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 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, approximately 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.
Muller
Y L
YL
Departments of Pediatrics and Physiology, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, 1501 N. Campbell Ave., Tucson, AZ 85724, USA.
Collins
J F
JF
Bai
L
L
Xu
H
H
Ghishan
F K
FK
D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D008322:Mammals; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D051381:Rats / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D017923:Sodium-Hydrogen Exchangers / Q000096:biosynthesis / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; C513110:Slc9a2 protein, rat; D017923:Sodium-Hydrogen Exchangers; D014157:Transcription Factors