Molecular cloning, gene organization and expression of rainbow trout (Oncorhynchus mykiss) inducible nitric oxide synthase (iNOS) gene

A full-length inducible nitric oxide synthase (fNOS) gene has been sequence d for the first time outside the mammals, and the gene organization compare d with that already determined for human iNOS. While there are some differe nces from the human gene, overall the exons show remarkable conservation in sequence and organization. As in human, the trout iNOS gene has 27 exons, with 18 of the trout exons being identical in size with the equivalent huma n exons. The cofactor-binding domains are found in the same exons and in so me cases are absolutely conserved. Differences include the start of the ORF in exon 3 instead of exon 2, resulting in a deletion at the 5' end of the trout iNOS protein. Exon 27 also shows a large difference in size and altho ugh the trout exon is larger this is due to the length of the 3'-UTR. Sever al non-mammalian features are notable, and include a conserved potential gl ycosylation site in chicken and fish, and an insertion at the boundary of e xons 20 and 21 in fish. The intron sizes in trout were generally much small er than in human iNOS, making the trout iNOS gene approximately half the si ze of the human gene. Analysis of RNA secondary structure revealed two regi ons with complementarity, which could interfere with reverse transcription. Using a trout fibroblast cell line (RTG-2 cells), it was shown by reverse transcriptase (RT)-PCR that virus infection was a good inducer of iNOS expr ession. However, when using a combination of Superscripts (R) II for revers e transcription and primers at the 5' end of the gene only very weak produc ts were amplified, in contrast with the situation when primers at the 3' en d of the gene were used, or ThermoScript (R) -derived cDNA was used. The im pact of such results on RT-PCR analysis of iNOS expression in trout is disc ussed.