REGULATION OF FOLATE-MEDIATED ONE-CARBON METABOLISM

叶酸介导的一碳代谢的调节

• 批准号: 2133623
• 负责人: DEAN R APPLING
• 金额: $ 6.81万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-01 至 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2133623
• 关键词: complementary DNA; folate; gel mobility shift assay; gene expression; genetic manipulation; genetic regulatory element; genetic transcription; laboratory rat; molecular cloning; nuclear magnetic resonance spectroscopy; nuclear runoff assay; transcription factor; vitamin metabolism

My immediate goals are: (1) to isolate clones for the rat gene encoding C1-THF synthase; (2) to use an in vitro transcription system to analyze regulatory elements of the C1-THF synthase gene in rat; and (3) to determine the extent and the control points of compartmentation of folate coenzymes and their activated one-carbon units between the cytoplasm and mitochondria. The health-relatedness of this project stems from the ubiquitous nature of folate-mediated one-carbon metabolism in all cells, and the critical role played by C1-THF synthase in folate interconversions. The experimental design and methods used to accomplish the specific aims involve (1) the use of rat C1-THF synthase cDNA clones as probes for the isolation of the rat gene encoding C1-THF synthase; (2) characterization of the C1-THF synthase gene by sequence analysis, and deletion analysis of the 5' flanking region to define promoter and other regulatory sequences; (3) identification of trans-acting transcription factors using a combination of gel mobility shift, footprinting and in vitro transcription assays; and (4) analysis of the intercompartmental pathway of folate-mediated one-carbon metabolism in yeast using a combination of biochemical, molecular genetic, and NMR techniques.

我的近期目标是：（1）分离编码大鼠基因的克隆 C1-THF合酶； (2)利用体外转录系统进行分析 大鼠C1-THF合酶基因的调控元件； (3) 至 确定叶酸的划分程度和控制点 辅酶及其在细胞质和细胞质之间的活性一碳单位 线粒体。 该项目与健康的相关性源于 叶酸介导的一碳代谢在所有细胞中普遍存在， 以及 C1-THF 合酶在叶酸相互转化中发挥的关键作用。 用于实现特定目标的实验设计和方法 涉及 (1) 使用大鼠 C1-THF 合酶 cDNA 克隆作为探针 分离编码C1-THF合酶的大鼠基因； (2) 表征 通过序列分析和缺失分析来确定C1-THF合酶基因 5'侧翼区域定义启动子和其他调控序列； (3) 使用组合鉴定反式作用转录因子 凝胶迁移率变化、足迹和体外转录测定；和（4） 叶酸介导的一碳分子的室间途径分析 酵母中的代谢结合了生化、分子遗传学、 和核磁共振技术。