We have recently succeeded in transforming mitochondria in living yeast cells with exogenous DNA using the """"""""biolistic"""""""" (biological ballistic) technique, which is the bombardment of cells with microprojectiles coated with DNA. The proposed research is intended to refine the technique, to develop a set of convenient vectors and recipient strains for transformation of yeast mitochondria, and to exploit these developments to study selected problems in mitochondrial molecular biology and biochemistry. Vectors and recipient strains will be constructed to allow selection of transformants, the maintenance of intramitochondrial autonomous expression vectors, and to introduce modified mitochondrial DNA sequences into homologous sites in the mitochondrial genome. The function and sequence requirements of conserved mitochondrial transcriptional promotors and putative origins of replication will be examined. These data will not only contribute to the design of better mitochondrial transformation systems, but will advance our understanding of the expression and transmission of the mitochondrial genome and provide important in vivo correlates to in vitro data, which has been impossible up to now. A systematic analysis of the mitochondrial-encoded cytochrome b protein will be carried out to understand further, ligand binding and other functional domains of the protein by site-directed mutagenesis. Finally, we propose to transform two nuclear-encoded genes encoding complex III polypeptides into mitochondria and to determine the ability of their protein products to be functionally incorporated into complex III.
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