Mitochondria are one of two organelles in eukaryotic cells that contain DNA. The entire sequence of mtDNA as determined for several eukaryotes shows a remarkable conservation of organization. The genome encodes its own tRNAs and rRNAs as well as the genes for 13 proteins. Higher eukaryotes are obligate aerobes, although some mutations in mtDNA do permit survival and have recently been associated with hereditary muscular disorders. The overall schemes for replication and transcription of mtDNA have been defined in recent years. These processes require the products of large numbers of nuclear genes for such proteins as DNA polymerase gamma, mtRNA polymerase and a host of accessory factors. Most of these proteins remain poorly characterized. We particularly lack details of the way in which the cell coordinates the activities of these proteins to result in balanced transcription and replication of mtDNA. In order to study this control, we have been interested in studying a system in which mitochondrial nucleic acid synthesis is under developmental control. Therefore, we have begun studies of the control of mtDNA replication and transcription in the oocytes of the African frog Xenopus laevis. During oogenesis, nuclear DNA replication is suspended while the cell accumulates as much mtDNA as 50,000 somatic cells. We have substantially purified the DNA polymerase gamma and mtRNA polymerase from oocytes. We propose to continue studies of these enzymes and to extend studies of accessory proteins. We have partially purified a mitochondrial transcription factor and determined the sequences required for mitochondrial promoter activity in vitro. Experiments are proposed to define the mechanism of transcription initiated by the mtRNA polymerase and its dissociable factor in more detail. We have also purified two single stranded DNA binding proteins and propose experiments to study the effects of these proteins and of other accessory proteins, such as DNA helicase, on the activity of DNA polymerase gamma on defined templates. Other experiments are proposed to study the mechanism of mtDNA replication in crude mitochondrial lysates. It has been suggested that replication of mtDNA is primed by transcription by the mtRNA polymerase initiated at light strand promoters. The broadly based approach described here, involving studies of both the transcription and replication machinery, should help to further define this model. Additional experiments are proposed to study the synthesis of several of the proteins required for transcription and replication. Polyclonal antisera specific for mitochondrial proteins will be used to determine the cellular content of these proteins during oocyte development and early embryogenesis. This approach may be able to determine whether the lack of replication and transcription of mtDNA during early embryogenesis is due to a relative lack of the polymerases. Efforts will also be made to clone several of the genes for proteins involved in mitochondrial nuclei acid synthesis. This will not only provide the primary sequence of these proteins, but will provide hybridization probes to study the developmental control of expression of the genes for the mitochondrial proteins.
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