ABSTRACT
The organization and expression of endogenous mouse mammary tumor virus (MMTV) proviruses in normal and neoplastic C3Hf/Ki tissues were examined. MMTV-containing EcoRI, HindIII, BamHI and PstI restriction fragments of C3Hf/Ki DNA were identical to those of C3H/StWi DNA. The full-length endogenous MMTV Units Ia (Mtv-7), II (Mtv-8), III (Mtv-9) and IV (Mtv-10), in addition to the subgenomic endogenous MMTV Units I (Mtv-6) and IX (Mtv-14), were germinally transmitted in C3Hf/Ki DNA. The previously uncharacterized Mtv-7 was contained in EcoRI fragments of 16.7 and 11.7 kbp. The endogenous MMTV Unit V (Mtv-1), which is responsible for virus production and mammary tumorigenesis in C3Hf/He mice, was absent from C3Hf/Ki DNA. The 9.0 kb gag-pol, the 3.8 kb env and the 1.7 kb LTR MMTV RNA transcripts were present in C3Hf/Ki mammary glands. MMTV proviruses, in addition to the endogenous C3Hf/Ki MMTV complement, were not detected in C3Hf/Ki mammary tumor DNA. The DNA organization and RNA expression of the putative mammary proto-oncogene regions int-1 and int-2 were also examined in C3Hf/Ki mammary tumors. The int-1 and int-2 regions did not appear rearranged, amplified, or expressed in C3Hf/Ki mammary tumors. These studies indicate that MMTV proviral activation of the int proto-oncogenes is not necessary for C3Hf/Ki mammary tumorigenesis.
Subject(s)
Mammary Neoplasms, Experimental/microbiology , Mammary Tumor Virus, Mouse/genetics , Oncogenes , Animals , Cell Transformation, Viral , DNA, Neoplasm/genetics , DNA, Viral/genetics , Female , Gene Amplification , Gene Expression Regulation , Genes, Viral , Mammary Neoplasms, Experimental/genetics , Mice , Mice, Inbred C3H/genetics , Mice, Inbred C3H/microbiology , Translocation, GeneticABSTRACT
The organization and expression of germinally transmitted mouse mammary tumor virus (MMTV) proviruses in C3Hf/HeSed mouse tissues were examined. Digestion with the restriction enzymes EcoRI, BamHI, and HindIII and hybridization with cloned probes specific for the long terminal repeat and the 5' and 3' regions of the MMTV genome revealed three full-length (units Ib, II, and V) and two subgenomic (units I and IX) MMTV proviruses in C3Hf/HeSed mouse germ line DNA. The EcoRI fragments (15.0 and 5.7 kilobase pairs [kbp]) that contained unit Ib were previously described as separate, subgenomic MMTV proviruses. The methylated state of each full-length MMTV provirus was examined in DNA from C3Hf/HeSed mouse livers, spleens, mammary glands, and mammary tumors by digestion with EcoRI or BamHI in combination with the methyl-sensitive restriction enzymes HhaI or HpaII. Unit Ib contained HhaI- and HpaII-sensitive sites in spleen, mammary gland, and mammary tumor DNA but was completely methylated in liver DNA. Units II and V contained HhaI- and HpaII-sensitive sites in mammary gland and mammary tumor DNA, but the sites were extensively methylated in spleen and liver DNA. The HhaI-sensitive sites were mapped to the 5' end of the 5' and 3' long terminal repeats of each full-length MMTV provirus. C3Hf/HeSed mouse tissue RNA was examined for MMTV transcripts. Mammary glands contained MMTV RNA species of 9.0, 3.8, and 1.7 kb. Mammary tumors contained high levels of the 9.0- and 3.8-kb transcripts but lacked the 1.7-kb species. A very low level of the 3.8-kb MMTV transcript was present in spleens. Livers lacked detectable MMTV RNA. These results implicate mammary tissue as the site of unit V activation in the formation of MMTV virions.
Subject(s)
Genes, Viral , Liver/microbiology , Mammary Glands, Animal/microbiology , Mammary Neoplasms, Experimental/microbiology , Mammary Tumor Virus, Mouse/genetics , Spleen/microbiology , Animals , Base Sequence , DNA Restriction Enzymes , Female , Methylation , Mice , Mice, Inbred Strains , Nucleic Acid Hybridization , RNA, Viral/geneticsABSTRACT
Murine mammary tumor virus (MMTV) RNA expression, DNA organization and DNA demethylation were examined in BALB/c B-lymphoid cell lines produced by transformation with the Abelson murine leukemia virus (AbMuLV). The MMTV DNA sequences in AbMuLV B cell lines, based on restriction mapping with EcoRI, PstI, BglII, BamHI and SacI and molecular hybridization with cloned probes of the MMTV LTR, gag-pol or env gene regions, were identical to the germinally-transmitted MMTV DNA complement of BALB/c mice. Several AbMuLV B cell lines expressed MMTV poly(A+)-RNA at detectable levels. MMTV poly(A+)-RNA for the env gene, 3.8 kb, and the long terminally redundant (LTR) region, 1.7 kb, were detected in some AbMuLV B cell lines. MMTV DNA sequences in the AbMuLV B cell lines were at least partially sensitive to digestion by the methylation-sensitive restriction endonucleases HhaI and HpaII. HhaI-sensitive sites were present in Units I, II and III of the germinally-transmitted MMTV DNA and were localized specifically near the 5' end of the 5' and 3' LTRs of both Units II and III. HpaII-sensitive sites were localized near the 3' end of the 3' LTRs of Units II and III, and at a cellular site 2.1 kbp 5' to the 5' LTR. These observations demonstrate that the germ line MMTV DNA sequences of BALB/c mice are expressed in cells of B lymphocyte origin, and suggest a correlation between MMTV RNA expression and selective demethylation in the LTR regions of germinally-transmitted MMTV DNA sequences.
