Data from a variety of organisms including mammals indicates that genetic recombination not only plays a role in altering the genome by classical genetic exchange during meiosis but recombination can also impact normal and abnormal gene expression. By exploiting the techniques of recombinant DNA and DNA-mediated gene transfer, we will study homologous recombination between closely-linked, or duplicated, chromosomal sequences in cultured mammalian cells. Using appropriate plasmid vectors, cell lines will be derived that contain integrated in the genome a single pair of different mutant forms of a selectable marker such as the Herpes simplex virus thymidine kinas gene, that flank a dominant selectable marker (a bacterial gene conferring drug resistance). Recombinants produced from interactions between the defective gene pairs can be detected by selection. The recombinants can be analyzed by molecular hybridization to infer the types of recombination events taking place. Both reciprocal and nonreciprocal events can be recovered in this system. Different orientations of the recombining gene pair (e.g. as direct vvs. inverted repeats) will be examined to: 1) better substantiate the nonreciprocal nature of the majority class of recombination products observed previously, 2) determine whether nonreciprocal and reciprocal events are associated, and 3) determine the relative contribution of intrachromatid vs. sister chromatid events to the overall rate of intrachromosomal recombination. The plasmid recombination vectors will also be used to search for recombination mutants amongst a collection of DNA repair mutants. To better understand the molecular mechanisms of gene conversion-like events, a role for RNA-intermediates in the gene conversion process will be assessed. So-called """"""""reverse genetic"""""""" approaches using cloned DNA sequences will be employed in an attempt to define specific proteins involved in homologous recombination in mammalian cells.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Mammalian Genetics Study Section (MGN)
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Yale University
Schools of Medicine
New Haven
United States
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