Early lymphoid development in the thymus is dependent on gene rearrangement, apoptosis and clonal expansion of lymphoid precursors. We have recently cloned a novel helicase family member from an early lymphocyte library. Gene products of helicase family members regulate developmental gene expression, as well as genome stability and DNA repair. The helicase is lymphoid specific expressed, with a peak of expression during gene rearrangement of early lymphocytes. The helicase gene is a single copy gene in mouse and human. It is localized on mouse chromosome 19 and human chromosome 10q23-24, which is an area of chromosomal breakpoints in some human leukemias. We characterized the genomic organization of the open reading frame in mice. We are currently attempting to express the helicase protein in a baculovirus system to determine its function in vitro. In order to test its role in lymphoid development in vivo we are generating a """"""""knockout mouse"""""""". We are currently transfecting a targeting construct in embryonal cells. The Ku protein is essential for gene rearrangement as well as general DNA repair of broken double stranded DNA. We have examined the interaction of the two Ku subunits (Ku80 and KU70) with the yeast-two-hybrid system as well as in an in vitro translation system. We determined the minimal functional interaction domain of Ku80 that consists of only 28 amino acids. The region is highly conserved between different species. It shows no homology to other known protein-interaction domains. We are currently expressing Ku mutants in a baculovirus system in order to determine other functional domains necessary for ATP binding or helicase activity. We also have generated stable transfectants of Ku80 mutants in human cell lines to determine the regions necessary for radiation responses and gene rearrangement. AIDS TITLE: N/A
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