The breast and ovarian cancer susceptibility gene, BRCA1, has been cloned and shown to encode a zinc-finger protein of unknown function. Mutations in BRCA1 account for at least 80% of families with both breast and ovarian cancer, as well as some non-familial sporadic ovarian cancers. The loss of wild-type BRCA1 in tumours of individuals carrying one nonfunctional BRCA1 allele suggests that BRCA1 encodes a tumour suppressor that may inhibit the proliferation of mammary epithelial cells. To examine the role of BRCA1 in normal tissue growth and differentiation, and to generate a potential model for the cancer susceptibility associated with loss of BRCA1 function, we have created a mouse line carrying a mutation in one Brca1 allele. Analysis of mice homozygous for the mutant allele indicate that Brca1 is critical for normal development, as these mice died in utero between 10 and 13 days of gestation (E10-E13). Abnormalities in Brca1-deficient embryos were most evident in the neural tube, with 40% of the embryos presenting with varying degrees of spina bifida and anencephaly. In addition, the neuroepithelium in Brca1-deficient embryos appeared disorganized, with signs of both rapid proliferation and excessive cell death.

Plasmids have been constructed in which the Escherichia coli alkaline phosphatase promoter and signal sequence have been fused to the staphylococcal nuclease gene to promote the high-level expression and secretion of this gene product in E. coli. We determined that the first amino acid residue after the signal sequence can determine whether this protein was processed and exported to the periplasmic space. Fractionation and protease accessibility studies were used to show that the export-defective, nuclease precursor is internal to the cytoplasmic membrane barrier of the cell. Furthernore, this export defect was suppressed in a strain containing a prUi mutation. These findings are novel in that this region of the polypeptide chain has been implicated in processing but not export and that priA mutations have not been previously known to suppress such defects.While engineering the high-level expression and secretion of the nuclease from Staphylococcus aureus Foggi in Escherichia coli, we noticed that the first amino acid after the signal sequence can determine whether staphylococcal nuclease was processed and exported to the periplasmic space. Furthermore, thiS export defect was suppressed in a strain containing a prlA mutation (4). These findings are novel since mutations adjacent to the processing site generally affect signal peptide cleavage but not export (8,16), and prlA mutations have been shown to suppress defects in the hydrophobic core region of the signal sequence (3, 4) but not defects adjacent to the processing site. Therefore, we characterized this phenomenon in greater detail, and the results are described below.To promote the high-level expression and secretion of staphylococcal nuclease in E. coli, plasmids have been constructed in which this gene lacking its own signal sequence (18) was fused to the E. coli alkaline phosphatase promoter and signal sequence (7). pFOG403 is a pBR322 derivative plasmid containing a precise fusion of these elements (Fig. 1). To maintain an amino acid sequence adjacent to the processing site similar to that found in the alkaline phosphatase precursor, oligonucleotide-directed mutagenesis was performed on pFOG403 to insert the triplet CGG encoding an arginine residue between the alkaline phosphatase signal sequence and the staphylococcal nuclease gene (Fig. 1). This procedure resulted in the creation of pFOG406. The details of these plasmid constructions will be described elsewhere (D. Shortle, manuscript in preparation).Strain MC4100 (F-AlacUJ69 araD136 relA rpsL thi) (10) or its isogenic derivative SE6004 containing the prlA4 mutation t3) was used as a host for pFOG403, pFOG406, or pBR322. Overnight cultures grown in L broth were diluted 50-fold into MOPS (morpholinopropanesulfonic acid) medium (14) containing 1.0% glucose, 0.15% vitamin-free casein hydrolysate, 0.0001% thiamine, and 0.1 mM KH2PO4 and grown at 37°C with shaking for 7 h to allow induction and accumulation of the plasmid-encoded staphylococcal nuclease. For radiolabeling protein, strains were grown for 4 ...