CLONING AND PARTIAL CHARACTERIZATION OF THE MOUSE GLUTAMINE-FRUCTOSE-6-PHOSPHATE AMIDOTRANSFERASE (GFAT) GENE PROMOTER

Glutamine:fructose-6-phosphate amidotransferase (GFAT) is the enzyme t hat is rate limiting in the synthesis of glucosamine and hexosamines. Glucosamine has been proposed to contribute to the glucotoxicity of di abetes. Evidence that the gene encoding GFAT is transcriptionally regu lated prompted us to clone and characterize its promoter. The position of the mouse GFAT promoter relative to the translational start site w as located by primer extension and found to be 149 bp upstream of the translational start site. A 1.9 kb Sad fragment of the GFAT gene was f ound to contain the promoter and 88 bp of sequence downstream of the t ranscriptional start site. This promoter segment could drive expressio n of a luciferase reporter gene, could confer correct transcriptional initiation to the reporter and could confer the EGF-responsiveness pre viously observed in the native gene. The mouse GFAT promoter lacks a c anonical TATA box and has several GC boxes within a highly CC-rich reg ion. Deletional analysis of the promoter indicated that a proximal ele ment extending to -120 relative to the transcriptional start site coul d confer reporter expression at a level of 57% of the 1.9 kb construct . Detailed analysis of this proximal region by DNase I footprinting, e lectrophoretic mobility shift assays and site-directed mutagenesis ind icated that Spl binds to three elements in this proximal promoter segm ent and plays a vital role in regulation of transcription from this ge ne.