Molecular Characterization of the SLC19A2 Promoter

SLC19A2 启动子的分子表征

• 批准号: 6896106
• 负责人: JACK C REIDLING
• 金额: $ 4.89万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-02 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6896106
• 关键词: DNA binding protein; biological transport; developmental genetics; gastrointestinal system; gel mobility shift assay; gene expression; gene mutation; genetic promoter element; genetic transcription; genetically modified animals; immunocytochemistry; laboratory mouse; postdoctoral investigator; protein localization; protein structure function; species difference; thiamine; transcription factor; transfection; vesicle /vacuole

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.

描述（由申请人提供）：饮食中硫胺素是人类必不可少的营养。硫胺素在肠中的摄取已经进行了充分的研究，但是摄入过程的分子机制和调节尚不清楚。最近，硫胺素转运蛋白是从几个人体组织克隆的（SLC19A2）。我们停用了小肠和大肠中该转运蛋白的存在。为了了解基因表达水平的转运蛋白的调节，我们克隆了5'调节区域，并使用荧光素酶报告基因系统确定了活动所需的最小区域。我们能够在肠道CACO-2细胞中表现出高水平的克隆假定启动子的活性，其活性大约是肝脏HEPG2细胞的两倍。使用基于Web的计算机程序在最小区域中检测到了几个顺式调节元素。我们计划在体外进一步表征推定的启动子区域，并使用凝胶移位和超移分析，突变分析和功能测定法，以确切确定哪种转录调节蛋白结合了推定的顺式元素。我们也希望在体内表征推定的启动子。为了彻底检查启动子，我们建立了带有启动子荧光素酶构建体的转基因小鼠系。我们将首先检查小鼠中启动子活性的组织分布，重点是肠。先前的工作已经确定，包括维生素在内的养分运输过程受到明确的发育调节。关于在开发过程中对硫胺素摄取过程的可能调节以及是否包括启动子调节的可能调节。这些问题将在本提案中解决。