Maturational Regulation of Lactase Gene Transcription

乳糖酶基因转录的成熟调控

• 批准号: 6368901
• 负责人: ERIC SIBLEY
• 金额: $ 7.85万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-15 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6368901
• 关键词: DNA footprinting; deoxyribonuclease I; developmental genetics; enzyme activity; enzyme induction /repression; gastrointestinal system; gel mobility shift assay; gene deletion mutation; genetic promoter element; genetically modified animals; laboratory mouse; laboratory rat; lactase; reporter genes; transcription factor; transfection /expression vector

DESCRIPTION (provided by applicant): The broad objective of this proposal is to enhance the transition of the investigator to an independent and productive status in the area of molecular and developmental biology of the gastrointestinal tract. The research goal of this proposal is to define in greater detail the molecular mechanisms regulating the maturational decline in intestinal lactase gene transcription. Intestinal lactase is the enterocyte disaccharidase responsible for digestion of lactose, the primary carbohydrate in milk. Lactase activity is maximal prior to weaning and then declines significantly during maturation. Decreased lactase activity combined with excessive milk consumption results in symptoms of carbohydrate malabsorption in most mature mammals, including humans. We have characterized several DNA regulatory elements and transcription factors (GATA-4,-5,-6 and Cdx-2) involved in regulating lactase gene transcription in intestinal cells. In addition, we have determined that a 2.0-kilobase fragment of the lactase promoter directs appropriate spatio-temporal expression in the small intestine of transgenic mice. Our hypothesis, based on this data, is that the maturational decline in lactase expression is mediated by differential interaction between this 2.0 kb promoter region DNA and specific nuclear transcription factors. Specific research objectives, therefore, are aimed at characterization of cis regulatory elements and identification of nuclear proteins interacting with those cis elements. We aim to characterize the lactase DNA regulatory elements by analyzing expression of additional genomic deletions linked to a reporter gene and expressed in transgenic mice. This analysis will be facilitated by the application of methods to allow for in vivo photonic detection of reporter gene expression. We will identify specific nuclear proteins interacting with lactase gene fragments using DNase I footprint and gel shift assays. We aim to functionally characterize the proteins by altering their expression in cell culture and in transgenic mice and by assaying for transcriptional activity. The continued research experience, supplemented with mentored advice and seminars in the fields of molecular and developmental biology, will facilitate the investigator's development as an independent and productive scientist performing research on the molecular biology of intestinal development.

描述(由申请人提供)： 这项建议的广泛目标是促进 在分子领域处于独立和富有成效的地位的研究者 以及胃肠道的发育生物学。的研究目标 这项提议是为了更详细地定义分子机制 调节肠道乳糖酶基因转录的成熟性下降。 肠道乳糖酶是负责消化的肠道细胞二糖酶。 乳糖是牛奶中的主要碳水化合物。乳糖酶活性最高的优先 到断奶，然后在成熟期显著下降。乳糖酶降低 运动和过量的牛奶摄入会导致 包括人类在内的大多数成熟哺乳动物的碳水化合物吸收不良。我们有 鉴定了几种DNA调控元件和转录因子 (GATA-4、-5、-6和CDX-2)参与调节乳糖酶基因转录 肠道细胞。此外，我们还确定了一个2.0千碱基的片段 乳糖酶启动子的启动子引导适当的时空表达 转基因小鼠的小肠。基于这些数据，我们的假设是 乳糖酶表达的成熟下降是由差异介导的 该2.0kb启动子区域DNA与特异性核的相互作用 转录因子。因此，具体的研究目标旨在 顺式调控元件的表征和核的鉴定 与这些顺式元件相互作用的蛋白质。我们的目标是描述 分析附加基因组表达的乳糖酶DNA调控元件 与报告基因相关的缺失，并在转基因小鼠中表达。这 将通过应用允许在体内进行的方法来促进分析 光子检测报告基因的表达。我们将确定具体的 利用DNase I与乳糖酶基因片段相互作用的核蛋白 足迹分析和凝胶移位分析。我们的目标是从功能上描述 通过改变蛋白质在细胞培养和转基因小鼠中的表达 并通过分析转录活性。 持续的研究经验，辅以指导建议和 分子和发育生物学领域的研讨会将促进 这位研究人员作为一名独立而多产的科学家的发展 开展肠道发育的分子生物学研究。