The long-term objectives of this application are l) to fully analyze the structure and function of the mitochondrial termination factor (mTERF) in human cells; 2) to elucidate the functions of the seven mitochondrial DNA (mtDNA)-encoded subunits of the human respiratory chain NADH dehydrogenase; and 3) to understand the rules that govern the complementation and segregation behavior of mtDNA in human cells. Specifically, the present proposal aims at cloning the cDNA(s) and the gene(s) encoding the human mTERF, to investigate in vivo and in vitro the mechanism and control of mTERF function in mtDNA transcription termination and initiation or elongation, and to analyze the regulation of synthesis and metabolic properties of mTERF in relationship to the physiological or pathological conditions of the cell. In addition, it is planned to analyze at the structural, functional and biosynthesis levels artificial and natural mutations affecting the mtDNA-encoded subunits of the respiratory NADH dehydrogenase, and to investigate the pathway of assembly of this enzyme complex in normal and mutant human cell lines. Finally, it is planned to investigate the factors that control the complementation, selection and segregation of mutant mtDNA and wild-type mtDNA coexisting in the same cell, and to determine the quantitative relationship between copy number of mtDNA, on one hand, and mitochondrial gene expression and respiratory capacity, on the other, in human cells in culture. The achievement of the above aims will have significant implications for understanding the regulation of mitochondrial gene expression, for elucidating the role of the mitochondrial genome in controlling the function of a key enzyme in the cell energetic metabolism, as well as for understanding some fundamental features of mammalian mitochondrial genetics. In another context, these studies are expected to provide insights into the pathogenetic mechanisms of mtDNA mutations causing disease in man, into the establishment and transmission of these mutations and in general, into the inheritance and evolution of mtDNA.
