BMP-2 REGULATED GENES DURING ENDOCHONDRAL BONE FORMATION

本提案的目的是鉴定和表征在软骨内骨形成中起作用的新蛋白。要解决的假设是，尽管BMPs和Cbfa1是在软骨内骨形成中起关键作用的因素，但其他蛋白质包括生长因子、转录因子、受体和激酶也参与了这一过程。我们选择的模型是MLB13MYC克隆17细胞系，用BMP-2处理后获得成骨细胞的分子标记。利用差分显示PCR技术，我们鉴定出三个早在8小时就被证实受rhBMP-2调控的产物。其中两个产物已被克隆和测序，在目前可用的数据库中不对应任何已知基因。由于在大多数ddPCR产物中所代表的基因的远端3'非翻译区在物种之间的保守性较差，并且通常没有提交到数据库中，因此这些产物仍有可能代表已知基因。获取编码区信息，确认其是否为新基因，如果为新基因，则通过cDNA文库筛选获得全长序列。基于对推导出的氨基酸序列的分析，将进行实验来表征蛋白质的推测功能(例如：（证实了一种假定的激酶的功能并使其激酶结构域发生突变）及其在软骨内骨形成中的作用。

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.