Molecular Characterization of the SLC19A2 Promoter

描述（由申请人提供）：膳食硫胺素是人类必需的营养素。肠道中硫胺素的摄入已得到很好的研究，但摄入过程的分子机制和调节尚未得到很好的理解。最近，已从几种人体组织中克隆了一种硫胺素转运蛋白（SLC 19 A2）。我们成功地确定了这种转运蛋白在小肠和大肠中的存在。为了试图理解转运蛋白在基因表达水平上的调节，我们克隆了5 '调节区，并使用荧光素酶报告系统建立了活性所需的最小区域。我们能够在肠Caco-2细胞中显示出高水平的克隆推定启动子活性，在肝HepG 2细胞中的活性约为其两倍。使用基于网络的计算机程序在最小区域中检测到几个顺式调节元件。我们计划进一步表征推定的启动子区域在体外使用凝胶位移和超位移测定，突变分析，和功能测定，以确定到底是什么转录调控蛋白结合推定的顺式元件。我们还希望在体内表征推定的启动子。为了彻底检查启动子，我们建立了携带启动子荧光素酶构建体的转基因小鼠系。我们将首先研究小鼠中启动子活性的组织分布，重点是肠道。以前的工作已经确定，包括维生素在内的营养物质的运输过程经历了明确的发育调节。没有什么是已知的可能调控硫胺素摄取过程中的发展，这是否包括启动子调控。这些问题将在本提案中得到解决。

DESCRIPTION (provided by applicant): Dietary thiamine is an essential nutrient for humans. The uptake of thiamine in the intestine has been well studied but the molecular mechanism and regulation of the uptake process is not well understood. Recently a thiamine transporter has been cloned from several human tissues (SLC19A2). We deafly established the presence of this transporter in the small and large intestine. In an attempt to understand the regulation of the transporter at the level of gene expression we cloned the 5'regulatory region and using a luciferase reporter system established the minimal region required for activity. We were able to show high levels of activity of the cloned putative promoter in intestinal Caco-2 cells with approximately twice that activity in liver HepG2 cells. Several cis-regulatory elements were detected in the minimal region using web based computer programs. We plan to further characterize the putative promoter region in vitro using gel shift and super-shift assays, mutational analysis, and functional assays to establish exactly what transcriptional regulatory proteins bind the putative cis-elements. We wish to also characterize the putative promoter in vivo. To thoroughly examine the promoter we established transgenic mouse lines carrying the promoter luciferase constructs. We will first examine the tissue distribution of the activity for the promoter in the mouse with an emphasis on the intestine. Previous work has established that transport processes of nutrients, including vitamins, undergo clear developmental regulation. Nothing is known about possible regulation of the thiamine uptake process during development and whether this includes promoter regulation. These issues will be addressed in this proposal.