EXTRACELLULAR MATRIX GENE EXPRESSION IN CORNEA

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The primary goals of the work proposed here are: 1) to examine the structure of the corneal endothelial short-chain collagen (CESCC) which is synthesized by rabbit corneal endothelial cells, and 2) to establish the structural and evolutionary relationship between the gene(s) encoding this collagen and genes that code for other short-chain collagens (Types IX, X, XII); 3) to examine the assembly and secretion of the CESCC molecule in the highly organized extracellular matrices of the cornea; 4) to examine the expression of the CESCC in the cornea during embryonic development; and 5) to analyze the modulated expression of the CESCC molecule in an in vitro model of posterior collagenous layer (retrocorneal fibrous membrane). To accomplish these goals, we will use a combination of DNA cloning/sequencing and protein chemistry. cDNA coding for the CESCC molecule will be constructed from rabbit corneal endothelial cell RNA. Primary structure of this molecule will be determined by nucleotide sequence analysis of isolated clones. The gene(s) coding of the CESCC molecule will be isolated from rabbit genomic libraries. Synthetic peptides will be made from the nucleotide sequence of rabbit cDNA. Specific antibodies will be generated against these peptides. Such antibodies, together with specific DNA probes, will be used to examine the synthesis of the CESCC molecule and the expression of its gene(s) in corneal development as well as in cultured endothelial cells. The study should provide information about the structure and functional role of a novel short-chain collagen of the cornea and the mechanisms regulating its synthesis. Such information should prove invaluable for understanding the assembly of the normal extracellular matrices of the cornea and alterations due to disease.