BMP-2 REGULATED GENES DURING ENDOCHONDRAL BONE FORMATION

BMP-2 在软骨内骨形成过程中调控的基因

• 批准号: 6207542
• 负责人: FRANCESCA GORI
• 金额: $ 4.63万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-15 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6207542
• 关键词: analog; bone development; bone metabolism; bone morphogenetic proteins; cartilage metabolism; cell line; developmental genetics; differential display technique; gene induction /repression; nucleic acid sequence; osteoblasts; protein localization; protein protein interaction; protein structure function; serial analysis of gene expression; transcription factor

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.

该提案的目的是识别和表征新型蛋白质，这些蛋白质在内向骨骨形成中起作用。要解决的假设是，尽管BMP和CBFA1是在软骨内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）也有助于这一过程。我们选择解决此假设的模型是MLB13MYC克隆17细胞系，当用BMP-2处理时，该系列会获得成骨细胞的分子标记。使用差分显示PCR，我们已经确定了三种产品，这些产品最早在8小时内已被RHBMP-2调节。这些产品中的两种已被克隆和测序，并且与当前可用的数据库中的任何已知基因都不相对。由于在大多数DDPCR产物中代表的基因的远端3'未翻译区域在物种之间的保守性很差，而且通常不提交数据库，因此这些产物仍然可能代表已知基因。将获得编码区域信息以确认它们代表新基因，如果是这样，将通过筛选cDNA文库获得全长序列。基于对推导的氨基酸序列的分析，将进行实验，以表征蛋白质的假定功能（例如，确认了假定的激酶的功能并突变其激酶结构域）及其在内侧骨骨形成中的作用。