This is a competing continuation request for another five years of research support. The major focus of the proposed studies is upon the structure, function and biological expression of macromolecules of the extracellular matrix, examining the protein and carbohydrate structure of laminin and the gene structure for the core protein of chondroitin sulfate proteoglycan. Laminin is isolated from the EHS tumor of mice and the specific chondroitin sulfate core protein gene under study is that of chicken embryo sternal cartilage. In the case of laminin, the objectives are to characterize the molecular anatomy of that glycoprotein, describing the relationships of the polypeptide subunits in the entire molecule as well as the relationships of the oligosaccharides to the polypeptides. The subunits will be isolated and their compositions and structures determined, including the location and structures of the major carbohydrate substituents. The interactions between the subunits in the molecule will be examined by selective dissociation of disulfide linkages and by immuno-electron microscopy, using specific antibodies to each subunit. In the case of the gene which encodes for core protein, the cDNA structure will be determined, and genomic clones will be isolated from a library of chicken DNA, followed by determination of the sequence of the genomic DNA. Parallel studies of core protein mRNA expression in a well-characterized differentiating system, the chick embryo limb bud, will be done as well as determination of the copy number of the gene in chicken tissues. A human genomic library will also be screened with either the chicken cDNA probe or genomic chicken DNA. These two projects encompass the scientific disciplines of biochemistry and molecular biology, utilizing contemporary methods to discern protein structure, carbohydrate structure, and gene structure. The health-relatedness of the research applies to the general area of acquired and hereditary connective tissue diseases.
