Maturational Regulation of Lactase Gene Transcription

描述（由申请人提供）： 这项建议的广泛目标是加强 在分子生物学领域取得独立和富有成效的地位 和胃肠道发育生物学。的研究目标 这一建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下降。 肠乳糖酶是负责消化的肠细胞二磷酸酶 乳糖是牛奶中的主要碳水化合物。乳糖酶活性最大优先 到断奶，然后在成熟过程中显著下降。乳糖酶降低 活动加上过量的牛奶消费导致的症状 碳水化合物吸收不良在大多数成熟的哺乳动物，包括人类。我们有 表征了几种DNA调控元件和转录因子 （加塔-4，5，6和Cdx-2）参与调节乳糖酶基因转录， 肠细胞此外，我们还确定了一个2.0-脱氢酶片段， 的乳糖酶启动子指导适当的时空表达， 转基因小鼠的小肠。基于这些数据，我们的假设是 乳糖酶表达的成熟性下降是由分化的 该2.0 kb启动子区DNA与特异性核内 转录因子因此，具体的研究目标是 顺式调控元件的表征和核转录因子的鉴定 与这些顺式元件相互作用的蛋白质。我们的目标是描述 乳糖酶DNA调控元件通过分析其它基因组的表达 与报告基因相连的缺失并在转基因小鼠中表达。这 将通过应用允许体内分析的方法来促进分析 报告基因表达的光子检测。我们将确定具体 利用DNA酶I与乳糖酶基因片段相互作用的核蛋白 足迹和凝胶位移测定。我们的目标是功能特征， 通过改变蛋白质在细胞培养物和转基因小鼠中的表达， 和通过测定转录活性。 持续的研究经验，辅以指导性建议， 在分子和发育生物学领域的研讨会，将促进 研究者作为一个独立和多产的科学家的发展 进行肠道发育的分子生物学研究。

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.