The primary goals of the work proposed here are 1) to establish the structure of a disulfide-bonded short collagen found in hyaline cartilage, 2) examine the biosynthesis of this collagen and determine the location of it within cartilage extracellular matrix, 3) determine the structure of the genes that encode the polypeptides of the collagen, and 4) study the expression of these genes in different tissues during embryonic development. To accomplish these goals, we will use a combination of protein chemistry and DNA cloning/sequencing. The structure of the disulfide-bonded collagen will be determined by sequencing of long cDNAs isolated from libraries of cDNA clones synthesized from chick embryo sternal cartilage and chondrocyte mRNAs. In addition, limited sequencing of DNA isolated from chick genomic libraries, will be used to obtain primary structure information in cases where cDNA sequences are difficult to obtain. The biosynthesis of the protein will be studied using chick embryo sternal chondrocytes in culture. The short collagen isolated from such cultures will be used for peptide mapping studies to provide support for the sequence information obtained by DNA sequencing. Purified protein will also be used for the generation of antibodies. Such antibodies will be used to localize the protein in cartilage (and possibly other tissues) by light and electron microscopical immunocytochemistry. The structure of the genes encoding the polypeptides of the disulfide bonded short collagen will be studied by DNA sequencing and electron microscopical R-loop mapping. The expression of these genes will be analyzed by using gene and cDNA probes for in situ hybridization of chick embryo tissues as well as for quantitative analyses of mRNAs during chick development by Northern blotting and RNA dot hybridization on nitrocellulose filters. The long term objective of these studies is to understand the structure of short collagens as it relates to their function, the modulated expression of their genes during normal embryonic development and alterations in disease states such as osteoarthritis. In addition, through the isolation and characterization of short collagen genes we hope to obtain data relevant to the question of the evolutionary relationship of these genes to the genes that code for interstitial (Type I, II, III) collagens.
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