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：(改编自研究人员摘要)纤维蛋白原 分子是在肝脏中合成的，是三个紧密结合的产物 连锁和协调控制的基因。纤维蛋白原生物合成 当肝细胞暴露在两种调节剂下时，增加2-10倍 分子；一种由激活的巨噬细胞产生的多肽，称为HSF/IL-6， 和糖皮质激素。HSF通过引起一种 增加每个纤维蛋白原基因的转录，这种刺激是 加上类固醇。启动这一过程的分子细节 转录活性增加尚不清楚。获取新信息 在这条调控途径上，这个时候就特别有希望了。这个 调节肽SF)已被克隆，重组HSF可在 足够的数量和纯度。此外，响应性细胞模型已经被 和克隆策略足够精确，以定义这两个 顺式作用元素，并鉴定和生化表征 参与交易因素(S)。基本的实验策略 这里描述的遵循公认的程序来创建一系列 基因5‘调控区的缺失，将它们与 报告基因，并将其导入反应细胞。程序 通过以下方式大大简化了生成特定DNA片段 使用聚合酶链式反应。SF/IL-6将添加到Response 转导特效记录器基因载体的细胞 以确定HSF/IL6反应启动子/增强元件。一旦成为 含有反应元件的缺失突变体已被鉴定，它 将被测序。应答基因的核苷酸序列 启动子/增强位点将用于合成寡核苷酸 然后将其耦合到固体基质中。这种特殊的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。这个 特定的人类免疫缺陷病毒应答交易因子(S将被分离和 生化特征的，包括部分氨基酸序列。这些 序列将被用来构建用于鉴定的寡核苷酸 交易蛋白的一个cDNAs。从以下方面获得的信息： 本提案中详细描述的实验将提供新的知识 关于这种重要的凝血蛋白是如何在基因上调节的 水平。