REGULATION OF FOLATE-MEDIATED ONE-CARBON METABOLISM

我的近期目标是：(1)分离大鼠编码基因的克隆 C1-THF合酶；(2)体外转录系统分析 大鼠c1-四氢呋喃合成酶基因的调控元件； 确定叶酸区隔的程度和控制点 胞质和胞质之间的辅酶及其活化的一碳单位 线粒体。这个项目与健康相关的原因是 叶酸介导的一碳代谢在所有细胞中普遍存在， 以及C1-四氢呋喃合成酶在叶酸相互转化中的关键作用。 为达到特定目的而采用的实验设计和方法 涉及(1)使用大鼠c1-四氢呋喃合成酶cdna克隆作为探针 大鼠编码C1-四氢呋喃合成酶基因的分离；(2)鉴定 C1四氢呋喃合成酶基因的序列分析和缺失分析 5‘侧翼区，定义启动子和其他调控序列；(3) 结合多种方法鉴定反式作用转录因子 凝胶迁移率变化、足迹和体外转录检测；以及(4) 叶酸介导的单碳室间途径分析 利用生物化学、分子遗传学、 和核磁共振技术。

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.