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0022-538X/79/11-0483/14$02.00/0

Organization of

Mouse Mammary Tumor

Virus-Specific DNA

Endogenous to BALB/c Mice

J. CRAIGCOHEN,`* JOHNE.MAJORS,2 ANDHAROLDE. VARMUS2

Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana70112,1 and Department of Microbiology, University of California, San Francisco,

California941432

Received forpublication13 June 1979

We used restriction endonucleases to prepare physical maps of the mouse

mammary tumorvirus(MMTV)-specificDNAendogenous to the BALB/c mouse

strain. The mapping was facilitated by the DNA transfer procedure, using

complementary DNAsspecific for the wholeandforthe 3' terminusof MMTV

RNA todetect fragmentscontaining viral sequences. The strategies used for the

arrangementof fragments into physicalmapsincludedsequential digestionswith

twoorthreeenzymes; preparativeisolationof EcoRIfragments containing viral

sequences;andcomparisons of virus-specific fragments derived from theDNAof

severalmousestrains. MostoftheMMTV-relatedDNAin theBALB/cgenome

is organized into two units (II and III) which strongly resemble proviruses acquireduponhorizontal infectionwithmilk-bornestrainsof MMTV and other

retroviruses. These units contain approximately 6.0 X 106 Mr of apparently

uninterruptedviral sequences, they bear redundant sequences totaling at least

700 to 800 base pairs at their termnini, and the terminal redundancies include

sequencesderived from the 3' end ofMMTVRNA. UnitsIIand III are closely

relatedinthat they share 12of14recognitionsites forendonucleases,butcellular

sequencesflankingunits II and III aredissimilarbythis criterion. Theremainder

of the MMTV-related DNA endogenous to BALB/c mice is found in a single subgenomic unit (unit I) withacomplexity ofca. 2x 106 Mr; thestructure ofthis unit has not been further defined. These results support the hypothesis that

endogenousproviruseshave beenacquired byinfection ofgerminal tissues with

MMTV. Thephysical maps arealso usefulforidentifyingtheMMTV genomes

endogenous to BALB/c mice in studies of the natural history of mammary

tumorigenesis.

DNArelatedtothegenomesof retrovirusesis

endogenoustonormal animals fromseveral

spe-cies (1, 34, 35), but the origin, structure, and function of suchvirus-specific DNA are

uncer-tain. Inparticular, the organization of

endoge-nousviral DNA has notbeen defined because

only

recently have techniques become available

to permit the physical mapping of sequences

occurringinlow numbers ofcopies in the

eucar-yoticgenome.

Afewcopies ofDNArelatedtothe genome of

mouse mammary tumor virus (MMTV) are

foundendogenouslyininbred strains ofmice, in

most wildmice, and in Asian mice (9, 18, 22, 24,

36).Genetically transmitted viruses with

onco-genic activitycanberecovered from some inbred

mousestrains(3, 20,23),indicatingthatatleast

some endogenous viral DNA must encode a

complete viral genome and be arranged as a

template suitable for transcription. In some

strains from which infectious virus hasnotbeen

recovered, most, if not all, of the genetic

infor-mation inoncogenic milk-borne viruseshas been

identified inendogenous MMTV DNA by

mo-lecular hybridization (18, 22), and the

endoge-nous viral DNA is often also transcribed into

RNA (19, 37). In these cases and others,

how-ever, little is known about the

organization

of

endogenous MMTV DNA.

Wehaverecently presented evidencefavoring

the hypothesis thatthe endogenous proviruses

ofMMTV wereacquired by

multiple,

independ-ent, andrelativelyrecentinfections of

germinal

tissues (5). Feral mice vary markedly in the

numbers of copies of MMTV DNA and the

positionsof viral DNA within the hostgenomes,

andinbred strains of miceappeartohave

inher-ited various combinations of viral DNA in a

manner suggesting that independentinsertions

of viral DNA have

segregated

likestable

genetic

markersduringinbreeding.

If endogenous proviruses were, in fact,

ac-483

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484 COHEN, MAJORS, ANI) VARMUS

quired byindependent infections ofgermlines,

theymightbeexpectedtoresemble structurally

theprovirusesfound in cells infected by

horizon-tally transmitted retroviruses. Proviruses

ac-quired byexperimentalinfectioncanbe

accom-modated at many sites in cellular DNA (4, 14,

16, 26, 28, 31), but they have similar features:

they are coextensive with unintegrated viral

DNA (and therefore approximately colinear

withviral RNA); theyareapproximately5 x 106

to 6X106Mrinsize; and they bearlarge terminal

redundancies composed of sequences derived

fromboth the 3' and 5' termini of viral RNA (4,

14, 16, 26, 28).

Inthis report,weused restriction

endonucle-ases to analyze the MMTV DNA endogenousto

onestrainof inbred mice (BALB/c)from which

noinfectious MMTV has been isolated in order

todetermine whether suchDNAisorganizedin

a fashionsimilar to that describedforproviruses

known to be introduced by infection. We

over-came thecomplexity of analyzing multiple units

of viral DNA in asingle cell by isolating EcoRI

fragments containingviral DNA before

second-ary digestions, and we used hybridization

re-agentsspecificforthe3'endof MMTVRNA to

search forterminal redundancies inthe

endog-enous viral DNA. The resultingphysical maps

revealthattheendogenous MMTV DNAinthis

strain is contained within threeunits located at

different positions in the host genome. Two of

these unitscloselyresemble MMTVproviruses

acquired byinfection withrespectto their com-plexity and terminal redundancies; the third unit

containsonlya portion of the MMTV genome.

Thus, the structure of most of theendogenous

MMTV DNA inBALB/cmice conforms tothe

hypothesis that this DNA was introduced by infection.

MATERIALS AND METHODS

DNA extraction. The liverfrom a male BALB/c mouse(Simonson Laboratories, Gilroy,Calif.)was ho-mogenized in DNA extraction buffer (0.02 M Tris-chloride [pH 8.11-0.01 M EDTA-0.1 MNaCl), using a motor-driven, Teflon-glass Dounce homogenizer. Afterdeproteinizationwithself-digested pronase and sodium dodecyl sulfate (SDS) (1 mg/ml and 1.0%, respectively) at 37°C for 12 h, DNA was extracted twice with equalvolumes ofphenol-chloroform (2:1). The preparation was dialyzed extensively against 5 mM Tris-chloride (pH 7.4)-0.1 mM EDTA before analysiswithrestriction endonucleases.

Restriction endonuclease digestion of DNA. EcoRI restriction endonucleases was a generous gift from P. Greene; PstI waspurified from Providencia stuartii by the procedure of Greene et al. (11); and

BglII and HpaI were obtained from New England

Biolabs (Beverly, Mass.). EcoRIdigestions were per-formed in 0.1 M Tris-chloride(pH7.4)-0.05 M

NaCl-0.005 M

MgC92-0.05%

Nonidet P-40. The reaction buffer for PstI included 0.006 M Tris-chloride (pH 7.2), 0.006 M

MgC92,

0.05MNaCl,0.006M 2-mercap-toethanol, and100jugofgelatinperml.HpaIdigestion buffer consisted of0.01MTris-chloride(pH 7.5),0.01 M MgCl2, 0.006 M KCl, and 0.001 M dithiothreitol. ReactionswithBgllI wereperformedin 0.006 MKCl, 0.01MTris-chloride(pH 7.4),0.01MMgCl2,and 0.001 Mdithiothreitol. Reactionswere monitored for com-pletenessbyadding either lambdabacteriophageDNA

(forEcoRI andBglll) or form Iofplasmid pBR313

(for PstI and HpaI) to a portion of the digestion mixture. A 5- to 10-fold enzyme excess was used in each reaction volume of0.1to 0.5ml, andinallcases reactionswereshown to becomplete by analysis of marker DNAsbygelelectrophoresis.

Gel electrophoresis, DNA transfer, and hy-bridization. After restriction endonuclease digestion, DNAsamples wereelectrophoresed in0.8% agarose (Seakem) gels containing 0.04 M Tris-acetate (pH 8.15),0.02Msodiumacetate, 0.018 MNaCl, and0.002 M EDTA(12).Bromophenol bluewasaddedas track-ing dye, andelectrophoresiswasperformedat 40 to 70

Vforatimesufficientfor thedyetomigrate12 to 18

cm.Ethidium bromide (2.5jig/ml)was usedtostain gels afterelectrophoresis for visualization of marker

DNAs (HindIll-cleavedlambdabacteriophage DNA

labeledwith32Pby nicktranslationwithEscherichia

coliDNApolymeraseI;15).Preparative fractionation

of DNAwasperformed under similar conditions, but thebuffer didnotincludeNaCl.Aspecial electropho-resisapparatus(17)wasusedtoprovide for discontin-uouselectroelutionfrom a0.8%agarosegel2cmthick and6 cmlong. Seventy fractionswere collected and analyzed individually by gelelectrophoresis and the DNA transfer-hybridization procedure of Southern (30).

For transfer of DNA to nitrocellulose sheets, we used the methoddescribed by Southern(30).Briefly, gels were treated with denaturing solution (0.5 N NaOH-1.5MNaCl)for 1 hand thenwithneutralizing buffer (0.5MTris-chloride[pH 7.0]-1.5M NaCl)for 3to 5h. DNA transfer wasaccomplishedbycapillary actionfromthegeltothe filterwith6xSSC (SSC =

0.15MNaCl-0.015M sodium citrate) for24 to 48h,

and thefilterwasdriedat80°Cinvacuofor2h. To detect virus-specific DNAbyhybridization, fil-terswereincubated with32P-labeledMMTV comple-mentary DNA (cDNA) synthesizedbyusingcalf thy-musoligomersasprimers (cDNArep; 16) or oligodeox-ythymidylic acid[oligo(dT)]asprimer(cDNA,3)with either sucrosegradient-purifiedvirion RNA(10 to 20

S)oroligo(dT)-cellulose-selectedMMTV RNA(10 to

20S; 33),respectively. SpecificityofviralcDNA's was evaluatedbyhybridization to filters with restriction endonuclease-digested unintegrated MMTV DNA (29). The annealing mixture included 3x SSC, 50% formamide, 0.05 M HEPES buffer (N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonicacid; pH7.0), 200

jg

of yeast RNA per ml, 50jig of sheared and denatured salmon sperm DNA per ml,1 x 106to2x 106cpmof

[32P]cDNA,andDenhardtbuffer (0.02% each ofbovine serum albumin, polyvinylpyrrolidone, and Ficoll; 6). Filters were incubated for12hat41°C with annealing mixlackingcDNAand then with 1 to 2 ml ofannealing J. VIROL.

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mix with cDNAfor 48 to 72 h. After incubation, filters werewashed for 1 h in 2x SSC at roomtemperature; in0.1xSSC-0.1% SDS at 50°C for 90 min; and with multiple rinses at roomtemperature with 0.1x SSC-0.1%SDS and then0.1x SSC only. After air drying, the filters wereexposed to KodakRP-Royal X-Omat film at -70°C in the presence of Dupont Cronex Lightning Plus screens (32). Reannealing filters with different cDNA's after initial analysis required that

filters be placed in Seal-a-meal food container bags

withannealing mixcontaining 1 x 106 to 2 x106cpm/ ml and incubated for 30 min in a 68°C water bath before incubation at 41°C for 48 to72h.

Nomenclature. To simplify identification of

BALB/crestriction endonuclease fragments contain-ingMMTV DNA, we have named them by letter and enzymein order ofmolecular weight, indicating the Mrx 106 in parentheses [e.g., EcoRI-A(10.0) is the largest EcoRI fragment containing MMTV-specific sequencesand hasanMr of10.0x

1061.

RESULTS

Restriction fragments of BALB/c DNA

containing

MMTV-specific sequences. By

annealing virus-specificcDNA tocellularDNA, we estimated that BALB/c mice contain

ap-proximately fivetosixcopiesofviral DNA per

diploid

genome (4, 22).Theextentof annealing

ofvirus-specificcDNAandRNA tonucleic acids

fromuninfectedcells indicatedthat most, if not

all, of the sequencesin thegenomes of horizon-tally transmitted MMTV were present in the germ lines of inbred strains. Analysis of the

kinetics andextentofannealing, however, does notindicate whether the mass ofvirus-specific

DNA isorganizedinto afewprovirus-likeunits or widely dispersed throughout the mouse

ge-nome in manydisordered,subgenomicunits.

An initial appraisal of this problem can be

obtained by digestionofcellularDNA with

var-iousrestriction endonucleases,followed by iden-tification of fragments containing

MMTV-spe-cific sequences with molecular hybridization. Enzymes chosen for this survey were among

thosepreviously testedfortheir ability tocleave viralDNAsynthesized afterinfection by MMTV

strains transmitted in the milk ofGR or C3H

mice (see Fig. 7A): EcoRI, which recognizes a

single site in the DNA of MMTV(C3H) or

MMTV(GR); PstI, which recognizes five sites;

BgIII, which cleaves at two sites; and HpaI,

whichdoesnotcleave such DNA (29). We made

the provisional assumption, later shown to be

correct, that suchenzymeswould also beuseful

forgenerating a

simple

physical map of

endog-enous MMTVDNA,althoughattheoutset we

did not know whether the maps ofendogenous

DNAwould bear anyrelationship to the maps

ofviral DNAsynthesizedafter horizontal

infec-tion. We also assumed, based upon annealing

experiments noted above,that cDNA's

prepared

from the RNA of milk-borne

MMTV(C3H)

would detectmostorall of the sequencespresent

within the units ofendogenous MMTV DNA.

Again, the results of mapping studies shown

below support the validity of this assumption, thoughminor differenceswould nothave been detected. The cDNA'sused

represented

either the entire genome of

MMTV(C3H)

(cDNA,p,

generated by random priming with oligomers of calf thymus DNA)orthe200 to 300nucleotides

atthe 3'terminus of viral RNA

[cDNA3,

pre-pared by priming

templates

of

polyadenylated

viral RNA with oligo(dT)]. cDNA3 detects

se-quencesthat are redundantatthe ends of

un-integrated linear and

integrated

MMTV(C3H)

and

MMTV(GR)

DNA (4, 29).

Occasionally,

minor bands were found on

single

autoradi-ograms but were not

reproducible

in

multiple

analyses (e.g., bandat3.4inFig.4,lane

C);

these

were not considered in the

generation

of the

maps.

Digestion ofBALB/c DNA with EcoRI

pro-duced five

virus-specific fragments,

all ofwhich

annealed with both of the

MMTV-specific

cDNA's (Fig. 1A and 1E; Table 1; 4, 5). This result suggested that the endogenous MMTV

DNA is not widely scattered in small units throughoutthe mouse genome. Itwouldaccord

well

with the estimates of copy number from association kinetics

if,

for

example,

two

proviral

elements had a single internal EcoRI site and the third lacked an internal site. In this case,

fourfragments wouldbe

generated

fromthefirst

two proviruses, and all four

fragments

would anneal with

cDNA3,

assuming

the

proviruses

bore terminal redundancies that included

se-quencesfromthe 3' end of viral

RNA;

the third

proviral

element would be

contained

within the

fifth

fragment.

Digestion ofBALB/cliverwithPstI (Fig. 1B and 1F; Table 1) yielded a larger number of virus-specific fragments,aswould have been

pre-dicted from therestriction map of

horizontally

transmitted MMTV DNA. Moreover, some of

these fragments have the same molecular

weights (e.g., 1.1 x

106

and 0.9 x

106

Mr)

as

fragments derived from

internal

regions of

MMTV(C3H) and MMTV(GR), suggesting that

homologousregionsmight be present in

endog-enousproviruses. HpaI,ontheotherhand,

gen-eratedfragments

smaller

than would havebeen

expected ifit had failed to cleave provirusesof

standard size (ca. 5.9x

106

Mr; Fig. 1Cand1G),

implying either that the endogenous MMTV

DNA contained recognition sites for HpaI or

thatsomeof the DNAwas

organized

in

subge-nomicunits.

BglII

alsogeneratedamodest

num-VOL. 32,1979

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486 COHEN, MAJORS, ANI) VARMUS

*

I;

FIG. 1. MMTV-specific fragments obtained from BALBIcliverDNAbydigestionwith various restric-tionendonucleases. Samples (10,ug) of high-molecu-lar-weight DNA extracted from the liverofa male BALB/c mouse were digested with the designated

restriction endonuclease as describedin Materials

andMethods. Thesamplesweresubjectedto electro-phoresisinan0.8% agarosegel,and DNAfragments

weretransferredtonitrocellulosesheets(30), hybrid-izedwith 32P-labeledMMTV-specificcDNArep(lanes A-D)orcDNA3 (lanes E-H), anddetectedby auto-radiography. A HindIII digest of lambda bacterio-phage 32P-labeledDNA(not shown)wasincludedto

obtain theMrx 10' valuesindicatedontheleft.A andE,EcoRIdigests;B andF, PstIdigests; Cand G, HpaI digests;D andH,BglII digests.

ber ofvirus-specific fragments (Fig. 1D and 1H)

without any obvious relationship to the BglII

fragmentsexpected from provirusesacquiredby

infection.

Although definitive deductions would have

beenprematureatthisstageof theanalysis, the

variations inintensity of bands and the

discord-ances between annealing with cDNA3 and

cDNArep could clearly be helpful in ordering

fragments into a restriction map. Forexample,

a large fragment detected readily with cDNA3'

but poorly with cDNArep [e.g., PstI-A(3.4) and

Pstl-D(1.2)] wouldprobably contain mostly host

DNA linked to the end of a provirus; on the

otherhand,asmall fragment thatannealed

ex-tremelyefficiently with either cDNA [e.g.,

PstI-H(0.5)] might be derived from more than one

unit ofendogenousviral DNA.

Preparation of fractions of

EcoRI-cleaved

BALB/c DNA containing

separated

virus-specific

fragments.

Cleavage

of

BALB/c

liver

DNA withvarious enzymesindicated that

rela-tivelysimple patternsofvirus-specific fragments couldbe obtained, but thepatternsseemedtoo

complicated

to

permit

construction of

physical

maps forindividual unitsofendogenousMMTV

DNA bydirect

sequential

digestions. We

there-fore elected to separate on a preparative scale

some ofthe EcoRI fragments ofvirus-specific DNA before attemptingto chartmore detailed

mapsby secondary cleavages. The EcoRI

frag-ments were chosen because each fragment

seemed likelytoprovide uswith alargeportion

or

all

ofanendogenous element, basedupon the

tentativeinterpretation offeredinthepreceding section. One obvious limitation ofthe choiceof

theEcoRI fragmentswastheinherent

difficulty

ofseparating

EcoRI-C(5.0)

and

EcoRI-D(4.7).

Five

milligrams

ofDNAfromBALB/c livers

wasdigestedwith

EcoRI

andsubjected to

pre-parative

electrophoresis

in a thick, short 0.8%

agarose gel, using an apparatus equipped for

timed

collection

ofDNAfragmentsexitingfrom

TABLE 1. Virus-specific fragments from restriction endonucleasedigests ofBALB/cDNA

Frag- M Detectedwith:

Enzyrne mentment Mr 06

cDNAr,p

cDNA3

EcoRI A 10.0 + +

B 6.0 + +

C 5.0 + +

D 4.7 + +

E 4.0 + +

PstI A 3.4 - +

B 3.3 +

-C 3.1 +

-D 1.2 - +

E 1.1 +

-F 1.0 + +

G 0.9 + +

H 0.5 + +

I 0.2 - +

HpaI A 4.4 + +

B 4.2 + +

C 1.1 - +

D 0.9 - +

E 0.6 + +

BglII A 4.0 + +

B 3.5 + +

C 2.8 + +

D 2.5 + +

E 1.3 + +

F 1.2 +

-a These v-alueswereobtainedbycomparison ofthe data shown inFig. 1 with HindIII digests of32P-labeled

lambda bacteriophage DNA electrophoresed in par-allellanes.

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the end of the gel (see Materials and Methods).

Seventy fractions were then analyzed by

re-peated electrophoresis, DNA transfer, and

mo-lecular hybridization for their content of

virus-specific fragments. Four pools (subsequently

calledI, II,III,and IV) were made from fractions

found to contain the EcoRI fragments

previ-ously identified in digestions of total BALB/c

liverDNA (Fig. 2). Pool I contained the

EcoRI-E(4.0) fragment, apparently uncontaminated

with othervirus-specific fragments; poolII

con-tained theEcoRI-C(5.0) andEcoRI-D(4.7)

frag-ments,contaminated with asmallamountof the

EcoRI-E(4.0) fragment; pool III contained the

EcoRI-B(6.0) fragment,

minimally

contami-nated withsmallerfragments (notvisible in the

test shown in Fig. 2C); and pool IV contained

A

B

C

D

E

.

t0

.

5.0

4.7

4.0

FIG 2 Fractionated EcoRI

fragments

of

MMTV-specificDNA

fr-om

BALBIcmouse liver DNA. Five

milligrams ofBALB/cliver DNAwasdigestedwith EcoRI andsubjectedtopreparative electrophoresis through a 0.8% agarosegel on a

fr-actionating

gel

electrophoresisapparatus (seeMaterials and

Meth-ods). Seventy

fr-actions

were obtained by collecting

electroelutedmaterialat

fr-equent

intervals,and each

fr-action

wasanalyzed by gel electrophoresis, transfer

to nitocellulose sheets, and hybridization with

MMTVcDNAp.

Fourpoolsweremade

from

multiple

fr-actions

which containedvirus-specificEcoRI

fr-ag-ments. A sample of each pool was reanalyzed to evaluatehomogeneity.MrX 10'6foreachviral

fr-ag-ment is indicatedon the rightand wasdetermined

by including32P-labeledHindjjjlaMbdaDNA in a

parallel lane (not shown). A,poolI; B,pool

1I;

C,

pool III; D, poolIV; E,

unfr-actionated

EcoRI-di-gestedBALE/cliver DNA.

the EcoRI-A(10.0) fragment. The amount and

purity of these fragmentsweresufficientto

pro-ceed withsecondarydigestions. In some cases,

however, fragments apparently derived from

secondary digestion of contaminating (usually smaller) EcoRI fragments complicated the anal-ysis, butthese could generally be identified as

contaminants without majordifficulty (see, for

example, fragments of2.8 x106 and2.6x106Mr

inFig.3Corthe fragment of1.4 x106MrinFig.

4B).

Secondary cleavages of isolated EcoRI fragments of MMTV DNA.Secondary diges-tions of the DNA in the four pools of EcoRI fragments with PstI, HpaI, andBglII (Fig.3-5;

Table 2) permitted us to make several

deduc-tions importanttothe construction of restriction endonucleasemapsforthe MMTV DNA

endog-enous toBALB/c mice. The following guidelines

were helpful in sorting out the complex data obtained inthese experiments.

First, fragmentsthat arenoveltothe

sequen-tial digestion products (i.e., notpresent among

products of digestion with the second enzyme

alone; Fig. 1andTable 1)mustbepositionedat an end of the EcoRI fragment used for the secondarydigestion.Forexample, PstI digestion of EcoRI-C(5.0) and EcoRI-D(4.7) generated major, novel fragments of2.8 x

106

and 2.6 x

106

Mr (Fig. 3B); the appearance of these two

fragments indicated that EcoRI sitesmust be

present in PstI-B(3.3) and PstI-C(3.1), as

con-fixned by the absence of these two PstI

frag-ments in all sequential digestions with EcoRI

and PstI (Fig. 3; also see below). Similar

argu-ments apply to the fragments of1.4 x

106,

3.0

x

106,

and 1.2x106 Mr derived by HpaI cleavage

ofDNAfrom pools I, II, and III, respectively, andtothe fragments of0.8x

106

and approxi-mately0.3x 106 Mr obtained byBglII digestion ofDNAfrompoolsI, II, and III.

There is a significant possible exception to

this firstrule, in that prior fractionation of the EcoRI

fragments

may

permit

detection of

frag-mentsthat have insufficient amountsof

virus-specific sequence to be observed in digests of

unfiactionated

DNA. There

is,

for

example,

no

otherway to

explain

the

relatively

faint bands representing fragments of1.5x

106

and1.3x

106

Mr observed after

HpaI digestion

of

EcoRI-0(5.0)

andEcoRI-D(4.7) (Fig. 4B).Infact, these fragmentsappear tobecomposed

largely

of cel-lularsequences(seebelow), making detection of the minor virus-specific sequences difficult in

unfractionatedDNA.

Second, when PstI, HpaI, and

BglII

fragments

ofMMTV DNA(Table 1)arealso foundamong

products of secondarydigestions of EcoRI

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488 COHEN, MAJORS, AND) VARMUS

.a.

*.: ;.'.;:'t :a

::.:::: *: :.:: !.

:::.

::

..

':

:...

*: :.:.

+ '".1

..::..

*s *..,'..:. :'.

....:

Pf%.

.:

# At #iiQ

FIG. 3. Products of PstI digestion of separated EcoRIfragmentsof BALB/cmouseliverDNA. Sam-plesof each poolofEcoRIfragment (seeFig. 2)were digested with PstI,electrophoresedon0.8%oagarose gels, and transferredto nitrocellulose sheetsfor hy-bridizationwith either[32P]cDNArep (lanes A-D)or

[32P]cDNA:s (lanes E-H). HindIII-digested

32P-la-beled lambda bacteriophage DNA (notshown) was

includedasmarkertoobtaintheMrX1o-6shownon theleft.AandE,pool I;B andF, pool II;C andG, pool III;D andH, poolIV.

ments(Table2), thesefragmentsarelikelytobe

positionedinternallyin therelevantEcoRI

frag-ment. However, it is possible to generate by

thesedoubledigestionsnewfragments,bounded

by anEcoRI site, whichfortuitously comigrate

with products of digestion with the second

en-zymealone. For example, thePstI-EcoRI

frag-ments of0.5 x 106are likelytobe amixture of

PstI-H(0.5) and theproductsof EcoRIdigestion

ofPstI-B(3.3) andPstI-C(3.1) (Fig. 3Band3F).

Thisinference is substantiatedin alater section

(see Fig. 8).

Third, fragmentslargerthan 0.3 x

106

Mrthat

are detected principally or solely with cDNA3

N [e.g., PstI-A(3.4) or PstI-D(1.2)] mustbe

com-N 4 posedmostly ofcellularDNA, since

cDNArep

is

capable of detecting fragments as small as 0.3

X106Mr,whicharelikelytobecomposedmostly

or entirely of viral sequences (see Fig. 5A and

5C).

Fourth, the total molecular weight of

virus-0. 9

n

:f, S jKfa s

.

a

z ffi

::::

:.'i: ::

.._....':7'....'.

*..je'';.,.:.

U:s3: :; : ..

:3t;i.ir!s-

-.i i;

'::*: ....:...'.'i:'?

..;.' '.'^:'...

....?e::

....,.,',

.... i,....eN.:: ::.:.:.:.''S5?. B. :.s.'.2@ ?$'

!g;:;;gj;jE...;,.;..

4

t = asit

Q _ -X.

;e-4,'t.,...:

,,4..:}i::.:8:''

:t.}

'-'.8':'4::;

.. ..::.; § + ... ... :. ::.:..

::.:

.: :...

.. ..

.; ..

..

..:

...e.

.::

.:

FIG. 4. Products of HpaI digestion ofseparated EcoRIfragmentsfromBALB/cliver DNA.HpaI was used todigestsamples ofpoolsofEcoRIfragments derivedfromBALB/cliver(Fig. 2); the products were analyzed with

cDNAr,,

(lanesA-C) andcDNA3(lanes D-F) as described in thelegend toFig. 1. Numbers and arrows on theleftindicate theMrx10-'of viral fragmentsasdetermined by inclusion of radiolabeled HindIIIfragmentsoflambda DNA (notshown) in a parallel lane. A and D, poolI; B and E, pool II; C andF,pool III.

i

I

.J. VIROL.

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[image:6.507.111.451.72.555.2]
(7)

MMTV DNA 489

cD

NArep

A

B

C

3.5_

2.5 -e

1.2-0.8

--*

0.3

-4

cDNA3

[image:7.507.55.253.78.478.2]

D

E

F

FIG. 5. Products ofBglII digestion ofseparated EcoRIfragments fromBALB/cliver DNA.Samples ofthe variouspools ofEcoRIfragments shown in Fig.2weretreatedwithBglII, subjectedto electro-phoresisin0.8%agarosegels, transferredto nitrocel-lulose sheets, and analyzed by hybridization with eithercDNAp(lanesA-C)orcDNA3(lanesD-F).Mr

X 10-6 indicated onthe left are basedon

bacterio-phage lambda DNA markers included in adjacent

lanes(notshown).AandD, poolI;B andE, poolII; C andF, poolIII. Thefaintbandin lane Catthe position ofca. 3.2 x 106 Mr was not observed in repeatedtestsand hasthereforenotbeen considered furtherin theconstructionofmaps.

specific fragments derived from the secondary

digestions provides an upper limit for the

amountof viralDNA ineachEcoRI fragment.

Forinstance, there canbe no morethan 2.8 x

106Mr of viral DNAin theEcoRI-E(4.0),based

upon the sum of the fragments generated by

secondary digestionwith PstI (Fig.3A). Usingtheseprinciplesand theircorollariesas

guides, it was possibleto draw several

conclu-sions about the viral DNA in the five EcoRI

fragments and,in somecases,toestablish maps

forPstI, HpaI, andBgIII sites within theviral

andcellular DNA in thosefragments.

First, EcoRI-A(10.0) contains less than2.5 x

106

Mr

ofviralsequences, and the EcoRI sites

appeartobelocatedincellular DNA outsidethe

viral DNA (Fig. 3Dand3H). Thus, thisEcoRI fragment harbors a subgenomic unit of viral

DNA which we have termed unit I. Limited recovery ofEcoRI-A(10.0) during fractionation proceduresand the unusualstructure of unit I have compromised further characterization. However, previous data

concerning

the

segre-TABLE 2. Virus-specific fragments from secondary restriction endonuclease digests ofpools of EcoRI

fragments of BALB/c DNA

Pool Detectedwith:

of EcoRI

Enzyme EcoRI frag- M,x 10'

frag- ments cDNA,p cDNA3' ments

PstI I E(4.0)b 1.2 - +

1.1 +

-0.5 + +

II D(4.7) 2.8 +

-C(5.0) 2.6 +

-0.5 + +

0.2 - +

III B(6.0) 3.4 - +

1.1 +

-0.5 + +

IV A(10.0) 1.0 + +

0.9 + +

HpaI I E(4.0) 1.4 +

0.9 - +

0.6 + +

II D(4.7) 3.0 + +

C(5.0) 1.5 +

-1.3 +

-III B(6.0) 3.7 - +

1.2 +

-0.6 + +

BglII I E(4.0) 1.3 + +

1.2 +

-0.3 +

-II D(4.7) 3.5 + +

C(5.0) 0.8 +

-III B(6.0) 2.5 + +

1.2 +

-0.3 +

-aThese

values

were obtained fromHindIII-digested

32P-labeledlambdabacteriophageDNA

electrophore-sesin aparallellane.

bMr X 10-6 of EcoRI

virus-specific fragments

in

designatedpool.

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490 COHEN, MAJORS, ANI) VARMUS

gation of endogenous MMTV proviral DNA

in-dicatedthattheEcoRI-A(10.0) fragment

segre-gated as anindependent unit (5).

Second, each of the remaining four EcoRI

fragments contains up to 3 x 106to 4 X 106Mr

ofviral sequences, including sequences specific

tothe 3'end of MMTV RNA.

Third, the products of secondary digestions

indicate that EcoRI-B(6.0) and EcoRI-E(4.0)

areeach derived from one of two units of viral

DNA (units II and III) that also yield

EcoRI-C(5.0) and EcoRI-D(4.7). As one example of evidence for this conclusion, the fragments

gen-erated by EcoRI cleavage of HpaI-A(4.4) and

HpaI-B(4.2)mustbe the 3.0x106Mrfragments

in theHpaI digest ofEcoRI-C(5.0) and

EcoRI-D(4.7) (Fig. 4B) and the 1.4 x 106and 1.2 x 106

Mr fragments in the HpaI digests of

EcoRI-E(4.0) and EcoRI-B(6.0), respectively (Fig. 4A

and4C).Inother words, these data locate EcoRI

sites within HpaI-A(4.4) andHpaI-B(4.2).

Sim-ilar arguments can be made about the location

ofEcoRI sites withinPstI-B(3.3) andPstI-C(3.1)

andwithinBglII-F(1.2).

Fourth, within each of the four EcoRI

frag-mentsfrom thesetwounits, variousHpaI, PstI, andBglIl fragments unaffectedby EcoRI diges-tioncanbearranged (see Table2anddiscussion

ofFig.7,below). Theorderingwasbasedinitially

upontherelationshipof fragment size and rela-tive efficienciesofannealingwith cDNArep and

cDNA3,asdiscussedearlier. Since the

complex-ityof viral sequenceswasconsiderably lessthan

the Mr of each EcoRI fragment (see second

conclusion, above) and since eachEcoRI

cleav-age appeared to affect only one PstI, BglII, or

HpaI fragment (seethirdconclusion,above),we

deduced that the viral sequenceswere

approxi-mately contiguous at one end of each EcoRI

fragment. Theproposalsfor overlappingsetsof

PstI, BglII, and HpaI fragments (see Fig. 7)

werefurther validatedby additional sequential

digestionspresentedinalater section.

Fifth,EcoRI-B(6.0) andEcoRI-E(4.0) appear

to be derived from closely related regions of

MMTVprovirusesasindicated by commonPstI

fragments (1.1 x 106 and 0.5 x 106Mr; Fig. 3A

and3C) andBglIIfragments (0.6x

106

Mr; Fig.

5Aand5C).

Usinggenetic segregationof endogenous

viral DNA to facilitate mapping. To

deter-mine which ofthefoursmallerEcoRIfragments

came from the same proviral elements, we

ex-ploited our recent finding that endogenous

MMTV DNA has been segregating like stable

genetic elements during the inbreeding of

con-temporarymousestrains (5). By analysisof

en-donucleasedigests ofDNA from severalstrains

among the progeny ofa cross between a Bagg

albino (forerunner of BALB/c) and a DBA

mouse, wefound thatindependentlysegregating

elements generated pairs of EcoRI fragments

[e.g.,EcoRI-B(6.0) and EcoRI-D(4.7),or

EcoRI-C(5.0) and EcoRI-E(4.0)]; PstI-C(3.1) was also

foundtobe associatedwith the presence of the

former pair of EcoRI fragments. Pertinent

ex-amples of such resultsare illustrated in Fig. 6.

AnEcoRIdigest of liver DNA fromaC3H/HeJ

mouse (lane F) contained EcoRI-C(5.0) and

EcoRI-E(4.0), but lacked EcoRI-B(6.0) and

EcoRI-D(4.7). The PstI digest of C3H/HeJ

DNA did not containPstI-C(3.1), which would

beexpectedtoproduce as strong a band as PstI-B(3.3); however, there was a less readily detected PstIfragment of ca. 3.1 x

106

Mr derived from anendogenousprovirus unique to the C3H/HeJ

mouse (lane B). These observations indicate

thatEcoRI-B(6.0) and EcoRI-D(4.7)form a sin-gle proviral unit, which we call unit II, and

EcoRI-C(5.0) and EcoRI-E(4.0) form another

proviralunit, called unit III. They also confirm that the PstI-B(3.3) fragment is from unit II and the PstI-C(3.1) fragment is from unit III. The

.0

FIG. 6. Comparison of restriction endonuclease patterns of MMTV-specific DNA in BALB/c and C3H/HeJmouseliver DNAs. A 10-,igsampleofDNA extractedfromthe liverofeitheramaleBALB/cor

a male C3H/HeJ mouse was digested with PstI,

HpaI, or EcoRI and analyzedfor MMTV-specific fragments with

cDNA,

asdescribedinthelegendto Fig. 1. M,x 106 ofsignificant viralfragments are indicated to the leftofeachpanelandwere deter-minedfrom HindIII-digested lambda[32P]DNA in-cluded inparallellanes(not shown). PstIdigests of BALB/c (A)andC3H/HeJ (B) DNAs; HpaI digests ofBALB/c (C) andC3H/HeJ (D) DNAs; EcoRI di-gestsofBALB/c (E)andC3H/HeJ (F)DNAs.

eJ. VtIROL,.

OWN.

AL,--11.

)w 40,

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additional HpaI site inone of thesetwo

provi-ruses can be mapped by comparison of HpaI

digestionsof DNAs from these two strains (lanes

Cand D). TheHpaI-A(4.4) fragment is common

tobothBALB/c andC3H/HeJ DNAs,

indicat-ing that the HpaI-B(4.2) fragment is derived

from unit III. [The minor fragment of

approxi-mately4.2 x106Mrin C3H/HeJ could represent

either a sequence unique to this strain or a

comigrating fragment from HpaI-B(4.2) in

di-gestsofBALB/cDNA and derivedfromunitI.]

Testing proposed physical maps of en-dogenous MMTV DNA with additional se-quential digestions. With the aid of the ge-neticarguments presented in the previous

sec-tion, it ispossibletoorganizeall ofthe fragments

obtained by secondary PstI, BglII, and HpaI digestionsofEcoRI fragmentsBthrough E (Fig.

3-5)intophysicalmapsofunits II and III (Fig.

7). Theonly arrangement fullyconsonant with

therulesgoverning interpretationof the

double-digestion productsindicates that these units

con-tain approximately 6.0 x

106

Mr of apparently

continuousviralsequences, withsequences from

the 3' end of viral RNA positioned near both

ends of both units. In the proposed terminal redundancies, therearethreesites (two PstI and one HpaI) that have the same relationship to

eachotheratboth endsof both units; this finding

supports the evidence for redundancies from

annealings with

cDNA3-,

suggests that the

ter-minal redundanciesin units IIandIII are very

similar, and shows that thesize of the redundant

sequence is atleast 700 to 800bases. The

ori-entation of themapswithrespect toviral RNA

was basedupon the evidence (discussed below

inrelationtoFig.9) that

cDNA3-

detectedonly

the 100 to 200 nucleotidesadjacentto

polyade-nylic acidatthe3' end of viral RNA. Thus, we

placed to theright the ends of unitsII and III

yielding fragments thatwere composed largely ofcellular DNA and annealed efficiently with

cDNA3.

In these constructions, the two units

appearclosely related in that they sharemany

restriction sites within the proposed viral

se-quences, but the sites in flanking regions of

cellular DNA are different. Thus, units II and IIIdifferonly withrespect to oneadditional site

forPstI and one for HpaI in unit III, with 12

sitesheld incommon;inflanking cellular DNA,

however, allsites except aBglII site to theleft

of viral DNA are independently positioned in

relation to the viral DNA of units II and III.

(The similarpositions of theBgII sitemay be

coincidental,butfurtherstudyof thehomology

of the cellular sequences flanking these

provi-rusesiswarranted.)

To challenge the proposed maps, we per-A

MMTVIC3H) DNA

F3 R

F3-.I

_w p

P Pp P P

s CELLDNA.-PROVIRAL DNA CELLDNA

I

H1

UNITSU

H? H

I

1

HH H

R || | R

I? H H HH

l l

Al l

I p p0

MrX10-6

H

R

[image:9.507.261.455.70.338.2]

9 10 11 12 13

FIG. 7. Physicalmapsofrestrictionendonuclease

siteson unintegrated MMTV(C3H) DNA (panel A;

29)andontwounitsof endogenousMMTVDNA in the BALB/cmousestrain(panel B).Datapresented

inFig. Ithrough6and Tables1 and 2are

summa-rizedaslinearmapsof bothviralandflanking cell sequencesindesignatedunitsIIand IIIof endoge-nousMMTV DNA. Therelativeorientation of

unin-tegratedMMTV DNAto viralRNA isindicatedby the RNA molecule at the top of panelA and the relativepositions of viral and cellularsequences are

indicatedat the topof panelB; the boxes indicate terminally redundant sequences which include

se-quencesfrom the 3'endofviralRNA. The scaleat

the bottomindicatesMrX10-6.Specificcleavage sites

forEcoRI(R),PstI(P), BglII (B),andHpaI (H) are

indicatedfor unintegratedandendogenous (unitsII

andIII)MMTVDNAs.Questionmarks indicatesites notmappedon aspecificunitofMMTV DNA.

formed additional sequential digestions with

various combinations of the fourenzymesused

in thisstudy (Fig. 8 and9). Several features of themapswereconfirmedbythese tests.

First, the datainFig.3suggested,butdidnot

prove,that EcoRIcleavedPstI-B(3.3) and

PstI-C(3.1) from units II and III to generate

frag-mentsof 0.5 x 106Mrfrom theright-handside

of theEcoRI site; these fragmentspresumably

comigrated with PstI-H(0.5), which is derived

from the terminally repeated structure and

hence from all EcoRI fragments from units II

andIII.SincethemapspredictthatPstI-H(0.5),

MMTV(C3H) RNA

(A)n

Iu0 1p p p p

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(10)

492 COHEN, MAJORS, AND VARMUS

Second, neither the 0.5 x 106 nor 0.4 x 106

fragments inFig. 8, lanes B andF, annealed with

cDNA3' (datanotshown), indicatingthatHpaI

digestion removed the portion of PstI-H(0.5) capable of reacting with this reagent. This result

supports the proposed orientation of the maps

with respecttoviral RNA bydemonstratingthat

cDNA3' is specific foronly100to200nucleotides

atthe 3' end of viral RNA.

Third, the experiments in Fig. 8 also show, as

predicted by the maps, that PstI-E(1.1) from

unit II cleaved byHpaI toproduce a fragment

of 0.95 x 106Mr (laneB); PstI-E(1.1) fromunit

III was cleaved at an additional HpaI site to

yield a fragmentof 0.6 x 106Mr (lane F). (The newfragmentsof0.1 x 106 to 0.15x106Mr tobe

expected afterdigestion with EcoRI, PstI, and

HpaIwerepresumablytoosmalltobe detected

with ourmethods.)

-9

4

wi+;i''''+

+~

ot

1*

i,

r!:

:---

*

04A

....

urn

4.

U:

FIG. 8. Virus-specificproductsof digestionof

sep-arated EcoRIfragments from BALB/c liver DNA withtworestrictionenzymes.PoolscontainingEcoRI fragments(seeFig. 2)weredigestedwith either PstI (A, C, and E) orPstI andHpaI (B, D, andF) and analyzed asdescribed inFig. 1 with

[32P]cDNArep-Mrx10-6ofviralfragmentsareindicatedontheleft

and determined by inclusion of HindIII-digested

lambda[32P]DNA. DNA frompool Idigestedwith PstI (A) or PstI andHpaI (B); DNA from pool II digestedwith PstI (C) orPstI andHpaI (D);DNA from pool IIIdigestedwith PstI(E)orPstI andHpaI

(F).

but not the 0.5 x 106 Mr fragments from the

double digestion,should be cleaved with HpaI,

the products of PstI digestion ofEcoRI-E(4.0)

andEcoRI-B(6.0) weresubjected to redigestion

with HpaI (Fig. 8, lanes A, B, E, and F). As

predicted, the relative intensity of the bands in

theregion of0.5 X

106

Mrwasdiminished after

theHpaIdigestion, andnewfragments of 0.4 x

106

Mrweregenerated (compare lanesA and E

[image:10.507.62.256.64.428.2]

withlanesBandF).

FIG. 9. Virus-specific products of BALB/c liver DNA digested with two restriction endonucleases. Ten-microgram samples ofliver DNA weredigested sequentially with two restriction enzymes and ana-lyzedwithcDNArrepasdescribed in thelegendtoFig. 1.MrX10-6ofviralfragmentsweredeterminedfrom HindIII-digested 32P-labeledlambda bacteriophage DNA and are indicated to theleft ofeachpanel.The enzymes used were:(A)PstI;(B)PstI andEcoRI; (C) PstI andHpaI; (D)EcoRI andBglII; (E) BglII.

J. VIROL.

2-6

.1,

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4'10'

Z.,V,

.:.

:4."';.1... V.,:

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It. .,:... S.:.A:..

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[image:10.507.264.458.276.567.2]
(11)

Fourth, themapspredict that the products of

double digestion with PstI and EcoRI should

includetwofragments from the left side of each

provirus, oneof2.8x 106or2.6x 106Mrwhich

isnot susceptibletorecleavage with HpaI and

oneof 0.5 x 106Mr [PstI-H(0.5)]which bearsa

site for HpaI.Sinceweproposethat the ends of

units II and Ill areterminallyredundant, the0.5

X 106 Mr fragments and their HpaI cleavage

products should be identical to those derived

from the right side of units II and III. All of

these predictions are clearly fulfilled, using

EcoRI fragments B and E from the right sides

ofunitsIIand III (Fig. 8, lanes A, B, E, and F)

and EcoRI fragments C and D from the left

sides(Fig. 8, lanes C and D).

Fifth,severalof theresultsillustratedin Fig.

8wereconfmed and extended by redigestion of

PstI fragments inunfractionated BALB/c DNA

witheitherEcoRIorHpaI (Fig.9,lanesA-C).

Thus,EcoRIcleaved onlyPstI-B(3.3)and

PstI-C(3.1) of those PstI fragments detectable with

cDNA,p;

the products of the double digestion

were2.8x 106, 2.6x106, and 0.5 x 106 Mr. HpaI

didnotcleavewithinPstI-B(3.3)orPstI-C(3.1),

butgeneratedfragmentsof 0.95 x 106, 0.6x 106,

and 0.4 x 106Mr bydigestion of PstI-E(1.1) and

PstI-H(0.5).

Sixth, the maps for units II and III predict

that BglII-F(1.2) represents not one but two

fragments which are adjacent in the internal

region of both proviruses. One of the

BglII-F(1.2) fragments from each unit should be

cleaved by EcoRI to produce fragments of 0.8

x

106

and 0.3 x 106 Mr; the otherBgIII-F(1.2)

fragmnents should remain intact. Thesepredicted

resultswere observed afterredigestion of BglII

fragments ofunfractionatedBALB/c DNA with

EcoRI (Fig. 9, lanes D and E). The relative

intensity of the band at 1.2 x 106Mr was

de-creased, and the appropriate new bands

ap-peared. EcoRI also cleaved BglII-A(4.0); this

fragmentmust be derived from unit I, but the

products of the secondary digestion with EcoRI

havenotbeenidentified.

DISCUSSION

In this report,weusedrestriction

endonucle-ases to prepare physical maps of the

MMTV-related DNA endogenous to the genome of

BALB/c mice.Themajor conclusionwedrew is

that mostof the MMTV DNA in theBALB/c

genomeisorganized into twounits (II andIII)

which resemble the proviruses acquired after

infection withretroviruses, i.e.,structures with

ca. 6.0x 106Mrofviral DNAcontaining

termi-nally redundant sequences derived from the

end(s) of viral RNA. A small amount of viral

DNAin BALB/c DNA isalsopresent in aunit ofsubgenomic size (unitI).

Thisstudy representsthe firsteffortto

deter-minethestructure ofendogenousviral DNAby

directphysical mapping procedures. The

tech-nical approach required the use ofagarose gel

electrophoresis,

the DNA transfer procedure

(30),and molecularhybridization with virus-spe-cific, radiolabeled cDNA's. These methods al-lowed detection of the

small

number of

virus-specific restriction fragments without purifica-tion of the viral DNAfromtheca.

106-fold

excess

of cellular DNA. The mapping procedure

in-cluded conventional

sequential

digestions with

variouscombinations oftwo orthree

endonucle-ases (Fig.8and 9).To

minimize

thecomplexity ofanalyzing multiple units withina single

cel-lulargenome,wealsousedpreparative

fraction-ation (17) of thelarge

EcoRI

fragments bearing MMTV sequencesbeforesomeof thesecondary digestions with other enzymes (Fig. 2-5). The use ofhybridization reagent specific for a few hundred bases at the 3' end of MMTV RNA

(cDNA3)was

particularly

helpful, sincewe were

abletoshow that thissequence wasrepeatedat

bothends of unitsIIandIII,asit isinproviruses

acquired

by infection (4, 14, 26). Furthermore,

the low complexity of cDNA3

allowed

us to

detectcertainfragments that containeda

small

amount ofviral DNA linked to

cellular

DNA.

Themostunconventionalaspect of the

mapping

procedure involved the use ofmouse genetics. We have shown elsewhere that endogenous MMTV proviruses have

segregated

like stable genetic elements during inbreeding (5), andwe

deduced thatsome restriction

fragments

must

have come from the same endogenous unit of viral DNAby showing that the appearance of those fragments was coordinate in digests of DNA from variousstrains (Fig.6;5).

These strategies for physical mapping were

adequate foranunambiguous ordering of

restric-tion fragments in units II and III and their immediately flanking

cell

sequences.

Neverthe-less,moredetailed analysis

will

benecessary to

exclude the presence of

small

regions of DNA

unrelated to MMTV (e.g.,

cellular

intervening sequences) within thestructuresdefined andto

determine theprecise length and

origins

of

se-quencesrepeatedatthe ends oftheendogenous proviruses. InMMTV DNA introduced by

in-fection, sequences fromboth the 3' and 5' ends

ofviral RNA are

terminally

repeated as a

se-quence of ca. 1,200 bases (29; J. E. Majors,

unpublished data). We showed here that

se-quences from the 3' end of MMTV RNA are

presentatbothends of units II and III (asbased upon

annealings

withcDNA3')and that the

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494 COHEN, MAJORS, AND VARMUS

minal redundancies are at least 700 to 800 base

pairs in length (as based upon PstI and HpaI

recognition sites repeated at both ends).

How-ever, we have not as yet used cDNA transcribed

from the 5' terminus of viral RNA to analyze

these redundancies, nor have we determined the

precise length of the redundant sequence.

Pre-sumably these and other refinements can now

beapproached afteramplificationofendogenous

proviruses inprocaryotic systems.

Since no endogenous MMTV strains have

been recovered from BALB/c mice, we were

obliged to use cDNA'spreparedfrom the RNA

of milk-borne MMTV. We then assumed that

these cDNA's would detect all the restriction

fragments generated from each unit that

con-tained endogenous DNA related to the genomes ofmilk-borneMMTV. Withinthesensitivity of the analysis, this assumption appears to have been valid, since we did not find anomalous

reductions in thecombined molecularweightsof

virus-specific fragments,usingmultipleenzymes tocleave endogenousDNA. It ispossible,

how-ever, that the unit we have denoted as

subge-nomic(unit I)is aconventionallysizedprovirus,

onlypart of which ishomologoustomilk-borne

strains of MMTV. The size of the other two

units(ca. 6.0x 106 Mr)is notsignificantly

differ-ent fromthe size ofproviruses

acquired by

in-fection withmilk-borne virus (ca. 5.9 x 106 Mr;

29), and we wereabletodetectallthefragments

derivedfrom units II andIII with our

hybridi-zationreagent. These findings support our

pre-viousclaims thatmost, if notall,of thegenomic

sequences of milk-borneviruses havehomologs

inthe DNA of inbred mice(4, 22, 25).However,

the locations ofrestriction sitesfor

HpaI, PstI,

andBglIIwithin theendogenousproviruses

dif-fer appreciably from the location of sites for

those enzymes within the DNA of milk-borne

strainsofMMTV (4, 29),suggestingthat

signif-icantdifferences in base sequences are

distrib-uted along the genomes. It is possible that

re-gionsof greatest difference accountfor the

evi-dence thatendogenousMMTV DNA may lack

sequences presentinmilk-borne viruses (7, 18);

the results presented here, however, argue

against the notion that the genomes of

milk-borne viruses carry genetic elements of

signifi-cantcomplexity totally unrepresentedin

endog-enousproviruses.

Our finding that most endogenous MMTV

DNA is organized into structures resembling

conventional proviruses acquired by infection

offers further support for our contention that

endogenous proviruses of MMTV were acquired

intherelatively recent past,longafterspeciation

bymultiple, independent infections ofgerminal

tissues (5). Our argument waspreviously based

upon a demonstration of the heterogeneity of

wild mice with respect to the number ofcopies

andsites ofintegration ofendogenous MMTV

DNA. Acomplementary analysisofendogenous

MMTV DNA in inbred micesuggested that the

heterogeneityof wild mice was not due to ahigh frequencyofdeletions, transpositions, or

ampli-fication ofendogenousproviral DNA,since such

DNA hassegregatedlike stableelements during

inbreeding. Support for this hypothesis has

come from similar studies of otherendogenous

viruses (2, 8a, 13) and from the demonstration

that the fourendogenousMMTV provirusesin

theA/HeJstrain mouse arelocatedon atleast

three chromosomes (21). The present report

shows that most of the endogenous MMTV

DNA in BALB/c mice has the structure to be

predicted for a provirus acquired by infection,

rather than for sequences that have evolved

directly from cellulargenes. Ofcourse, the pres-ent results, of themselves, donot indicate how

or when theproviruses mighthave been

intro-duced into the germline.

The resultspresented here have an

interme-diatepractical implication in thatthey provide

several markers (inthe form of restrictionsites)

with which to identifythe MMTV genomes

en-dogenoustoBALB/c andtodifferentiate them

from the genomes ofother strains of MMTV,

both horizontally and genetically transmitted.

We have already exploited the differences in

patterns ofrestriction fragments from

endoge-nous MMTV DNA and milk-borne MMTV

DNA in ordertoassay organsof BALB/c mice

forinfectionbyvirusingested via foster nursing with C3H mice(4). V. L.Morriset al. (personal

communication) have used a similar approach

to show that MMTV occasionally recovered

from tumors ofBALB/cmiceisnot endogenous

to those mice and was presumably introduced

by inadvertent infectionwithvirusstrains trans-mitted in the milk of females with high tumor

incidence.

We chose to perform these initial mapping

studies of endogenous MMTV DNA with

BALB/c mice for at least three reasons: (i) the

units of viral DNA appeared relatively low (4,

22);

(ii)

the strain is extremely susceptible to

milk-borne viruses and hence widely used to study virus-induced mammary cancer (3, 20, 23);

and

(iii)

there isvery limited expression of

en-dogenous viral DNA, even in the lactating

mam-maryglands of multiparous animals or in

mam-mary tumors arising in uninfected animals (8, 19, 37). For the last reason, we supposed that

this strain might be the most likely to lack

MMTVDNA organized into conventional

pro-J. VIROL.

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http://jvi.asm.org/

(13)

viral structures. In other mouse strains that lack MMTV in the milk, there is evidence for pro-duction of infectious virus late in life (3, 20, 23) or for synthesis of viral RNA similar in size to the MMTV RNA identified in infectedcells (27), thus suggesting the presence of endogenous pro-viruses that resemble viral DNA acquired by infection with MMTV. It would be of obvious interest to define the factors that impair the expression of the two apparently complete pro-viruses endogenous to BALB/c mice (units II andIII).

ACKNOWLEDGMENTS

Wethank J. M. Bishop,S. Hughes, and D. Robertson for useful discussions of these data and the manuscript and Y. W. Kan for use of instrumentation essential to the completion of this work.

Thisstudy was supported by Public Health Service grants CA 12705,CA 19287, and CA 21454 and by American Cancer Society grant VC 70. J.C.C. was supported by a fellowship from the National Institutes of Health (grant CA 04471) and by grant IN-133 from the American Cancer Society.

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Figure

FIG.1.phoresisBALBIcphageBALB/clar-weightrestrictiontionA-D)andradiography.wereizedobtainandG, HpaI MMTV-specific fragments obtained from liver DNA by digestion with various restric- endonucleases
FIG. 3.plespoolgels,EcoRIdigested[32P]cDNA:sthebridizationincludedbeled Products of PstI digestion of separated fragments ofBALB/c mouse liver DNA
FIG. 5.positionphoresisphagefurtherrepeatedFig.eitherofEcoRIClanesluloseX 10-6 and the Products of BglII digestion of separated fragments from BALB/c liver DNA
FIG. 6.patternsfragmentsextractedgestsHpaI,aFig.C3H/HeJBALB/cminedofcludedindicated male BALB/c Comparisonof restriction endonuclease of MMTV-specific DNA in BALB/c and mouse liver DNAs
+3

References

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