Salivary Gland Gene Transcription and Organogenesis

这项研究的目的是确定唾液腺 建立和维持发育程序的转录因子 是下颌下腺特有的与其他发育器官雅阁 系统，它提出了许多转录因子，介导 细胞特异性基因在成熟腺体中的表达也将起到调节作用， 在器官形成过程中的作用。因此，计划是开始（具体 目的1）通过鉴定介导DNA结合转录因子 激肽释放酶基因转录增强子的下颌下活性 家族，它们是分化的导管细胞的产物， 下颌下腺这将需要定义最小的功能增强子 然后通过DNA酶I足迹法绘制核蛋白结合位点， 体外和基于计算机的搜索已知的共有序列， 转录因子家族足迹和共识的相关性 结合位点将通过分析突变位点的影响来确认 增强剂的活性。候选DNA结合转录因子 将通过筛选表达模式提示 它们可能在唾液腺基因表达中起作用， 在成熟和发展中国家存在的因子家族的其他成员 通过RT-PCR方案（特定目标2）检测下颌下腺。个人 下颌下因子将与潜在的增强子结合位点相匹配 通过它们所针对的家族的已知共有结合序列 归属感将通过测试是否 因子结合的核苷酸序列要求与序列匹配 增强子中元件的活性要求以及因子是否 可以通过转染细胞中的元件激活报告基因。最后， 候选因子的作用将通过强制表达 显性阴性和组成型活性嵌合形式的因子， 能够在培养中形成形态发生的下颌下胚（具体目标3）。

The goal of the proposed research is to identify salivary gland transcription factors that establish and maintain the developmental program specific for the submandibular gland. In accord with other developing organ systems, it is proposed that many transcription factors that mediate cell-specific gene expression in the mature gland will also play a regulatory role in the process of organogenesis. Therefore, the plan is to begin (Specific Aim 1) by identifying the DNA-binding transcription factors that mediate the submandibular activity of the transcriptional enhancers of the kallikrein gene family, which are products of the differentiated ductal cells of the submandibular gland. This will require defining minimal functional enhancers and then mapping nuclear protein binding sites by DNase I foot-printing in vitro and computer-based searches for known consensus sequences of transcription factor families. The relevance of the footprints and consensus binding sites will be confirmed by analyzing the effects of mutating the sites on the activity of the enhancer. Candidate DNA-binding transcription factors will be identified by screening known factors whose expression patterns suggest they may play a role in salivary gland gene expression and by identifying additional members of factor families present in the mature and developing submandibular glands by an RT-PCR protocol (Specific Aim 2). Individual submandibular factors will be matched with potential enhancer binding sites through the known consensus binding sequences of the family to which they belong. The factor identification will be verified by testing whether the nucleotide sequence requirements for factor binding match the sequence requirements for activity of the element in the enhancer and whether the factor can activate a reporter gene through the element in transfected cells. Finally, the role of candidate factors will be tested by forced expression of dominant-negative and constitutively active chimeric forms of the factors in submandibular rudiments capable of morphogenesis in culture (Specific Aim 3).