Molecular cloning and expression of an avian macrophage nitric-oxide synthase cDNA and the analysis of the genomic 5'-flanking region.

We report the first nonmammalian inducible nitric-oxide synthase (NOS) cDNA obtained from chicken macrophages. It exhibits an open reading frame encoding 1,136 amino acid residues, predicting a protein of 129,648-Da molecular mass. The deduced NOS protein sequence showed 66.6%, 70.4%, 54.2%, and 48.7% sequence identity to mouse and human inducible NOS and to two constitutive NOSs from rat brain and bovine endothelium. Overall, NOS appears to be a moderately conserved protein. Northern analysis showed that chicken iNOS mRNA is approximately 4.5 kilobases (kb), a size similar to mammalian inducible NOS. Analysis of 3.2 kb of 5'-flanking sequence of the chicken iNOS gene showed a putative TATA box at 30 base pairs (bp) upstream of the transcription initiation site. The functional importance of the upstream region was determined by transient expression of deletion constructs. An endotoxin regulatory region was located exclusively within 300 bp upstream of the transcription initiation site. This is in contrast to the two distinct sites identified in the mouse macrophage NOS promoter. Transcription factor binding sites such as NF-kappaB, PEA1, PEA3, and C/EBP were identified. Using a NF-kappaB inhibitor, we showed that NF-kappaB is indeed involved in the induction of chicken iNOS gene by lipopolysaccharide. Our results suggest that NF-kappaB is a common regulatory component in the expression of both mammalian and nonmammalian iNOS genes.