The specific aims of this research program are: 1) to investigate by in vivo experiments the mechanism and regulation of differential gene expression, in particular of that of rRNAs and mRNAs, in human mitochodria; the significance of the complete light strand transcription and its relationship to the heavy strand transcription and to the primer synthesis for heavy strand DNA synthesis; the control of mitochondrial DNA transcription and RNA processing by mitochondrial translation products and by nucleocytoplasmic components; 2) to develop in vitro systems utilizing isolated organelles or crude extracts or purified enzymes for the study of the interrelationship between the nucleocytoplasmic compartment and mitochondria and for the dissection of mtDNA transcription and RNA processing; 3) to investigate the nature of the proteins encoded in the unidentified reading frames of human mtDNA; 4) to investigate the nature of the proteins associated with human mtDNA near the origin or replication; and 5) to isolate the specific cDNAs and nuclear genes for the mitochondrial RNA polymerases(s), some of the mitochondrial ribosomal proteins and the cytoplasmic subunits of cytochrome C oxidase for the purpose of studying the organization of these genes, the mechanism and regulation of their expression and the control that they exert on various phases of mitochondriogenesis. In the in vivo experiments, HeLa cells will be subjected to long-term labeling, pulse-labeling or pulse-chases with RNA precursors under a variety of conditions and the labeling behavior of various mitochondrial RNA species will be analyzed. In the in vitro experiments, RNA synthesis and processing will be investigated in isolated mitochondria, or in sub-mitochondrial fractions in the absence or presence of added mtDNA template, and the parameters, requirements and controlling elements of the reactions will be analyzed. Mitochondrial RNA polymerase(s) and RNA processing enzymes will be isolated and characterized. Antibodies against synthetic peptides will be used to identify the products of the unidentified reading frames of human mtDNA.
