The goal of these studies is to characterize and elucidate the biological function of two new collagen chains that were identified by analysis of human cDNA clones. One clone, which was isolated from a placenta library, has a 2.1 kb insert. It encodes 709 amino acids that correspond primarily to a collagenous region containing 13 interruptions of 2-45 amino acids. Two long interruptions include multiple consensus sequences for attachment of glycosaminoglycan moieties; other interruptions are characterized by the presence of potential asparagine-linked acceptor sites suggesting that this collagen chain may be extensively glycosylated. Antisera prepared against a synthetic peptide identified a 125 kd protein sensitive to bacterial collagenase. Hybridization of the clone to human fibroblast RNA revealed a moderately abundant RNA of 5.3 kb and two lower molecular weight RNAs, whereas at least six equally represented transcripts are present in bovine cells. This collagen has been named alphal (XV). The second clone, which was identified from a rhabdomyosarcoma library, has a 0.6 kb insert. It codes for short noncollagenous sequences flanking four collagenous domains separated by three interruptions. This collagen, which appears to be encoded by a very large and rare mRNA in tumor and fibroblast cell lines, has been designated type RH.
Aims of this proposal are to determine the complete primary structure of the previously unidentified collagens from the DNA sequence, to screen custom-made fibroblast and rhabdomyosarcoma libraries for clones with related sequences, to define the chromosomal localization of the genes, and to establish linkage with neighboring genes and disease markers. In situ hybridization will be employed to investigate the in vivo distribution of the type XV and RH transcripts relative to other collagens. To determine the expression of the proteins, peptide-specific antisera will be purified by affinity chromatography and used to localize the collagens by immunoperoxidase staining. In both studies, bovine fetal tissue will be examined together with rat tissue obtained at different stages of development. Two additional aims are specific for type XV collagen. One is to define the origin of the multiple human and bovine RNA transcripts by S1 mapping, primer extension, and PCR amplification of discrete regions. The second is to determine whether type XV collagen is a proteoglycan and/or glycoprotein. This will be accomplished using specific endoglycosidases, SDS-PAGE, and Western blotting of the protein synthesized in cultured cells and tissue. In the next five years, these efforts should result in considerable progress towards ascertaining the nature of types XV and RH collagens, including their possible role in inherited or acquired diseases of connective tissue.
Showing the most recent 10 out of 93 publications
