The primary goals of the work proposed here are: 1) to examine the evolutionary history of the Type X collagen gene and to establish its relationship to other genes encoding proteins with collagenous domains, 2) to examine the biosynthesis of the molecule, and its potential role in the physiological removal of cartilage during endochondral ossification, 3) to examine the expression of Types II and X collagens during embryonic development and maturation of cartilage, and 4) to determine the molecular mechanisms of the high level of expression of Type X collagen in hypertrophic cartilage. To accomplish these goals, we will use a combination of DNA cloning/sequencing and protein chemistry. Type X collagen genes will be isolated from rat and human genomic libraries and their structure, as determined by nucleotide sequence analysis, will be compared to that of the chicken gene which we have isolated. Nucleotide sequences for chicken, rat, and human Type X collagens will be used to synthesize peptides. These will be used as haptens to generate specific antibodies. Such antibodies, together with specific DNA probes, will be used to study the synthesis of Type X collagen and the expression of the Type X gene during normal and abnormal cartilage development. Isolated cDNA and genomic clones will allow studies of the molecular mechanisms responsible for the expression of Type X collagen by hypertrophic chondrocytes. Nuclease hypersensitivity and methylation in and around the Type X gene will be mapped in expressing and non-expressing chondrocytes. The transient expression of the cloned genes will be studied in cultured cells, and we will assay for the binding of nuclear proteins to sequences within and flanking the gene. The long term objective of these studies is to understand the biogenesis and cellular regulation of extracellular matrices during normal and abnormal development. The studies on Type X collagen should provide information about the function and mechanisms controlling the synthesis of a specific collagen in a connective tissue undergoing morphogenetic destruction. They may also provide more general insight into the synthesis of extracellular matrix components by chondrocytes in normal cartilage and in disease states such as rheumatoid arthritis.
Showing the most recent 10 out of 152 publications
