Evolution and gene expression analysis of inducible nitric oxide synthase (iNOS) in fish
In this thesis, the full-length iNOS cDNA was sequenced from a cartilaginous fish, small spotted catshark (Scyliorhinus canicula), which had 77% amino acid (aa) similarity to chicken iNOS, and 73% aa similarity to trout iNOS. Expression studies in vivo also indicated that in S. canicula iNOS expression was strongest in the spleen. In addition, two full-length iNOS coding regions were sequenced from a bony fish species, goldfish (Carassius auratus). The two transcripts were designed iNOSa and iNOSb, with 93.5% aa similarity to each other and 93.4 and 92.0 % aa similarity to carp respectively. An in silico study to find iNOS and other NOS sequences from vertebrates and invertebrates in genomic and cDNA databases discovered full-length iNOS coding regions from several vertebrate species, including the western clawed frog (Xenopus tropicalis) and two distinct anciently duplicated genes from the zebrafish (Danio rerio). Partial or full-length NOS genes were also found in two species of urochordate (sea squirts, Ciona sp.), honey bee (Apis mellifera) and constitutive NOS genes from several vertebrates. Expression studies using the promoter region of rainbow trout (Oncorhynchus mykiss) iNOS in a luciferase reporter gene vector indicated that bacterial lipopolysaccharide (LPS) was a good inducer of iNOS expression in vitro, and required as little as 208bp of 5’ flanking region and intron 1. The promoter region of goldfish iNOS was sequenced, and had similar transcription factor binding sites to those of iNOS genes from other species.