The mechanism of the self-splicing class II mitochondrial intron, oxi3 I5g, of the yeast, Saccharomyces cerevisiae will be studied using genetic, biochemical and recombinant DNA methods. Mitochondrial splicing defective mutants of that intron will be isolated. Their in vivo phenotypes and sequence alterations will be determined and their activity under in vitro self-splicing conditions determined. Pseudorevertants of them will be isolated and analyzed similarly. Further alterations of the intron sequence will be made using in vitro mutagenesis methods both to confirm and extend insights to functionally important sequences gained from studies of the in vivo mutants and to provide a more complete view of those sequences. A lacZ fusion gene, in which the expression of B-galactosidase activity in E. coli cells will depend on intron splicing, will be constructed and investigated as a means of enhancing the efficiency of screening in vitro mutations of the intron. Nuclear genes involved in the in vivo splicing of oxi3 I5g will be identified using nuclear suppresors of mitochondrial splicing defective mutants and pet- mutants. Major emphasis will be given to the development of an in vitro assay for mitochondrial proteins which participate in the splicing of class I and class II introns; representative introns of each class will be studied. Ribonucleoprotein particles (RNPs) from mitochondria will be characterized and tested for their ability to promote the splicing of endogenous pre-mRNAs. RNA-free protein samples, either isolated directly from mitochondria or obtained upon nuclease treatment of isolated RNPs, will be fractionated and tested for their ability to promote the splicing of exogenous model pre-mRNAs. Extracts from both mitochondrial and nuclear mutants will be used to identify specific proteins involved in splicing.
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