I. In vitro genetic studies of mitochondrial DNA. A vast amount of descriptive data has been accumulated recently concerning the mitochondrial DNA (mtDNA) genomes of human and mouse cells. The complete DNA sequences, locations of heavy and light strand replication origins and map positions of stable transcripts will be known at the inception of this proposed research. In each genome, an "origin region" containing less than 200 base pairs of the 16 kb genome contains sites of initiation of both RNA synthesis and RNA-primed DNA replication. I propose to use this background information to develop in vitro genetic techniques to elucidate DNA sequences required for initiation of DNA and RNA synthesis. Recombinant DNA methods will be used to construct mutagenized DNA templates that may support synthesis of novel products distinquishable from endogenous mitochondrial nucleic acids. These mutagenized DNAs will be added to extracts of isolated mitochondria to assay their ability to support initiation of RNA or DNA synthesis. II. Studies of mitochondrial nucleic acids during cell differentiation. The dependency of mitochondria upon nuclear genes is a central problem of mitochondrial biogenesis. In many examples of cellular differentiation, mitochondrial nucleic acid metabolism may vary only quantitatively in response to varied needs for oxidative phosphorylation. However, the conversion of presumptive adrenal cortical cells to actively secreting, non-dividing cells involves extensive changes in mitochondrial morphology and a reported increase in mtDNA synthesis concomitant with a cessation of nuclear DNA synthesis. The altered mitochondrial morphology and induction of nuclear-coded, intramitochondrial steroid hydroxylases may involve qualitative changes in mtRNA synthesis. I propose to study mtDNA and mtRNA synthesis during differentiation of mouse Y-1 cells in tissue culture.