| The goals of this research were: (1) to identify and characterize apoptosis-related genes and/or proteins in the channel catfish, and (2) to identify antiviral genes (e.g., IFN and TNFalpha) and determine their role in antiviral immunity. Reactivity with three proteins (approximate mol wts 70, 37, and 15 kDa) was seen when lysates from catfish cell lines were screened by immunoblotting using a mouse anti-human Fas ligand (FasL) monoclonal antibody. Treatments that induce FasL in mammalian systems induced expression of the 37 kDa protein in catfish T cells. At the molecular level, Fas receptor (FasR), FADD, and caspase 8 were identified. FADD was constitutively expressed in all cell lines tested and in PBL, whereas FasR and caspase 8 were expressed in all cell lines except for a FasL-positive fibroblast cell line. In contrast to FasL, expression of FasR and caspase 8 was inversely proportional to cell density.; A TNFalpha-like gene was identified in catfish by PCR. Phylogenetic analysis clustered catfish and other fish TNF sequences together, but apart from mammalian TNFalpha and beta genes, supporting the suggestion that TNFalpha and beta genes separated after the divergence of mammals and teleosts. Expression studies demonstrated that catfish TNFalpha mRNA was present in all tested tissues from an unstimulated fish. Moreover, catfish TNF was constitutively expressed in actively proliferating, but otherwise unstimulated, macrophage and T cell lines. TNF expression was upregulated in PBLs, T cells, and macrophages by PMA/calcium ionophore treatment.; Catfish IFN-1 was identified by screening a catfish EST library. Since Southern analysis indicated that several copies of IFN existed in the catfish genome, attempts to identify additional transcripts were made. PCR identified catfish IFN-2 and -3 from UV-CRV infected CCO cells and catfish IFN-4 from B cells. The catfish sequences average ∼70% similarity to each other and ∼42% similarity to zebrafish and salmon IFN. Catfish IFN was upregulated following infection of CCO cells with UV-CRV or exposure to dsRNA. Upregulation of catfish IFN mRNA was accompanied by the appearance of an antiviral factor in the culture medium. Finally, recombinant catfish IFN-1, synthesized in COS-7 cells, was shown to have antiviral activity in CCO cells. |
