TRANSCRIPTIONAL REGULATION OF FIBRINOGEN BIOSYNTHESIS

纤维蛋白原生物合成的转录调控

• 批准号: 3361648
• 负责人: GERALD M FULLER
• 金额: $ 21.86万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-04-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3361648
• 关键词: affinity chromatography; biological signal transduction; complementary DNA; fibrinogen; genetic enhancer element; genetic promoter element; genetic transcription; interleukin 6; liver cells; macrophage; molecular cloning; mutant; nucleic acid sequence; oligonucleotides; phosphorylation; polymerase chain reaction; protein biosynthesis; protein purification; proteins; reporter genes; transcription factor; transfection

DESCRIPTION: (Adapted from investigator's abstract) The fibrinogen molecule is synthesized in the liver and is the product of three closely linked and coordinately controlled genes. Fibrinogen biosynthesis increases 2-10 fold when the hepatocyte is exposed to two regulatory molecules; a polypeptide, made by activated macrophages called HSF/IL-6, and glucocorticoids. HSF exerts its stimulatory signal by causing an increase in transcription of each fibrinogen gene, and this stimulation is augmented by the steroid. The molecular details that initiate this increased transcriptional activity is not known. Gaining new information on this regulatory pathway is particularly promising at this time. The regulatory peptide SF) has been cloned, and recombinant HSF is available in sufficient amounts and purity. Also, responsive cell models have been established, and cloning strategies are sufficiently precise to define both the cis-acting elements and to identify and biochemically characterize the transacting factor(s) involved. The basic experimental strategies described herein follow well established procedures of creating a series of deletions in the 5' regulatory region of the gene, linking them to a reporter gene and transfecting them into a responsive cell. The procedures to generate specific DNA fragments has been significantly simplified by using the polymerase chain reactions. SF/IL-6 will be added to responsive cells transfected with specifically constructed recorder gene vectors in order to identify HSF/IL6 responsive promoter/enhancement elements. Once a deletion mutant containing the responsive element has been identified, it will be sequenced. The nucleotide sequence of the responsive promoter/enhancement site will be used to make a synthetic oligonucleotide which will then be coupled to a solid matrix. This specific DNA affinity resin will aid in the purification of the specific nuclear proteins that bind to and control the transcription of the fibrinogen genes. The specific HSF responsive transacting factor(s will be isolated and biochemically characterized including a partial amino acid sequence. These sequences will be used to construct oligonucleotides for the identification of a cDNA of the transacting protein. Information gained from the experiments described in detail in this proposal will provide new knowledge on how this essential blood clotting protein is regulated at the gene level.

描述：（改编自研究者的摘要）纤维蛋白原 分子在肝脏中合成，是三个密切相关的产物 连锁和协调控制的基因。 纤维蛋白原生物合成 当肝细胞暴露于两种调节剂时，增加 2-10 倍 分子；一种由活化的巨噬细胞产生的多肽，称为 HSF/IL-6， 和糖皮质激素。 HSF 通过引起 每个纤维蛋白原基因的转录增加，这种刺激是 由类固醇增强。 引发这一现象的分子细节 转录活性的增加尚不清楚。 获取新信息 目前，这一监管途径尤其有希望。 这 调节肽SF）已被克隆，重组HSF可用于 足够的量和纯度。 此外，响应细胞模型已 已建立，并且克隆策略足够精确来定义两者 顺式作用元件并鉴定和生化表征 涉及的交易因素。 基本实验策略 本文中描述的遵循创建一系列的既定程序 基因 5' 调控区的缺失，将它们与 报告基因并将其转染到响应细胞中。 程序 生成特定 DNA 片段的过程已大大简化 使用聚合酶链式反应。 SF/IL-6 将添加到响应式 用专门构建的记录基因载体转染的细胞 为了鉴定 HSF/IL6 响应启动子/增强元件。 一旦一个 含有响应元件的缺失突变体已被鉴定，它 将被测序。 响应的核苷酸序列 启动子/增强位点将用于制备合成寡核苷酸 然后将其耦合到固体基质。 这种特定的 DNA 亲和力 树脂将有助于纯化特定的核蛋白 结合并控制纤维蛋白原基因的转录。 这 特定的 HSF 反应因子将被分离并 生化特征包括部分氨基酸序列。 这些 序列将用于构建寡核苷酸以进行鉴定 反式蛋白的cDNA。 获得的信息来自 本提案中详细描述的实验将提供新知识 关于这种重要的凝血蛋白如何在基因上受到调节 等级。