Molecular characterization of type I and type II interferons and identification of interferon stimulated genes in channel catfish

Multiple species of type I interferon (IFN) have been identified in channel catfish, CF (Ictalurus punctatus). Herein we extend those studies and report the genomic organization and promoter elements of CF type I IFN genes, the identification and characterization of type II IFN genes, and the identification of IFN stimulated genes (ISG) in response to a synthetic dsRNA (poly[I:C]).;First, sequence analysis of the CF IFN-alpha1 gene revealed the presence of several putative regulatory elements similar to IRF-1/-2, IRF-3/7, ATF-2/c-Jun, NFKB, ISRE and GAS. These motifs are hallmarks of mammalian IFN and ISG promoters. Additionally, sequence analysis showed that the exon/intron organization of CF IFN-alpha1 was similar to that present in other fish IFN genes, while PCR/Southern blot analysis identified a single BAC clone that contained all CF Type I IFN genes.;Secondly, as with zebrafish and the green spotted pufferfish, two CF IFN-gamma genes were identified and shown to be closely linked but dissimilar in sequence. One of them (IFN-gamma2) encodes two distinct messages that likely arose via alternative splicing at two closely spaced splice donor sites within the first intron. Sequence analysis indicates that CF IFN-gamma genes contain the hallmarks of authentic IFN-gamma including: a conserved nuclear localization site at the C terminus (CF IFN-gamma2 only), an IFN-gamma signature sequence, six putative helical regions within the mature protein, potential glycosylation sites, and multiple mRNA instability motifs within the 3' untranslated region. In addition, expression studies showed, for the first time in any lower vertebrate, that fish NK and T cells synthesize IFN-gamma and are consistent with results in mammalian systems where T cells and NK cells are the major sources of type II IFN production.;Finally, screening of CF EST libraries and microarray analyses identified several IFN stimulated genes some of which are homologues of those found in mammalian systems. Furthermore, their expression was induced after the treatment of clonal catfish lymphocytic and fibroblastic cell lines with poly[I:C].;Collectively, these results indicate that catfish possess multiple copies of Type I/II interferons and that these proteins likely play key roles in activating the innate immune response in channel catfish.