No abstract

By radioimmunoassay (RIA) mammary tumour virus (MTV) antigens were detected in individual milk samples of C3Hf mice, (female BALB/c X male C3Hf)F1 mice and (female C3Hf X male BALB/c)F1 mice; milk samples of BALB/c mice were negative. In the segregating backcross I population, female BALB/c X male (female BALB/c X male C3Hf) viral antigens were found in the milk of 93 out of 169 mice (55%). In the Bc II population (daughters of Bc I mothers and BALB/c fathers) two groups were distinguished. In the first group, derived from positive Bc I mothers, 55 out of 110 mice (50%) had detectable levels of viral antigens in the milk. In the second group, progeny of negative Bc I mothers, 1 mouse out of 47 was positive. These data are consistent with the assumption that one dominant gene is responsible for the presence of viral antigens in the milk of C3Hf mice. This gene (Mtv-1) seems to be linked with the albino locus situated on chromosome 7; the recombination percentage was about 29. In the first experiment with Bc I mice a significant difference was found between the tumour ages of the mice with virus-positive milk and of the mice with virus-negative milk: all mice (18) with viral antigens in the milk developed mammary tumours at an age ranging from 7 to 18 months, whereas in only 7 out of 16 mice with virus-negative milk were mammary tumours found before the age of 21 months. Viral antigens were detectable (by RIA) in the tumours of mice of both subgroups; however, the amounts (mU/mg tumour) were significantly lower in the tumours derived from mice with virus-negative milk. Although MTV-L of C3Hf mothers could be transmitted to BALB/c mice by foster-nursing, viral antigens could not be detected in milk samples collected prior to the third lactation period; thus an influence on the data of extrachromosomally transmitted MTV-L is unlikely.

By immunodiffusion assay (ID-test) milk samples of mice of several strains and of F1-hybrids of the GR strain were tested for the presence of mammary tumour virus (MTV) antigens. The results clearly demonstrated that the presence of viral antigens in the milk of the first lactation period is restricted to mice harbouring endogenous MTV-GR. Viral antigens were detectable in about 50% of the milk samples collected during the first (occasionally the second) lactation periods of mice of the segregating backcross I (Bc I) populations: DBAfX(DBAfXGR), AKRX(AKRXGR), BALB/cX(BALB/cXGR) and C57BLX(C57BLXGR), indicating that one dominant gene is responsible for the presence of viral antigens in the milk of GR mice. The proposed gene symbol is Mtv-2. Milk samples from female mice of three different Bc II populations were tested for the occurrence of viral antigens. In the first Bc II: [BALB/cX(BALB/cXGR)]XBALB/c 33 out of 51 mice, descending from ID-positive mothers, had ID-positive milk and only one out of 71 mice, which were the progeny of ID-NEGATIVE Bc I mothers, was ID-positive. These results may be influenced by the MTV transmitted extrachromosomally via the milk of the mother. The two other Bx II populations were derived from Bc I fathers, either [BALB/cX(BALB/cXGR)] or [(BALB/cXGR)XBALB/c] f and BALB/c females. The results obtained with these Bc II populations suggested that 6 Bc I fathers were heterozygous for Mtv-2. Since the segregation ratio (60:29) in the Bc II population (progeny of these 6 Bc I male) deviates significantly from the expected 1:1 ratio, one may assume extrachromosomal transmission of MTV via the seminal fluid of the father to the progeny. A close correlation was found between the presence of MTV antigens in the milk and the occurrence of both early mammary tumours after hormone treatment and spontaneous mammary tumours before the age of 13 months. These results suggest that the early appearance of mammary tumours in the GR strain and the early expression of MTV antigens in the milk appear to be controlled by the same genetic factors.

The distribution of the normal differentiation antigen Thy 1 and the mammary tumor virus (MTV)-induced antigens or antigen complexes MLm and MLr were studied in mouse mammary gland cells, mammary tumor cells, and other cell types, by use of ascites leukemia cells of the GR mouse strain as target cells in the cytotoxicity test. The Thy 1.2 antigen was detected by an AKR antiserum to C3Hf thymocytes. MLm was shown by a homologous C57BL antiserum to GRSL2 leukemia (absorbed in vivo in GR mice); MLr was detected by a rabbit heterologous antiserum (absorbed in vivo in C57BL or GR mice and in vitro with BALB/c milk) prepared against Tween 80- and ether-treated purified B particles. Sera from Sprague-Dawley rats bearing murine leukemia virus (MuLV)-producing syngeneic tumors were not cytotoxic or only slightly cytotoxic for GR leukemias transplanted in vivo, which indicated that MuLV-induced antigens were absent or present in very low quantity in such leukemias. The MLr and MLn antigens or antigen complexes were possibly identical to the mammary leukemia (ML) antigen, since they could be detected not only on GR but also on DBA/2 leukemia cells and since their distribution was exactly the same as that of MTV. Both the MLr and MLm antigens were present in purified B particles, and antigenic activity were present in purified B particles, and antigenic activity was enhanced by destruction of the purified virus particles. The antigens were about eightfold enriched in a preparation of B-particle envelopes, as shown by quantitative cytotoxicity absorption (CYTA) tests. Purified nucleoid fractions of B particles were only lightly positive for the antigen, probably due to envelope contamination. One dominant gene was responsible for the expression of MLr, as shown by CYTA tests with mammary glands of individual animals of segregating crosses between the GR strain with high mammary cancer incidence and strains with low incidence. This gene was closely linked with or was possibly identical to 1) the gene for cytoplasmic MTV gs antigen expression as seen by fixed cell immunofluorescence, and 2) the gene causing mammary tumors in the GR mouse strain.

In the mouse strain GR, the Mtv-2 gene controls the expression of large amounts of mammary tumor virus (MTV) antigens in the milk at first lactation. It also controls the early appearance of mammary tumors. We have investigated the number of MTV proviral sequences associated with this gene by nucleic acid hybridization between MTV [3H]cDNA and DNA from GR, BiO, and GR-Mtv-2-mice. BIO and GRMtv-2-mice lack Mtv-2 gene expression. The molecular hybridizations revealed that the DNA of GR mice contains 12 copies of MTV proviral sequences, whereas only 4 copies are present in the DNA of BlO and GR-Mtv-2-mice. We therefore conclude that the Mtv-2 gene in the GR mouse strain is associated with eight additional MTV proviral sequences. The four Mtv proviral sequences in the GR-Mtv-2-DNA might represent another MN gene in the GR mouse. Different amounts of MTV RNA are detected in mammary glands at first lactation of BiO and GR-Mtv-2-mice, even though both contain four copies of MTV proviral sequences. This indicates a difference between these two mouse strains either in the regulation of expression of these MTV proviral sequences or in the location of these sequences in the murine genome. Mtv-1 gene is linked to the presence of very low amounts of MTV antigens in the milk of the first lactation and to the appearance of mammary tumors in this strain relatively late in life (average, 13 months). In the GR mouse strain, another dominant gene, Mtv-2, controls the expression of large amounts of MTV antigens in the milk of the first lactation and the early appearance of mammary tumors (all before age 13 months), (3). It furthermore has been shown (4) that the numbers of MTV proviral copies in the genomes of the GR and C3Hf mouse strains are different. The DNA of C3Hf mice contained 3 or 4 MTV proviral copies per haploid genome, whereas the GR genome contained approximately 16 copies.The present study focuses on the relationship between the number of MTV proviral sequences in the cellular genome of GR mice and the Mtv-2 gene. Therefore a comparison was made between the number of MTV proviral copies in the DNA of somatic (liver) cells in mouse strains GR, C57BL, and a GR mouse lacking the Mtv-2 gene (GR-Mtv-2-). The GR-Mtv-2-strain was developed by introducing genetic material of the C57BL mouse into the GR mouse strain as described by v. Nie et al. (6). We report here that the Mtv-2 gene is associated with approximately eight MTV proviral copies per haploid genome, and that the GR mouse strain carries four additional MTV proviral sequences that are probably similar to another Mtv gene. The effect of different numbers of MTV proviral sequences on the expression of MTV RNA is discussed. MATERIALS AND METHODSViruses. The propagation and purification of the MTV virus of the Mm 5mt/cl cell line have been described (7). Rauscher leukemia virus was obtained from plasma of BALB/c mice infected with Rauscher leukemia virus. The virus was purified by equilibrium centrifugation in sucrose. Mice: GRS/A (GR) and C57BL/10 JA (B10) mi...