Isolation of Genomic Targets for the Suspected DNA-Binding Protein BRCA1

摘要

The purpose of this project was to immunopurify genomic DNA fragments bound by BRCAl protein in vivo. Likely isolated DNA fragments were to be tested in a reporter system to determine if they confer BRCAl - dependent expression, and analyzed in gel shift assays and DNase I footprinting experiments to determine if they were bound by BRCAl in vitro. We have made significant gains in achieving the objectives of this project during the past (second) year of funding. Our accomplishments include: 1) the construction of a library of immunopurified DNA fragments cloned into a luciferase reporter vector; 2) identification of at least three independent clones that appear to switch on reporter gene expression when BRCAl is over-expressed; 3) sequence analysis of the aforementioned clones which has revealed that one of them (clone 3-9) contains an unusual repeat structure containing binding sites for multiple known transcription factors; 4) evidence, based upon Southern hybridization, that DNA fragments conferring BRCAl regulation contain related sequences. Further characterization of identified clones and complete screening of our library is expected to yield important new information on the function of the crucial BRCAl protein.