MOLECULAR-CLONING AND CHARACTERIZATION OF THE RAT NHE-2 GENE PROMOTER

To understand the molecular mechanisms underlying NHE-2 regulation in the mammalian kidney and intestine, we cloned and sequenced 5.6 kb of the 5'-flanking region of the rat NHE-2 gene. DNA sequence analysis re vealed multiple putative cis-acting regulatory elements including SP1, CK, NFY-CBF, Tant, GCN4, and one progesterone and several retinoic ac id response elements. The upstream sequence lacked TATA and CAAT boxes , but contained a high G/C rich region within the first 300 bp. A sing le transcriptional initiation site was identified by primer extension in rat kidney and small intestine, similar to 103 bp upstream of the p reviously identified 5'-end of the rat NHE-2 cDNA. Various regions of the promoter (from [-]5567 to [+]105 bp) were tested for their ability to drive expression of the luciferase reporter gene in transiently tr ansfected murine Inner Medullary Collecting Duct (mIMCD-3) cells. Resu lts demonstrated that [-]289, [-]1271 and [-]2630 bp constructs showed promoter activity that was significantly higher than the negative con trol construct (20-fold). These results also demonstrated that basal c is-acting elements are contained within [-]289 bp of the transcription al start site. However, the functional activity of the [-]5567 bp cons truct was not significantly different from the negative control, sugge sting that a negative regulatory element may be present between [-]263 0 and [-]5567 bp of the promoter region. (C) 1998 Elsevier Science B.V . All rights reserved.