Transcriptional Regulators of Exocrine Pancreatic Develo

描述(由申请人提供)： 这项拟议的研究的目标是识别胰腺转录。 建立和维持针对中国的发展计划的因素 胰腺的外分泌组织室。我们将从(具体目标1)开始 鉴定介导腺泡细胞的DNA结合转录因子 PTF1-P48基因转录增强子的活性 腺泡特异性基因转录激活因子。为此，我们将定义 最小功能增强子，然后通过以下方式定位核蛋白结合位点 DNA酶I在体外的足迹和基于计算机的进化搜索 特定转录因子特有的保守结合序列 家人。我们将确认足迹的相关性和具有约束力的共识 通过分析突变位点对酶活性的影响 增强剂。我们将确定候选的DNA结合转录因子 通过筛选七大转录因子的全部成员实现胰腺外分泌 在发育中和成熟的外分泌胰腺中表达的家族 RT-PCR方法(特异性目标2)。我们将匹配个体胰腺 通过已知共识获得具有潜在P48增强子结合位点的因子 它们所属家族的结合序列。我们将核实匹配情况 通过检测因子和增强子位置之间的核苷酸序列是否 绑定的要求与中活动的顺序要求匹配 增强子，以及该因子是否可以通过 在转基因细胞中的元素。我们将通过考试来筛选更多的候选人 它们的表达是否发生在胚胎阶段，与 发育调节作用。最后，我们将测试候选人的角色 强迫表达显性-负性和结构性活跃的因素 胚胎胰腺组织中因子的嵌合形式能够 培养中的形态发生(特定目标3)。

DESCRIPTION (Provided by Applicant): The goal of the proposed research is to identify pancreatic transcription factors that establish and maintain the developmental program specific for the exocrine tissue compartment of the pancreas. We will begin (Specific Aim 1) by identifying the DNA-binding transcription factors that mediate the acinar cell activity of the transcriptional enhancer of the gene for PTFl-p48, a critical transcriptional activator of acinar specific genes. To do this we will define the minimal functional enhancer and then map nuclear protein binding sites by DNase I footprinting in vitro and computer-based searches for evolutionarily conserved binding sequences characteristic of specific transcription factor families. We will confirm the relevance of the footprints and consensus binding sites by analyzing the effects of mutating the sites on the activity of the enhancer. We will identify candidate DNA-binding transcription factors of the exocrine pancreas by screening all members of seven major transcription factor families for expression in the developing and the mature exocrine pancreas by an RT-PCR protocol (Specific Aim 2). We will match individual pancreatic factors with potential p48 enhancer binding sites through the known consensus binding sequences of the family to which they belong. We will verify the match between factor and the enhancer site by testing whether the nucleotide sequence requirements for binding match the sequence requirements for activity in the enhancer and whether the factor can activate a reporter gene through the element in transfected cells. We will screen additional candidates by examining whether their expression occurs at an embryonic stage consistent with a developmental regulatory role. Finally, we will test the role of candidate factors by forced expression of dominant-negative and constitutively active chimeric forms of the factors in embryonic pancreatic rudiments capable of morphogenesis in culture (Specific Aim 3).