Endogenous mouse mammary tumor virus DNA is distributed among multiple mouse chromosomes

VIROLOGY

92, 46-55 (1979)

Endogenous

Mouse Mammary Tumor Virus DNA Is Distributed Multiple Mouse Chromosomes

among

VINCENT L. MORRIS,*,’ CHRISTINE KOZAK,** J. CRAIG COHEN,Y PETER R. SHANK,Y PAUL JOLICOEUR,S FRANK RUDDLE,O AND HAROLD E. VARMUSt *Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A SC1; **Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 2901& TDepartment of Microbiology, University of California, San Francisco, California 9.&l.& &Institute de RecLrches, Cliniques de Montreal, 110 West Pine Avenue, Montreal, Quebec H2W lR7; and SDepartment of Biology, Yale University, New Haven,

Connecticut

16520

Accepted September 14, 1978

We have examined the distribution of endogenous mouse mammary tumor virusspecific DNA in the genome of A/HeJ mice by using molecular hybridization and restriction endonucleases to analyze DNA from mouse-hamster hybrid clones that segregate mouse chromosomes. We have found that MMTV sequences are located on at least three separate chromosomal pairs, including chromosome number four. INTRODUCTION

Although the mouse mammary tumor virus (MMTV) was originally discovered as a transmissible agent present in the milk of female mice with a high incidence of mammary tumors (Bittner, 1936; Nandi and McGrath, 1973; Bentvelzen, 1974), it has also been shown that mice (including feral strains of Mus musculus and the Asian mice, Mus caroli and Mus cervicolor) bear MMTV-related sequences normally in their genomes (Varmus et al., 1972; Gillespie et al., 1973; Parks and Scolnick, 1973; Michalides and Schlom, 1975; Morris et al., 1977). Closely related rodents, including Chinese hamsters, are devoid of MMTV-related DNA (Varmus et al., 1972, 1975; Gillespie et al., 1973; Bishop et al., 1974). Most laboratory strains of mice (including the A strain used in this study) in1 Author to whom requests for reprints should be addressed. 2 Present address: Department of Microbiology and Immunology, Tulane University, New Orleans, Louisiana 70112. 3 Present address: Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912. 0942~68w79/019946-10$02.09/0 Copyright 0 1979 by Academic F’ress, Inc. All rights of reproduction in any form reserved.

herit approximately six to eight copies of MMTV-related DNA per diploid cell, regardless of tumor incidence (Morris et al., 1977). Little is known about the origins and functions of these multiple copies, about the relationship of each to the genomes of horizontally or genetically transmitted strains of MMTV, or about their distribution among and organization within chromosomes. As one approach to these questions, we have examined the distribution of endogenous MMTV-specific DNA sequences among murine chromosomes. Combining the techniques of somatic cell hybridization, molecular annealing, and restriction endonuclease analysis, we have shown that endogenous MMTV specific DNA sequences present in A/HeJ mice are present on multiple chromosomes. MATERIALS

AND METHODS

Sources of virus, cells, and animals. Virus, purified from the milk of female strain Rlll mice (Lyons and Moore, 1965; Moore et al., 1973), was provided by Dr. Dan Moore (Hahemann Medical College, Philadelphia, Pa.). MMTV (C3H) derived 46

CHROMOSOMAL

DISTRIBUTION

from the Mm5mt/cl mammary carcinoma cell line (Fine et al., 1974) was provided by the Frederick Cancer Research Center (Bethesda, Md.). Chinese hamster ovary cells (CHO) used to prepare the hamster unique sequence [14C]DNA were supplied by Dr. David Martin (University of California, San Francisco). A/HeJ mice were obtained from Jackson Laboratories (Bar Harbor, Me.); Chinese hamsters were purchased from Chick Line Hamstery (Vineland, N. J.). Somatic cell hybrids. The Chinese hamster-mouse hybrid cell lines were produced by the fusion of A/HeJ mouse peritoneal macrophages and E36 cells (Gillin et al., 1972; Kozak et al., 1974, 1975). Cells were maintained in MEM supplemented with 10% fetal calf serum, penicillin (50 U/ml), and streptomycin (50 pg/ml). Primary hybrids were grown in HAT-selective medium (MEM with 10m4M hypoxanthine, 4 x 10e5M aminopterin, 1.6 x 10e5 M thymidine). Secondary clones were either isolated in HAT medium or backselected and maintained in MEM with 10 to 20 pg/ml 8azaguanine (Kozak et al., 1974, 1975). Each hybrid clone was characterized for the expression or loss of 18 mouse isozymes for which hamsters and mice have electrophoretically distinguishable forms (Nichols and Ruddle, 1973; Kozak et al. 1974, 1975; Kozak and Ruddle, 1976). Karyotypic analysis of 50 to 100 metaphase spreads was done by sequential staining procedures (Kozak et al., 1977). Six independently derived secondary clones which represent six independent fusion events were selected for the DNA-DNA annealing studies. Each of these clones contained different complements of mouse chromosomes. Hybrids were examined for expression of mouse isozymes (Nichols and Ruddle, 1973; Kozak and Ruddle, 1976) and chromosome constitution (Kozak et al., 1977) prior to expansion of the cell population for DNA extraction. In addition, as each clone was grown in culture for DNA extraction, a sample of cells was removed for isozyme analysis (Nichols and Ruddle, 1973; Kozak and Ruddle, 1976); in addition, a portion of the cells was frozen and later

OF MMTV

DNA

47

grown in culture (for approximately two to three generations) for analysis by chromosome banding techniques (Kozak et al., 1977). The average number of mouse chromosomes was determined for each hybrid by counting the number of chromosomes in each metaphase plate with brightly fluorescent centromeres after staining with Hoechst 33258 (Hilwig and Gropp, 1972). DNA extraction. DNA for annealing experiments was extracted as previously described (Morris et al., 1977). For restriction endonuclease experiments DNA was prepared as described in Cohen et al. (1979). Briefly, cells were dispersed in DNA extraction buffer (0.02 M T&-Cl, pH 8.1, 0.01 M EDTA, 0.1 M NaCI). Pronase (selfdigested for 2 hr at 37”) and SDS were added, with final concentrations of 1 mg/ml and l%, respectively, and the sample was incubated at 37” for 12 hr. DNA was deproteinized twice with equal volumes of phenol-chloroform (2:1), followed by extensive dialysis against 5 mM Tris-Cl, pH 7.4, 0.1 mM EDTA. Hybridization reagents and conditions. The 14C-labeled unique sequence DNA was prepared from Chinese hamster (CHO) cells as previously described (Morris et al., 1977). Two methods were used to generate DNA complementary to the RNA genome of MMTV (MMTV cDNA). (1) The endogenous DNA polymerase associated with detergent-activated virions (isolated from Rlll milk) was used to synthesize MMTV cDNA in the presence of actinomycin D (Ringold et al., 1975; Morris et al., 1977). Several preparations of MMTV cDNA made by this procedure protected 50 to 60% of MMTV 70 S RNA from digestion with pancreatic ribonuclease in 0.3 M NaCl after hybridization with a 15- to 50-fold excess of MMTV cDNA (Varmus et al., 1973, Medeiros, 1975). (2) Labeled MMTV cDNA was also synthesized in a reaction catalyzed by AMV DNA polymerase (supplied by J. Beard and the Office of Program Resources and Logistics, National Cancer Institute) using oligomers of calf thymus DNA as primers (Goulian, 1968). The cDNA was synthesized with 35 S RNA isolated from virions purified from the Mm5mt/cl cell line,

48

MORRIS

using reaction conditions described by Shank et al. (1978). Complementary DNA made with this technique has been shown to be representative of most, if not all, of the viral genome by its ability to detect all the restriction endonuclease fragments of viral DNA with an efficiency equal to that of iodinated viral RNA (Ringold et al., 1978). MMTV cDNA was isolated by modification of a procedure of Dr. S. Hughes (personal communication) as follows: After adding 50 pg of calf thymus carrier DNA, the reaction mixture was incubated with Pronase (0.5 mg/ml) and 1% SDS at 37” for 1 hr, extracted twice with phenol, and precipitated with ethanol. The nucleic acid was resuspended in 3 n-&Z EDTA and chromatographed on a G-50 Sephadex column (0.02 M Tris, pH 7.4, 0.3 M NaCl, 3 mM EDTA, 0.1% SDS). After 50 pg of calf thymus DNA was added, the DNA was precipitated with ethanol, resuspended in 10 mM EDTA, and incubated with 100 ,ug/ml pancreatic ribonuclease (Worthington) for 1 hr at 37”. We then treated the mixture again with Pronase and extracted it with phenol. After ethanol precipitation, the pellet was resuspended in 0.12 M phosphate buffer and single-stranded DNA was then separated from double-stranded DNA on a hydroxylapatite column (Morris et al., 1977). After adding 50 pg of calf thymus DNA, we concentrated the single-stranded cDNA by blowing air over the sample and chromatographed it on a G-50 Sephadex column as described above. Fifty micrograms of calf thymus DNA was added and the sample was again treated with Pronase and SDS and ethanol precipitated. We then resuspended the pellet in 3 mM EDTA. The cDNA bands broadly at 4 S in an alkaline sucrose gradient. The conditions of the annealing reactions (0.6 it4 NaCl, 68”) and assay of duplex DNA by fractionation on hydroxylapatite columns have been previously described (Morris et al., 1977). For analysis of the products of digestion by restriction endonucleases, the cDNA was synthesized by method (2), labeled with [32P]dCTP (New England Nuclear), and purified as described by Cohen et al. (1979). Briefly, the reaction mix was alkali-

ET AL.

treated (0.6 M NaOH, 37”, 2 hr), neutralized with HCl, chromatographed on a Sephadex G-75 column to remove unincorporated dCTP, and precipitated with ethyl alcohol. Restriction endonuclease analysis of DNA. High molecular weight DNA was completely digested with the endonuclease EcoRI (kindly provided by Drs. P. Greene and H. Boyer, University of California, San Francisco) in 0.1 M Tris-Cl, pH 7.4, 0.05M NaCl, 5 mM MgCl,, and 0.05% NP40. The reactions were monitored by the inclusion of pBR 313 DNA, which is cleaved once by EcoRI [see Shank et al. (1978)]. The DNA fragments were separated by electrophoresis in agarose gels, transferred onto nitrocellulose sheets, and annealed to 2 x lo6 to 4 x lo6 cpm of [32P]cDNA (2 x lo* to 4 x lo* cpm/pg) as previously described (Shank et al., 1978). After incubation, the filters were washed in 2x SSC (1 x SSC is 0.15 M sodium chloride, 0.015 M sodium citrate) at room temperature for 1 hr, incubated in 0.1 x SSC-0.1% SDS at 50”, rinsed with 0.1~ SSC-0.1% SDS and 0.1~ SSC at room temperature, and air-dried (Cohen et al., 1979). Filters were exposed at -70” to Kodak RP-Royal X-Omat film in the presence of DuPont Cronex “Lightening Plus” screens (Swanstrom and Shank, 1978). RESULTS

Karyology of the Mouse-Hamster

Hybrid

Clones

We have combined somatic cell hybridization, molecular annealing, and restriction endonuclease analysis to study the distribution of endogenous MMTV-specific DNA among murine chromosomes. Our experiments utilize Chinese hamster-mouse somatic hybrid cells which preferentially segregate mouse chromosomes (Kozaket a?., 1974, 1975). Clones of hybrid cells were isolated after the fusion of primary A/HeJ mouse macrophages and Chinese hamster lung cells of the established line, E36 (Gillin et al., 1972; Kozak et al., 1974, 1975). Hybrids obtained from this fusion always lose mouse chromosomes while retaining at least one complement of hamster

CHROMOSOMAL

DISTRIBUTION

OF MMTV

49

DNA

tion of mouse chromosomes in each line during propagation.

chromosomes (Gillin et al., 1972; Kozak et al., 1974, 1975). Several independently isolated hybrid clones were selected and grown in culture. The mouse chromosomes retained by each clone were identified both directly by chromosome banding techniques (Kozak et al., 1977) and indirectly by monitoring the expression of isozyme markers on 14 mouse chromosomes (Nichols and Ruddle, 1973; Kozak and Ruddle, 1976). Table 1 lists values for each chromosome in the six clones which proved useful for the analysis of the distribution of MMTV DNA. We measured frequencies at which the mouse chromosomes were observed both before and after propagation for DNA extraction (Table 1); both of these values must be considered in the subsequent analysis, since there were changes in the distribu-

MMTV DNA in Hybrid Clones as Measured by Annealing Kinetics We have determined the amount of virusspecific DNA present in the hybrid clones using the kinetics of annealing of [3H]DNA complementary to the MMTV RNA genome (MMTV cDNA) and cellular DNA (Britten and Kohne, 1968; Morris et al., 1977). We first measured the kinetics of annealing of viral cDNAs (see Materials and Methods for description) with artificial mixtures of A/ HeJ and Chinese hamster DNA (Fig. 1A); since these mixtures were composed of unfractionated cellular DNAs, the results resembled those to be expected if MMTV-re-

TABLE

1

KARYOLOGYOF MOUSE-HAMSTER SOMATICCELL HYBRIDS Mouse-hamster hybrid clone Chromosome No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X

10-2

6D3AZ

4, 0” 75, 30

B2b AZa23c

4B31AZ

42, 84

94, 40

60, 14

4E4AZ 1

13-2AZ

8, 14

6 4 88, 79

0 12, 0 25, 4 31, 54 44, 13 44, 0

17, 0

67, 53

88, 40

8, 0

13, 0

23, 44

62, 24

70, 55

44, 25

20, 3 45, 47

0, 75, 0, 0, 75,

23, 34 54, 45 30, 24

63, 33

6, 78 80, 94

4 52 4 44 52

30, 45

94, 50 31, 46 0, 79

-c

a The first number represents the percentage of cells with the given chromosome before expansion for DNA extraction; the second number refers to the observed frequency after the growth of the clone for DNA isolation. ’ Isozyme markers of mouse chromosome 4 were expressed, but no intact chromosome 4 was identified. c Expression of mouse hypoxanthine phosphorlbosyl transferase was detected, but not a-galactosidase; no intact X chromosome was observed.

MORRIS ET AL.

50

40-

IO-

15-

\

6. 20 IO’

o a\ .-5

I 3

102

IO4

IO5

Cot ( MolelsO~ L-‘. Set) FIG. 1. (A) Kinetic analysis of MMTV-specific DNA in reconstructed samples and mouse-hamster hybrid DNAs. Mixtures of various ratios of DNA extracted from A/HeJ mice and Chinese hamsters were annealed to [3H]cDNA synthesized in an endogenous MMTV DNA polymerase reaction, (see Materials and Methods). Symbols: (0) 0% mouse DNA; (0) 2% mouse DNA; (A) 5% mouseDNA; (A) 10% mouse DNA; (m) 25% mouse DNA; (0) 50% mouse DNA. (B) MMTV [3H]cDNA (made using the endogenous MMTV DNA polymerase) was annealed to DNA from mouse-Chinese hamster hybrid clones as described above and under Materials and Methods. The dashed lines represent annealing of MMTV [3H]cDNA and artificial mixtures of mouse (A/He.J) and Chinese hamster DNA (data from A); the numbers indicate the percentage of mouse DNA in each fraction. The symbols represent annealing of MMTV [aH]cDNA and DNA extracted from the following mouse (A/HeJ)-Chinese hamster (E36) hybrid cell clones: (0) 10-2; (A) 4E4AZl; (W) 13-2AZ, and (e) 6D3AZ. Chinese hamster unique sequence [WIDNA (prepared from CHO cells) was included in each annealing reaction in both (A) and (B) to serve as an internal standard. The annealing of the unique sequence DNA with the unlabeled DNA present in each reaction mixture followed expected hybridization kinetics (data not shown).

lated DNA were randomly and uniformly distributed among the 40 mouse chromosomes (i.e., approximately 0.35 copies per chromosomal pair or 0.18 copies per chromosome, assuming seven copies per diploid cell [Morris et al. (1977); Fig. 31.

For the experimental analysis, the proportions of mouse and hamster DNA in each hybrid clone were calculated from the chromosomal data shown in Table 1. The observed rates of annealing of MMTV cDNA to the DNA from each clone (Fig. 1B) were

CHROMOSOMAL DISTRIBUTION

51

OF MMTV DNA

TABLE 2 SUMWARY OF ANNEALING OF MOUSE-HAMSTER

HYBRID CLONE DNA AND MMTV cDNA

Extrapolated percentage of murine DNA per clone*

Clone

Observed percentage of murine DNA per diploid hybrid clone’

Endogenous cDNA=

Calf tbymus DNA primed cDNAd

10-Z 6D3AZ B2bAZa23c 4B31AZ 4E4AZl 13-2AZ

2-3 2-3 8 8-9 6 8-11

4 4 NT” NT 5 3

3 3 1 1 NT NT

a The percentage of mouse DNA per hybrid cell was determined from the karyology (Table 1). * The percentage of mouse DNA in a mouse-hamster hybrid clone which would be predicted by comparing the annealing of mouse-hamster hybrid cell clone DNA and MMTV [SH]cDNA with the standard annealing curves of MMTV [SH]cDNA and artificial mixtures of mouse and hamster DNA (Figs. 1 and 2). c cDNA was synthesized by detergent disrupted virions as described under Materials and Methods. d cDNA was synthesized by AMV DNA nolymerase, using oligomers of calf thymus DNA as primers and MMTV 70 S RNA-as template: e Not tested.

compared to the rates expected on the basis of the reconstruction experiments (Fig. 1A) for DNA with the estimated proportion of mouse DNA. A greater than expected rate of annealing would indicate that the chromosomes retained in that clone had greater than 0.18 MMTV DNA copies per chromosome, whereas a reduction in rate of annealing would indicate a relative paucity of viral DNA in those chromosomes. In the extreme cases, DNA from a clone which bore only those mouse chromosomes devoid of virus-specific DNA would fail to anneal with MMTV cDNA, and DNA from a clone which harbored a single mouse chromosome containing all the virus-specific DNA would appear to be 20-fold enriched for viral sequences. We first chose clone 6D3AZ for analysis by this procedure because it contained only one mouse chromosome, chromosome 4 (Table 1). Despite the low proportion (23%) of mouse DNA in clone 6D3AZ (Table 2), the annealing of MMTV cDNA to DNA from this clone conformed to the pattern expected for DNA composed of 3 to 4% murine DNA (Figs. 1B and 2; Table 2). Thus, chromosome 4 is enriched for MMTV-specific DNA.

Since the observed frequency of chromosome 4 varied from 0.6 to 0.14 during propagation of the cells, we cannot assign a precise value for the number of copies of viral DNA in this chromosome; however, using these values (Fig. lB, Table 2), we estimate that between 0.7 copy4 and 4.0 copies5of MMTVspecific DNA are present on chromosome pair 4 per diploid cell. Similar results were obtained from a second independently derived hybrid clone (10-2) which contained chromosome 4 as its only intact mouse chromosome after expansion of the clone for DNA extraction (Table 1). Using the data from this clone, we estimate that chromosome pair 4 contains 0.5 to 1.9 copies of MMTV-specific DNA (Figs. 1B and 2; Table 2). No significant differences were noted between results obtained with cDNA synthesized in an endogenous DNA polymerase reaction and with cDNA primed by oligomers of calf thymus DNA (see Materials and Methods and Table 2). * Calculation: (3%/2.5% x 0.35 copies per chromosome paYO.6). 5 Calculation: (4%/2.5% x 0.35 copies per chromosome pair/O. 14).

52

MORRIS ET AL.

Since these calculations were based upon a total of seven copies of MMTV DNA per diploid cell, all MMTV DNA sequences do not appear to be present on chromosome 4. This deduction was confinned by the detection of virus-specific DNA in clones B2bAZa23c, 4B31AZ, and 13-2 AZ, which lacked chromosome 4 by both karyotypic and isozyme analysis (Tables 1 and 2; Figs. 1B and 2). Thus MMTV DNA endogenous to A strain mice appears to be located on more than one chromosome. Analysis of DNA from Mouse-Chinese Hamster Clones with a Restriction Endonuclease

We have recently found that we can distinguish among the several copies of MMTV DNA endogenous to a single mouse strain by the use of restriction endonucleases (unpublished data of J.C. C. and H.E. V.). Thus, using EcoRI [which recognizes a single site in the proviruses acquired by in-

FIG. 3. Detection of MMTV DNA in hybrid clones after digestion with a restriction endonuclease. DNA extracted from the liver of A/HeJ mice (lane B) or from various mouse-hamster clones (lanes C-F) was digested with the enzyme EcoRI. After digestion, the DNA fragments were separated by electrophoresis in an 0.8% agarose gel and transferred to nitrocellulose sheets (Southern, 19’75)for hybridization with [3*P]cDNA (Cohen et al., 1979). DNA fragments containing MMTV DNA were visualized as bands by autoradiography, as described previously (Swanstrom and Shank, 1979). (A)HindIII digest of 3ZP-labeledlambda bacteriophage DNA. (B) AfHeJ liver DNA (10 pg). (C) Clone 10-2 (30 pg). (D) Clone 4E4AZl DNA (30 pg). (E) Clone B2bAZa23c DNA (30 pg). (F) Clone 4B31AZ DNA (30 pg).

fection with milk-borne MMTV (Shank et 19’78)]and the DNA transfer procedure (see Materials and Methods), we have identified seven large virus-specific fragments in digests of DNA from uninfected tissues of A/HeJ strain mice (Fig. 3, lane B). Preliminary mapping data and analogy with FIG. 2. Kinetic analysis of MMTV-specific DNA in fragments from the BALB/c strain (Cohen mouse hamster hybrids. MMTV-specific [3H]cDNA synthesized with calf thymus oligomers as primers (see and Varmus, paper in preparation) suggest Materials and Methods) was annealed to DNA from that six of these fragments each represent mouse-Chinese hamster hybrid clones. The dashed approximately half of an endogenous prolines and solid circles represent annealing of MMTV virus plus adjacent cellular DNA; the [3H]cDNA and artificial mixtures of mouse (A/HeJ) seventh and largest fragment (10 x lo6 and Chinese hamster DNA. The numbers indicate the MW) probably contains a subgenomic segpercentage of mouse DNA in each fraction. The ment of proviral DNA. These results are symbols represent annealing of MMTV [3H]cDNA and in good agreement with kinetic analysis, DNA extracted from the following mouse (A/HeJ)which indicates that about seven copies of Chinese hamster (E36) hybrid cell clones: (0) 10-2; (W) 6D3AZ; (A) 4B31AZ; and (A) B2bAZa23c. Chinese MMTV DNA are endogenous to each diploid hamster unique sequence [W]DNA was also included cell in A/HeJ mice (Morris et al., 197’7). Regardless of the detailed structure of in each annealing reaction, as described in the legend the viral DNA, the distribution of the fragto Fig. 1. al.,

CHROMOSOMAL

DISTRIBUTION

ments in EcoRI digests of DNA from the hybrid clones (Fig. 3, lanes C-F) strongly supports and extends the results obtained by analysis of the same clones with conventional hybridization kinetics (Figs. 1 and 2). Thus the EcoRI digest of DNA from clone 10-2, which contains only mouse chromosome number 4, showed only two virusspecific fragments (3.2 and 4.6 x lo6 MW) (Fig. 3, lane C). In contrast, the EcoRI digest of DNA from clone, 4E4AZ1, which lacks chromosome 4 but contains several other chromosomes, included all five of the fragments present in the digest of A/HeJ liver DNA but missing in the digest of 10-2 DNA (Fig. 3, lanes B, D). Analysis of DNA from two other clones (B2bAZa23c and 4B31AZ) was complicated by the diffuse background and by several light bands (at 2.0,2.5,3.8, and 8 x lo6 MW). These bands may indicate the presence of previously undetected homologs of MMTV DNA in hamster DNA but they have not been further investigated. It is apparent, however, that the only identifiable fragment present in these two digests as well as in digests of A/HeJ strain DNA in approximately molar yields is the fragment of 10 x lo6 MW (Fig. 3, lanes E and F). The analysis of DNA from hybrid clones with EcoRI therefore supports our conclusions that endogenous MMTV DNA is distributed over multiple chromosomes, since chromosomes that yield at least three sets ofEcoR1 fragments appear to have segregated independently in the generation of the clones. The fragments of 3.2 and 4.6 x lo6 MW cosegregate and are probably derived from a single provirus on chromosome 4. Therefore chromosome pair 4 would presumably contain two copies of MMTV DNA in a diploid cell. The fragment of 10 x lo6 MW is found independently of the others and, based upon the karyology presented in Table 1, could be derived from any of several chromosomes. The other four fragments presumably represent two proviruses whose location cannot be precisely assigned; however, they are likely to be located on either or both of the two chromosomes (15 and 17) which are present in clone 4E4AZl but not in clones B2bAZa23c or 4B31AZ.

OF MMTV

DNA

53

DISCUSSION

Using a combination of somatic cell hybridization, molecular annealing, and digestion with restriction endonucleases, we have shown that the endogenous MMTV DNA sequences in A/HeJ mice are present on at least three separate pairs of mouse chromosomes. Specifically, chromosome pair 4 contains two copies of MMTV DNA. Four copies of MMTV proviral sequences are likely to be located within chromosome pairs 15 and 17. An additional unit of viral DNA segregates independently, although its location is ambiguous. The results obtained by kinetic analysis of DNA extracted from the mouseChinese hamster clones (Figs. 1 and 2; Table 2) are in reasonable agreement with the data obtained by analysis with restriction endonucleases (Fig. 3). In addition, all of the EcoRI fragments containing virusspecific DNA found in digests of A/HeJ liver DNA were also observed in digests of DNA from hybrid clones. This suggests that most or all of the endogenous MMTV DNA in the A/HeJ genome is represented in the hybrid clones we have studied. The techniques described in this paper should eventually facilitate the mapping of the MMTV or MuLV DNA present endogenously in the mouse onto specific chromosomes in a variety of strains with high and low tumor incidence. In fact, using similar procedures, the distribution of the DNA of exotropic murine leukemia virus endogenous to A/HeJ mice has also been determined (Jolicoeur et al., manuscript in preparation). These viral DNA sequences have been found on multiple murine chromosomes, including chromosome 4. The latter result is of particular importance since the Fv-1 locus which regulates virus expression has also been mapped on chromosome 4 (Rowe and Sato, 1973); however, the relationship of the Fv-1 locus and the MuLV and MMTV sequences on chromosome 4 has not yet been determined. Two additional proviruses of murine RNA tumor viruses have also been mapped to specific mouse chromosomes. The Akv-1 locus, which appears to encode an endogenous provirus for the AKR strain of MuLV, has been located on chromosome 7

54

MORRIS ET AL.

(Chattopadhyay et al., 1975). Also, Jaenisch and coworkers have succeeded in inserting a Moloney MuLV provirus into the DNA of single mouse blastomeres at the 4 to 8 cell preimplantation stage (Jaenisch, 1976). The subline of mice derived from this infection was subsequently shown to have Moloney MuLV-specific sequences on chromosome 6, using the techniques of somatic cell hybridization and molecular annealing (Jaenisch, 19’76;Jaenisch et al., 19’78). Our results and those of Jolicoeur and coworkers offer an interesting contrast with the results obtained in studies of the multiple copies of feline leukemia virus and RD-114 virus-related DNA endogenous to normal cat cells. By measuring virus-specific DNA in progeny of crosses and backcrossesbetween cats and ocelots, Benveniste and Todaro (1975) concluded that the multiple copies of each type of viral DNA segregated together and that each type of viral DNA must be linked in a single chromosome. This difference may imply the existence of several mechanisms by which animals may acquire multiple copies of virusspecific DNA. Using an analysis with restriction endonucleases similar to that illustrated in Fig. 3, it has recently been shown that individual feral mice and various inbred mouse strains differ greatly with respect to the MMTVspecific DNA sequences present in their genomes (Cohen and Varmus, manuscript in preparation). These findings imply that the endogenous proviruses were acquired by independent infections of the germ line of individual progenitor mice and have since segregated during the development of inbred strains. Proviruses acquired in this fashion would be predicted to be unlinked in the mouse genome, in accordance with our findings in the studies reported here. ACKNOWLEDGMENTS

We thank Drs. D. Moore, D. Martin, P. Greene, H. Boyer, and J. Beard for providing the materials mentioned in the text. MMTV (C3H) and AMV DNA polymerase were graciously supplied by the office of Program Resources and Logistics, National Cancer Institute. We thank Dr. J. M. Bishop for helpful discussions and Dr. David Baltimore, in whose laboratory a portion of this work was begun, for his active

support. We also acknowledge the excellent technical assistance of Janet Vlasschaert. This project was supported by grants from the Medical Research Council of Canada (MA 5970) and the National Cancer Institute (CA 21311) to V.M., from the National Cancer Institute (CA 19237)to H.E.V., from N.I.H. Grant 09966 to F. R., and from the National Cancer Insitute of Canada to P.J. Work performed in Dr. David Baltimore’s laboratory was supported by grants from the American Cancer Society (VC-4G) and the National Cancer Institute (CA-14051). J.C.C. was supported by a fellowship from NIH(CA 044’71). P.R.S. was supported by a National Cancer Institute training grant 5TOlCAO5303. REFERENCES

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