Molecular cloning, characterization, and expression of the chicken heme oxygenase-1 gene in transfected primary cultures of chick embryo liver cells

Abstract

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-1s, 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, H 2O 2, transition metals and 12- O-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.