Abstract
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 revealed multiple putative cis-acting regulatory elements including SP1, CK, NFY-CBF, Tant, GCN4, and one progesterone and several retinoic acid response elements. The upstream sequence lacked TATA and CAAT boxes, but contained a high G/C rich region within the first 300 bp. A single transcriptional initiation site was identified by primer extension in rat kidney and small intestine, ~ 103 bp upstream of the previously 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 transfected murine Inner Medullary Collecting Duct (mIMCD-3) cells. Results demonstrated that [-]289, [-]1271 and [-]2630 bp constructs showed promoter activity that was significantly higher than the negative control construct (20-fold). These results also demonstrated that basal cis- acting elements are contained within [-]289 bp of the transcriptional start site. However, the functional activity of the [-]5567 bp construct was not significantly different from the negative control, suggesting that a negative regulatory element may be present between [-]2630 and [-]5567 bp of the promoter region.
Original language | English (US) |
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Pages (from-to) | 314-319 |
Number of pages | 6 |
Journal | Biochimica et Biophysica Acta - Gene Structure and Expression |
Volume | 1442 |
Issue number | 2-3 |
DOIs | |
State | Published - Nov 8 1998 |
Keywords
- (Rat)
- Molecular cloning
- NHE-2 promoter
- Sodium hydrogen antiporter
ASJC Scopus subject areas
- Structural Biology
- Biophysics
- Biochemistry
- Genetics