The action of aldose reductase has been implicated as an etiological factor in diabetic complications including cataract, retinopathy, and neuropathy. Treatment of diabetic patients with aldose reductase inhibitors has been proposed as a means of delaying or preventing such pathologies. However, aldose reductases isolated from various human tissues have different kinetic and structural properties. The genetic mechanisms leading to this enzyme heterogeneity are not understood. The long-term objective of the proposed research is to delineate the structural and genetic properties of aldose reductase. Aldose reductase will be purified to apparent homogeneity from bovine lenses. Peptide fragments of the enzyme will be isolated by high performance liquid chromatography and electrophoresis, and will be subjected to amino acid sequence analysis. Portions of the primary amino acid sequence will be used to design and construct synthetic oligonucleotide probes for identifying recombinant DNA clones containing the aldose reductase gene sequence. Complementary DNA (cDNA) will be synthesized from bovine lens messenger RNA, and will be used to construct DNA libraries in plasmid and bacteriophage expression cloning vectors. Antibodies to purified aldose reductase will be prepared in rabbits, and will also be used to isolate aldose reductast cDNA clones. Such DNA clones will be characterized by nucleotide sequence determination, and the primary amino acid sequence of the final aldose reductase gene product will be deduced from the cDNA sequence. Aldose reductase gene fragments will be isolated from genomic DNA libraries. Nucleotide sequence analysis of these gene fragments will be used to define the structural organization (intron-exon distribution) of the aldose reductase gene, and to identify genetic elements (promoters and enhancer sequences) which may affect its expression. Qualitative and quantitative aspects of aldose reductase gene expression in bovine and human tissues will be studied through RNA blot hybridization analysis. Aldose reductase gene expression in normal and diabetic human tissues will also be analyzed through RNA hybridization.
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