Amyloid A protein deposits in tissues cause the clinical manifestations of reactive systemic amyloidosis secondary to inflammatory disorders. The genes coding these proteins, and the inducible synthesis of the proteins, and provide insight into the pathophysiology of amyloidosis as well as the regulatory mechanisms of transcription. The specific experimental proposals outlined below concern a family of three murine genesserum amyloid A genes (SAA)-that are transcribed by hepatocytes in response to leukocytic hormones released during inflammation. Normally these genes are transcriptionally quiescent but following induction with endotoxin the mRNA levels increase nearly 2000 fold. The following goals are: 1) To define which nucleic acid sequences in the SAA genes are essential for regulated expression. Portions of the genes including promoter regions will be joined to the coding domain of the bacterial chloramphenicol acetyltransferase(CAT) gene. Transcription will be assayed enzymatically following transfection and stable transformation of a hepatocyte cell line in which SAA expression is inducible. Deletion mutations will be generated to localize enhancer and/or inducible elements in the genes. The putative promoters will be fused to the human growth hormone structural gene and introduced into fertilized mouse ova; transgenic studies may help to identify tissue-specific DNA regulatory sequences. Changes in chromatin structure detected by nuclease hypersensitivity may locate regulatory regions. 2) To identify SAA DNA binding proteins in induced and uninduced cellular extracts by polyacrylamide gel DNA binding and in vitro transcription assays. 3) To study the consequences of SAAprotein overproduction in transgenic mice. In particular, the necessity of concommitant inflammation in the pathogenesis of amyloidosis will be determined. Site-directed mutagenesis of the SAA transgene may identify domains in SAA that are amyloidogenic. A program of study of relevant biochemistry and molecular biology courses is also proposed as well as participation in Genetics Clinics. Long-term goals of the investigator include contributing to our knowledge and treatment of hereditary disorders at a molecular level.
