REGULATION OF SERUM AMYLOID A PROTEIN SYNTHESIS

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DESCRIPTION: (Adapted from the investigator's abstract). The goal of the proposed research is to elucidate the molecular mechanism of induction of serum amyloid A (SAA) protein synthesis in liver. SAA protein is an acute phase protein and is the precursor of amyloid protein A (AA), the main protein constituent of amyloid fibrils deposited in tissues in the disease associated with chronic inflammation e.g., rheumatoid arthritis, juvenile chronic arthritis. During periods of inflammation SAA protein synthesis in liver increases dramatically which has been found to be due to increased transcription of this gene. The objective is therefore to identify the regulatory elements of the SAA gene and the molecular events involved in this inducible biosynthetic process. To achieve these objectives investigators intend to specifically pursue the following sequences: (1) identification of DNA-binding domains that are required for the transcriptional activation; (2) determining the sequence of the regulatory regions; (3) characterization of these regions using a reporter chloramphenicol acetyl transferase (CAT) gene. Specific DNA binding domain, where transcription factor(s) bind to influence transcription of the gene, will be determined by DNA- protein gel retardation assay using fragments of the SAA genomic DNA. Identity and sequence of the binding domain will be determined by methylation interference assay. This regulatory structural element (s) will be characterized by ligating these sequences to an easily identifiable chloramphenicol acetyl transferase gene, whose transient expression in a transfected primary culture of hepatocytes will be followed in presence and in absence of SAA inducer (interleukin-1, interleukin-6). The potential importance of this project is to gain insight into cellular control mechanism of SAA gene induction. These studies will enable the formation of the basis for future work regarding the regulation of SAA biosynthesis in patients with disease which predisposes to amyloidosis.