Our long-term goal is to define the molecular which determine the physiologic and pathologic levels of hemostatic protein synthesized in the liver. We plan to begin by analyzing the """"""""acute phase reaction."""""""" Maintenance of hemostasis requires regulatory mechanisms which can restore the blood coagulation factors to physiological levels when they become depleted. An experimental example of depletion is that produced by defibrinogenation. There is a co- ordinate 10-fold increase in the levels of the mRNAs coding for alpha, beta and r-fibrinogen and subsequently the peptides themselves. This response is known to be mediated at least in part by Hepatocyte Stimulating Factor (HSF), an inter-cellular messenger synthesized in monocytes, and Kupfer cells. Our goal is to utilize molecular biological approaches to analyse this relationship. We have constructed a set of adenovirus """"""""delivery"""""""" vectors for transferring molecularly modified fibrinogen genes into cells in culture. The chimeric viruses have the """"""""promoter"""""""" region of the r-fibrinogen gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene, and their ability to enter hepatocytes in culture was confirmed. A number of deletional mutants of the fibrinogen promoter have been constructed. Studies with these mutants suggests that cis-acting elements act both positively and negatively to control fibrinogen synthesis in hepatocytes. During the course of this project we plan to 1) identify and characterize the genomic DNA sequences required to transcribed and regulate fibrinogen mRNA levels in response to HSF, by the systematic construction and analysis of adenovirus and retrovirus shuttle vectors containing specific natural and synthetic fragments of the fibrinogen genes. Once putative regulatory domains have been identified, we shall confirm the function in vivo using transgenic mice. 2) identify the nuclear factors which regulate the transcription of the fibrinogen genes using gel retardation assays, DNA footprinting and in vitro transcription. 3) clone the cDNA for the Hepatocyte Stimulating Factor using a eukaryotic expression vector system. This will allow for subsequent characterization of the factor, localize the tissues where it is expressed, and begin examination of the mechanisms controlling its production in the """"""""acute phase reaction"""""""".
