Identification of oxygen responsive elements in the 5'-flanking region of the human glutathione peroxidase gene.
The -1389 to +588 region of the human genomic glutathione peroxidase gene (hgpx1) was amplified using the polymerase chain reaction. This DNA fragment was cloned and sequenced, and various deletion constructs derived from the hgpx1 5'-flanking region were fused to the chloramphenicol acetyltransferase gene. These reporter genes were analyzed in transient transfection assays using primary cultured human ventricular cardiomyocytes obtained from patients with tetralogy of Fallot. Two distinct regions upstream from the transcription start site, which was determined using S1 nuclease analysis, were identified to be responsive to the oxygen tension in culture (pO2 values of 150 or 40 mm Hg). Methylation interference footprinting assays revealed proteins closely apposed to two sequences located at -1232 to -1213 and -282 to -275. We have designated these oxygen responsive elements ORE1 and ORE2, respectively. Gel mobility shift assays using double-stranded oligonucleotides corresponding to each site have demonstrated the formation of specific complexes using both cultured human cardiomyocyte and HeLa nuclear extracts. ORE1 and ORE2 bind disparate proteins with equal precision as bound complexes could be competed away with identical sequences but not with either the other ORE or an unrelated sequence. Insertion of these oxygen responsive elements into a reporter gene governed by a SV40 promoter similarly regulated chloramphenicol acetyltransferase activity according to the oxygen tension in culture.
Cowan
D B
DB
Department of Clinical Biochemistry, Toronto Hospital, Ontario, Canada.
Weisel
R D
RD
Williams
W G
WG
Mickle
D A
DA
D001483:Base Sequence; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D005979:Glutathione Peroxidase / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008745:Methylation; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010100:Oxygen / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic
D009838:Oligodeoxyribonucleotides; D005979:Glutathione Peroxidase; D015500:Chloramphenicol O-Acetyltransferase; D010100:Oxygen