Although mitochondrial DNA (mtDNA) encodes only 13 proteins, these are vital for survival. MtDNA replication and transcription depend entirely on the coordinated synthesis of nuclear gene products, including mitochondrial DNA polymerase (DNA pol (y), RNA polymerase and a set of essential factors. MtDNA accumulates to high levels in mammalian oocytes and this is even more pronounced in amphibian oocytes, where one cell contains as much mtDNA as 100,000 somatic cells. The African frog, Xenopus laevis, provides a model system in which to study the regulation of replication and transcription of mtDNA. This system offers the opportunity to study factors that control mtDNA replication, since replication occurs at high rates in immature oocytes and is inactive in early embryos. We propose to study the biosynthesis of two important proteins in mtDNA replication. One of these is DNA (y), which is also of interest because it is a target for toxicity of several anticancer and antiviral drugs. The second is the HMG-box transcription factor, mtTFA, that also serves as an abundant DNA binding protein in mitochondria. MtTFA is of special interest since its abundance varies in direct proportion to that of mtDNA in human patients with tissue-specific depletion of mtDNA, which is typically a fatal genetic disease. We propose to determine whether the abundance of mtTFA and DNA pol (y) is proportional to the rate of mtDNA synthesis and to use microinjection techniques to determine the effects on mtDNA replication of manipulating the quantity of either protein in oocytes. We will also perform functional studies to identify important sequences in the promoters for these genes in both oocytes and somatic cells to determine whether there are tissue-specific factors that augment the expression of these genes in oocytes.
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