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Am J Physiol Renal Physiol 282: F898-F909, 2002. First published December 18, 2001; doi:10.1152/ajprenal.00268.2001
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Vol. 282, Issue 5, F898-F909, May 2002

Genomic organization of the 5' end of human beta -ENaC and preliminary characterization of its promoter

Christie P. Thomas, Randy W. Loftus, Kang Z. Liu, and Omar A. Itani

Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242-1081

The mRNA for the beta -subunit of the epithelial Na+ channel (beta -ENaC) is regulated developmentally and, in some tissues, in response to corticosteroids. To understand the mechanisms of transcriptional regulation of the human beta -ENaC gene, we characterized the 5' end of the gene and its 5'-flanking regions. Adaptor-ligated human kidney and lung cDNA were amplified by 5' rapid amplification of cDNA ends, and transcription start sites of two 5' variant transcripts were determined by nuclease protection or primer extension assays. Cosmid clones that contain the 5' end of the gene were isolated, and analysis of these clones indicated that alternate first exons ~1.5 kb apart and ~ 45 kb upstream of a common second exon formed the basis of these transcripts. Genomic fragments that included the proximal 5'-flanking region of either transcript were able to direct expression of a reporter gene in lung epithelia and to bind Sp1 in nuclear extracts, confirming the presence of separate promoters that regulate beta -ENaC expression.

amiloride; gene regulation; transcription start sites; RNA splicing; gel mobility shift assay; beta -subunit of the epithelial sodium channel


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