The hypothesis of this work is that the extracellular matrix of bone both facilitates mineral nucleation and serves as the suprastructure for mineral deposition. The identification of bone specific matrix components, determination of their quantitative expression, the temporal regulation of their expression, and their assembly will provide a crucial understanding of the mechanisms of mineralization. Phosphoproteins have been identified as a possible matrix component that facilitates and controls mineralization in bone. IN order to further examine phosphoproteins' role in mineralization two specific phosphoproteins will be characterized at the protein level and subsequently cDNAs and genomic clones will be identified and analyzed. Their expression, levels of regulation and mechanisms of regulation will be analyzed during both in vivo bone development and in vitro development in a mineralizing osteoblast culture system. Two other genes associated with bone development (alkaline phosphatase and osteocalcin) and the general group of bone specific cDNAs, identified by subtractive hybridization screening, will be isolated. These genes will be similarly analyzed as the phosphoproteins. Their expression will also be examined during both in vitro and in vivo development and mineralization. These experiments will provide information on the role of phosphoproteins during bone development and mineralization. The comparative analysis of several different bone specific genes will provide a more complete view of the processes of cellular differentiation, and help identify the interaction of several bone specific proteins and the crucial molecular events of their quantitative and temporal expression during bone development and mineralization.
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