The research proposed in this application is aimed at understanding genetic recombination in a simple eucaryotic organism, baker's yeast. Yeast provides a powerful system for the analysis of recombination because it provides a superb genetic system in addition to molecular biology and biochemistry. The focus is on meiotic recombination, with particular attention given to the question of how exchange begins. There are three major areas of research proposed. First, we will continue studies of a DNA sequence (hotspot) near the HIS2 gene which appears to be involved in the initiation of meiotic exchange. We have cloned the HIS2 gene and surrounding region and have located the hotspot to about one kb. We will continue that localization by the analysis of the effect of both insertions and deletions (made in vitro) on unselected gene conversion in homologous chromosomes. While that will remain an important part of our approach, we also will examine alternative assay systems (e.g., the production of prototrophs from heteroallelic pairs) to see if they reflect what happens in chromosomes. Both the entire region and the hotspot fragment alone will be moved to other regions in the genome to verify what sequences are important and also to determine whether chromosomal context plays a role. Second, we plan to use a selective system to isolate mutations in genes required for the initiation of meiotic recombination. Normally, Rec-mutants produce inviable meiotic products; this approach takes advantage of a meiotic mutant which bypasses meiosis I (spo13) and a known Rec-mutant (rad52) to create a selection. We have already shown that the addition of an early block will allow a rad52 spo13 strain to produce viable spores. After isolation, these mutants will be studied to help define the pathway of meiotic recombination; interesting genes will be cloned. In addition, we are continuing our work using two different approaches to study meiotic functions (rad50 revertants and a yeast gene that complements lambda exo-). Third, the hotspot region will be examined to determine whether functions bind to or interact with it in vivo. We will examine UV light (""""""""photofootprinting"""""""") and DMS in meiotic cells in the presence and absence of meiotic Rec- mutations. In addition, nuclei will be examined for sensitivity to DNase I. Finally, nuclear extracts will be used to examine binding by electrophoretic mobility assays. As controls for all these experiments, mitotic cells will also be examined.
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