BMP-2 REGULATED GENES DURING ENDOCHONDRAL BONE FORMATION

这项建议的目的是识别和表征在软骨内骨形成中发挥作用的新蛋白质。需要解决的假设是，尽管BMP和Cbfa1是在软骨内骨形成中发挥关键作用的因子，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，当用BMP-2处理时，它获得了成骨细胞的分子标记。利用差异显示聚合酶链式反应，我们已经鉴定了三个早在8h就被证实受重组人骨形态发生蛋白-2调控的产物。其中两个产物已经被克隆和测序，与目前可用的数据库中的任何已知基因都不对应。由于基因的远端3‘非翻译区在大多数ddPCR产物中存在，在物种之间保存不佳，通常不提交数据库，因此这些产物仍有可能代表已知的基因。将获得编码区信息，以确认它们代表新基因，如果是，将通过筛选cDNA文库获得全长序列。基于对推导出的氨基酸序列的分析，将进行实验，以确定该蛋白的假定功能(例如，确认假定的激酶的功能并突变其激活域)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.