Salivary Gland Gene Transcription and Organogenesis

唾液腺基因转录和器官发生

• 批准号: 6923633
• 负责人: RAYMOND J. MACDONALD
• 金额: $ 29.64万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-15 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6923633
• 关键词: DNA binding protein; DNA footprinting; acinar cell; binding sites; computer assisted sequence analysis; developmental genetics; embryogenesis; gene expression; genetic enhancer element; genetic mapping; genetic transcription; histogenesis; in situ hybridization; laboratory mouse; nuclear proteins; nucleoproteins; polymerase chain reaction; protein structure function; salivary glands; submandibular gland; tissue /cell culture; transcription factor

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).

这项拟议的研究的目标是确定建立和维持发育程序的唾液腺转录因子。 专门针对颌下腺的。与其他发育中器官一致 系统中，许多转录因子被认为是介导 细胞特异性基因在成熟腺体中的表达也将起到调节作用 在器官发生过程中的作用。因此，计划将开始(具体 目的1)通过鉴定DNA结合的转录因子，介导 激肽释放酶基因转录增强子的颌下活性 家族，它们是分化的导管细胞的产物 颌下腺。这将需要定义最低限度的功能增强器 然后通过DNase 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识序列 转录因子家族。足迹与共识的关联性 结合位点将通过分析突变位点的影响来确定 关于增强剂的活性。候选DNA结合转录因子 将通过筛选其表达模式表明 它们可能在唾液腺基因表达中发挥作用，并通过识别 在成熟和发展中存在的其他因子家族成员 颌下腺采用RT-PCR方法(特异性目标2)。个体 下颌下因子将与潜在的增强子结合位点相匹配 通过它们所到的家族的已知共识结合序列 归属感。因素识别将通过测试是否 因子结合的核苷酸序列要求与序列匹配 对增强剂中元素活性的要求以及该因子是否 可以通过该元件激活转基因细胞中的报告基因。最后， 候选因素的作用将通过强制表达 中因子的显性-负性和结构性活性嵌合形式 能够在培养中形成形态的下颌下原始组织(特定目标3)。