The long term goal of the proposed research is to define in molecular and cellular terms the processes underlying the recombination and genetic behavior of the yeast mitochondrial genome. Particular emphasis will be placed on the analysis of novel recombination events and the identification and characterization of key components that participate in those processes. The present proposal focuses in three major areas of investigation: 1) a protein involved in group I intron intron mobility and splicing, 2) mobile GC-rich palindromes (GC clusters) and 3) the cell biology of mitochondrial genetics. Studies of mobile introns focus on detailed biochemical and reverse genetics studies of a bifunctional protein encoded by the open reading frame (ORF) of intron 4alpha of the cytochrome oxidase subunit I gene. aI4 alpha is one of two known mobile introns of the yeast mitochondrial genome. Its ORF product is both an endonuclease required for intron mobility and a latent maturase required for RNA splicing. We will characterize the interaction of the purified aI4 alpha encoded endonuclease, I-SceII, with DNA and define further its recognition site. We will use newly developed methods of mitochondrial transformation to define important domains of that protein required for endonuclease and maturase activities. We will test the hypothesis that endonuclease and maturase activities share domains of the protein. We will test a model that activation of the latent maturase activity requires an interaction between the aI4 alpha-encoded protein and a mutant form of a nuclear- encoded mitochondrial leucyl tRNA synthetase. Mitochondrial transformation and transmission genetics will be used to evaluate the donor and recipient sequence requirements for GC cluster mobility and the role of in vivo double strand breaks that occur at the borders of those elements. Mutants affecting the mobility of GC clusters will be sought. And finally, we will use fluorescence microscopy and reagents that we have developed to follow in situ the mixing of mitochondria and mtDNAs in zygotes and in heteroplasmic haploid cells and their segregation to emerging buds. Those experiments are designed to establish the rules governing those processes, which must ultimately determine the observed recombination patterns of mtDNA. Mutant screens will be undertaken aimed at defining nuclear genes that participate in mitochondrial segregation.
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