CHARACTERIZATION OF THE 5'-FLANKING REGION OF OK CELL-TYPE-II NA-P-I COTRANSPORTER GENE

The renal type II Na-P-i cotransport is the rate-limiting step in prox imal tubular phosphate (P-i) reabsorption. Among the different ''proxi mal tubular'' cell lines, this transporter seem only to be expressed i n opossum kidney cells (OK cells). We have isolated the 5'-flanking re gion of the ok-Npt2 gene (OK cell type II Na-P-i cotransporter) includ ing exons 1-3 and containing a TFIID site (TATA box), a GCCAAT site, a n AP1 site, and two microsatellite GGAA repeats. Major transcription i nitiation sites were determined by primer extension and rapid amplific ation of 5' cDNA ends (5'-RACE). A 327-bp fragment containing the TFII D and GCAAT element was driving the downstream luciferase reporter gen e in homologous transfection assays. Slightly reduced promoter activit y was observed with a 198-bp fragment containing the GCAAT element; sh orter fragments were without activity. Promoter activity (327-bp fragm ent) could also be observed in transfections into HeLa cells but not i n U937 human macrophage cells, MCT mouse kidney cortex cells, and MDCK cells. Different ''physiological'' stimuli known to be associated wit h altered proximal tubular Na-P-i cotransport activity are without eff ect on transcriptional activity in above homologous transfection exper iments.