MOLECULAR PATHOLOGY OF AFIBRINOGENEMIA

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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.