MOLECULAR PATHOLOGY OF AFIBRINOGENEMIA

我在这项建议中的目标是更全面地了解 协调的基因表达和多链蛋白的组装和功能 使用凝血因子纤维蛋白原作为模型系统。纤维蛋白原是一种 多链蛋白在肝细胞中的合成和组装 由单拷贝基因产生的多肽。基因的反应是协调的 对更多这种蛋白质的需求，使纤维蛋白原成为一个有吸引力的 研究协调表达的一般机制的系统 几个基因。肝细胞内功能性纤维蛋白原的形成 使其成为研究多肽结构特征的理想系统 对于多链蛋白的组装是必不可少的。这个 突变基因的可用性，例如来自无纤维蛋白原血症患者的基因 谁的缺陷程度未知，将允许与 正常基因和重要区域的定义 纤维蛋白原的生产、组装或功能。 这种比较的第一步是检查功能能力 分离出的正常基因。我已经克隆了人类的基因和cDNA 正常受试者的纤维蛋白原。我将使用它们来完成开发 表达全长人类的细胞培养系统 在允许评估基因的条件下的纤维蛋白原基因 形成信使核糖核酸或蛋白质的能力。转录能力将是 在大多数生理条件下对同种肝细胞进行检查 基因导入产生纤维蛋白原大鼠肝癌的环境 台词。对于蛋白质功能和组装的研究，我将努力提高 Rous肉瘤或SV40对正常纤维蛋白原基因表达的影响 启动子-增强子元件在上游。这类基因的转染具有 被特定的实验室构建的突变所改变，将使我能够 确定干扰正常组装的多肽区域 纤维蛋白原。或者，对由Defined产生的纤维蛋白原的研究 转基因的正常基因和改变基因的组合可以在 不产生纤维蛋白原的肝癌或其他非肝细胞。 利用这些转染系统和核苷酸序列分析，基因 从无纤维蛋白原血症患者中分离出来的研究将确定 这种出血障碍的分子基础，因此，重要的区域 在正常的基因表达或蛋白质组装中。

My goal in this proposal is to understand more fully mechanisms of coordinated gene expression and multichain protein assembly and function, using the clotting factor, fibrinogen, as a model system. Fibrinogen is a multichain protein produced and assembled in liver cells from 3 polypeptides produced by single copy genes. The genes respond coordinately to a demand for more of this protein, making fibrinogen an attractive system to study general mechanisms underlying the coordinated expression of several genes. The formation of functional fibrinogen within hepatocytes makes it an ideal system to examine structural features of polypeptides that are essential for the assembly of multichain proteins. The availability of mutant genes, such as those from afibrinogenemic patients in whom the level of the defect is unknown, will allow a comparison with normal genes and the definition of regions that are important for the production, assembly or function of fibrinogen. The first step in such a comparison is to examine the functional capability of the isolated normal genes. I have cloned the genes and cDNAs for human fibrinogen from normal subjects. I will use them to complete development of cell culture systems that express introduced full length human fibrinogen genes under condition that permit an assessment of the gene's ability to form mRNA or protein. Transcriptional competence will be examined under the most physiologic conditions in an homologous liver cell environment by transfecting genes into fibrinogen-producing rat hepatoma lines. For studies of protein function and assembly, I will try to enhance the expression of normal fibrinogen genes by placing Rous sarcoma or SV40 promoter-enhancer elements upstream. Transfection of such genes which have been altered by specific laboratory constructed mutation would allow me to identify polypeptide regions which interfere with the normal assembly of fibrinogen. Alternatively the study of fibrinogens produced by defined combinations of transfected normal and altered genes can be conducted in non-fibrinogen-producing hepatoma or other non-hepatic cells. Using these transfection systems and nucleotide sequence analysis, genes isolated from afibrinogenemic patients will be studied to define the molecular basis of this bleeding disorder and, thereby, regions important in normal gene expression or protein assembly.