MOLECULAR PATHOLOGY OF AFIBRINOGENEMIA

我在这个建议中的目标是更全面地了解 协调基因表达和多链蛋白组装和功能， 使用凝血因子纤维蛋白原作为模型系统。 纤维蛋白原是 在肝细胞中产生和组装的多链蛋白质， 由单拷贝基因产生的多肽。 基因会协调反应 对这种蛋白质的需求，使纤维蛋白原成为一种有吸引力的 系统研究协调表达的一般机制， 几个基因。 肝细胞内功能性纤维蛋白原的形成 使其成为检测多肽结构特征的理想系统 对多链蛋白质的组装至关重要。 的 突变基因的可用性，例如来自无纤维蛋白原血症患者的突变基因 在其中的缺陷的水平是未知的，将允许比较， 正常的基因和区域的定义是重要的， 纤维蛋白原的产生、组装或功能。 这种比较的第一步是检查功能能力 分离的正常基因。 我已经克隆了人类的基因和cDNA 正常人的纤维蛋白原。 我会用它们来完成开发 表达引入的全长人源化的细胞培养系统 纤维蛋白原基因的条件下，允许评估基因的 形成mRNA或蛋白质的能力。 转录能力将是 在最生理条件下在同源肝细胞中检查 纤维蛋白原基因转染大鼠肝癌的实验研究 线 对于蛋白质功能和组装的研究，我将尝试增强 通过放置Rous肉瘤或SV40来表达正常纤维蛋白原基因 上游的启动子-增强子元件。 转染这样的基因， 被特定的实验室构建的突变所改变， 鉴定干扰正常组装的多肽区域， 纤维蛋白原。 或者，研究纤维蛋白原产生的定义 转染的正常和改变的基因的组合可以在 不产生纤维蛋白原的肝细胞瘤或其它非肝细胞。 使用这些转染系统和核苷酸序列分析， 将研究从无纤维蛋白原血症患者中分离的 这种出血性疾病的分子基础，因此，重要的区域 正常基因表达或蛋白质组装。

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.