COORDINATE REGULATION OF FIBRINOGEN GENE TRANSCRIPTION

主要的凝血蛋白纤维蛋白原在肝脏中合成 并以三种不同的多聚体形式分泌到血液中 亚基，命名为A α、B β和γ。 的总体目标 本提案中描述的研究旨在确定 编码三种纤维蛋白原亚基的独立基因是协调的， 监管. 基本的实验策略是使用强大的基因 在纤维蛋白原中产生精确突变的转染方法 基因调控区在体外和分析功能的后果 将DNA导入培养的肝细胞后， 将使用来自蛙非洲爪蟾的高度纯化的肝细胞， 其中纤维蛋白原的合成是由肾上腺 类固醇糖皮质激素 本提案特别侧重于 编码γ亚基的基因，具体目标如下：1） γ纤维蛋白原上游调控区的分离 亚基基因; 2）将γ基因DNA导入初级爪蟾 通过转染和糖皮质激素诱导的肝细胞 转录; 3）产生缺失和点突变， 转录调控区及其作用分析 在转染的肝细胞中的转录; 4）蛋白质的鉴定 与关键DNA调控元件的相互作用;和50发展 一个体外转录系统， 转录控制的基础相互作用。

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.