We propose to investigate two related aspects of mammalian mitochondrial molecular biology. First, we will establish the extent, origin, and potential function of intracellular mitochondrial DNA (mtDNA) sequence heterogeneity, with particular emphasis on a sequence involved in controlling gene expression which we have shown is heterogeneous in tissue. We will compare the magnitude and distribution of mtDNA sequence polymorphs as a function of adult and fetal tissue in the dairy cow. Potential nuclear effects will be assessed by a similar analysis as a function of bovine lineage, breed, and artiodactyl species. The clonal origin of mtDNA heterogeneity will be tested by studying tissue culture cells cloned from bovine, human, and mouse lines. Finally, the relative template activity of individual polymorphs will be studied in vitro using mtRNA polymerase. Second, to better understand mtDNA sequences controlling initiation of transcription and replication, we will analyze the in vitro functon of artiodactyl mtRNA polymerases on homologous and non-homologous mtDNA templates. We will finish determining the D-loop region sequence of ten artiodactyls, accurately locate; the termini of D-loop region RNA and DNA, partially purify several artiodactyl mtRNA polymerases and size in vitro transcription products to correlate with in vivo RNA initiation sites. This study will define what sequence features regulate mtRNA polymerase initiation. It will also afford a unique analysis of the co-evolution of a DNA sequence and its interactive protein over a relatively short evolutionary span, but one in which significant DNA sequence change has occurred.
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