Betts, Jonathan Charles; (1991) Structure and expression of the human serum amyloid A gene family. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Serum amyloid A (SAA) proteins comprise a family of phylogenetically conserved major acute phase reactants. To delineate the structure of the human SAA genes extensive Southern analysis was carried out using SAA probes. Multiple hybridizing fragments were detected with a range of restriction enzymes. Restriction fragment length polymorphisms (RPLPs) were identified for Bg1I, HindIII, NcoI and PstI which were inherited in a Mendelian fashion for the former two enzymes. Three contiguous clones isolated from a human λEMBL3 genomic library spanned 30kb, and contained a single SAA gene predicted to encode apoSAA1β. SAA1β was approximately 4kb long with 4 exons and showed >90% sequence homology to the previously characterized SAA2β in exon, intron and 5'-controlling regions. A fourth λ clone encoded an allele of SAA2β, namely SAA2α with HindIII, NcoI and PstI polymorphic sites. A third novel SAA gene (SAA4) was isolated from a cos202 cosmid library and mapped 10kb downstream of SAA2. These studies demonstrated that the human SAA gene family comprises four discrete loci. The effects of inflammatory mediators on gene expression were studied following transient transfection of SAACAT reporter genes into HepG2 cells. Similar responses were obtained for SAA1 and SAA2 constructs following treatment with IL1β, IL6 and TNFα. Whereas IL1 acts via the NFκB site in the SAA promoter, IL6 was shown to require both an intact NFκB site and a putative IL6 responsive element (IL6RE) upstream for full induction. In vitro bandshift and interference footprinting studies demonstrated binding of novel nuclear factors from IL6 treated HepG2 cells to both the NFκB region and an NFIL6-like recognition sequence at the IL6RE. IL1 and IL6 activated SAACAT synergistically. Functional studies indicated that this synergism was mediated through the NFκB site and in part through the putative IL6RE.

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