The proposed research represents an integrated genetic and biochemical approach to mtDNA recombination in Saccharomyces cervisiae. Particular emphasis will be given to the analysis of the molecular features of non-reciprocal recombination at the 21S rRNA locus. Evidence will be sought for the suggestion from preliminary data that an open reading frame within a 1.1 kb intron of one of the 21S rRNA alleles encodes a trans-acting factor required for non-reciprocal recombination. We posit that this factor, perhaps with other proteins that may be associated with it, introduces a site specific double-strand cut in the intron-less 21S allele and that this double-strand cut is an intermediate in the non-reciprocal exchange. This proposition will be examined by the analysis of mutants of both alleles of the 21S gene, in vitro biochemical studies to reproduce aspects of the in vivo phenomenon, immunological approaches to identify and characterize the putative ORF product, and by comparisons of the properties of non-reciprocal exchange at the 21S locus in S. cerevisiae with reciprocal recombination occurring at other loci in the mitochondrial genome. Additional studies are proposed to take advantage of methodology we have developed to introduce productively into S. cerevisiae cells, the mitochondrial genomes of various industrial species that harbor useful polymorphisms and genetic properties. These studies include measurements of the gradient of flanking sequence co-conversion associated with intron transfer and how sequence variations in the 21S rRNA gene in strains which lack the intron, but yield atypical outputs in heteropolar crosses, affect recombinational events there. Finally, studies are proposed to develop methods to obtain and characterize nuclear mutants with defects in general mtDNA recombination and to initiate studies on the molecular mechanisms of var1 recombination--the only other locus on the yeast mitochondrial genome that is known to undergo non-reciprocal exchange.
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