The primary goal of the work proposed here is to establish the structure of the propeptides of two genetic types of procollagens, Type I and Type II. Type I procollagen, the biosynthetic precursor of Type I collagen, is synthesized by many tissues including skin, tendon and bone. The Type II procollagen is the precursor of Type II collagen in hyaline cartilage, nucleus pulposus, intervertebral discs and sclera. The complete amino acid sequence of the carboxyl and amino propeptides of proAlpha1(I), proAlpha2(I) and proAlpha1(II) chains will be determined. In addition, we will attempt to determine the structure of the """"""""signal"""""""" sequences at the amino termini of the initial translation products of the three kinds of proAlpha-chains. To accomplish these goals, we will use a combination of peptide and DNA sequencing. The propeptides will be isolated from organ cultures of chick embryo tendons and sterna. The DNA will include cDNA complementary to propeptide regions of Type I and Type II-specific mRNA, purified and amplified by molecular cloning in E. coli strain HB101, as well as fragments of the proAlpha(I), proAlpha(2), and proAlpha1(II) genes isolated from genomic libraries. We will continue to work mostly with chick procollagens. However, in a comparison of propeptide structures among different animal species we will also characterize the proppeptides of human Type II procollagen. This will be done by isolation of human Type II collagen cDNA and gene clones and nucleotide sequencing of such clones. The long-term objective of these studies is to understand the structure of procollagen propeptides as it relates to their possible functions (intracellular folding of procollagen molecules, transport and secretion of procollagen, collagen fiber-formation and possibly regulation of collagen biosynthesis). In addition, through the isolation and characterization of the Type II procollagen genes we hope to build a basis for future studies of the modulated expression of this gene in normal development and its altered expression in disease states that involve hyaline cartilage such as osteoarthritis.
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