of Work: The substantial advancements in TAR cloning in the Chromosome Stability Group provided for development of a separate, independent Gene Isolation Unit that devotes much of its efforts to refining TAR cloning approaches. With the GIU, we identified yeast genes controlling human DNA stability in yeast artificial chromosomes. Various repair and recombination deficient strains were developed that provide safe repositories of human DNA; the most stable are rad52 mutants. The systems for studying YAC stability are being utilized to understand mechanisms of recombination in human DNA. In a collaboration with Ron Walters, we initiated the development of libraries from the fish Xipophorous, since fish are excellent genetic models for DNA repair and tumor suppressor control in complex organisms. TAR cloning of cDNAs is being refined to examine expressed genes from various cancer tissues using our yeast functional assays. We found TAR cloning of p53 cDNA from cell lines to be an accurate process. TAR cloning is also being developed and utilized for the isolation of human cDNAs from various tumor tissues. These genes, such as p53, are being cloned into systems that allow the functional evaluation of possible mutants and polymorphisms as part of a study with the Taylor laboratory at NIHES. We have established that this approach provides for highly accurate cloning based on the genetic effects of cloned cDNA from normal tissue. With this baseline, we are pursuing additional modifications to improve TAR cloning accuracy and efficiency into yeast plasmids and chromosomes. - Chrosome Mapping, Chrosomes, Yeast Artificial, DNA Repair, Saccharomyces Cerevisiae
