COORDINATE REGULATION OF FIBRINOGEN GENE TRANSCRIPTION

主要的凝血蛋白纤维蛋白原是在肝脏中合成的。 并以三种不同的多聚体的形式分泌到血液中 亚基，指定为Apha、Bbeta和Gamma。该计划的总体目标 这项建议中描述的研究是为了确定如何表达 编码三个纤维蛋白原亚基的单独基因是协调的 受监管的。基本的实验策略是利用强大的基因 在纤维蛋白原中产生精确突变的基因转染法 基因调控区的体外实验及功能后果分析 将DNA导入培养的肝细胞后的这些变化。 将使用来自非洲爪蛙的高度纯化的肝细胞， 其中纤维蛋白原的合成由肾上腺戏剧性地诱导 类固醇，糖皮质激素。这项提案特别关注 伽马亚基的基因编码，具体目的如下：1) γ-纤维蛋白原上游调控区的分离 亚基基因；2)将伽马基因DNA导入非洲爪哇 糖皮质激素诱导性肝细胞的转染性和示范性 转录；3)产生缺失和点突变 转录调控区域及其对基因表达的影响分析 转基因肝细胞的转录；4)蛋白质的鉴定 与关键DNA调控元件的相互作用；以及50个发展 用于解剖特定分子的体外转录系统 转录控制的潜在相互作用。

The major blood-clotting-protein, fibrinogen, is synthesized in the liver and secreted into the bloodstream as a multimer of three different subunits, designated Aalpha, Bbeta, and gamma. The overall goal of the research described in this proposal is to determine how expression of the separate genes coding for the three fibrinogen subunits is coordinately regulated. The basic experimental strategy is to use the powerful gene transfection approach to generate precise mutations in the fibrinogen gene regulatory regions in vitro and analyze the functional consequences of these alterations after introducing the DNA into cultured hepatocytes. Highly purified hepatocytes from the frog Xenopus laevis will be used, in which fibrinogen synthesis is dramatically induced by the adrenal steroids, glucocorticoids. This proposal focuses specifically on the gene coding for the gamma subunit, with the following specific aims; 1) Isolation of the upstream regulatory region of the gamma fibrinogen subunit gene; 2) Introduction of the gamma gene DNA into primary Xenopus liver cells by transfection and demonstration of glucocorticoid-inducible transcription; 3) Generation of deletion and point mutations in the transcriptional regulatory region and analysis of effects on transcription in transfected liver cells; 4) Identification of protein interactions with critical DNA regulatory elements; and 50 Development of an in vitro transcription system for dissecting the specific molecular interactions underlying control of transcription.