The fibrinogen molecule is synthesized in the liver and is the product of three closely linked and coordinately controlled genes. Fibrinogen biosynthesis increases 2-10 fold when the hepatocyte is exposed to two regulatory molecules; a polypeptide, made by activated macrophages called HSF/IL-6, and glucocorticoids. HSF exerts its stimulatory signal by causing an increase in transcription of each fibrinogen gene, and this stimulation is augmented by the steroid. The molecular details that initiate this increased transcriptional activity is not known. Gaining new information on this regulatory pathway is particularly promising at this time. The regulatory peptide SF) has been cloned, and recombinant HSF is available in sufficient amounts and purity. Also, responsive cell models have been established, and cloning strategies are sufficiently precise to define both the cis-acting elements and to identify and biochemically characterize the transacting factor(s) involved. The basic experimental strategies described herein follow well established procedures of creating a series of deletions in the 5' regulatory region of the gene, linking them to a reporter gene and transfecting them into a responsive cell. The procedures to generate specific DNA fragments has been significantly simplified by using the polymerase chain reactions. SF/IL-6 will be added to responsive cells transfected with specifically constructed recorder gene vectors in order to identify HSF/IL6 responsive promoter/enhancement elements. Once a deletion mutant containing the responsive element has been identified, it will be sequenced. The nucleotide sequence of the responsive promoter/enhancement site will be used to make a synthetic oligonucleotide which will then be coupled to a solid matrix. This specific DNA affinity resin will aid in the purification of the specific nuclear proteins that bind to and control the transcription of the fibrinogen genes. The specific HSF responsive transacting factor(s will be isolated and biochemically characterized including a partial amino acid sequence. These sequences will be used to construct oligonucleotides for the identification of a cDNA of the transacting protein. Information gained from the experiments described in detail in this proposal will provide new knowledge on how this essential blood clotting protein is regulated at the gene level.
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