A series of respiration-deficient Chinese hamster cell mutants isolated and characterized by our laboratory will be used as starting materials in attempts to isolate and clone nuclear genes coding for proteins in the inner mitochondrial membrane. The initial emphasis is on the genes for succinate dehydrogenase (SDH) and genes affecting complex I of the electron transport chain (NADH-CoQ reductase). Much is to be learned about basic mechanisms in the biogenesis of mammalian mitochondria. We are interested in the coordinate expression and regulation of several nuclear genes, and the detailed mapping of these genes of mammalian chromosomes. Precursor polypeptides made in the cytoplasm have to be recognized, transported across the outer and possibly inner mitochondrial membrane, and then assembled in the inner membrane. Signals on the cytoplasmic precursor polypeptides have to be elucidated, and pathways of post-translational processing have to be worked out. Our approach makes use of the mutants in selections for a complementing, wild-type gene from human cells. The human genes are introduced by somatic cell fusion after irradiation of the human parental cells to introduce extensive chromosome breakage. Thus, in the final hybrid cells, only fragments of the human chromosome I (for SDH), or the X chromosome (for complex I) will be retained. Libraries from such hybrids with less than .1% of the human genome will be constructed and screened for vectors (phage Lambda) with human DNA inserts. In the further analysis a variety of approaches will be tried. Comparisons of independently-derived hybrids will narrow the choice. We have, or are in the process of making, antisera against SDH subunits, which will be used in the identification of genes subcloned into expression vectors and placed into bacteria. Knowledge of a portion of the amino acid sequence of the 70K subunit of SDH will also be exploited; a) to make a specific antiserum, and b) to make synthetic oligonucleotide probes for screening our cloned sequences. With the help of specific antisera against SDH peptides, we also plan to examine further a mutant in our collection which is almost totally defective in mitochondrial protein synthesis and has morphologically abnormal cristae.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033752-05
Application #
3283704
Study Section
Mammalian Genetics Study Section (MGN)
Project Start
1984-08-01
Project End
1989-07-31
Budget Start
1988-08-01
Budget End
1989-07-31
Support Year
5
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Type
Schools of Arts and Sciences
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Au, H C; Scheffler, I E (1998) Promoter analysis of the human succinate dehydrogenase iron-protein gene--both nuclear respiratory factors NRF-1 and NRF-2 are required. Eur J Biochem 251:164-74
Au, H C; Scheffler, I E (1997) A respiration-deficient Chinese hamster cell line with a defect in mitochondrial protein synthesis: rapid turnover of some mitochondrial transcripts. Somat Cell Mol Genet 23:27-35
Cereghino, G P; Scheffler, I E (1996) Genetic analysis of glucose regulation in saccharomyces cerevisiae: control of transcription versus mRNA turnover. EMBO J 15:363-74
Oostveen, F G; Au, H C; Meijer, P J et al. (1995) A Chinese hamster mutant cell line with a defect in the integral membrane protein CII-3 of complex II of the mitochondrial electron transport chain. J Biol Chem 270:26104-8
Cereghino, G P; Atencio, D P; Saghbini, M et al. (1995) Glucose-dependent turnover of the mRNAs encoding succinate dehydrogenase peptides in Saccharomyces cerevisiae: sequence elements in the 5' untranslated region of the Ip mRNA play a dominant role. Mol Biol Cell 6:1125-43
Au, H C; Ream-Robinson, D; Bellew, L A et al. (1995) Structural organization of the gene encoding the human iron-sulfur subunit of succinate dehydrogenase. Gene 159:249-53
Saghbini, M; Broomfield, P L; Scheffler, I E (1994) Studies on the assembly of complex II in yeast mitochondria using chimeric human/yeast genes for the iron-sulfur protein subunit. Biochemistry 33:159-65
Leckschat, S; Ream-Robinson, D; Scheffler, I E (1993) The gene for the iron sulfur protein of succinate dehydrogenase (SDH-IP) maps to human chromosome 1p35-36.1. Somat Cell Mol Genet 19:505-11
Schmidt, D M; Saghbini, M; Scheffler, I E (1992) The C-terminus of the succinate dehydrogenase IP peptide of Saccharomyces cerevisiae is significant for assembly of complex II. Biochemistry 31:8442-8
Lombardo, A; Cereghino, G P; Scheffler, I E (1992) Control of mRNA turnover as a mechanism of glucose repression in Saccharomyces cerevisiae. Mol Cell Biol 12:2941-8

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