Gal-32 is a Chinese hamster lung (CHL) cell mutant that is unable to grow in galactose due to a nuclearly encoded, recessive mutation affect mitochondrial (mt) protein synthesis. The goals of this proposal are to determine the molecular basis of the mutations and to clone a wild type gene that corrects the mutation. The mitochondrially synthesized subunits of NADH dehydrogenase and cytochrome c oxidase are significantly reduced in Gal-32 compared to the wild type; however, the mitochondrially synthesized subunits of ATPase are not drastically affected. In contrast to the protein levels, the mRNA and tRNA steady state levels are elevated to the mutant compared to the wild type. The differential reduction in mitochondrially encoded proteins in Gal-32 is the result of decreased translation of specific mRNAs, not increased protein degradation. In order to obtain information about the initiation, elongation, and termination steps in protein synthesis as well as the sequestering of mRNAs in particles, mitochondrial polysomes will be examined using sucrose gradient centrifugation and hybridization to complementary mtRNA probes. The size and modification of tRNAs and aminoacylation of tRNAs will be examined on one-dimensional and two-dimensional polyacrylamide gels. If differences are observed in Gal-32 compared to the wild type on one- or two- dimensional polyacrylamide gels, the 5'- and 3'-ends of tRNAs and tRNA modification will be pursued. Activity of mt translation factors, mt ribosomes, inner membrane association of mt RNAs, and polyadenylation of mt proteins will be measured. A wild type gene that corrects the Gal-32 mutation will be cloned using gene transfer. These studies will help to elucidate the regulation and factors involved in mitochondrial gene expression and may provide a model system for human patients with analogous deficiencies in the mitochondrial respiratory complexes.

Project Start
1998-08-01
Project End
1999-07-31
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
28
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Howard University
Department
Type
DUNS #
056282296
City
Washington
State
DC
Country
United States
Zip Code
20059
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