| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1990: ¥200,000 (Direct Cost: ¥200,000)
Fiscal Year 1989: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
1. A generalized deficiency in the mitochondrial enzyme. Ornithine aminotransferase (OAT : EC 2.6.1.13), is the hallmark of gyrate atrophy (GA), a hereditary degenerative disease of the choroid and retina of the eye that leads to blindness. A human OAT cDNA, previously constructed and characterized in our laboratory, and anti-human OAT antibody were used as probes to examine the OAT gene, mRNA and protein of GA patients. A blot analysis of the genomic DNAs, RNAs, and proteins of 14 GA patients identified a case with a partial heterozygous deletion of the functional OTA gene, no detectable OAT mRNA, and a barely detectable level of OAT antibody-reactive protein. The rest of the cases showed grossly normal OAT gene, mRNA, and variably reduced levels of OAT protein.
2. We then analyzed another case of GA more precisely. Southern analysis indicated the functional gene to be grossly intact. Northern analysis of his OAT mRNA demonstrated only half the normal level of OAT message, suggesting e
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xpression of only one of the two alleles of the OAT gene. A functional assay of the expressed OAT mRNA by in vitro translation and immunoprecipitation with antihuman OAT antibody indicated synthesis of an OAT protein from the message. The expressed message was cloned and sequenced and was shown to contain a single base change from C to T, resulting in an amino acid codon change from CAT (histidine) to TAT (tyrosine) at position 319 in the translated OAT protein. The mutant precursors were tested in an in vitro mitochondrial transport/ processing system. The results indicate that the mutant OAT precursor from the gyrate atrophy patient can be transported to the mitochondria but is minimally processed there.
3. We localized ornithine aminotransferase in human ocular tissues using immunocytochemical procedures. In the retina, ganglion cells and some amacrine cells were immunoreactive. Pigmented granules made it difficult to identify immunoreactive products in the iris, pigmented epithelium of the ciliary body, choroid, and retinal pigment epithelium. Our findings suggested that ornithine aminotransferase plays an important role in ornithine metabolism in these ocular tissues. Less
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