TRANSCRIPTIONAL REGULATION OF FIBRINOGEN BIOSYNTHESIS

描述：（改编自研究者摘要） 分子是在肝脏中合成的，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因子时， 分子;一种多肽，由激活的巨噬细胞产生，称为HSF/IL-6， 和糖皮质激素。 HSF通过引起一种刺激信号来发挥其刺激作用。 增加每个纤维蛋白原基因的转录，这种刺激是 类固醇的作用 启动这个过程的分子细节 增加的转录活性是未知的。 获取新信息 在这个调控途径上的作用在这个时候特别有希望。 的 调节肽SF）已被克隆，重组HSF可在 量和纯度。 此外，反应性细胞模型已经被 建立，克隆策略是足够精确的定义， 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 本文所述遵循创建一系列 在基因的5'调控区的缺失，将它们连接到一个 报告基因，并将其导入应答细胞。 的程序 产生特异性DNA片段的方法已经大大简化， 使用聚合酶链式反应。 SF/IL-6将被添加至应答 用特异性构建的记录基因载体转染的细胞， 以鉴定HSF/IL 6应答启动子/增强元件。 一次 已经鉴定了含有应答元件的缺失突变体， 将被排序。 反应性的核苷酸序列 启动子/增强位点将用于制备合成寡核苷酸 然后将其偶联到固体基质上。 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。 的 将分离特异性HSF响应性交易因子， 生物化学特征包括部分氨基酸序列。 这些 序列将用于构建用于鉴定的寡核苷酸 反式作用蛋白的cDNA。 资料来自 本提案中详细描述的实验将提供新的知识 这种重要的凝血蛋白是如何在基因中被调节的 水平

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.