus

isnotpresentinthegenomeofGR-Mtv-27mice

(Fig. 5). TheGR-Mtv-2- line originatedby cross-ing GR mice withastrain (C57BL) exhibitinga

lowmammary tumorincidence (25). GR-Mtv-2

mice have a very low incidence of mammary

tumors,donottransmitamilk-bornevirus, and

have fewer endogenous MuMTV proviral

se-quencesthanGR mice(12,25). Thus,ourresults

suggest that the genetic locus responsible for

mammarytumorsinGR mice is the GRprovirus (GR-MTV-2) thatwe found amplified inthese

tumors.Since all GRmammary tumorsthatwe

haveexamined exhibitamplification of this pro-virus,weinfer that GR-MTV-2amplification is critical in GRmammarytumorigenesis.

The majority of the restriction enzymes we

haveusedcleave the MuMTV provirusesone or two times and, thus, should give rise to

high-molecular-weight DNA fragments containing

bothproviraland theflankingmousesequences.

WefindthatMuMTV-specific,

high-molecular-weight fragments can be detected in tumor DNAs after restriction enzyme-Southern blot analysis (Fig. 2). The sizes of these fragments

varyfromtumor totumor,suggesting thatthey

arenotinternalfragmentsof the MuMTV pro-virus, but rather are the provirus-mouse

se-quences. The number of proviral integration

sites in the mouse genome is apparently large,

possiblyin thehundreds,andnositeappearsto

be preferred (6). Thus, our finding of specific

proviral-mousesequences suggeststhat thevast

majority of cellsinthetumor masscontain the

amplified provirusinthesame integrationsite.

This impliesthat thetumor mass contains the

progeny ofa unique cell (monoclonality) or a

limited number of subsets of cells (limited pol-yclonality). Others have concluded that GR

tu-mors areclonalonthebasis ofhonnonal

respon-siveness (21). In addition, restriction mapping

data have suggested a clonal origin for C3H

milk-virus-induced BALB/c mammary tumors

(6).

The variability in the provirus-mouse

frag-ments seenfromtumor to tumor (Fig. 2) rules

outthepossibilityofsimplechromosomal

dupli-cation.Instead,the-additionalprovirus(es)must

beintegrated into other sites in themouse

ge-nome.Whether thisoccursbya processof

pro-viralexcision-reintegrationorby superinfection

ofmammarycells by virions is unknown.

Our finding of proviral amplification in GR

mammary tumorsstrengthens the correlationof

MuMTV gene amplification with mammary tumorigenesis. Geneamplificationmayresultin aselectiveadvantagefor the cell (18). Selection

ofone or a few cellshaving amplified proviral

sequences would also account for the

(postu-lated) clonal origin of thetumor. Theselective advantage conferred upon the cell may be re-lated to the integration site of the amplified provirusor to anincrease inMuMTV mRNAor geneproduct(s), orboth.In any event,proviral

gene amplification, asobserved in

MuMTV-in-ducedmurinemammary tumorsand other

on-cornavirus-related tumors (5, 10, 23), deserves seriousconsiderationas afundamental process leadingtoneoplastic transformation.

ACKNOWLEDGMENTS

Wethank L. J. T.Youngfortechnicalassistanceand D. Freer for densitometerscans.

This work wassupported by Public Health Service grant5 RO1CA21454awardedbytheNational Cancer Institute.

LITERATURE CITED

1.Andervont, H. B., and W. J. McE:leney. 1939. The influence of foster nursing upon the incidence of

spon-taneousbreast cancer in strainC3Hmice. PublicHealth Rep. 54:1597-1603.

2. Bentvelzen, P. 1968. Resistance to small amounts of Bittnermammarytumorvirus inoffspring of C57BL female mice with the virus. J.Natl. CancerInst.41: 757-765.

3. Bentvelzen, P. 1974. Host-virus interactions inmurine

mammarytumorigenesis.Biochim.Biophys.Acta355: 236-259.

4.Bentvelzen, P.,J. H. Danam,P. Hageman, and J.

Calafat. 1970. Genetic trn on ofviruses that incitemammarytumor inmice. Proc. Natl. Acad. Sci. U.S.A. 67:377-384.

5. Berns, A., and R. Jaenisch 1976. Increase of

AKR-specificsequences intumortissues of leukemic AKR mice. Proc.Natl. Acad.Sci.U.S.A.73:2448-2452. 6. Cohen,J.C.,P.R. Shank,V. LMorris,R.Cardiff,

andH.E.Varmus.1979.Integrationof the DNA of mouse mammarytumorvirus in virus-infected normal andneoplastictissue of themouse.Cell 16:333345.

7. Cohen,J.C., and H. E. Varmus. 1979. Endogenous

mammarytumorvirusDNA varies amongwild mice and segregates during inbreeding. Nature (London)

278:418-423.

8. Dion,A.S.,U.L.Heine,A.A.Pomenti,J.Korb,and G. H. Weber. 1977. Electrophoretic analysisofthe molecular weight of murine mammary tumor virus RNA.J.Virol. 22:822-825.

9.Gross-Bellard, M,P.Oudet,and P.Chambon.1973.

Isolation ofhigh-molecular weightDNA from

mam-malian cells. Eur. J. Biochem.36:32-38.

10. Jaenisch,R.1976.Germ lineintegrationandMendelian transmon of the exogenousMoloneyleukemia virus.

Proc.Natl. Acad.Sci.U.S.A.73:1260-1264.

11. Michalides,R., and J. Schlom. 1975.Relationshipin nucleic acid sequencesbetweenmousemammarytumor

http://jvi.asm.org/

(6)

virus variants. Proc. Natl.Acad. Sci. U.S.A. 72:4635-4639.

12. Michalides, R., L. Van Deemter, R. Nusse, and R. VanNie. 1978. Identification of the Mtv-2 gene respon-sible for theearly appearance ofmammarytumors in the GR mouseby nucleic acid hybridization. Proc. Natl. Acad. Sci. U.S.A.75:2368-2372.

13. Michalides, R.,G.Vlahakis,and J.Schlom. 1976.A

biochemical approachtothestudyof thetransmission of mouse MTV inmousestrainsRIII and C3H. Int. J.

Cancer 18:105-115.

14. Moore, D.H.,C. A.Long,A. B.Vaidya,J.B.Sheffield,

A. S. Dion,and E. Y.Lasfargues. 1979.Mammary

tumorviruses. Adv. Cancer Res. 29:347418. 15.Morris, V. L., E. Mederios, G. M. Ringold,J. M.

Bishop, and H. E. Varmus. 1977. Comparison of mouse mammarytumorvirus-specific DNA in inbred, wildand asian mice, andintumorsand normal organs from inbred mice.J. Mol. Biol. 114:73-91.

16. Murray,K., and N. E. Murray. 1975.Phage lambda receptor chromosomes for DNA fragments made with restrictionendonucleaseIIIofHaemophilusinfluenzae andrestriction endonuclease Iof Escherichia coli. J. Mol. Biol.98:551-564.

17. Myers,J.C.,S.Spiegelman,and D. L. Kacian.1977.

Synthesis of full-length DNA copies of avian myelo-blastosis virus RNA inhigh yields. Proc. Natl. Acad. Sci. U.S.A.74:2840-2843.

18.Nowell, P. C. 1976.The clonal evolution oftumor cell populations. Science 194:23-28.

19. Rigby,P.W.,M.Dieckmann,C.Rhodes,and P.Berg.

J. VIROL.

1977.Labellingdeoxyribonucleic acid tohigh specific activity in vitroby nick translation with DNA polym-eraseI.J.Mol. Biol. 113:237-251.

20. Ringold,G.M.,E.Y.Lasfargues,J.M.Bishop,and H. E. Varmus. 1975.Production ofmousemammary

tumor virusby cultured cells in the absence and pres-ence ofhormones: assay by molecular hybridization. Virology 65:135-147.

21. Sluyser,M.,T.Nouwen,J. Hilgers,and J.Calafat.

1977. Levels of mammary tumorvirus in

hormone-de-pendent and -independent mouse mammary tumor

cells.Cancer Res.37:1986-1990.

22. Southern, E. M. 1975.Detection ofspecific sequences amongDNAfragments separated by gel electrophore-sis. J.Mol. Biol. 38:503-517.

23. Steffen,D., S. Bird, W. P. Rowe, and R. A. Weinberg. 1979. Identification of DNA fragments carrying eco-tropic proviruses of AKR mice. Proc. Natl. Acad. Sci. U.S.A.76:4554-4558.

24. Taylor,J. M.,R. lilmensee, and J.Summers. 1976.

Efficient transcription of RNA into DNA by avian sarcomaviruspolymerase.Biochim.Biophys. Acta 442: 324-330.

25. VanNie, R., andJ.deMoes. 1977. Development of a congeneicline of the GRmouse strain without early mammary tumors. Int. J. Cancer20:588-594. 26. Varmus, H. E., J. M. Bishop, R. C.Nowinski, and N.

H. Sarkar. 1972.Mammarytumorvirusspecific nu-cleotide sequences inDNA ofhigh and low incidence

mouse strains. Nature (London) New Biol.

238:189-190.

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