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We identified mouse mammary tumor proviral loci in the AKR/J, C3H/HeJ, C57BL/6J, C57L/J, DBA/2J, and SWR/J inbred mouse strains and determined their segregation patterns in the AKXD, AKXL, BXD, BXH, and SWXL recombinant inbred strain sets. Two new Mtv loci, Mtv-29 and Mtv-30, were identified. Mtv-30 was genetically mapped to chromosome 12. Additionally, two previously identified Mtv loci, Mtv-14 and Mtv-23, were genetically mapped to chromosome 4 and chromosome 6, respectively.

Mice in breeding colonies of feral Mus musculus brevirostris (Azrou, Morocco), M. m. musculus (Studenec, Czechoslovakia), and M. m. molossinus (Fukuoka, Japan) were found to lack the mouse mammary tumor virus (MMTV-alpha) proviral genome in their germ line. MMTV-alpha proviral genomes have been found in all inbred strains of M. musculus by using high-stringency nucleic acid hybridization conditions. We conclude that feral mice in these colonies are heterozygous for a limited number of MMTV-alpha proviral genomes and that those lacking them arose as a result of random chromosomal segregation. All mice in another breeding colony of feral M. m. musculus (Sladeckovce, Czechoslovakia) lack MMTV proviral genes. By relaxing the conditions of nucleic acid hybridization, MMTV-related sequences (designated MMTV-beta) were detected in restricted cellular DNA from MMTV-negative mice and all other inbred strains and feral species of the genus Mus. The apparent ubiquity of the MMTV-beta DNA sequences in the genus Mus and the lack of variation in the pattern of restriction fragments containing these sequences within a species distinguishes them from MMTV-alpha. These results suggest that the MMTV-beta DNA sequences either are the evolutionary progenitors of the infectious MMTV genome or represent an accumulation of evolutionarily divergent MMTV-alpha insertions into the mouse germ line.

A specific probe for detecting ecotropic murine leukemia virus sequences was constructed by cloning a 500-base-pair DNA segment, corresponding to a portion of the env region of the AKR ecotropic virus, in a pBR322/Escherichia coli K-12 host/vector system. This probe was used to screen the cellular DNAs of six inbred strains of mice for the presence of ecotropic retroviral DNA sequences by the Southern blot hybridization procedure. Three copies of ecotropic viral DNA were detected in AKR/N (a high-ecotropic virus strain) and two were found in BALB/c (a low-ecotropic virus strain) DNAs. As expected, no sequences reactive with this probe were found in NFS mouse DNA (a virus-negative strain). However, cellular DNA sequences that reacted strongly with the ecotropic-specific DNA probe were detected in certain NZB, C57L, and 129 mice (all virus-negative strains). In contrast to the reactive sequences in AKR and BALB/c, the reactions were chiefly associated with EcoRI segments that were subgenomic in size.

Proviruses of the mouse mammary tumour virus (MMTV) endogenous to normal mice can be identified by molecular hybridisation and distinguished using restriction endonucleases. Feral mice display marked heterogeneity with respect to the number of copies and the sites of insertion of endogenous MMTV-specific DNA, with occasional mice apparently free of MMTV DNA. Several different MMTV proviruses present in laboratory mice have segregated like stable, independent genetic elements during the inbreeding which followed a cross between Bagg albino and DBA mice 60 years ago. The results favour the hypothesis that endogenous proviruses have been established by multiple, independent infections of germ cells rather than by somatic mutation of ancestral proviruses or of cellular genes.

A comparative study has been made of the susceptibility of fibroblastic cells to transfection by DNA from C57BL/Ka mouse lines producing either fibrotropic or thymotropic retroviruses. DNA isolated from fibroblasts that release a B-ecotropic, fibrotropic virus, BL/Ka(B), was found to transfect fibroblasts of Fv-1bb genotype with release of virus similar to BL/Ka(B). Fv-1nn fibroblasts were also susceptible but expression was delayed, and xenotypic mink lung cells were refractory. In contrast, DNA prepared from a murine T-cell lymphoma line producing a B-ecotropic, thymotropic virus failed to transfect mouse fibroblasts though it transfected a nonproducer T-cell lymphoma line. The data suggest that the Fv-1 and differentiation-specific restriction mechanisms operate at different molecular levels.

We studied endogenous type C virus-related sequences of mice by annealing Moloney murine leukemia virus DNA to agarose gel blot transfers of uninfected mouse cell DNA which had been cleaved with restriction enzymes. We found that many of the endogenous murine leukemia virus-related sequences in mice consist of two organizational classes that are integrated into many different loci. Both of these classes resemble standard murine leukemia virus proviral DNA in both size and sequence organization. All lines of inbred mice examined contained both organizational classes, as did feral isolates of Mus musculus domesticus. However, a related Asian subspecies, Mus musculus molossinus, contained different organizational classes of endogenous murine leukemia virus-related sequences. Among inbred strains and feral isolates of M. musculus domesticus, the murine leukemia virus-related sequences were present at different loci. This suggested that most of these sequences were acquired relatively recently during subspeciation and inbreeding.

Eight recombinant DNA clones of endogenous murine leukemia virus (MuLV)-related DNA sequences have been isolated from a lambdaphage genomic library of Balb/c mouse DNA. Each clone contains LTR (long terminal repeat) and gag-related sequences, as well as 5' cellular DNA sequences. The virus-related sequences in each clone show an organization similar to that of integrated proviruses; those clones with the greatest length of MuLV-related sequences also contain pol and env gene-related sequences. One clone appears to contain an intact endogenous provirus. Unique cellular DNA segments from three of these clones were subcloned and used as specific "integration site" hybridization probes. Restriction fragment-length polymorphisms (RFLPs) were observed for these integration sites in the DNA of a number of different inbred mouse strains. One provirus-containing fragment was observed only in Balb/c mice while two others were observed in some but not all of the inbred mouse strains tested. Further restriction enzyme mapping of these three loci in the genomic DNA of Balb/c and C3H/HeJ or C57BL/6 mice indicated that the observed RFLPs were due to the presence of proviral DNA sequences in the Balb/c strain at these three integration sites which were lacking in the other mouse strains. The strain distribution of these three provirus insertions suggests that the BE 1 and 7 proviruses were widely, although not universally, present among the progenitors of modern inbred mouse strains, while the BE 16 provirus may be a recent addition to the genome of Balb/c mice.

We have derived hybridization probes from analogous 100-base-pair segments located within the N-terminal region of gp70 coding sequences which differentiate xenotropic from mink cell focus-forming (MCF)-related murine leukemia virus (MuLV) DNAs. The MCF probe annealed to the integrated proviruses of all six MCF MuLV isolates tested; the xenotropic probe hybridized to the DNAs of all four xenotropic proviral isolates examined. No cross-hybridization was observed, and neither probe reacted with the env segments of amphotropic or ecotropic MuLV DNAs. Southern blot analysis of HindIII- or EcoRI-digested genomic DNAs from a variety of inbred laboratory mice demonstrated the presence of more MCF- than xenotropic MuLV-related segments in every strain tested.

Utilizing a single-stranded [3H]DNA probe highly representative of AKR viral 70S RNA, we have performed association kinetics experiments with cellular DNA in vast excess from 3 high-, 5 low- and 4 non-virus-yielding mouse strains. Our hybridization studies indicate that in the strains so far tested, the complete genome of the AKR-type MLV is present in the DNA of the embryos of both high- and low-virus-yielding mouse strains, while DNA of non-virus strains contains only a part of the genome. Furthermore, at least two populations of virus-specific DNA sequences can be identified (more abundant and less abundant species) according to their rate of association. Low-virus-yielding mouse strains contain a smaller number (1-2 copies) of the less abundant species, and thus a lower number of complete viral genome than do high-virus strains (3-4 copies). Non-virus-yielding strains are lacking these less abundant sequences in their genome. DNA from wild Mus musculus also contained viral sequences, the sample tested showing association kinetics identical to the non-virus-producing strains. Thus there is a good correlation between completeness of the AKR-type MLV genome in cellular DNA and the capacity of the cells to release AKR-type MLV. Mice of a non-virus-yielding strain made partially congenic for the AKR virus-inducing locus Akv-1 contained the complete virus genome, confirming that this locus consists of structural genes of the virus.

Five mouse mammary tumor virus proviruses and their flanking cellular DNA sequences have been cloned from a transplanted C57BL/6 (B6) T-cell lymphoma containing additional copies of mouse mammary tumor virus DNA. Characterization of these proviruses and their flanking DNA indicates that B6 lymphomas contain many newly integrated mouse mammary tumor virus copies synthesized by a mechanism(s) which generates polymorphism or deletions or both.

BXH-2 recombinant inbred mice spontaneously produce a B-tropic murine leukemia virus (MuLV) beginning early in life and have a high incidence of non-T-cell lymphomas. These traits are not characteristic of the progenitor strains (C57BL/6J and C3H/HeJ) or of 11 other BXH recombinant inbred strains. Since B-tropic virus expression may be causally related to the high incidence of lymphoma in this strain, we have analyzed the ecotropic MuLV DNA content of both normal and lymphomatous tissues of BXH-2 mice. Southern analysis and hybridization with an ecotropic MuLV DNA-specific probe showed that DNA of normal BXH-2 tissues contained both parental N-tropic MuLV proviruses but lacked endogenous B-tropic MuLV DNA sequences. In addition, none of 116 F1 hybrid mice derived from male BXH-2 mice spontaneously produced ecotropic MuLV early in life. These results suggest that the B-tropic virus is horizontally transmitted in BXH-2 mice. Southern analysis of DNA from tumor tissues of 12 BXH-2 mice showed that amplification of ecotropic-specific DNA sequences had occurred in lymphomatous tissues of 3 mice and suggested that these tumors were monoclonal. The number of newly acquired proviruses, which appeared to be structurally nondefective and integrated at different sites, varied from one to three copies. Since lymphomatous tissues from only 3 of 12 mice examined carried additional detectable ecotropic proviruses, these results suggest that amplification of ecotropic MuLV DNA sequences is not required for maintenance of transformation in BXH-2 lymphomas.

Characterization of endogenous ecotropic Akv proviruses in DNA of low and high leukemic mouse strains revealed the presence of one to six copies of the Akv genome per haploid genome equivalent integrated in the germ line. Low leukemic strains analyzed so far contained only one complete copy of the Akv proviral DNA. The site of integration varied among strains, although genetically related strains often carried the Akv proviral gene in the same chromosomal site. The different substrains of the AKR mouse displayed the presence of variable numbers (two to six) of Akv genomes. In all substrains one Akv genome was present in an identical chromosomal site; this locus probably comprised the progenitor genome. Closely related substrains had several Akv proviral DNAs integrated in common sites. The accumulation of Akv genomes in the germ line of the AKR/FuRdA strain is likely the result of independent integration events, since backcross studies with the Akv-negative 129 strain showed random segregation of all six proviral loci. The AKR/Cnb strain carried a recombinant provirus in the germ line. This provirus resembled in structure the AKR mink cell focus-forming viruses, which are generated by somatic recombination during leukemogenesis. Therefore, the germ-line amplification of Akv proviral DNAs occurs most likely through infection of embryonic cells by circulating virus.

We have examined the spleen DNA of individual mice of the RFM/Un strain for evidence of re-integration of the endogenous ecotropic provirus in radiation-induced and spontaneous neoplasms. The ecotropic env specific probe detects only a single 19 kb EcoRI or a single 7.0 kb HindIII fragment in all DNA preparations from normal tissues of RFM mice, corresponding to the endogenous provirus. Additional DNA restriction fragments containing the ecotropic virus (eco) specific sequence, corresponding to somatically acquired provirus, are detected in two out of five spleen DNA samples from animals with myeloid leukemia and one of three with thymic lymphoma. In addition somatically acquired eco-specific fragments are also detected in greater than 85% of DNA samples from reticulum cell sarcomas, a late occurring spontaneous hematopoietic neoplasm in this mouse strain. These results are consistent with a 'promoter/enhancer insertion' model of leukemogenesis involving the endogenous ecotropic provirus and are of particular interest since the RFM/Un mouse possesses a locus that restricts exogenous infection of cells by the endogenous virus.