Molecular cloning and characterization of the rat NHE-2 gene promoter

Yunhua Li Muller, James F. Collins, Liqun Bai, Hua Xu, Fayez K. Ghishan

Research output: Contribution to journalArticle

23 Scopus citations

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 languageEnglish (US)
Pages (from-to)314-319
Number of pages6
JournalBiochimica et Biophysica Acta - Gene Structure and Expression
Volume1442
Issue number2-3
DOIs
StatePublished - Nov 8 1998

Keywords

  • (Rat)
  • Molecular cloning
  • NHE-2 promoter
  • Sodium hydrogen antiporter

ASJC Scopus subject areas

  • Structural Biology
  • Biophysics
  • Biochemistry
  • Genetics

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