The overall goal of the research is to understand the developmental processes which take place in the avian embryonic limb. In this system we propose to examine the synthetic activity of the chondrocytes during the establishment of the extracellular matrix of cartilage by normal embryos and by embryos whose cartilage is abnormal owing to a genetic lesion. We will investigate the transcriptional activity in differentiating chondrocytes, in chondrocytes whose differentiation is arrested in the protodifferentiated state, and in those that are caused to undergo """"""""de-differentiation"""""""". We will also investigate changes in chromatin structure which accompany changes in transcriptional activity. These measurements will be done using four cDNA probes containing coding regions for cartilage-specific proteins (Type II collagen, 54 k Da matrix protein, cartilage proteoglycan core protein, and link protein) and two cDNA probes containing coding regions for non-cartilaginous proteins (Type I Alpha1 and Alpha2). With the proteoglycan core protein cDNA probes we will determine the genetic basis for the core protein defect in the cartilage mutant, nanomelia. We will study the relationship between structure and function of the components which make up the link stabilized proteoglycan aggregates of cartilage by generating monoclonal antibodies which can identify the sites on proteoglycan core protein which interact with link protein and hyaluronic acid, and those on link protein which interact with core protein and hyaluronic acid. The monoclonal antibodies will also be used to identify polypeptides synthesized by bacteria which were transformed with expressing plasmids containing cDNA inserts which code for different regions of core protein and link protein. In this manner the functional domains of the proteins will be identified and their physical limits will be determined by mapping the extent of the expressing cDNA inserts.
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