The goal of this project is to identify and analyze the cellular functions which are required specifically for meiosis in the yeast, Saccharomyces cerevisiae. In particular, we are focusing on the analysis of the SPO11 gene of yeast which is required for recombination and proper chromosome segregtion during meiosis. A general system has been developed to isolate meiosis specific genes of yeast for which mutants are available. Using this system, the SPO11+ wild type gene has been isolated following transformation and complementation of a spo11-1 mutant with a total genome clone bank. The function of the cloned gene has been examined during a detailed analysis of the complementation of the spo11-1 mutant by the isolated SPO11+ gene. The structure of the cloned gene has been determined by restriction enzyme analysis and subcloning. A restriction fragment of 2200 bases containing the SPO11+ gene has been isolated and its DNA sequence determined. An open reading frame of 398 amino acids has been identified as the tentative coding sequence of the SPO11 gene product. The candidate coding sequence predics a 45 kilodalton basic protein. We are attempting to directly identify the SPO11 gene product by expression of the genetically engineered yeast gene in E. coli systems. The function of the cloned gene is being further characterized by in vitro mutagenesis of the cloned DNA and by substitution of the chromosomal SPO11+ gene by the in vitro engineered constuctions.
