The proposed research addresses the behavior of chromosomes during meiosis in S. cerevisiae. Pairing, synapsis and recombination between homologous chromosomes are unique and fundamental aspects of meiosis which distinguish it from a mitotic cell cycle and which make possible the reduction in ploidy essential for sexual reproduction in both lower and higher eukaryotes. The long term goal of this research is a molecular understanding of chromosome pairing and synapsis, and the relationship of recombination to these processes. Several different approaches to this problem will be pursued. (1) Analysis of the RAD50 gene will continue; this gene is required meiotic recombination and chromosome synapsis as well as for recombinational repair of double strand breaks in vegetative cells. Projects include: detailed characterization of interesting non-null alleles, biochemical analysis of wild type and mutant proteins, and immunocytological analysis of wild type and mutant strains. Also, second site revertants of certain rad50 mutations will be isolated in order to identify other genes involved in chromosome pairing. (2) Analysis of the MRE1 gene will continue; this gene is required specifically in meiosis for recombination and chromosome synapsis. Molecular characterization, immunological localization, and detailed characterization of non-null alleles are the immediate priorities. Comparison between MRE1 and RAD50 will also provide insight as to the relationship between meiosis-specific and non-meiosis-specific functions. (3) The detailed genetic nature of a meiotic reciprocal recombination hot spot will be investigated. Certain double strand breaks seen at the hot spot will be mapped and examined for covalent linkage to a protein; preliminary investigations into the possibility of an in vitro assay for breaks will be made. This hot spot will be used to identify additional types of DNA alterations relevant to chromosome pairing and recombination and to characterize meiosis-specific changes in chromosome structure. (4) Purification of synaptonemal complexes will continue. Purified complexes will provide individual polypeptides for immunocytological analysis and """"""""reverse genetics"""""""" and will also be useful substrates for immunological probing with antibodies to proteins identified by """"""""forward genetics"""""""". (5) An effort to develop a biochemical assay for chromosome pairing will be initiated. (6) Mutations which cause a lethal block meiotic prephase will be identified using new genetic assays and methods that we have recently developed for this purpose. Very few such mutations currently exist. These mutations should help to define the pathway of events during this stage of meiosis. (7) Abundantly expressed meiosis-specific genes with interesting phenotypes will be identified by a molecular approach, underway, involving cDNA cloning of meiotic messages, insertion mutagenesis of cloned cDNAs, and generic screening of insertions after transformation into yeast. Genes for major structural proteins, i.e. SC components, should be preferentially recovered; new meiosis-specific structures could also be identified.
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