We wish to continue our studies on nuclear-cytoplasmic interactions in the biogenesis of mitochondria. Three major areas of investigation will be undertaken. 1. The ribosomal protein and ribosome assembly - We will examine the role of the S. cerevisiae motochondrial DNA gene product, varl protein, in the assembly of small (38S) mitochondrial ribosomal subunits. Polyclonal and nonoclonal antibodies to nuclear-encoded 38S subunit proteins will be used to determine if the synthesis and import of these proteins into mitochondria is coordinated with the synthesis of varl and the small (15S) mitochondrial ribosomal RNA. Preliminary evidence indicates that the varl cognate in the yeast Torulopsis glabrata is a product of the nuclear genome. The varl gene will be identified and characterized in this organism and used to attempt transformation of S. cerevisiae varl mutants. This would allow for the eventual systematic molecular analysis of the function of varl in mitochondrial protein synthesis and ribosome assembly. 2. RNA processing - The regulation of mitochondrial gene expression by post-transcriptional processing activities will be investigated for the varl system. Using defined RNA substrates synthesized in vitro, we propose to identify and characterize RNA processing enzymes responsible for the in vivo post-transcriptional generation of varl transcripts. 3. Mitochondrial DNA sequences in the yeast nucleus - Using cloned mtDNA probes, we will determine the extent to which the yeast nuclear genome contains integrated sequences homologous to mtDNA. In particular, we will examine the nuclear representation of mitochondrial DNA exon versus intron sequences. We will characterize the organization of those sequences homologous to mtDNA and determine if they are randomly dispersed or clustered in the nuclear genome and their extent of association with yeast transposable (Ty) elements. These studies bear directly on the evolution of mitochondrial genomes and the origin of unique intervening sequences, and interorganellar genetic flux.
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