IDENTIFICATION OF 5'-FLANKING SEQUENCES THAT AFFECT HUMAN PANCREATIC CHOLESTEROL ESTERASE GENE-EXPRESSION

Pancreatic cholesterol esterase (CEL) is shown to play a significant r ole in cholesterol metabolism. As the hydrolytic property of CEL is im portant for transport of lipid esters, the extent of its expression is an important factor in the metabolism of lipids. Therefore, to identi fy the elements that modulate the transcription of its mRNA, we obtain ed several cosmid clones carrying the CEL gene. From one of these cosm id clones a 6.5-kb SmaI fragment that hybridizes to the 5' untranslate d region of CEL cDNA was subcloned. Primer extension and S1 protection assays revealed that the 5' untranslated region is relatively short ( only 20 nucleotides long). An analysis of the 5' flanking sequence rev ealed typical TATA and CCAAT boxes that impart tissue specificity. Fur ther, consensus sequences of several cis elements described earlier co uld also be detected in this region. To identify the promoter sequence s, various deletion constructs of the 5' region were made using polyme rase chain reaction. These constructs were subcloned into a bacterial plasmid vector carrying chloramphenicol acetyltransferase (CAT) as the reporter gene and transfected into HepG-2 cells. CAT activity in the cell homogenate of the transfected cells was measured 48 h after trans fection. Results showed that the promoter activity of human pancreatic CEL mRNA is in a large segment of 5' flanking sequences spanning the -10 and -930 nucleotides of its gene.