Details

Autor(en) / Beteiligte

• Brody, Lawrence Craig

Titel

The functional consequences of mutations at the ornithine-delta-aminotransferase locus

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

1991

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• Ornithine-$\delta$-aminotransferase (OAT) catalyzes the conversion of ornithine and $\alpha$-ketoglutarate to $\Delta\sp1$-pyrroline-5-carboxylate (P5C) and glutamate. This reaction acts as a direct link between the urea cycle, proline and glutamate biosynthesis and the tricarboxylic acid cycle (via P5C). The cDNAs for rat and human OAT have been cloned. The chromosomal location and organization of the human OAT structural gene and related pseudogenes are known. A deficiency of OAT causes the autosomal recessive blinding disorder gyrate atrophy of the choroid and retina (GA). Using a variety of mutation detection techniques, we have defined the molecular defect in 20 OAT alleles associated with GA. Using site-directed mutagenesis and calcium phosphate mediated DNA transfection, nine of these mutations have been expressed in cultured cells. Biochemical characterization of the expressed proteins demonstrates that the mutations found inactivate OAT and thus cause GA. Functional inactivation is inferred in eight others encoding mRNAs incapable of producing full-length OAT protein due to deletion, nonsense and frameshift mutations. Missense mutations associated with GA provide a valuable resource for identification of amino acid residues important to the structure and function of OAT. This work combines a genetic approach with biochemical and phylogenetic analysis to identify functionally important components of the OAT protein. The biochemical characterization of these mutations and a compilation of the mutant alleles at this locus are reported. The ability to carry out in vivo inhibition of this enzyme would add to our understanding of its function and provide a model system for studying GA. OAT active-site mutants were produced using site-directed mutagenesis and tested for their ability to inactivate OAT in vivo. These mutant subunits were shown to produce a dominant negative effect when expressed in the presence of wildtype subunits in a co-transfection assay. A model of inhibition based on protein subunit interactions is proposed. Inhibition of OAT in an intact organism would produce an animal model of GA. To test the ability of these active-site mutants to function in this capacity several lines of mice were established carrying OAT active-site mutant transgenes. Characterization of transgene transmission and expression is presented.