Mitochondrial biogenesis in animal cells involves the coordinate expression of a large number of nuclear genes coding for varied tissue specific and generally occurring mitochondrial proteins and a limited number of genes of the 16 kbp mitochondrial genome. Despite intensive efforts, however, the molecular mechanisms involved in the coordinate expression of the two genetic systems have not yet been elucidated. Recent results in our laboratory showed the possibility that three of the mitochondrial genome coded proteins are transported to the nucleus as possible regulatory signals. Based on this, the objective of this grant proposal is to undertake a detailed characterization of the three proteins transported to the nucleus with respect to their nuclear DNA binding characteristics, mode of regulation of nuclear genes. It is proposed to continue the present line of studies on the characterization of observed induction of nuclear genes coding for varied mitochondrial proteins like cytochrome oxidase, ATPase, RNA polymerase, etc. by long-term exposure of cells to mitochondrial translation inhibitors. One of the objectives is to characterize the high affinity binding sites for the three mitochondrial proteins on the nuclear chromosomal DNA and determine their physical relationship or proximity to genes coding for the known mitochondrial proteins using cDNA cloning, in situ hybridization to G-banded chromosomes and mapping of genomic clones. Finally, the precise regulatory roles of the three proteins and the nature of gene(s) or gene clusters under their influence will be assessed using in vitro transcription in isolated nuclei and in HeLa cell extracts with added DNA templates. The genomic areas showing high specificity for the proteins will be sequenced to gain insight on the mode of regulation by the three mitochondrial proteins.
