REGULATION OF SYNTHESIS OF BLOOD COAGULATION FACTOR XIII

凝血因子 XIII 合成的调节

• 批准号: 3087220
• 负责人: LAURIE J WEISBERG
• 金额: $ 6.76万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-04-01 至 1990-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3087220
• 关键词: bacteriophage M13; blood coagulation disorders; complementary DNA; congenital blood disorder; fibrin stabilizing factor; fibrinolysis; fibrinolytic agents; genetic library; genetic manipulation; genetic mapping; genetic regulation; genetic translation; human tissue; liver cells; messenger RNA; molecular cloning; nucleic acid chemical synthesis; nucleic acid structure; tissue /cell culture

The goal of this project is to characterize the genetic regulation of synthesis of the a-chain of Factor XIII, the clotting factor essential for covalent stabilization of the fibrin clot. An existing human hepatocyte cDNA library in lambdaGTll will be screened with antibodies against the Factor XIII a-chain, which is the enzymatically active subunit. If screening with antibodies is unsuccessful, cyanogen bromide fragments will be purified and sequenced. Based on amino acid sequences with limited codon degeneracy, oligonucleotide probes will be constructed to screen the library. The cloned cDNAs for Factor XIII a-chain will be characterized by restriction enzyme mapping and hybrid-selected in vitro translation. If the cloned cDNA is incomplete, full-length Factor XIII a-chain DNA will be synthesized using a neucleotide restriction fragment as a primer for extension with purified poly(A+) mRNA and reverse transcriptase. Using the Southern blot technique, the gene structure in normal individuals and in patients with cogenital deficiency of Factor XIII will be analyzed. Restriction fragments of full-length cDNA will be inserted into the sequencing vector, bacteriophage M13, and overlapping DNA inserts will be sequenced. A variety of possible physiologic mechanisms for regulating synthesis of Factor XIII a-chains will be studied. Using cDNA probes, the mRNA from cultured rat hepatocytes will be analyzed after incubation of intact cells with various components of the activated coagulation and fibrinolytic systems: fibrin degradation products, a-chains of the intermediate and active forms of plasma Factor XIII, Factor XIII b-chains, thrombin, plasmin, and coagulation Factors IXa and Xa. Similarly, the effects of defibrination or induction of fibrinolysis in animals on Factor XIII a-chain mRNA in liver tissue will be determined. The knowledge obtained from these studies will contribute to understanding the genetic regualtion of Factor XIII synthesis and eventually to understanding the coordination of synthesis of two gene products, the Factor XIII a- and b-chains.

该项目的目标是描述 合成因子XIII的a链，因子XIII是一种必需的凝血因子 纤维蛋白凝块的共价稳定。 现有的人类肝细胞 将用抗GT 11的抗体筛选GT 11中的cDNA文库。 因子XIII a链，其为酶活性亚基。 如果 用抗体筛选不成功，溴化氰片段将 进行纯化和测序。 基于氨基酸序列， 密码子简并性，将构建寡核苷酸探针以筛选密码子简并性。 图书馆 因子XIII α链的克隆cDNA将通过以下表征： 限制性内切酶图谱和杂交选择体外翻译。 如果 克隆的cDNA是不完整的，全长因子XIII a链DNA将被 使用核苷酸限制性片段作为引物合成， 用纯化的poly（A+）mRNA和逆转录酶延伸。 使用 Southern杂交技术检测了正常人和非正常人的基因结构， 将分析先天性因子XIII缺乏的患者。 将全长cDNA的限制性片段插入到 测序载体，噬菌体M13和重叠DNA插入物将被 测序 多种可能的生理机制调节合成的 将研究因子XIII α链。 使用cDNA探针， 培养的大鼠肝细胞将在孵育完整细胞后进行分析 与激活凝血和纤溶的各种成分 系统：纤维蛋白降解产物，中间体的α链和 血浆因子XIII的活性形式，因子XIII b-链，凝血酶， 纤溶酶和凝血因子IXa和Xa。 同样， 在动物中对因子XIII的纤维蛋白溶解的脱髓鞘或诱导 将测定肝组织中的α-链mRNA。 从这些研究中获得的知识将有助于理解 因子XIII合成的遗传调节， 了解两种基因产物合成的协调， 因子XIII a链和b链。