The overall goal of this project is to understand the mechanism of replication and expression of vertebrate mitochondrial DNA (mtDNA) as it relates to the regulation of organelle biogenesis and the interplay between organelles and nuclear genomes that must occur to ensure a correct number of functional mitochondria in cells.
The specific aims of this proposal are directed toward an understanding of how mtDNA is replicated and expressed at the level of protein transactions with the genome and protein/smRNP-mediated RNA processing events, and the nature and regulation of the nuclear genes for these activities. This requires the dissection of the components required for these fundamental biological events in order to identify relevant control regions and the enzymes and elements that interact with them. Mitochondria are central to the production of cellular energy and the mitochondrial genome is known to be of altered topology in malignant cells. With regard to mtDNA replication, the two origin regions will be featured. The commitment to a genomic replication event occurs by transcriptional priming in the displacement-loop (D-loop) of mtDNA with second-strand synthesis initiating later in time and at considerable distance from the D-loop. Each origin presents a different opportunity with regard to basic enzymology and specific questions, one example being the phenomenon of replication termination exclusive to the D-loop. Transcription-related investigations will exploit our current in vitro systems and move from regulatory sequence identification to protein and ribonucleoprotein purification and characterization; this is the case for both initiation and termination. A major aim will be the understanding of the nuclear genes encoding components of the recently discovered mitochondrial ribonucleoproteins. This involves the genetic elements for both the RNA and protein components of these activities; our first experiments will focus on the RNA component(s) because we have the required data to pursue it.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Physiological Chemistry Study Section (PC)
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Stanford University
Schools of Medicine
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Shadel, G S; Buckenmeyer, G A; Clayton, D A et al. (2000) Mutational analysis of the RNA component of Saccharomyces cerevisiae RNase MRP reveals distinct nuclear phenotypes. Gene 245:175-84
Wang, J; Wilhelmsson, H; Graff, C et al. (1999) Dilated cardiomyopathy and atrioventricular conduction blocks induced by heart-specific inactivation of mitochondrial DNA gene expression. Nat Genet 21:133-7
Wiesner, R J; Hornung, T V; Garman, J D et al. (1999) Stimulation of mitochondrial gene expression and proliferation of mitochondria following impairment of cellular energy transfer by inhibition of the phosphocreatine circuit in rat hearts. J Bioenerg Biomembr 31:559-67
Van Dyck, E; Clayton, D A (1998) Transcription-dependent DNA transactions in the mitochondrial genome of a yeast hypersuppressive petite mutant. Mol Cell Biol 18:2976-85
Larsson, N G; Wang, J; Wilhelmsson, H et al. (1998) Mitochondrial transcription factor A is necessary for mtDNA maintenance and embryogenesis in mice. Nat Genet 18:231-6
Lee, D Y; Clayton, D A (1998) Initiation of mitochondrial DNA replication by transcription and R-loop processing. J Biol Chem 273:30614-21
Shadel, G S; Clayton, D A (1997) Mitochondrial DNA maintenance in vertebrates. Annu Rev Biochem 66:409-35
Larsson, N G; Oldfors, A; Garman, J D et al. (1997) Down-regulation of mitochondrial transcription factor A during spermatogenesis in humans. Hum Mol Genet 6:185-91
Shang, J; Luo, Y; Clayton, D A (1997) Backfoot is a novel homeobox gene expressed in the mesenchyme of developing hind limb. Dev Dyn 209:242-53
Larsson, N G; Barsh, G S; Clayton, D A (1997) Structure and chromosomal localization of the mouse mitochondrial transcription factor A gene (Tfam). Mamm Genome 8:139-40

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