genetic mapping of endogenous mouse manmmary tumor

Vol. 40, No. 3JOURNAL OF VIROLOGY, Dec. 1981, p. 735-7440022-538X/81/120735-10$02.00/0

Genetic Mapping of Endogenous Mouse Manmmary TumorViruses: Locus Characterization, Segregation, and

Chromosomal DistributionVICKI L. TRAINA,I* BENJAMIN A. TAYLOR,2 AND J. CRAIG COHEN'

Department ofMicrobiology and Immunology, Tulane Medical Center, New Orleans, Louisiana 70112,1 andThe Jackson Laboratory, Bar Harbor, Maine 046092

Received 15 June 1981/Accepted 6 August 1981

The restriction endonuclease EcoRI has been used to study the inheritance ofstrain differences in endogenous mouse mammary tumor virus DNA sequences.This enzyme, which cleaves at only one site within the nondefective viral genome,generates DNA fragments containing mouse mammary tumor virus sequenceswhich vary in size according to the locations of EcoRI restriction sites in theflanking mouse sequences, thereby defining unique integration sites of the viralgenome. Recombinant inbred strains of mice have been used to study theinheritance of these DNA fragments which hybridize to mouse mammary tumorvirus ,DNA sequences. The results define 11 segregating units consisting of 1 or2 fragments. These units were shown to segregate among the recombinant inbredstrains, and in some instances linkage was established. Two units were shown tobe linked on chromosome 1. Another unit was mapped to chromosome 7, whichis presumably identical to the previously defined genetic locus Mtv-1. One othermouse mammary tumor virus locus was tentatively assigned to chromosome 6.The results are consistent with the view that integration of mouse mammarytumor virus can take place at numerous sites within the genome, and onceinserted, these proviruses appear to be relatively stable genetic entities.

Mammary carcinomas in the mouse are asso-ciated with infection by a retrovirus, mousemammary tumor virus (MMTV; 2, 7, 23, 26),which synthesizes a double-stranded DNA in-termediate that integrates into the host genome(7, 30, 32). Formation of this provirus (the inte-grated form of viral DNA) is an essential step inthe replication of the virus (30,41). In mice, twodistinct routes of virus transmission result in thepresence of MMTV sequences within the hostchromosome. Virus is transmitted horizontallyvia the milk of infected female mice to theiroffspring. In this case the virus and viral prod-ucts can be found in infected tissues, the mam-mary glands, and associated tumors. All inbredmouse strains, even though they may not pro-duce infectious virus, bear MMTV-related se-quences in their genomes normally and transmitthem genetically (vertical transmission; 11, 21,25). These endogenous, genetically transmittedMMTV sequences can be found not only ininbred mice, but also in most wild and Asianmice of the genus Mus (25). Endogenous provi-ruses analyzed by restriction endonucleasedigestion and molecular hybridization varied notonly in number, but also in chromosomal posi-tion among the mouse strains tested (8, 24).

These viral sequences are thought to have beenacquired by multiple, independent, and rela-tively recent infection events of the germ line,since considerable variability was noted amonginbred mice, and some feral mice lack endoge-nous MMTV proviruses entirely (8).

In this study the specific chromosomal loca-tion of the MMTV proviruses is examined as aninitial step in evaluating the possible relation-ship of integration sites within the mouse ge-nome and the oncogenic potential of geneticallytransmitted virus. The BALB/c mouse strain isthe only strain in which endogenous provirusesare fully characterized by restriction endonucle-ase analysis and molecular hybridization (6).Most of the MMTV DNA in the BALB/c ge-nome is organized into two units (i.e., virus-spe-cific loci), II and III, which resemble the provi-ruses acquired from exogenous infection, i.e.,structures with approximately 10 kilobases (kb)of viral DNA containing long terminal repeatstranscribed from the ends of the virion RNA (7,29, 32). Other than units II and III, a third virus-specific EcoRI fragment is present. However,this fragment is 16.7 kb in size and contains onlya portion of the MMTV genome. This subge-nomic fragment segregates independently and

735

736 TRAINA, TAYLOR, AND COHEN

alone represents the proviral unit I. In additionto the BALB/c strain, the endogenous provi-ruses of the CBA-C3H family of inbred micewere characterized and shown to segregate asstable, independent genetic elements followingexpected Mendelian rules (8). Assignment ofEcoRI and PstI restriction endonuclease frag-ments into specific genetic units of MMTV wasbased on cosegregation patterns. A maximum ofsix units (I through VI) are present in the ge-nomes of the CBA-C3H and BALB/c mousestrains. Unit II was present in all inbred strainstested. Units I, III, and IV were polymorphicamong the strains tested and could be charac-terized completely. Units V and VI representsimple groupings as the cosegregation data ofthese remaining fragments were limited.Recombinant inbred (RI) strains of mice were

employed in this study to evaluate provirus lo-cation on mouse chromosomes. These strainswere developed by systematic inbreeding begin-ning with the F2 generation of a cross betweentwo preexisting progenitor strains (38). Impor-tantly, in these strains many genetic loci havebeen identified and mapped to specific chromo-somes (38). Genetic mapping of units ofMMTVDNA was accomplished in this study by corre-lation of the segregation patterns of MMTV-specific DNA with other genetic markers amongthe many RI strains.

MATERIALS AND METHODSMouse strains. Two sets of RI strains and their

progenitor strains, obtained from the Jackson Labo-ratory, Bar Harbor, Maine, were used in this study.The BXD and BXH RI strains were derived fromcrosses of the C57BL/6J strain with strains DBA/2Jand C3H/HeJ, respectively (38, 39). All of the RIstrains had attained more than 30 generations of in-breeding and therefore were expected to be homozy-gous at more than 99% of the loci that distinguish theprogenitor strains. Male mice were fasted overnightand killed by CO2 asphyxiation, and their livers were

excised, blotted to remove excess blood, frozen at-80°C, and then shipped on dry ice to New Orleansfor analysis.DNA extraction and restriction endonuclease

digestion. DNA was extracted from the livers of malemice as described previously (6). Briefly, liver homogenates were incubated with sodium dodecyl sulfateand proteinase K to deproteinate the sample followedby phenol-chloroform (2:1) extraction and ethanol pre-cipitation of the DNAs by spooling on a glass rod.DNAs were resuspended in 5 mM Tris-hydrochloride(pH 7.4)-0.1 mM EDTA for storage before restrictionendonuclease digestion.DNA was cleaved with the EcoRI restriction en-

donuclease (Bethesda Research Laboratories) by us-

ing a 5- to 10-fold enzyme excess in 0.1 M Tris-hydro-chloride (pH 7.4)-0.05 M NaCl-0.005 M MgC12-0.05%Nonidet P-40 at 37°C. Reactions were judged complete

on the basis of lambda bacteriophage DNA includedin the reaction and visualized by ethidium bromidestaining and UV irradiation.

Gel electrophoresis, DNA transfer, and hy-bridization. After restriction endonuclease digestion,DNA samples were electrophoresed (1 to 2 V/cm) inhorizontal 3- to 6-mm-thick slab gels of 0.8% agarose(Seakem) in buffer containing 0.04 M Tris-acetate (pH8.15)-0.02 M sodium acetate-0.018 M NaClI-.002 MEDTA (12). DNA was transferred to nitrocellulosesheets by a modification of the procedure originallydescribed by Southern (34). Virus-specific fragmentswere detected by hybridization with 1 x 106 to 2 x 106cpm of 32P-labeled MMTV cDNA per ml synthesizedin an in vitro reverse transcriptase reaction with calfthymus DNA oligomers as primers (18). The annealingbuffer included 3x SSC (lx SSC is 0.15 M NaCl plus0.015 M sodium citrate), 50% formamide, 0.05 M N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH7.0), 10% dextran sulfate, 200,ug of yeast RNA per ml,50 yg of alkali-sheared and denatured salmon spermDNA per ml, and Denhardt solution (0.02% each bo-vine serum albumin, polyvinylpyrrolidone, and Ficoll)(9). The filters were incubated for 2 to 3 h at 41°C inannealing buffer without dextran sulfate and cDNAwith 5x Denhardt solution. After the initial prean-nealing step, filters were incubated with the annealingmix described above for 16 to 24 h. After incubationfilters were sequentially washed in 2x SSC at roomtemperature for 1 h, 0.1x SSC-0.1% sodium dodecylsulfate at 50°C for 1 to 2 h, 0.1x SSC at room temper-ature; and 2x SSC with 20 Mg of proteinase K per mlat 37°C for 1 to 2 h. Filters were air dried and exposedto Kodak XR film at -70°C in the presence of DuPont Cronex Lighting Plus screens (37).Brain mitochondrial malic enzyme activity in

RI strains. The BXD and BXH RI strains were typedfor the Mdr locus, which controls the level of mito-chondrial malic enzyme in mouse brains. Brains fromtwo males of each RI strain were tested for mitochon-drial malic enzyme levels by the method of Bernstein(5). Each strain was analyzed in duplicate, and high-and low-activity progenitor strain controls were in-cluded in each day's test.

RESULTSSegregation of virus-specific EcoRI frag-

ments among RI mouse strains. The endog-enous MMTV proviruses of the C57BL/6J,C3H/HeJ, and DBA/2J mouse strains were re-defined by noting which pairs of virus-specificfragments cosegregated among the BXD andBXH sets ofRI strains. DNA extracted from theuninfected liver of each RI strain was cleavedwith the restriction endonuclease EcoRI, an en-zyme with only one cleavage site in the viralgenome (7, 32). Therefore, the size of the virus-specific fragments would be determined by thelocation of EcoRI cleavage sites in the flankingcellular sequences (7, 8). A variety of virus-spe-cific fragments were generated among the mousestrains tested. For this study, cosegregating frag-

J. VIROL.

GENETIC MAPPING OF MMTV PROVIRUSES

ments once identified were considered tenta-tively as a single proviral genetic unit.DNA fragments produced by EcoRI digestion

were separated by agarose gel electrophoresisand transferred to nitrocellulose sheets, and vi-rus-specific fragments were identified by molec-ular hybridization and autoradiography (seeabove). Figure 1 shows a representative sampleof the virus-specific fragments obtained fromparental and RI strain liver DNA. The distri-bution of these EcoRI fragments among the RIstrain is presented in Tables 1 and 2. Twelvevirus-specific fragments ranging from 1.7 to 16.7kb were found to have segregated randomlyamong the 26 BXD and 11 BXH RI mousestrains.

Analysis ofthe segregation patterns ofMMTVproviruses is complicated by the observationthat fragments of similar size can be associatedwith more than one unit of endogenous MMTVDNA. Earlier studies with the CBA-C3H familyof inbred mice had shown that EcoRI producesa 10-kb fragment from both units III and IV (8).Since both of these units were present in theDBA/2J parent (Fig. 1; Table 1; and reference8), one would expect a 10-kb fragment to bepresent when either the 7.8- or 11.7-kb fragment,the additional portion of units III or IV, respec-tively, was observed. The BXH strains werederived from the C3H/HeJ parent which lacksunit IV, and thus only the 7.8-kb virus-specific

A B C D E F G H I

15ooU;P~* 4F Wv

4-5

58 - w,.R.*.

45 r..:/ l*

fragment would be expected to cosegregate withthe 10-kb EcoRI fragment. This relationship wasmaintained in all of the DNAs from the BXHstrains; however, in the DBA/2J-derived BXDstrains, there were several inconsistencies. Insome of the BXD strains (i.e., BXD-12, -15, -18,-19, -20, -23, and -31) the 11.7- and 7.8-kb frag-ments were absent, yet a 10-kb fragment per-sisted. Of the unassigned fragments none consis-tently cosegregated with the 10-kb fragment.Thus, the presence of the 10-kb fragment as asubgenomic element distinct from units IH andIV was indicated. Its parental origin is not ap-parent from our data. The lack of any subge-nomic 10-kb fragments in the BXH strains sug-gests that this fragment was not derived fromthe C57BL/6J progenitor, which again suggeststhat it may be derived from the DBA/2J parentin the BXD strain. However, the limitation ofnumber of RI strains may explain this observa-tion. Therefore at this time, the parental deri-vation of the 10-kb fragment which we nowdesignate as new unit X is unknown.

Other fragments previously grouped in unitsV (5.8, 6.5, and 4.5 kb) and VI (15 and 1.7 kb)were examined for inconsistencies in their seg-regation patterns among the RI strains of inbredmice. This resulted in the redefinition of thesetwo units into four units (V, VI, Vm, and IX) ofindependently segregating MMTV proviruses.Of the previously described unit V fragments,

I K I. M N 0 Pi b.7

t ^ _ _ ~~~~85~7.8F_ j _ . ~~~~~~~~~~~x h7

-a. 6--i

4.5

i4 7

i.

1I 7 -+ .I

FIG. 1. MMTV-specific fragments obtained from parental and RI strain liver DNA by EcoRI digestion.Samples (10 pg) ofhigh-molecular-weight DNA were extracted from the livers ofmale mice of the designatedinbred and RI strains and digested with the restriction endonuclease EcoRI as described in the text. Thesamples were subjected to electrophoresis in an 0.8% agarose gel, and DNA fragments were transferred tonitrocellulose sheets (34), hybridized with 3P-labeled MMTV-specific cDNA, and detected by autoradiogra-phy. A HindIII digest oflambda bacteriophage 3P-labeledDNA (not shown) was included to obtain the DNAfragment size values in kb as indicated above. Lanes: A, DBA/2J; B and I, C57BL/6J; C, BXD-1; D, BXD-2;E, BXD-5; F, BXD-6; G, BXD-8; H, BXD-1I; J, C3H/HeJ; K, BXH-10; L, BXH-11; M, BXH-6; N, BXH-7; 0,BXH-3; P, BXH-2.

VOL. 4(), 1981 737


TABLE 1. Distribution ofMMTV-specific EcoRI fragments in BXD RI mouse strainsaProgenitorEcoRI Proento BXD mouse strain

fragment(kb) B D 1 2 5 6 8 9 11 12 13 14 15 16 1819 20 21 22 23 24 25 27 28 29 30 31 32

16.7 - + - - +--+ ++.- - - - - - - -15.0 - + - + - + + + + - - + + + - + - - + - + + - - + - + +11.7 - + - + +--+-+.- - - - - - - -10.0 + + + + + + + + + + + + + + + + + + + + + + + + + + + +9.0 - + + - + - - + - + - + + + + + - + + + - + + + + + - +8.5 + + + + + + + + + + + + + + + + + + + + + + + + + + + +7.8 + - I---------------6.7 + _ + + + + _ + + _ + + + _ _ _+ + _+ _ _ _+ + + _6.5 - + -----+ . ----------------5.8 - + + ++++++ - + + -+ + + - + + + + + + +4.5 - + - + . - - - - - +- - - --++----1.7 - + +--+--+- - I +1 + +

The presence or absence of virus-specific EcoRI fragments (indicated by + or -) was determined fromdigestions of DNA extracted from the liver of two individual male mice of each strain.

b Progenitor inbred mouse strains C57BL/6J (B) and DBA/2J (D).

TABLE 2. Distribution ofMMTV-specific EcoRI fragments in BXH mouse strainsa

EcoRI Progenitor BXH mouse strainfragment sri

(kb) B H 2 3 4 6 7 8 10 11 12 14 19

16.7 - + + - + + - + - + + + +10.0 + - + + + + - + - + - - +8.5 + + + + + + + + + + + + +7.8 + - + + + + - + + +6.7 + + + + + + + + + + + + +6.5 - + + + + + - + + + + + +5.8 - + + - + - - - - + + + +4.5 - + + + + + - + + + + + +1.7 - + - - - + + + +

a See footnote a of Table 1.bProgenitor inbred mouse strains C57BL/6J (B) and C3H/HeJ (H).

the 6.5- and 4.5-kb EcoRI fragments consistentlycosegregated in all RI strains, but varied withrespect to the 5.8-kb fragment (Table 1). Unit Vwas used to designate this MMTV genetic unitderived from the 6.5- and 4.5-kb fragments. The15-, 9-, 5.8-, and 1.7-kb fragments were not foundto cosegregate with each other or any of theother virus-specific fragments; therefore, theywere considered independent, subgenomic,MMTV genetic elements, although the 15.0-kbfragment might represent a provirus lacking anEcoRI site.The segregation patterns observed in the

BXD and BXH RI strains define 10 distinctMMTV units. The segregation ratios were con-sistent with single-gene inheritance for most ofthe MMTV units, with the exceptions beingunits VII, VIII, and IX in the BXD strains andunit V in the BXH strains (Tables 3 and 4). Thedeviation from 1:1 segregation of unit VIII in theBXD strains was only of borderline significance.

The most striking exception to 1:1 segregation(P < 0.001) was unit VII. This unit, contributedby DBA/2J, was present in 23 of the 26 strains.However, unit VII, contributed by C3H/HeJ,segregated normally in the BXH strains, beingpresent in 6 of 11 strains. Thus, abnormal seg-regation of unit VII was restricted to the BXDset of RI strains. Unit II was the only unit of the10 that was present in all three progenitor strainsand, as expected, was found in all of the RIstrains.Chromosomal distribution of MMTV

DNA. It is possible to define the chromosomallocation of several MMTV proviruses by relatingthe distribution of specific units of viral DNA toisozyme and other chromosomal markers in theRI strains of inbred mice. Comparisons of the RIstrain distribution patterns of genetic units ofMMTV DNA and genetic markers present inindividual strains are presented in Tables 5through 10. Cosegregation of an MMTV proviral

J. VIROL.

GENETIC MAPPING OF MMTV PROVIRUSES 739

TABLE 3. Segregation of endogenous MMTVproviruses among BXD mouse strainsa

Provi- Progenitor BXD mouse strain X2 test ofr

EcoRI frag- strain"uns ment(kb)_--l2 6 1111:ratiounit B D 1 2 5 6 8 9111213 14 15 16 18 1920 21 22 23 24 25 27 28 29 30 31 32

I 16.7 - - - - - - - - - - - - - - - - - - P >0.25++II 8.5, 6.7 + + + + + + + + + + + + + + + + + + + + + + + + + + + +

III 10.0, 7.8 + - + + + + - + + - + + - + - - - + + - + - - - + + - - P > 0.25rIV 11.7,10.0 - + - + +--+-+.- - - - - - - - - - P > 0.50cV 6.5, 4.5 - + - + + + + _ _ _ _ + + _ + _ _ + + p >..10CVI 15.0 - + - + + + + + - + + + - + - - + + + _ + + + P > 0.25cVII 5.8 - + + + + + + + + + + + + + +- + + +- + + + + + + + P < 0.001VIII 9.0 - + + _ + _ _ + _ + _ + + + + + _ + + + _ + + + + + - + P = 0.05rIX 1.7 - + +- - +- --+- +-- - - +- - +-- +.- - P < 0.02X 10.0 ± ± ±I+±± +±+ ++±1±i± + ± ± ± ± ± ±++±,

a The presence or absence of particular proviruses (indicated by + or -) was determined by examining EcoRIdigestions of DNA from two male mice of each strain by the DNA transfer method. The inability to determinethe presence or absence of provirus due to comigration is indicated by ±.

b See footnote b of Table 1.C Not significant.

TABLE 4. Segregation of endogenous MMTVproviruses among BXH mouse strainsa

Provi Progenitor BXH mouse strains 2teat of 1:1Proun EcoRl frag. ________________________

unit ment (kb) ratiounit B H 2 3 4 6 7 8 10 11 12 14 19

I 16.7 - + + - + + - + - + + + + P >0.10II 8.5, 6.7 + + + + + + + + + + + + +

III 10.0, 7.8 + - + + + + - + - + - - + P > 0.25cV 6.5, 4.5 - + + + + + - + + + + + + P < 0.01

VII 5.8 - + + - + - - - - + + + + P > 0.75cIX 1.7 - + - - - + + + - + - - -- P > 0.25ca See footnote a of Table 1.b See footnote b of Table 1 and footnote b of Table 2.c Not significant.

unit with a particular marker indicates theirassociation on a specific chromosome.

In general the MMTV units defined in thisstudy segregate independently of each other,indicating that the proviruses are inserted atmultiple sites widely distributed throughout themouse genome. An exception to the general ob-servation is the high degree of concordance be-tween units I and IV in the BXD strains. In only3 of 26 strains, BXD-2, -9, -22, did these twoproviruses demonstrate recombination. Further-more, both of these markers showed strong link-age with other markers on the distal portion ofchromosome 1. Unit IV showed close linkagewith the Mls locus (three recombinants among24 RI strains). Unit I showed close linkage withthe Eph-I locus (one recombinant among 26 RIstrains). The distribution of alleles at this clusterof loci is shown in Table 5. Since any arrange-ment of these two MMTV proviruses involvesapparent double crossovers, one cannot defini-tively order these two MMTV proviruses withrespect to the other loci. The order shown in

Table 5 is only one of several possible arrange-ments. Surprisingly, in the BXH strain, unit I,as defined by the 16.7-kb EcoRI fragment, showsfree recombination with several markers in thissame region of chromosome 1. Since unit I isinherited from strains DBA/2J and C3H/HeJ inthe BXD and BXH RI strains, respectively, the16.7-kb fragment must be located at unlinkedsites in these two inbred strains. The C3H-CBAfamily of mouse strains was derived from cross-ing the Bagg albino stock with the DBA stock,the ancestors ofthe BALB/c and DBA/2 strains,respectively. Since modem BALB/c mice alsopossess a 16.7-kb fragment, one might suspectthat the C3H/HeJ strain inherited its 16.7-kbfragment from its Bagg albino ancestor. Alter-nately, one might hypothesize a genetic trans-location to explain the results. On the basis ofthe distinct genetic inheritance ofunit I in DBA/2J, we propose to designate it unit Ia. Unit I ofC3H/HeJ could not be assigned to any specificchromosome.Unit V showed linkage to two chromosome 7

VOL. 40, 1981


markers (Tam-i and Mdr) in the BXD RIstrains, and with one of these (Mdr) in the BXHRI strains. The inheritance of unit V and variouschromosome 7 markers is shown in Tables 6 and7. There are only five recombinants among the26 BXD strains between unit V and Tam-i, asignificant departure from independence (P <

0.005). Combining data from the BXD and BXHRI strains, there are nine recombinants among37 strains between unit V and Mdr, a gene thatregulates the brain level of mitochondrial malicenzyme. The Mdr locus typing of the RI strains(previously unpublished) was clear-cut; RIstrains could be readily assigned to high-activ-

TABLE 5. Linkage of unit Ia and unit IV with chromosome 1 markers in the BXD RI strainsa

Locus or BXD RI strain Source orunit 1 2 5 6 8 9 11 12 13 14 15|17 18 19 20 21 22 23 24 25 27 28 29 30 31 32 reference

b- B B D B B D D BD B B B B B B B D B D D D D D DN ND 2x x

Eph-1 B B D B D D DBBBBBBBBBDBDDDDDD B D 20x

Unitla B B D B D D D B B B B B B B B B B B D D D D D D B D Tables3x x and 4

MIS BD D BD D DBB BB B BBB B BD DDDD DDNDNDND1Ox x x

UnitIV B D D B D B D B B B B B B B B B D B D D D D D D B D Tables3x and 4

Ly_9C B D D B D D D B B B B B B B B B DB DiD D DB DNDN D 19a The letters B and D are used as genetic symbols for alleles inherited from the progenitor strains C57BL/6J

and DBA/2J, respectively. Regions where crossovers have resulted in recombination in the different BXD RIstrains are noted by x. ND, Not done.

b No symbol has been assigned to this locus, which controls a kidney protein of approximately 24,000 daltons(28).

'Ly-9 is the provisional locus symbol for a surface glycoprotein of lymphocytes termed LgplO0 (19).

TABLE 6. Linkage between unit Vprovirus and chromosome 7 markers in the BXD RI strains'Mouse BXD strain

Locusor-.unit 1 2 5 6 8 9 11 1213141516181920212223242527282930 31 32 Sourceorref-

erenceGpi-1 B D B D B BB D B DD B B B B B B|B BBD|D!B|B D D 45

x x x x xTam-i B D D D B B B D B D B B B B B B D B B D B D B B D D 33

x x x x xUnit V B D B B B B B D B D D B B B B D D B B D B B B B D D Table 3

x x x x x x x xMdr B B D B B D B D D D D B D B B B B B D D B B B B D D Thispaper

xHbb B B D BB D B D D D D B|D B B B|B B D D B|D|B IB|D|D| 45aSee footnote a of Table 5.

TABLE 7. Linkage between unit Vprovirus and chromosome 7 in the BXH RI strainsaMouse BXH strainLocus or Source or referenceunit 2 3 4 6 7 8 10 11 12 14 19

Unit V H H H H B H H H H H H Table 3x

Mdr H H H H B H H H H B H This paper, 5x x x x

Hbb B B H H B H H H B B B 36, unpublished dataa The letters B and H are used as genetic symbols for alleles inherited from the progenitor strains C57BL/6J

and C3H/HeJ, respectively. Regions where crossovers have resulted in recombination in the different BXH RIstrains are denoted by x.

J. VIROL.


ity (DBA/2J and C3H/HeJ) or low-activity(C57BL/6J) classes. The observed frequency ofrecombination between Mdr and Hbb is consist-ent with published linkage data which placeMdrapproximately 2 centimorgans distal to the al-bino locus. Using formulas for estimating recom-bination frequencies and their standard errorsfrom RI linkage data, we can place unit V be-tween Tam-1 and Mdr with the following dis-tances: Tam-1-6.8 ± 3.8 centimorgans-unitV-9.6 ± 4.4 centimorgans-Mdr. Van Nie et al.(42, 43) have identified a gene (designated Mtv-1), present in the C3Hf and DBAf strains, thatdetermines the presence ofMMTV viral antigenin milk. This gene exhibited 29 and 20% recom-bination with the albino locus in crosses withC3Hf and DBAf, respectively. Although thisamount of recombination is somewhat greaterthan would be expected between unit V andalbino, it nonetheless seems probable that unitV and Mtv-1 are identical.

Unit IX showed a significant association withthe Lyt-2 lymphocyte alloantigen locus on chro-mosome 6 (P < 0.005; Tables 8 and 9). Amongthe 37 BXD and BXH strains there were ninerecombinants between unit IX and Lyt-2 forestimated recombination frequency of 0.096 ±

0.044. There are two more recombinants withthe y-glutamyl cyclotransferase locus (Ggc), sothe presumed gene order is unit IX-Lyt-2-Ggc. The orientation of this gene triplet withrespect to the centromeres is unknown. Al-

though the unit IX-Lyt-2 linkage is formallysignificant, this assignment should be consideredas tentative, pending confirmatory data.The strain distribution patterns for the five

remaining proviral units which segregate in theBXD and BXH RI strains are shown in Tables10 and 11. These patterns did not exhibit signif-icant linkage with any of the genetic markersavailable. Units VII and VIII appear to be as-sociated with each other, however (five recom-binants among 26 BXD strains; P < 0.005). Themeaning of this association is unclear due to theabnormal segregation of unit VII.

DISCUSSIONNomenclature proposal for MMTV ge-

netic elements defined by restriction en-donuclease digestion of mouse DNA. In thepresent and past papers (6, 8) characterizing theendogenous proviruses of inbred strains of mice,we have designated individual genetic elementsas units with arbitrarily assigned Roman numer-als. The data presented in Table 5 link a specificset of virus-specific fragments produced by re-striction endonuclease digestion and a previ-ously defined genetic locus, Mtv-1 (42). There-fore, it seems appropriate at this time to aban-don our system of Roman numerals and applythe previously established alphanumerical sys-tem. This system as applied to available datafrom restriction endonuclease studies is pre-sented in Table 12.

TABLE 8. Linkage between unit IXprovirus and chromosome 6 markers in the BXD RI strainsa

Locus or Mouse BXD strainsu.it.Sourceunit 1 2 5 6 8 9 11 12 13 14 15 16 18 19 20 21 22 23 24125 27 28 29130 31 32

Unit IX D B B D B B D B D B B B B D B B D B B D B B B B B B Table 3x x x x x

Lyt-2 D B D D D B D B D B B B B D B D D B D D D B B B B B Tulchin andx x x x x Taylorb

Ggc B B B D D B D B D B B B B D BVD D B B D D D B D B B TulchinandI- I. -~ - .. - -- - - Taylor'

a See footnote a of Table 5.bTuilchin and Taylor, unpublished data.

TABLE 9. Linkage between unit IXprovirus and chromosome 6 markers in the BXH RI strainsaMouse BXH strain

Locus or unit Source2 3 4 6 7 8 10 11 12 14 19

Unit IX B B B H H H B H B B B Table 2x x x x

Lyt-2 B B B B B B B B B B B Tulchin and Taylorbx

Ggc H B B B B B B B B B B Tulchin and Taylorba See footnote a of Table 7.b Unpublished data.

741VOL. 40, 1981


TABLE 10. Strain distribution patterns of additional MMTVprovirus sequences in the BXD RI strainsa

Mouse BXD strainUnit

1 2 56 8911112113114151161819 2021222324 25 2728129 30 3132

III BB BB DB B D B B D B D D B B B D B D D DIB B D DVIB D BD DD D B B D D D B D B B D B D D B B D B D DVII DDDDDD D D B D D D D D B D D D B D D D D D D DVIII D B D B B D B D B D D D D D B D D D B D D D D D B D

a See footnote a of Table 5.

TABLE 11. Strain distribution patterns ofadditional MMTVprovirus sequences in the BXH

RI strains'Mouse BXH strain

Unit2 3 4 6 7 8 10 11 12 14 19

IH B H H B H B H H H HIII B B B B H B H B H H BVII H B H B B B B H H H H

a See footnote a of Table 7.

TABLE 12. Proposed nomenclature for MMTVgenetic loci

MMTV provirus nomencla-ture EcoRI frag Chromo-

ments (kb) some loca-Alphanumeri- Roman nu- tion

cal locus meral unit

Mtv-1 V 6.5, 4.5 7Mtv-2 None 11.0, 6.9 18bMtv-3c None NDd?Mtv-4e None NDMtv-5f None NDMtv-6 I 16.7Mtv- 7 Ia 16.7 1Mtv-8 II 8.5, 6.7 ?Mtv-9 III 10.0, 7.8 ?Mtv-10 IV 11.7, 10.0 1Mtv-11 VII 5.8 ?Mtv-12 VI 15.0 ?Mtv-13 VIII 9.0 ?Mtv-14 IX 1.7 6Mtv-15 X 10.0 ?a Reference 22.'Van Nie, unpublished results.c Reference 27.d ND, Not done'J. Hilgers, personal communication.f Reference 16.

Mtv-1, -2,, and -3 are genetic loci describedpreviously by using classical genetic methods (4,17, 42, 43). The data presented in Tables 6 and7 would indicate that unit V is identical to Mtv-1; thus, it is designated as such. Restrictionendonuclease fragments synonymous with theMtv-2 locus have been recently described (15,

22). Correlative molecular data are not yet avail-able for the Mtv-3 locus. The Mtv-4 locus in theSHN strain controls high levels of virus expres-sion and high early mammary cancer incidence(J. Hilgers, personal communication). The Mtv-5 locus in the SL/NiA mouse strain controlsmoderate levels of MTV expression (unpub-lished data); however, it is not involved in mam-mary cancer (16). In Table 12, Roman numeraldesignations have been enumerated with an al-phanumerical system from Mtv-1 to Mtv-15;thus, nomenclature continuity is maintained asthe transition from genetic to molecular defmi-tion of endogenous MMTV genetic elementsprogresses.Origin and genetic stability of endoge-

nous MMTV proviruses. The distribution ofendogenous MMTV proviral DNA observed inthe BXD and BXH RI strains has resulted in aredefinition of segregating units of MMTV se-quences. In most instances normal Mendelianratios and independent assortment were ob-served. Two MMTV units were linked to eachother on chromosome 1, although three recom-binants were observed, indicating that the twoproviruses are several centimorgans apart. Un-expectedly, unit I, which is linked to chromo-some 1 markers in the BXD RI strains, segre-gated independently of chromosome 1 markersin the BXH strains, indicating that the provi-ruses generating the 16.7-kb fragment in DBA/2J and C3H/HeJ are nonallelic and unlinked. Itis possible that these two proviruses are inte-grated in distinct sites which coincidentally gen-erate similar EcoRI fragments. Another possi-bility is that viral integration occurs preferen-tially in specific sequences which are present inmultiple sites in the mouse genome. A thirdexplanation is that the virus, once integrated,was translocated or transposed to another chro-mosome. It is of interest that Howe et al. (13)have reported the nonallelic inheritance of alymphocyte alloantigen (Lyb-4) in these sametwo strains.

One possible explanation of the abnormal seg-regation of unit VII in the BXD RI strains (23+; 3 -) is that these sequences are represented

J. VIROL.


in two or even three locations in the DBA/2Jgenome. The alternative is that selection hasfavored the fixation of the DBA/2J unit VII,possibly as the result of selection acting on somelinked locus. A 10-kb fragment was observed inthree independently segregating MMTV provi-ruses (units III, IV, and X). This also could beinterpreted as evidence that viral integrationfavors certain sites that are represented in dif-ferent parts of the genome. Further restrictionanalysis is needed to test this possibility. Theavailability of RI strains with reduced numbersofMMTV proviruses could be useful for such ananalysis. Another question is to what extent thevarious endogenous MMTV proviruses are ex-pressed and what role they play in mammarycarcinogenesis.No novel fragments were detected among the

RI strains which might indicate a reinsertion inthe germ line after the RI strains were estab-lished. Reinsertion events appear to be relativelycommon for murine leukemia viruses in someviremic strains (31). In conclusion, our resultssupport the notion that MMTV sequences havebeen acquired by multiple integrations in thegerm line at numerous sites in the genome. Sincethese sites vary among inbred strains, they arepresumed to be of relatively recent origin. Onceintegrated, MMTV proviruses appear to be rel-atively stable and are transmitted from genera-tion to generation like other cellular genes.

ACKNOWLEDGMENTSWe thank T. Brown, R. Baumann, M. Higgins and C.

Baigent for their technical assistance.This work was supported by American Cancer Society

grant MV-65, IN-133, Cancer Society of Greater New Orleansgrant 106, Public Health Service grant GM 18684 from theNational Institutes of Health, and by Public Health Serviceresearch contract N01 CP33255 from the Division of CancerCause and Prevention, National Cancer Institute. The JacksonLaboratory is fully accredited by the American Associationfor Accreditation of Laboratory Animal Care.

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genetic mapping of endogenous mouse manmmary tumor

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