Summary A total of 80 primary human breast carcinoma DNAs were analysed for loss of heterozygosity (LOH) on the long arm of chromosome 6, using microsatellite markers whose location has been defined physically and by linkage analysis. Loss of heterozygosity was observed in 38 of 80 (48%) tumours that were informative for at least one locus. The analysis revealed partial or interstitial deletions of chromosome 6q. Detailed mapping of chromosome 6q in these tumour DNAs identified two and perhaps three commonly deleted regions. One of these is located between markers D6S251 and D6S252 (6q14 -q16.2), another between D6S268 and D6S261 (6q 16.3 -q23) and a third between D6S287 and D6S270 (6q22.3 -q23. 1).Keywords: breast cancer; deletion; chromsome 6q; tumour-suppressor gene Cytogenetic (Dutrillaux et al., 1990;Lu et al., 1993;Thompson et al., 1993; Trent et al., 1985Trent et al., , 1993 and molecular analyses (reviewed in Callahan et al., 1993) of primary human breast carcinomas have documented frequently occurring genetic alterations that take place during the evolution of tumour development. It is thought that these mutations either inactivate normal growth controls or give the tumour some selective advantage. At the molecular level, loss of heterozygosity (LOH) is the most frequent type of genetic alteration in primary human breast tumours . LOH at specific chromosomal loci has been taken as evidence for the presence of putative tumour-suppressor genes within the affected regions (Knudson, 1989). In sporadic primary human breast carcinomas LOH has been detected on at least 12 different chromosome arms . However only in the case of chromosome l7pl3 has the target gene for LOH (TP53) been identified (Hollstein et al., 1991). Generally one allele of the target gene is lost and the remaining allele contains a nonsense or missense mutation. The involvement of chromosome 6q in breast carcinomas has been noted in cytogenetic analysis of primary tumours (Dutrillaux et al., 1990;Lu et al., 1993;Thompson et al., 1993; Trent et al., 1985Trent et al., , 1993. Similarly, molecular analysis of primary breast tumour DNAs has shown that chromosome 6q is frequently affected by LOH (Devilee et al., 1991). In this report, we describe studies aimed at defining the location of putative tumour-suppressor gene(s) on Table I. The PCR products were diluted with loading buffer (95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol), heat denatured and rapidly cooled. Samples were run in pairs (tumour and lymphocyte PCR product from the same patient) on a denaturing gel (7% acrylamide, 32% formamide, 6 M urea, 1 x TBE) at a constant 30-35 W. After electrophoresis the gel was transferred to 3MM Whatman paper and autoradiography performed with Kodak X-Omat AR film at -70'C. When the signal of an allele in tumour DNA was less than 50% of intensity observed in matching normal DNA from a heterozygous patient, LOH was considered to have occurred (Bieche et al., 1993). ResultsPreliminary results obtained at 6 loci (D6S254,...

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Summary. The content of the two pyridine-nucleotide coenzymes, namely nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), has been determined in bull and rabbit spermatozoa, using the enzymic-fluorimetric procedure of Bassham, Birt, Hems & Loening (1959). It has been found necessary to wash spermatozoa thoroughly before extraction, so as to remove all traces of seminal plasma enzymes which otherwise would have degraded the coenzymes in the course of the analytical procedure. In epididymal spermatozoa a markedly higher content of pyridine-nucleotide coenzymes has been found, compared to that in ejaculated spermatozoa. Incubation experiments distinctly indicated a lability of the coenzymes in both types of spermatozoa. It has also been shown that a very short (1 min) incubation of epididymal spermatozoa with seminal plasma causes a definite decrease in the coenzyme content.

The frequency with which int-I and int-2 are rearranged in mouse mammary tumors by mouse mammary tumor virus (MMTV)-induced insertional mutagenesis is a consequence of the host genetic background. In 75 % of C3H mammary tumors, int-i is rearranged by MMTV insertion, whereas only 30% of BALB/cfC3H tumors contain a virus-induced rearrangement of int-1. This difference is significant (P < 0.005) and could not be accounted for by the potentially additive effect of the genetically transmitted Mtv-1-encoded virus in C3H mice. Similarly, MMTV-induced rearrangement of the int-2 gene in mammary tumors of the Rlll mouse strain (59%) occurred at a significantly (P < 0.025) higher frequency than in BALB/cfRlll (25%) mammary tumors. Moreover, in BALB/cfRlll mammary tumors, there is evidence that rearrangement of int-i and int-2 does not occur independently (P < 0.025). These results suggest that the long history of inbreeding for high tumor incidence of C3H and Rlll mouse strains has selected for the fixation of host mutations which either complement the action of the particular int gene or affect the sensitivity of specific subpopulations of mammary epithelium to infection by particular strains of MMTV.

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The C3H and RIII mammary tumor viruses (MTV) carried by BALB/cfC3H and BALB/cfRIII breeding females have been quantified and compared in milk samples, after partial purification with a sucrose density gradient. The samples were collected at identical times during the first 3 lactation periods from individual mice (6 per strain), standardized for age at delivery and size of litter. Milk samples from 6 MTV negative BALB/c controls have also been analyzed. Data for comparison are expressed in optical density units (ODU) and refer to the protein content of the whole milk using MTV-negative Balb/c milk as blank. The results have shown 1) an increase of MTV released through milk in each MTV-carrying female from the first (average ODU, 0.542) to the second (1,351) and third (2.105) lactation, 2) individual variations, and 3) a significant difference in release between C3H and RIII MTV, the latter being more than double (average ODU, 1.801) in respect to the former (0.847). The apparent discrepancy between these results and the bioactivity in BALB/c mice of C3H and RIII MTV, significantly lower for the latter, is discussed.

The bioactivities of C3H and RIII mammary tumor virus (MuMTV) in virgin female BALB/c mice differed. The average number of mammary hyperplastic alveolar nodules per mouse after noduligenic tests was 20.1 in BALB/cfC3H and 10.7 in BALB/cfRIII females. Spontaneous mammary tumor incidence after 20 months of observation was 47.5% in BALB/cfC3H and 14.6% in BALB/cfRIII females (P less than 0.01). The frequency of lung metastases in mammary tumor-bearing mice was 63.1% in BALB/cfC3H and 16.6% in BALB/cfRIII females (P less than 0.05), although the clinical duration of mammary tumors was the same. These data demonstrated a lower bioactivity of RIII MuMTV when compared to C3H MuMTV in BALB/c mice and suggested that the causative virus may control all the steps of mouse mammary tumor development, including metastasis.

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