Regulation of BRCA1 Function by Physophorylation.
Annual rept. 1 Sep 97-31 Aug 98,
TEXAS UNIV HEALTH SCIENCE CENTER AT SANANTONIO
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The purpose of the studies outlined herein was to map phosphorylation sites on the BRCA1 protein as well as study the effects of these modifications on BRCA1 function. In numerous metabolic labeling studies, it has proven impossible to incorporate sufficient 32-Phosphate into the protein to properly carry out phosphopeptide mapping studies. In order to determine the mechanism of aberrant subcellular localization of BRCA1 in breast cancer, a series of deletion mutants of the protein, all containing the nuclear localization sequences, were prepared as green fluorescent protein fusions, expressed in both normal and transformed human breast cell lines and identified by fluorescence microscopy. The results indicate that the amino terminus of the protein may contain a motif that must be modified before the protein translocates from the cytoplasm to the nucleus. The development of a BRCA1 somatic cell knockout breast epithelial cell line through targeted disruption was begun. Screening of 100 drug resistant colonies generated one homologously targeted clone. Unfortunately, screening of several hundred clones in a second round of targeting failed to detect targeting of the second allele, indicating that BRCA1 expression is required for survival in cell culture, at least in the cell line being targeted.
- Genetic Engineering and Molecular Biology
- Medicine and Medical Research