BMP-2 REGULATED GENES DURING ENDOCHONDRAL BONE FORMATION

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The objective of this proposal is to identify and characterize novel proteins that play a role in endochondral bone formation. The hypothesis to be addressed is that, although BMPs and Cbfa1 are factors that play a critical role in endochondral bone formation, other proteins including growth factors, transcription factors, receptors and kinases also contribute to this process. The model that we have chosen to address this hypothesis is the MLB13MYC clone 17 cell line, which when treated with BMP-2 acquires the molecular markers of an osteoblast. Using differential display PCR, we have identified three products which have been confirmed to be regulated by rhBMP-2 as early as 8 hours. Two of these products have been cloned and sequenced, and do not correspond to any known genes in the currently available data base. Since the distal 3' untranslated region of genes, represented in most of ddPCR products, is poorly conserved among species and often not submitted to data bases, it is still possible that these products represent known genes. Coding region information will be obtained to confirm that they represent novel genes and if so, full length sequence will be obtained by screening a cDNA library. Based on analyses of the deduced amino acid sequence, experiments will be performed to characterize both the putative function of the protein (eg., confirming the function of a putative kinase and mutating its kinase domain) and its role in endochondral bone formation.