Isolation and characterization of the human CLC-5 chloride channel gene promoter

The human CLC-5 chloride channel is expressed mainly in the kidney and its mutations cause Dent's disease (a familial renal tubular syndrome with hypercalciuria, tubular proteinuria, rickets, nephrocalcinosis, and eventual renal failure). To gain insight into the regulatory mechanism of CLC-5 expression, a genomic clone that contains the 5'-flanking region of the human CLC-5 gene was isolated and characterized. Two types of 5'-ends of cDNA were isolated by 5'-rapid amplification of cDNA ends, and one of them, approximately 2.1 kbp upstream of ATG-containing exon II, was first identified in human. The major promoter activity was detected in the 5'-flanking region of this newly identified exon Ia. The sequence of the proximal 5'-flanking region contained an activator protein (AP)-1-like site and cAMP-responsive element, but it lacked a TATA box, a GC-rich element, and an SP-1 site. Deletion analysis of the 5'-flanking region showed that the fragments containing the AP-1-like element (TGACTCC) positioned at -38 exhibited high promoter activities in CLC-5 expressing LLC-PK1 cells, but that further deletions not containing this AP-1-like element resulted in a great loss of luciferase activities. Gel-retardation analysis demonstrated the existence of a specific protein binding to this AP-1-like element in LLC-PK1 cells, which seemed to differ from an authentic AP-1. This study clarified the key element of the human CLCN5 promoter, and the mutation in this region could be the cause of Dent's disease.     

Isolation and characterization of the human catalase gene.

Catalase is a tetrameric hemoprotein which degrades H2O2. Recombinant phage clones containing the human catalase gene have been isolated and characterized. The gene is 34 kb long and is split into 13 exons. The precise size and location of the exons has been determined. In addition, essentially full length catalase cDNA clones have been isolated and sequenced and used to tentatively identify the 5'-end of the gene. This assignment, if correct, predicts that the region upstream of the gene does not contain a TATA box. This region is GC rich (67%) and contains several CCAAT and GGGCGG sequences which may form part of the promoter. Translation of the catalase mRNA appears to begin immediately upstream of the amino-terminal Ala residue of catalase.