Identification of ecotropic proviral sequences in high- and low-ecotropic-virus-producing mouse strains.

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Vol. 0022-538X/82/091038-08$02.00/0

Identification

of

Ecotropic

Proviral

Sequences in High-

and

Low-Ecotropic-Virus-Producing

Mouse

Strains

JANETL. MOORE1tANDHARDYW. CHAN2t*

Laboratory ofViralDiseases'andLaboratoryof Molecular Microbiology,2 National Institute of Allergy and

InfectiousDiseases, Bethesda,Maryland20205

Received 18February 1982/Accepted4 June 1982

The arrangement ofendogenous ecotropic retroviruses in selected high- and

low-ecotropic-virus-producing mouse strains was examined by Southern blot

hybridization analysis, usingan ecotropic retrovirus-specific DNA probe.

High-ecotropic-virus-producingmousestrains of the AKRfamily displayed

heterogene-ity withrespect tothe number ofcopies and the sites of insertion of endogenous ecotropic specificDNA. Thisdiversity was seen evenamong individuals ofthe same AKR subline. Contrastingly, individuals within the same

low-ecotropic-retrovirus-producing mouse strain showed no evidence of variability in their endogenous ecotropic proviral sequences. These results favored the hypothesis that germ line proviral reinsertion was responsible for the proviral sequence

heterogeneity observed inhigh-ecotropic-virus-producing mousestrains.

The endogenous type C viralgenomes in the

mouse are acomplexgroupof DNAs,

represent-ing a multicopy gene family. Some of the

se-quences are potentially infectious genomes of ecotropic andxenotropicmurineleukemia

virus-es (MuLVs), but the majority appear to be defective. The inducible, infectious viruses have been extensively characterized with regard to

theirbiological properties (19), but biochemical characterization of the endogenous chromo-somal sequenceshasbeen limited. Studies done with liquid hybridization between in vitro

syn-thesizedcDNAand mousecellular DNAs show

that partsof the MuLV genome are present in many copies, whereas other portions of the genome, namely, those that code for

type-spe-cific properties, are presentin smaller numbers

(4). Ecotropic MuLV-specific sequences fell

into threecategories with respect to copy

num-ber in the DNA of different strains of mice.

High-virus-producing mice such as AKR

showed 3 to 4copies per haploid genome,

low-virus-producingmice such as BALB/c showed 1 to 2, and ecotropic virus-negative strains

showed none. Xenotropic MuLV-specific

probesshowedlarger copy numbers (3 to 10 per

haploidgenome inall mice), but the number did not appear to correlate with the ease of

induc-tionof virus(14).

Studies involving blot hybridization

tech-tPresent address: Barbara Kopp Research Center,

Au-burn,NY13021, and Upstate Medical Center, State Universi-tyof New York, Syracuse, NY 13210.

tPresentaddress:SyntexCorp.,PaloAlto, CA 94304.

niques have confirmed and extended these

re-sults (1-3,22, 23). Probesprepared from whole viralgenomesshowallmouseDNAstocontain many copies of viral sequences, whereas more

type-specific probes prepared by absorption or

molecular cloning of subgenomic portions ofthe

virus have shown the env genedeterminants of ecotropic virusestobepresent in much smaller numbers (1, 3, 4, 17). In addition to providing

more precise quantitation, the blot analyses

al-lowed the identification of single-copy

provi-ruses in terms of the restriction sites in their

flanking sequences, the organization of the se-quences,and characterization of thediversity of the endogenous sequences (7).

We previously have presented preliminary results ofour studies ofendogenous ecotropic

MuLV sequences based on blot hybridization with a molecularly cloned env gene segment fromAKR ecotropic virus (1). In thisreport, we

extend these studiesto furthercharacterize the

differences inendogenous ecotropic proviruses

within and among various strains of laboratory

mice.

MATERIALS AND METHODS

Mice.AKR/N, AKR/J, BALB/c, C3H/He, C57BR,

CBA,C57BL/6, SEA/J, and DBA mice were obtained from the National Institutes of Health and the Jackson

Laboratory. The AKR/Boy mice were a kind gift of E.A.Boyse. The AKV congenic NFS mice were bred inthe laboratory of W. Rowe.

DNA extraction and purification. High-molecular-weight mouse liver DNAs were extracted and purified fromfresh tissue as previously described (1).

Restriction endonuclease digestion and gel

electropho-1038

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ENDOGENOUS ECOTROPIC MuLV SEQUENCES 1039

resis. All restriction enzymes used in these experi-ments wereobtained from New England Biolabs and used as specified by the supplier. Completeness of digestion of cellular DNAs was monitored by adding lambda DNA to a portion of the restriction digestion mixture and evaluating its cleavage by gel electropho-resis. Multiple fragments of digested cellular DNA were resolved byelectrophoresis in 0.7 or

1.0%1

neu-tralagarose gels as described previously (11, 12) and transferred to nitrocellulose sheets as outlined by

Southern (21).

Preparation of hybridization probe. The construction of the recombinant plasmid clone consisting of the 400-base-pair ecotropic-specific env segment from a cloned infectious AKR ecotropic MuLV (15) has been

previously described (1). The DNA was labeled by

nicktranslation (16) and hadspecific activities of 8 x

1 to12x 106cpm/gg.

Nuclekacidhybridization. Preincubation of nitrocel-lulose sheets was carried out asdescribedpreviously (5), and the filters were hybridized in sealed plastic

bags containing10 ml of 3x SSC, pH 7.4(Ix SSC is 0.15 M NaCl plus 0.015 M sodium citrate), lOx

Denhardt solution (1x Denhardt is 0.02% each of bovine serum albumin, Ficoll, and

polyvinylpyrrol-idone)(5),0.1%sodiumdodecyl sulfate, sheared salm-onsperm DNA (50g/ml),andlabeled DNA (3 x 106 cpm persheet) at 60°C for 16 to 36 h. Nitrocellulose membranes were washed as previouslydescribed(13), airdried, andexposed topreflashedKodakXR-2 film.

RESULTS

Because of the extensive polynucleotide se-quencehomology among ecotropic, xenotropic, and dualtropic MuLVs, we have prepared

enve-lope-specificprobes (1) that specifically

hybrid-ize to different classes of proviral DNA and

permitanexamination ofendogenous retroviral sequences. In aprevious report that described the construction and potential usesforan

eco-tropic envelope probe,wedemonstratedthatthe

copy number of endogenous ecotropic

provi-rusesand the arrangement of cellular sequences that flank integrated ecotropic proviral DNA

could bereadily ascertained (1). Suchanalyses

weregreatly facilitated by thediscoverythat the

restrictionenzyme mapsofecotropicproviruses

were virtually identical (3, 22; unpublished

data). Thus,the strategy outlinedpreviouslyof

using restriction endonucleases which cleave

ecotropicproviralDNAsasingletime(eitherto

the 5' or the 3' side of the 400-base-pair

eco-tropic envelope-specific segment) in

combina-tion withspecific MuLVprobestoevaluate the

organization of endogenous ecotropic

provi-ruses in AKR orBALB/c mice could be

safely

extended to all mouse strains because of the

nearly completeconservation of restriction en-zyme sites.Intheexperiments tobedescribed,

mouseliver DNAs weredigestedwithHindIII,

Sacl,orXbaI,which cleavethe

ecotropic

provi-ral DNA3.1,3.7,and 7.8kilobases(kb)from the

5' terminus,

respectively.

Since HindIII and

Sacl cut the provirus to the 5' side of the

ecotropic env-specific segment (5.7 to 5.1 kb from the 3' terminus), fragments that hybridize to theprobe will contain viral sequences includ-ing the 3' long terminal repeat (LTR) and flank-ingcellular sequences (Fig. 1). Conversely, cel-lular DNA fragments which hybridize to the ecotropic envelope-specific probe after XbaI digestion will contain 7.8

kb.

ofproviral DNA including the 5' LTR and adjacent cellular DNA sequences. Mouse liver DNAs were also hybrid-ized to the ecotropic MuLV-specific probe after digestion with EcoRI which does not cleave within the ecotropic provirus. Although diges-tion with EcoRI should also indicate the number of ecotropic proviruses present in a given cellu-lar DNA preparation, the very high-molecucellu-lar- high-molecular-weight cleavage productsgenerated were often difficult to resolve in the agarose gel systems employed.

Characterization of the ecotropic proviral DNAspresent in three colonies of AKR mice. The number of ecotropic proviruses in AKRJN,

AKR/J, and AKRIBoy mice was initially deter-mined by digesting preparations of mouse liver DNAwith HindIII (Fig. 2A). Cleavage of intact endogenous ecotropic provirus with HindIII generated a restriction fragment at least 5.7 kb in size (Fig. 1) that contained the 3' LTR and adjacent flanking cellular sequences. Digestion of mouse liver DNA prepared from an NFS mouse congenic for the Akv-1 locus with HindlIl yielded asingle 5.8-kbfragmentwhich

hybridized to the ecotropic env-specific probe (Fig. 2A, lane 9). Liver DNAs obtained from three different AKR/N (Fig.2A, lanes 1, 2, and

4),four differentAKR/J(Fig. 2A, lanes5 to8),

andtwo different AKR/Boy (Fig. 2A, lanes 10

and 11) mice andcleavedwith HindIII all

con-tained the 5.8-kb DNA fragment characteristic

oftheAkv-1 locus. Hindllldigestion of cellular

DNApreparedfromanNFSmousecongenicfor

the Akv-2 locus generated a 7.0-kb fragment

whichannealedtotheecotropicenvDNAprobe (Fig. 2A, lane 3). This same 7.0-kb cleavage productwas present in the three AKR/N (Fig.

2A, lanes 1, 2, and 4) DNAs and in the two

AKR/Boy (Fig. 2A, lanes 10 and

11)

DNAs.

However, a band comigrating with the 7.0-kb

HindlIl Akv-2fragmentwasabsent inoneAKR/ Nmousesampleandwasdetected inonlytwoof

fourAKR/J DNA

samples

(Fig. 2A, lanes 5 to

8).ForthetwoAKR/Boy (Fig. 2A,lanes 10 and

11)DNA, oneHindIII

fragment

appeared

tobe

shorter than 5.7 kb. This fragment probably represented apartially deleted

proviral

genome (i.e., PstIdigestionsof thetwoDNAsgenerated

aweakreactiveband ofabout 3.3kb,in addition to an8.2-kb bandwhich istypicalofacomplete

ecotropic viral sequence

[data

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Hind1II

-~~Si.7kb -i< .1 kb

78kb

[image:3.501.104.392.83.137.2]

Xba I

FIG. 1. Restriction enzymecleavagemap ofintegratedAKRecotropicMuLV. Thestraight line together with theclosed boxes(representingthe LTRsequence)is indicative of thelengthof the AKRecotropicviral genome. The openbox represents theportionof theenvgene thatwas subcloned inpBR322togenerate theecotropic MuLV-specificprobe (1). indicates theadjacentmousecellularsequence.Thehorizontalarrowsrepresentthe distance (inkb)between the enzymecleavagesites and the viral-cellular DNAjunctions.

Saclcan also be usedinconjunctionwith the ecotropic env probe to evaluate endogenous

ecotropic proviruses and cellular sequences which flank the 3' LTR sequence (Fig. 1). To

furtherinvestigatethepossibleabsenceor

alter-ation of theAkv-2locus insomeAKR/Janimals suggestedfrom the HindIIIdigestions (Fig. 2A),

mouseliver DNAswere cleaved with Sacland annealedto theecotropicenvprobeafter

trans-fertonitrocellular membranes. None of thefou.

AKR/J DNAs examined (Fig. 2B, lanes 3 to 6)

containedthe 6.2-kb SacIfragment

characteris-tic of the Akv-2 (Fig. 2B, lane 9) locus. On the otherhand,twoAKR/Boy(Fig. 2B, lanes 1 and 2) and one AKR/N (Fig. 2B, lane 7) DNAs containedthis DNA segment. In agreementwith the results obtained afterHindIII cleavage,

rep-resentatives of all three AKR colonies contained

1 2 3 4 5 6 7 8 9 10

23.5- W_e OW;}44

9.6-6.6-.

4.4-A

1 2 3 4 5 6 7 8 9

*11e < 1FV .I

:

:.

s.P -

A2-FIG. 2. EcotropicMuLV proviralDNAsequencesinindividualAKRmouse.(A)AKR/N (no. 7) (lane1),

AKR/N(no. 78) (lane2),NFS.Akv-2(lane 3), AKRIN(no. 79)(lane4),AKR/J(no. 66) (lane5),AKR/J(no.67)

(lane 6),AKR/J(no.42)(lane 7),AKRIN (no. 55) (lane 8),NFS.Akv-1 (lane9),AKR/Boy(no. 49)(lane10), and

AKR/Boy (no. 50) (lane 11)mouse liverDNAswerecleaved withHindIIIand analyzed by hybridization to

labeled ecotropic env MuLV DNA probe as described in the text. Arrows indicate the DNA fragments

correspondingtoAkv-land Akv-2genesthatarecharacteristic ofthe analogousgeneinNFS congenic mice(see

lanes 3and 9). The sizes(in kb) of HindlIl-cleavedXDNAfragmentsareindicatedatthe left.(B)AKR/Boy(no.

49) (lane 1),AKR/Boy(no. 50) (lane2), AKR/J(no. 66) (lane 3),AKR/J(no. 67) (lane 4), AKR/J(no.42) (lane5), AKR/J (no.55) (lane6),AKR/N(no. 7)(lane7), NFS.Akv-1 (lane8), and NFS.Akv-2 (lane9) DNAsweretreated exactly thesame asin (A)exceptfor digestionwithSacl.

-*-.A--1

J. VIROL.

1040 MOORE AND CHAN

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the8.8-kbSaclfragmentcharacteristic of

Akv-1

(Fig. 2B).

Liver DNAs from AKR mice were digested withXbaI and hybridized tothe ecotropic env

probe to evaluate the organization of cellular sequences flanking the 5' LTR sequence. One

AKR/N (Fig. 3, lane 7), four AKR/J (Fig. 3,

lanes3 and 6), and twoAKR/Boy (Fig. 3, lanes 1

and2) DNA preparationscontainedthe 12.5-kb

XbaI fragment associated with the

Akv-1

locus.

The 16-kb XbaI cleavage product characteristic

ofAkv-2 (Fig. 3, lane 8) was present in AKR/N

DNA (Fig.3, lane 7), clearlyabsent in twoAKR/ BoyDNAs (Fig. 3, lanes 1 and 2), and detected

intwooffourAKR/J DNAs(Fig. 3, lanes 3 to 6).

Digestion ofmouse liver DNAs with EcoRI,

whichdoes not cleave withinthe ecotropic pro-virus (23), followed by hybridization to the

eco-tropic envelope DNA probe, was the third

ap-proach used to examine the organization of

endogenous ecotropic proviral DNA in AKR

mice. AKR/N (Fig. 4, lanes 5 and 6), AKRIJ

(Fig. 4, lanes 3 and 4), and AKR/Boy DNAs

contained the 32-kbAkv-1EcoRIfragment. Con-versely, the 25-kb EcoRI fragment associated

with the Akv-2 locus (Fig. 4, lane 2) was only

detected inAKR/N (Fig. 4, lanes 5 and 6) DNA

preparations; itwas not seenin samplesof AKR/ J(Fig. 4, lanes 3 and 4)orAKR/Boy (Fig.4,lane

1) liver DNAs.

Ananalysis of proviral DNAfragments char-acteristic of the Akv-1 and Akv-2 loci andpresent

in liver DNAs from three AKR colonies is

summarized in Table 1. Mice from all three

coloniesinvariablycontained the Akv-1locus,as

1 2 3 4 5 6 7 8

23.S-"l 'A ---rAKV-2

12.3- v vAKf-1

9.6-FIG. 3. Hybridizationofendogenous MuLV-relat-ed DNA sequences to an ecotropic MuLV-specific

DNA probe. AKRIBoy (no. 50) (lane 1), AKR/Boy (no. 49)(lane 2),AKR/J (no. 55) (lane3),AKR/J(no.

42) (lane 4), AKR/J(no. 67)(lane5), AKR/J(no. 66) (lane 6),AKRIN(no.7)(lane 7),andNFS.Akv-2 (lane 8) DNAswerecleaved withXbaIandanalyzed byblot hybridization as described in the text. The arrows correspond to ecotropic-specific DNA fragments

found inNFS.Akv-1 and NFS.Akv-2 congenic mice.

Thesizes(in kb)ofHindIII-cleavedA DNAfragments areindicatedattheleft.

1 2 3 4 5 6

--*'-AKV-2

23.S;- * v ; ;

12.3- i _ _

9.6-FIG. 4. Reactivity of endogenous ecotropic provi-ral sequence with an ecotropic MuLV-specific DNA probe. AKR/Boy (no. 49) (lane 1), NFS.Akv-2 (lane2), AKR/J (no. 67) (lane 3), AKR/J (no. 66) (lane 4), AKR/ N(no. 7) (lane 5), and AKR/N (no. 78) (lane 6) DNAs were digested withEcoRI. Southemblot hybridization was performed as described in the text.

judged by thepresence of characteristicHindIII, Sacl, XbaI, and EcoRI fragments. The three

AKR/N animals examined also contained the

Akv-2 locus. On the other hand, AKR/J and

AKR/Boy animalsexhibited an inconsistent pat-tern with respect to the presence of authentic

Akv-2 cleavage products. For example, four

AKR/J liverDNA preparations did not contain

the Sacl or EcoRI fragment characteristic of

Akv-2,yetthreeof the four contained fragments

that comigrated with HindlIl orXbaI cleavage

products associated with the Akv-2 locus.

One possible explanation forthe inconsisten-cyis that the 16-kbXbaIfragments observed in

thetwo AKR/J animalswere in fact of slightly different molecular weights than the Akv-2 se-quence. This is a likely interpretation since

molecularweight resolution byagarosegel

elec-trophoresisis relativelypoor in this size range. Athirdlocus(ecotropicEnvlocus-3,Table1) containingsequencesreactivewith theecotropic

envelope probe wasconsistently detected in all

three AKR mouse colonies. This endogenous

proviral DNA segment was associated with

characteristic HindIII (22.5 kb; Fig. 2A), SacI

(5.3 kb; Fig. 2B), XbaI (12.3 kb; Fig. 3), and

EcoRI (13 kb; Fig. 4) fragments most readily

seen inAKR/N DNA which contains onlytwo

other(Akv-1 andAkv-2)reactive loci. A second

consistentfeature of thisanalysis wasthe exis-tenceofareactive6.0-kbHindlIl fragmentin all

AKR/J andAKRIBoyDNAs(Fig. 2A,lanes5 to

8, 10, and 11). It should benoted that the data

shown inFig. 2-4arecompatiblewith the pres-ence of3, 4or 5, and 7 copies ofendogenous ecotropic proviruses in AKR/N, ADR/J, and

AKR/Boymice, respectively. In allcases, PstI

digestionofAKRmouseliver DNAgenerateda

single 8.2-kb cleavage product which reacted

with theecotropic envprobe (datanotshown).

Evaluation of theendogenous ecotropic provi-VOL.43, 1982

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1042 MOORE AND CHAN

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++++ ++

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4z _~+ +

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ta00 +

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ENDOGENOUS ECOTROPIC MuLV SEQUENCES

ral DNA present in mouse strains expressing

ecotropic MuLVs at a low frequency. The ap-proach outlined in Fig. 1 and used to examine

AKRmouse strains was alsoemployedtostudy

ecotropic proviruses in low-virus-expressing mice. Incontrasttothepatterns observed with

AKR animals, most low-virus-expressing mice

contained only a single ecotropic env reactive DNA segment. As shown in Fig. 5A, the

eco-tropic envelope probe hybridized to a 7.0-kb

HindIII fragment present in restricted C3H/He

(lane 1), C57BR (lane 2), CBA (lane 3), C57BL

(lane 4), BALB/c (lanes 5 and 6), and SEA/J

(lane 7) DNAs. DBA mouse liver DNA

con-tained a single 6.5-kb HindIII ecotropic env

reactive(Fig.5A,lane 8). C57BR and SEA mice were theonlymembersofthis group containing

asecondcopy ofendogenous ecotropic proviral DNA after digestion with HindIII. A similar

simple blot hybridization patternwas observed

aftercleavageoflow-ecotropic-virus-expressing

mouseDNA withXbaI(Fig.SB).Inthis experi-ment, however, the ecotropic provirus present in C57 andBALB/c micecould bedistinguished; theecotropicenv probehybridizedto an11.5-kb XbaI fragment from C57BR (lane 1), C57BL/6 (lane 2), and C57BL/10 (lane 3) DNAs and to a

10.5-kb fragment from BALB/c (lanes 4 and 5) DNAs. As was the case after HindIIIcleavage, XbaIdigestion of SEA (lane6)DNA generated

tworeactive fragments;onecomigratedwith the

8.5-kb XbaI fragment found in the DBA (lane7) mouse,and the othercomigrated with the single endogenous ecotropicprovirus bandpresent in

BALB/canimals.

1 23 4 56 78

A

2 3 4 5 6 7

11.5- -*

8.5-.*:.;. 0

am1. _f

B

FIG. 5. Ecotropic MuLVsequences in inbred mice with lowincidence ofleukemia. The experiments were

performed in the same manner as described in the legends to Fig. 2-4. (A) HindlIl digestion of DNAs fromC3H/He (lane 1), C57BR (lane 2), CBA (lane 3), C57BL (lane 4),BALB/c (lanes 5 and 6),SEA/J(lane 7), and DBA (lane 8) mice. (B) XbaI-cleaved DNAs from C57BR (lane 1), C57BL (no. 6) (lane 2), C57BL (no. 10)(lane 3),BALB/c (lanes 4 and 5), SEA (lane 6), and DBA (lane 7) mice.

DISCUSSION

The most striking observation in the studies

presentedhereis the extentofheterogeneity in the number and location of endogenous

eco-tropic MuLV proviruses among different AKR

mice,notonlyamongdifferentsublinesbut also

among individuals of a single colony. These resultsareconsistentwiththosepreviously

pub-lishedby otherinvestigators (8, 17, 18, 25). Data

from ourlaboratory showed that AKR/N mice

generally contained three ecotropic

proviruses,

AKR/J

generally contained

four,

and

AKR/Boy

contained aboutseven.AllAKRmicecontained

a provirus

corresponding

to that in the Akv-1

congenic mice, but onlythe AKR/N mice

con-tainedaprovirus with all the characteristics of the Akv-2 locus, as was

pointed

out

previously

(20).

The question of whether AKR/J mice carry

Akv-2, meaning the chromosome 16 locus de-fined by the NFS congenics, is not clear from these studies. Some AKR/J mice showed

eco-tropic

env-specific

probe-reactive

HindIII or XbaI orboth restriction

fragments

comigrating

with those of Akv-2, but otherAKR/J mice did

not.Inthose DNAs with both HindIII andXbaI fragments like thoseofAkv-2, it is notpossible

to say if they are from the same or different

proviruses,becauseof themultiplicityof bands. Since thetwofragmentsare notuniformly pres-ent together, it seems most likely that they

representdifferentprovirusesand that their sim-ilarity in size to Akv-2-containing fragments is

fortuitous. Further Mendelian and biochemical studies arein progress to clarifythis

point.

In addition to Akv-1 and Akv-2

proviruses,

AKR/N DNAsconsistently showed athird eco-tropic-reactive provirus; its flanking sequence parameters with the various enzymescould be reliably inferred sincethe only other

uniformly

presentbandwasAkv-1.This third

provirus

was present in all mice of all three AKR

colonies,

andmustrepresentanoninducible

provirus

that,

like Akv-l, was present in AKR before the

separation ofAKR/J and AKR/N in the 1940s.

(Bothlineswerederivedfrom AKR stockatthe

Rockefeller Institute in the

1940s,

the N line

being carried initially by

Lloyd

Law

[6].)

This

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1044 MOORE AND CHAN

provirus couldrepresentthe "Akv-3" locus de-tected by its ability to induce antibody to MuLV,orpossiblytheAkvlp locus,detectedby its induction ofgs andGixantigens (9, 10).

Heterogeneity between individuals of the same colony wasseenwith all three AKR sub-lines. This was most evident with AKRIJ, in whichatleast four different patterns were seen among the eight mice tested with both HindIII

and XbaIdigestion. Ofthe three AKR/N mice, one showed an additional provirus, and one of thetwoAKR/Boy mice showedanextra provi-rusnotcarried by its sibling. These extracopies

wereoften less intense than theconstantbands, suggesting that they represent heterozygotic loci.

The heterogeneity ismost likely due togerm lineproviral reinsertions previouslyobservedin high Akv-1 congenic mice (20). This is

under-scored by the multiplicity of ecotropic provi-ruses seen in the other high-virus-producing strains, e.g., C3H/Fg, C58, and M. molossinus

(H. W. Chan, unpublished data),aswellas low-virus-producing strains showing little or no evi-dence of reinsertions. In fact, we were able to show that in related low-ecotropic-virus-produc-ingmousestrains, despite separation of perhaps 50years or300 generations, the identical provi-ralintegrations arestill retained.

Ifgermline proviral reinsertions indeedoccur in high-virus-producing mouse strains, the fre-quency ofreinsertion orthe ability to maintain the inserted proviral sequences must vary

among individual strains. For instance, AKR/N mice generally showed three ecotropic provi-ruses,whereasAKR/Boymice had aboutseven.

Thus, other hostgenesarelikely tobe involved in the control ofgermline proviral reinsertions.

Althoughgermline proviral reinsertion is one

possibleexplanation forourfinding, ourresults

can also be seen as evidence that retroviral

genomes undergo frequent genetic

rearrange-mentsuchastranslocation and amplification. In

many regards, MuLV proviral DNA is

remark-ably similar to the structure of bacterial and eucaryotic transposable elements(24). Whether

endogenous viral genomes can indeed

translo-cate to new genomic loci via transposition is difficulttoevaluate empirically. Suffice ittosay,

however, that such postulated ability to

trans-pose must have been lost among

low-virus-producingmouse strains.

ACKNOWLEDGMENTS

We thank MalcolmA.Martin andWallaceP. Rowe fortheir

interest andsupportof this work. LITERATURECITED

1. Chan,H. W., T. Bryan, J.L. Moore, S.P.Staal,W.P.

Rowe,andM. A. Martin. 1980. Identificationof ecotropic

proviralsequencesin inbredmousestrainswithacloned

subgenomic DNA fragment. Proc. Natl. Acad. Sci. U.S.A. 77:5779-5783.

2. Chattopadhyay, S. K., M. R. Lander, S. Gupta, E. Rands, andD. R. Lowy. 1981. Origin of mink cytopathic focus-forming (MCF) viruses: comparison with ecotropic and xenotropic murine leukemia virus genomes. Virology 113:465-483.

3. Chattopadhyay, S.K., M. R. Lander, E. Rands, and D. R. Lowy. 1980. Structure ofendogenous murine leukemia virus DNA in mouse genomes. Proc. Natl. Acad. Sci. U.S.A.77:5774-5778.

4. Chattopadhyay, S. K., D. R. Lowy, N. M. Teich, A. S. Levine, and W. P. Rowe. 1974. Qualitative and quantita-tive studies of AKR-type murine leukemia virus se-quences in mouse DNA. Cold Spring Harbor Symp. Quant. Biol.39:1085-1101.

5. Denhardt, D. T. 1966. A member filtertechnique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23:641-646.

6. Furth, J. 1978. The creation of the AKR strain whose DNAcontains the genome ofaleukemia virus. In H. C. Morse III(ed.), Origins of inbred mice. Academic Press, Inc., New York.

7. Groner, B.,and N.E.Hynes.1980. Number and location of mouse mammary tumor virusproviral DNA in mouse DNAof normal tissue andof mammarytumors.J. Virol. 33:1013-1025.

8. Herr, W.,and W.Gilbert. 1982. Germ-line MuLV reinte-grations in AKR/J mice. Nature(London)296:865-867. 9. IhIe, J. N., and D. R. Joseph. 1979. Serological and

virological analysis of NIH (NIH XAKR)mice: evidence for three AKR murine leukemia virus loci. Virology 87:287-297.

10. Ikeda, H.,W. P.Rowe,E. A.Boyse, E.Stockert,H. Sato, and S. Jacobs. 1976. Relationship of infectious murine leukemia virus andvirus-relatedantigensingenetic cross-esbetween AKR and Fv-1 compatible strains C57L. J. Exp. Med. 143:32-46.

11. Israel, M. A., H. W. Chan, W. P. Rowe,and M. A. Martin. 1979.Molecularcloning of polyoma virus DNA in Escherichia coli:plasmidvectorsystem.Science 203:883-887.

12. Israel,M.A.,D. F.Vanderryn, D. F.Meltzer, and M. A. Martin. 1980. Characterization of polyoma viral DNA sequencesin polyoma induced hamster tumor cell lines. J.

Biol.Chem. 255:3798-3805.

13. Jeffreys, A. J., and R. A. Flavell. 1977. Aphysical map of the DNA regions flanking rabbit B globin gene. Cell 12:429-439.

14. Lowy, D. R., S. K.Chattopadhyay, N. M. Teich, W. P. Row, and A. S. Levine. 1974. AKRmurine leukemia virus genome:frequencyof sequences in DNA of high-, low-, andnonvirus yielding mouse strains. Proc. Natl. Acad. Sci.U.S.A. 71:3555-3559.

15. Lowy, D. R., E. Rands, S. K. Chattopadhyay, C. F. Garron, and G. L. Hager. 1980. Molecular cloning of infectious integrated murine leukemia virus DNA from infected mouse cells. Proc. NatI. Acad. Sci. U.S.A. 77:614-618.

16. Maniatis, T., A.Jeffreys, and D. G. Kleld. 1975. Nucleo-tide sequence of therightward operator of phage X. Proc. Natl. Acad. Sci. U.S.A.72:1184-1188.

17. Quint, W., W. Quax, H. van der Putten, and A. Berns. 1981. Characterization of AKR murine leukemia virus sequences in AKR mouse substrains and structure of integrated recombinant genomes in tumor tissues. J. Virol. 39:1-10.

18. Quint, W., H. van der Putten, F.Janssen, and A. Berns. 1982.Mobility of endogenous ecotropicmurineleukemia viral genomes within mouse chromosomal DNA and

integrationof a mink cell focus-forming virus-type recom-binant provirus in the germ line. J. Virol. 41:901-908. 19. Rowe, W. P. 1978. Leukemia virus genomes in the

chro-mosomal DNA of the mouse. Harvey Lect. 71:173-192. 20. Rowe, W. P., and C. A. Kojak. 1980. Germ-line

http://jvi.asm.org/

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tions of AKV-1 congenic mice. Proc. Natl. Acad. Sci. U.S.A. 77:4871-4874.

21. Southern, E. M. 1975. Detection ofspecific sequences amongDNAfragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517.

22. Steffen, D., S. Bird, W. P. Rowe, and R. A. Weinberg. 1979. Identification of DNA fragments carrying ecotropic proviruses of AKR mice. Proc. Natl. Acad. Sci. U.S.A.

76:4554-4558.

23. Steffen, D., and R. A. Weinberg. 1978. The integrated

genomeof murine leukemia virus. Cell 15:1003-1010. 24. Temin,H. M. 1980. Origin of retroviruses from cellular

moveablegenetic elements. Cell 21:599-600.

25. Yoshimura, F. K., and M. Breda. 1981. Lack of AKR ecotropic provirus amplification in AKR leukemic

thy-muses.J.Virol. 39:808-815. VOL. 43, 1982

http://jvi.asm.org/