Aldose reductase has been implicated in the etiology of many diabetic complications including cataract, retinopathy, nephropathy, and neuropathy. The long-term objective of the proposed research is to identify molecular parameters which regulate the genetic expression of this important enzyme. Research effort will focus on three levels of human aldose reductase gene expression: 1) Isolation and characterization of aldose reductase mRNA, 2) molecular cloning and sequencing of the aldose reductase gene, and 3) analysis of aldose reductase gene expression in various normal and diabetic human tissues. 1) Aldose reductase mRNA will be isolated by affinity and immunological methods and characterized with respect to molecular size, translational efficiency under various conditions, and post-transcriptional modifications. 2) The structural and functional organization of the aldose reductase gene system will be determined through molecular cloning studies. Aldose reductase ds cDNA will be cloned into homopolymer-tailed plasmid vectors and also cloned into plasmid expression vectors. Human aldose reductase genomic DNA will be cloned into bacteriophage expression vectors. Recombinant expression vector libraries will be screened by newly developed immunological procedures. The DNA sequence of appropriate recombiant clones will be determined by chemical and/or dideoxy chain termination methods. The functional organization of aldose reductase genomic DNA will be analyzed for possible regulatory elements; promotor sequences, initiation sites for transcription and translation, mRNA splice sites and polyadenylation signal, and termination sites for translation. The aldose reductase amino acid sequence will be deduced from cloned cDNA and genomic DNA sequences. 3. The level of aldose reductase gene expression in normal and diabetic tissues will be studied at the mRNA and protein levels. Dot blot and Northern hybridization will be used to quantitate and characterize tissue-specific aldose reductase mRNA. Aldose reductase gene translation products will be determined by immunological methods as well as by enzyme kinetic analysis. Aldose reductase gene expression in tissue culture will be explored.
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