Isolation and characterization of kidney-specific ClC-K1 chloride channel gene promoter.
Isolation and characterization of kidney-specific ClC-K1 chloride channel gene promoter.
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肾脏特异性 ClC-K1 氯离子通道基因启动子的分离和表征。
DOI：
10.1152/ajprenal.1998.274.3.f602
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
F. Marumo
中科院分区：
文献类型：
--
作者：
S. Uchida;T. Rai;H. Yatsushige;Y. Matsumura;M. Kawasaki;S. Sasaki;F. Marumo
大鼠 ClC-K1 氯离子通道是 ClC 氯离子通道家族的肾脏特异性成员，仅在肾脏亨利氏襻的细升肢中发现。为了深入了解 ClC-K1 肾脏特异性表达的机制，分离了包含大鼠 ClC-K1 基因 5' 侧翼区域的基因组克隆。单个转录起始位点位于起始密码子上游 84 bp 处。近端5'侧翼区域的序列包含激活蛋白(AP)-3位点、糖皮质激素反应元件、几个AP-2位点和几个E-盒，但缺少TATA盒。为了功能性地表达启动子，将〜2.5-kb对5'-侧翼区域连接至荧光素酶报告基因并转染至内髓（IM）细胞（源自猿猴病毒40转基因小鼠内髓的稳定表达ClC-K1的细胞系）和不表达ClC-K1的细胞系。 IM 细胞中的荧光素酶活性比非表达细胞系中的荧光素酶活性高 7 至 24 倍，表明~2.5-kb 片段包含用于 ClC-K1 基因细胞特异性表达的顺式作用调节元件。删除分析显示，IM细胞中的这种细胞特异性启动子活性仍然存在于包含5'-侧翼区域的51bp的构建体中，但在-29构建体中丢失，清楚地证明从-51到-30的22bp在ClC-K1启动子的细胞特异性活性中具有主要作用。这些 22 bp 由富含嘌呤的序列 (GGGGAGGGGGAGGGGAG) 组成，凝胶延迟分析证明 IM 细胞中存在与该元件结合的特定蛋白质。这些结果表明，新的富含嘌呤的元件可能在 ClC-K1 基因启动子的活性中发挥关键作用。
The rat ClC-K1 chloride channel is a kidney-specific member of the ClC chloride channel family found exclusively in the thin ascending limb of Henle's loop in the kidney. To gain insight into the mechanism(s) of kidney-specific expression of ClC-K1, a genomic clone that contains the 5'-flanking region of the rat ClC-K1 gene was isolated. A single transcription start site was located 84 bp upstream of the start codon. The sequence of the proximal 5'-flanking region contained an activator protein (AP)-3 site, a glucocorticoid-responsive element, several AP-2 sites, and several E-boxes, but it lacked a TATA box. To functionally express the promoter, the ∼2.5-kb pair 5'-flanking region was ligated to a luciferase reporter gene and transfected into inner medullary (IM) cells, a stable ClC-K1-expressing cell line derived from the inner medulla of simian virus 40 transgenic mouse, and ClC-K1-nonexpressing cell lines. Luciferase activity was 7- to 24-fold greater in IM cells than those in nonexpressing cell lines, suggesting that the ∼2.5-kb fragment contained cis-acting regulatory elements for cell-specific expression of the ClC-K1 gene. Deletion analysis revealed that this cell-specific promoter activity in IM cells was still present in the construct containing 51 bp of the 5'-flanking region but was lost in the -29 construct, clearly demonstrating that the 22 bp from -51 to -30 have a major role in the cell-specific activity of the ClC-K1 promoter. These 22 bp consist of purine-rich sequence (GGGGAGGGGGAGGGGAG), and gel-retardation analysis demonstrated the existence of a specific protein(s) binding to this element in IM cells. These results suggest that the novel purine-rich element may play a key role in the activity of the ClC-K1 gene promoter.
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发表时间：
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影响因子：
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