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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029037-09
Application #
3276498
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1982-05-01
Project End
1991-04-30
Budget Start
1990-05-01
Budget End
1991-04-30
Support Year
9
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Type
Schools of Veterinary Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Carter, R S; Bhat, N K; Basu, A et al. (1992) The basal promoter elements of murine cytochrome c oxidase subunit IV gene consist of tandemly duplicated ets motifs that bind to GABP-related transcription factors. J Biol Chem 267:23418-26
Carter, R S; Avadhani, N G (1991) Cloning and characterization of the mouse cytochrome c oxidase subunit IV gene. Arch Biochem Biophys 288:97-106
Basu, A; Avadhani, N G (1991) Structural organization of nuclear gene for subunit Vb of mouse mitochondrial cytochrome c oxidase. J Biol Chem 266:15450-6
Basu, A; Avadhani, N G (1990) Nucleotide sequence of cDNA for nuclear encoded subunit Vb of mouse cytochrome-c oxidase. Biochim Biophys Acta 1087:98-100
Bhat, K S; Avdalovic, N; Avadhani, N G (1989) Characterization of primary transcripts and identification of transcription initiation sites on the heavy and light strands of mouse mitochondrial DNA. Biochemistry 28:763-9
Vijayasarathy, C; Bhat, N R; Avadhani, N G (1989) Intramitochondrial fatty acylation of a cytoplasmic imported protein in animal cells. J Biol Chem 264:7772-5
Kulkarni, G R; Kantharaj, G R; Fluellen, C et al. (1987) Stimulation of transcription and translation by aurin tricarboxylic acid in mitochondrial lysates from Ehrlich ascites cells. Biochem Biophys Res Commun 145:1149-57
Bhat, K S; Bhat, N K; Kulkarni, G R et al. (1985) Expression of the cytochrome b-URF6-URF5 region of the mouse mitochondrial genome. Biochemistry 24:5818-25
Bhat, N K; Avadhani, N G (1985) Transport of proteins into hepatic and nonhepatic mitochondria: specificity of uptake and processing of precursor forms of carbamoyl-phosphate synthetase I. Biochemistry 24:8107-13