A candidate tumor suppressor gene in human breast cancers.

We have isolated a candidate gene (designated Brush-1) located at 13q12-q13, proximal to the retinoblastoma gene (RB1). Brush-1 codes for a 4.7-kilobase mRNA expressed at high levels in normal breast epithelium but drastically reduced in 6 of 13 breast cancer cell lines. RB1 mRNA expression is at normal levels for 5 of these 6 lines suggesting a greater importance of Brush-1 for breast cancer. Four primary breast tumors which showed no loss of heterozygosity in the 13q13-q14 region demonstrated normal levels of mRNA for both Brush-1 and RB1. However, four additional primary tumors which displayed loss of heterozygosity for this region had markedly decreased levels of Brush-1 mRNA while maintaining the normal levels for RB1. This differential loss of Brush-1 mRNA expression for both primary tumors and breast cancer cell lines is the expected pattern for a breast tumor suppressor gene.

Loss of heterozygosity and p53 gene mutations in breast cancer.

Immunopositivity for the p53 tumor suppressor gene product was evaluated in 133 breast cancers and compared to loss of heterozygosity (LOH) at various chromosomal loci. The validity of p53 immunopositivity as an indicator for p53 mutations was verified using two molecular assays of p53 mutations: single stranded conformational polymorphism (32 cases) and/or direct sequencing (14 cases). Immunopositivity was highly specific for mutations, since all of 15 strongly immunopositive tumors (> 10% of the cells are positive) and seven of nine cases with borderline immunopositivity had mutations by molecular analysis but were somewhat lower in sensitivity, p53 mutations being also detected in three of 23 (13%) immunonegative cases. LOH was measured at loci on the following chromosomes (1p,q; 2p; 3p; 7q; 11p,q; 13q; 16q; 17p; 18p,q; and 22q) by Southern blotting, polymerase chain reaction amplification of restriction fragment length polymorphisms, or repetitive cytidine and adenine stretches (CA repeats). There was no association between p53 mutations and one measure of genomic instability, namely, high incidence of overall LOH. In contrast, p53 mutations strongly associated with LOH at two specific loci, 3p24-26 (P < 0.001) and 7q31 (P < 0.05). There was no association between p53 mutations and LOH at 17p (site of the p53 gene), suggesting that breast cancers often have only one defective allele of the p53 gene.

Molecular cloning, sequence analyses, and expression of complementary DNA encoding murine progesterone receptor.

Progesterone receptors exist in two molecular forms commonly designated as "A" and "B" forms, the relative proportion of which can vary among species. In murine tissues, progesterone receptor exists predominantly as the "A" form which, in mammary glands, is also under developmental regulation [Shyamala et al. (1990) Endocrinology 126, 2882-2889]. Therefore, toward resolving the molecular mechanisms responsible for the predominance of the "A" form of progesterone receptor in murine tissues and its developmental regulation, we have isolated, sequenced, and expressed the complementary DNA corresponding to the mouse progesterone receptor. Nucleotide sequence analysis revealed two in-frame ATG codons, such that the largest open reading frame beginning with the first codon could encode a polypeptide with an estimated molecular weight of 99,089, while the shorter open reading frame beginning with the second codon could produce a polypeptide with a calculated molecular weight of 81,829. The murine progesterone receptor had complete identity for the DNA binding domain of human and rabbit progesterone receptors and 99% homology with the chicken progesterone receptor; for the steroid binding domain, it had 96% homology with human and rabbit progesterone receptors and 86% homology with chicken progesterone receptors. Expression of the complete complementary DNA in Chinese hamster ovary cells yielded a protein which bound the synthetic progestin promegestone with an equilibrium dissociation constant of approximately 1 nM, and in Western blot analyses revealed both "A" and "B" forms of immunoreactive receptor.

Characterization of the AdhSL regulatory mutation in Drosophila melanogaster.

We describe the characterization of a previously reported control mutation, AdhSL, in the alcohol dehydrogenase gene of Drosophila melanogaster, which results in decreased production of ADH molecules and subsequently lower ADH activity in adults. We find that the regulatory element modifies ADH mRNA levels and acts cis on both ADH protein and mRNA. It is not promoter specific but is developmentally specific to the adult stage. The AdhSL allele carries a 4.5-kb insert approximately 3 kb 5' to the distal promoter. This new insertion may be responsible for the regulatory phenotype of AdhSL.

Molybdenum hydroxylases in Drosophila. III. Further characterization of the low xanthine dehydrogenase gene.

The biochemical effects of several newly induced low xanthine dehydrogenase (lxd) mutations in Drosophila melanogaster were investigated. When homozygous, all lxd alleles simultaneously interrupt each of the molybdoenzyme activities to approximately the same levels: xanthine dehydrogenase, 25%; aldehyde oxidase, 12%; pyridoxal oxidase, 0%; and sulfite oxidase, 2% as compared to the wild type. In order to evaluate potentially small complementation or dosage effects, mutant stains were made coisogenic for 3R. These enzymes require a molybdenum cofactor, and lxd cofactor levels are also reduced to less than 10% of the wild type. These low levels of molybdoenzyme activities and cofactor activity are maintained throughout development from late larval to adult stages. The lxd alleles exhibit a dosage-dependent effect on molybdoenzyme activities, indicating that these mutants are leaky for wild-type function. In addition, cofactor activity is dependent upon the number of lxd+ genes present. The lxd mutation results in the production of more thermolabile XDH and AO enzyme activities, but this thermolability is not transferred with the cofactor to a reconstituted Neurospora molybdoenzyme. The lxd gene is localized to salivary region 68A4-9, 0.1 map unit distal to the superoxide dismutase (Sod) gene.