Branched chain alpha-ketoacid dehydrogenase provides an excellent model by which to study the synthesis and assembly of mitochondrial multienzyme complexes. The proteins of this complex are encoded in the nucleus on separate genes and must assemble in a specific stoichiometry within the mitochondria. Inherited mutations in humans are known to affect the function of this complex. These mutations continue to be expressed in cells cultured from the patients and therefore can be used to study mutant and wild type gene expression and protein function. The plan of this project is to isolate cDNA and genomic clones for the various protein components. The nucleotide sequence for these DNA molecules will be determined and the wild type structures compared with mutant structures. This can easily be done using the polymerase chain reaction to amplify specific regions of the genes. Cells cultured from some individuals expressing defects in the branched chain dehydrogenase complex activity have been shown to lack one or another protein component of the complex. With clones for the missing component the cells can be transfected with the cloned DNA for the missing component and return of function can be determined. In this way we can begin to define the mutations at the gene level which give rise to Maple Syrup Urine disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK038320-05
Application #
3237633
Study Section
Biochemistry Study Section (BIO)
Project Start
1986-08-15
Project End
1994-07-31
Budget Start
1990-08-01
Budget End
1991-07-31
Support Year
5
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Emory University
Department
Type
Schools of Medicine
DUNS #
042250712
City
Atlanta
State
GA
Country
United States
Zip Code
30322
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Doering, C B; Danner, D J (2000) Expression of murine branched-chain alpha-keto acid dehydrogenase kinase. Methods Enzymol 324:491-7
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Doering, C B; Coursey, C; Spangler, W et al. (1998) Murine branched chain alpha-ketoacid dehydrogenase kinase;cDNA cloning, tissue distribution, and temporal expression during embryonic development. Gene 212:213-9
McConnell, B B; Burkholder, B; Danner, D J (1997) Two new mutations in the human E1 beta subunit of branched chain alpha-ketoacid dehydrogenase associated with maple syrup urine disease. Biochim Biophys Acta 1361:263-71
McConnell, B B; McKean, M C; Danner, D J (1996) Influence of subunit transcript and protein levels on formation of a mitochondrial multienzyme complex. J Cell Biochem 61:118-26
Lanterman, M M; Dickinson, J R; Danner, D J (1996) Functional analysis in Saccharomyces cerevisiae of naturally occurring amino acid substitutions in human dihydrolipoamide dehydrogenase. Hum Mol Genet 5:1643-8
England, B K; Greiber, S; Mitch, W E et al. (1995) Rat muscle branched-chain ketoacid dehydrogenase activity and mRNAs increase with extracellular acidemia. Am J Physiol 268:C1395-400

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