The goal of the proposed studies is to provide a better understanding of the regulation of the expression of the genes of connective tissue proteins, e.g., prolyl 4-hydroxylase (EC.1.14.11.2), collagens. Synthesis of collagens is developmentally regulated and involves several posttranslational modifications. Prolyl 4-hydroxylase is the key enzyme in collagen synthesis, since the presence of sufficient hydroxyproline residues is essential for collagen to assume triple-helical conformation. Therefore, prolyl 4-hydroxylase is a prerequisite for cells to synthesize functional collagen molecules. However, many pathogenestic conditions which are accompanied by increased prolyl hydroxylase activities frequently lead to the undesirable deposition of collagen fibers in tissues, e.g., formation of corneal scars, vitreous fibrosis, etc. A better understanding of the expression of prolyl hydroxylase genes will give insight into the synthesis of collagens. We recently isolated cDNA clones which encode portions of Alpha and Beta subunits of prolyl hydroxylase. To characterize cDNA clones, we will determine the DNA sequences of the cDNA's so that the amino acid sequences can be deduced. In addition, partial amino acid sequences of each of the CB-peptides of Alpha and Beta subunits will be determined. Thereafter, the cDNA clones can be unequivocally assigned to the subunits of prolyl 4-hydroxylase. The genomic DNA's will then be isolated from a chick DNA library by screening a genomic DNA library with 32p-labeled cDNA. The structure of the prolyl hydroxylase genes can then be analyzed to determine distribution of introns and exons. In further experiments the metabolism of prolyl hydroxylase will be examined in corneas of chick embryos and in cultured corneal cells under various conditions, e.g., """"""""crowding"""""""", subculturing. The levels of mRNA's of prolyl hydroxylase will be measured by slot-blot hybridization using specific cDNA's. Similar experiments will be performed to examine the regulation of collagen(I) synthesis by corneas of chick embryos and cultured corneal cells. Furthermore, the effects of retinoids on the synthesis of collagen by corneal epithelia of developing embryo will be evaluated. Finally, nuclear proteins in crowded corneal cells will be characterized.
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