서지주요정보
Transcriptional regulation of the Wilson disease gene(ATP7B) = 윌슨씨 유전자 (ATP7B) 의 전사조절 연구
서명 / 저자 Transcriptional regulation of the Wilson disease gene(ATP7B) = 윌슨씨 유전자 (ATP7B) 의 전사조절 연구 / Won-Jun Oh.
저자명 Oh, Won-Jun ; 오원준
발행사항 [대전 : 한국과학기술원, 2001].
Online Access 원문보기 원문인쇄

소장정보

등록번호

8012478

소장위치/청구기호

학술문화관(문화관) 보존서고

DBS 01021

SMS전송

도서상태

이용가능

대출가능

반납예정일

초록정보

Wilson disease (WD), an autosomal recessive inherited disorder of copper transport, is marked by impaired biliary excretion and incorporation of copper into ceruloplasmin. Clinical features of the WD are characterized by hepatic cirrhosis and neuronal degeneration, which result from toxic levels of copper that accumulate in the liver and brain, respectively. To investigate the molecular mechanisms regulating expression of the WD gene, we isolated, sequenced, and characterized ~1.3 kb of the 5'-flanking region of the WD gene from the human genomic library. The ~1.3 kb of the WD sequence directed high level of luciferase activity in HepG2 cells. Interestingly, sequence analysis showed that the 5'-flanking region contained four metal response elements (MREs)-two of four MREs are in reverse orientation- and six MRE-like sequences (MLSs), usually found in the metallothionein gene. It also contained a number of putative regulatory elements such as Sp1, AP-1, AP-2, and E-box, but lacked TATA box near transcription start site. The transcription start site was located at 335 base pairs upstream from the translation initiation site. Further successive 5'-deletion analyses suggested the important role of the 159-base pair region from -811 to -653, which includes MLS2 (-802 to -796) and MLS3 (-785 to -779), as a positive regulatory element during the expression of the WD gene. A negative element was also identified at region -1038 to -812. A protein-MLS complex was identified through electrophoretic mobility shift and competition assay using MLS2/MLS3 and HepG2 cell nuclear proteins. Among the four MREs, MREa plays the most important role in the transcriptional activation of the WD promoter. Electrophoretic mobility shift assays (EMSAs) using synthetic MREa and an oligonucleotide containing the binding site for transcription factor Sp1 revealed the presence of nuclear factors that bind specifically to MREa. Two MREa-binding proteins of 70- and 82-kilodaltons were purified using avidin-biotin affinity chromatography. Amino acid sequences of peptides from each protein were found to be highly homologous to the Ku proteins. Immunoblot analysis and EMSAs showed that the MREa-binding proteins are immunologically related to the Ku proteins. To study further the functional significance of these Ku related proteins in transcriptional regulation of the WD gene, we performed RNA interference (RNAi) assays using a Ku 80 inverted-repeat gene to inhibit expression of the Ku 80 gene in vivo. Results of the RNAi assays showed that expression of the Ku 80 protein was suppressed in transfected cells, which in turn lead to the suppression of the WD gene. We also found that WD promoter activity was decreased in Xrs 5 cells, which unlike the CHO-K1 cells, are defective in the Ku 80 protein. When Ku 80 cDNA was transfected in Xrs 5 and CHO cell, WD promoter activity only was recovered in Xrs 5 cell. In addition, deleted Ku 80 (ΔKu 80) acting as a negative dominant resulted in a decrease of WD promoter activity in HepG2 cell. Taken together, our findings suggest that the Ku 80 subunit is required for constitutive expression of the WD gene.

서지기타정보

서지기타정보
청구기호 {DBS 01021
형태사항 ix, 93 p. : 삽도; 26 cm
언어 영어
일반주기 저자명의 한글표기 : 오원준
지도교수의 영문표기 : Ook-Joon Yoo
지도교수의 한글표기 : 유욱준
수록잡지명 : Biochemical and biophysical research communications, v. 259, pp. 206-211(1999)
학위논문 학위논문(박사) - 한국과학기술원 : 생물과학과,
서지주기 Reference : p. 78-89
주제 Wilson disease
promoter
MRE(metal response element)
Ku protein
RNAi
윌슨씨 병
전사조절부위
스크리닝
QR CODE qr code