TRANSCRIPTIONAL REGULATION OF FIBRINOGEN BIOSYNTHESIS

纤维蛋白原生物合成的转录调控

• 批准号: 3361646
• 负责人: GERALD M FULLER
• 金额: $ 20.17万
• 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/3361646
• 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; natural gene amplification; nucleic acid sequence; oligonucleotides; phosphorylation; polymerase chain reaction; protein biosynthesis; protein purification; 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：（根据研究者的摘要改编）纤维蛋白原 分子在肝脏中合成，是三个紧密的产物 连接和协调控制的基因。 纤维蛋白原生物合成 当肝细胞暴露于两个调节状态时，增加2-10倍 分子；由激活的巨噬细胞制成的多肽，称为HSF/IL-6， 和糖皮质激素。 HSF通过导致 每个纤维蛋白原基因的转录增加，这种刺激是 被类固醇增强。 启动此的分子细节 转录活性增加尚不清楚。 获得新信息 目前，在这种监管途径上特别有希望。 这 调节肽SF）已被克隆，重组HSF可用 足够的数量和纯度。 另外，响应迅速的细胞模型已经 建立和克隆策略足够精确，可以定义这两个 顺式作用元素，并识别和生化表征 涉及的交易因子。 基本的实验策略 本文所述遵循完善的程序，创建一系列 在基因的5'调节区域中删除，将其连接到 记者基因并将其转染到响应性细胞中。 程序 生成特定的DNA片段已通过 使用聚合酶链反应。 SF/IL-6将添加到响应速度 用特定构造的录音机基因向量转染的细胞中 为了识别HSF/IL6响应启动子/增强元件。 一次 已经确定了包含响应元件的删除突变体， 将被测序。 响应性的核苷酸序列 启动子/增强站点将用于制造合成寡核苷酸 然后将耦合到实心矩阵。 这个特定的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响应式交易因子（s将隔离，并且 生化表征包括部分氨基酸序列。 这些 序列将用于构建寡核苷酸以识别 交易蛋白的cDNA。 从 本提案中详细描述的实验将提供新的知识 关于该基因上的必需血液凝结蛋白如何在基因上调节 等级。