Molecular cloning, characterization, and expression of the chicken heme oxygenase-1 gene in transfected primary cultures of chick embryo liver cells.
Using chick heme oxygenase-1 (cHO-1) cDNA as a probe, three independent clones were identified from screening a lambda FixII chick genomic library. Genomic Southern blots using this cDNA probe or a cHO-1 5' specific probe showed that cHO-1 is a single-copy gene. Based on restriction enzyme analysis, Southern blots, polymerase chain reaction analysis and DNA sequencing, it was confirmed that the three overlapping clones isolated cover the entire cHO-1 gene, as well as approximately 10 kb of the flanking regions on both ends. As with mammalian HO-1x, cHO-1 has five exons and four introns. Computer analysis of the DNA sequence obtained identified consensus sequences corresponding to numerous transcription factor recognition elements. These include AP-1, AP-2, NF-kB, C/EBP, c-Myc and a metal-responding element identified in the promoter region, and two Sp-1 elements in intron 1. Transient expression studies in transfected primary cultures of chick embryo liver cells showed that a CAT reporter gene construct containing 2.8 kb of the cHO-1 promoter region responded to sodium arsenite, H2O2, transition metals and 12-0-tetradecanoylphorbol 13-acetate, but not to heme. Studies with deletion mutants, consisting of various lengths of the cHO-1 promoter region, indicated that there are two regions important for sodium arsenite induction, one located between residues -1642 and -1293, and the second located in the first 263 base pairs of the cHO-1 promoter. DNA binding studies by electrophoretic mobility shift assay showed that nuclear protein isolated from primary cultures of chick embryo liver cells bound to the oligonucleotide probe containing an AP-1 element identified at -1573 to -1580. In addition, such binding was increased by cobalt or sodium arsenite treatment.
Lu
T H
TH
Department of Medicine, University of Massachusetts Medical Center, Worcester 01655, USA. Tze.Lu@ummed.edu
Lambrecht
R W
RW
Pepe
J
J
Shan
Y
Y
Kim
T
T
Bonkovsky
H L
HL
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002642:Chick Embryo; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D005091:Exons; D015870:Gene Expression; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics*; D051547:Heme Oxygenase-1; D007438:Introns; D008099:Liver / Q000166:cytology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017384:Sequence Deletion; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014162:Transfection
D018808:Transcription Factor AP-1; D004247:DNA; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1