1998 Fiscal Year Final Research Report Summary
Transcriptional regulation of kidney-specific genes
| Project/Area Number |
08457285
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Kidney internal medicine
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| Research Institution | Tokyo Medical and Dental University |
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Principal Investigator |
INASE Naohiko Tokyo Medical and Dental University, Scholl of Medicine Lectv, 医学部, 助手 (60262185)
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| Co-Investigator(Kenkyū-buntansha) |
UCHIDA Shinichi Tokyo Medical and Dental University, School of Medicine, Lecture, 医学部, 助手 (50262184)
SASAKI Sei Tokyo Medical and Dental University, Scholl of Medicine, Assoctate Prottese, 医学部, 助教授 (60170677)
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| Project Period (FY) |
1996 – 1998
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| Keywords | AQP-2 / AQP-3 / CLC-K1 / CLC-K2 / Promoter / CRE / GATA / purine-rich sequence |
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| Research Abstract |
Expression of aquaporin-2 (AQP2) is exclusively limited to kidney collecting duct cells, and this strictly limited expression could be mediated by transcription of the gene. The 5-flanking region of the AQP2 gene was characterized by a TATA box, two GATA consensus sequences, an AP-1 site, an AP-2 site, three E-boxes, and a cycle AMP-responsive element. A reporter gene assay performed in the 1st day of primary culture of IMCD cells showed that the 5' region up to -2.9 kb worked as a promoter. Deletion experiments showed that at least two regions, from -434 to -364 and from -153 to -84, contain negatively acting cis-elements. To evaluate the functional role of GATA motifs in AQP2 gene, we sought to isolate a GATA factor(s) expressed in collecting ducts. Two cDNAs encoding GATA factors were isolated from rat kidney (GATA-2 and -3). GATA motifs in the 5-flanking region of the AQP2 gene were functional cis-elements and that GATA-3 in collecting ducts may be one of the important regulators o
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f AQP-2 expression in vivo.
The rat CIC-K1 chloride channel is a kidney-specific member of the 010 chloride channel family found exclusively in the if-in ascending limb of Henle's loop in the kidney. To gain insight into the mechanism(s) of kidney-specific expression of CIC-K1, a genomic done that contains the 5'-flanking region of the rat CIC-K1 gene was isolated. The sequence of the proximal 5-flanking region contained an AP-3 site, a GRE, several AP-2 sites, and several E-boxes, but it lacked a TATA box.
Deletion analysis revealed that this cell-specific promoter activity in IM cells was still present in the construct containing 51 b p of-the 5'-flanking region but was lost in the -29 construct, dearly demonstrating that the-22 bp from -51 to -30 have a major role in the cell-specific activity of the CIC-K1-promoter. These 22 bp consist of purine-rich sequence (GGGGAGGGG-GAGGGGAG), 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 CIC-K1 gene promoter. Less
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