The animal oocyte is a highly differentiated cell in which a number of cellular organelles are accumulated for distribution to the cells of the developing embryo. The overproduction of mitochondria is particularly interesting, since this organelle contains a distinct genome. Thus, mitochondrial biogenesis requires the coordinated activity of the nuclear and mitochondrial genomes. The Xenopus (African frog) oocyte, a well-studied model for oogenesis, contains as much mitochondrial DNA (mtDNA) as 100,000 somatic cells. Transcription and replication of Xenopus mtDNA are accomplished by enzymes produced by nuclear genes, including the mitochondrial RNA and DNA polymerases. Assay methods will be developed to permit study of the transcription and replication of mtDNA using in vitro reactions. The nucleotide sequences required for control of both transcription and replication of Xenopus mtDNA will be determined. The RNA and DNA polymerases will be purified, as will a number of accessory proteins required for efficient nucleic acid synthesis, such as type I topoisomerase and mtDNA binding proteins. Antibodies raised against these proteins will be used to confirm their intramitochondrial localization in situ using immunofluorescence microscopy and to follow the rate of accumulation of the proteins during development of the oocyte. The antisera will also be used as probes to identify cDNA clones for the respective mRNAs in an expression library. These cDNA clones will be used to study the transcription of nuclear genes encoding these important regulatory proteins.
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