Characterization and expression analyses of anti-apoptotic Bcl-2-like genes NR-13, Mcl-1, Bcl-X1, and Bcl-X2 in Atlantic cod (Gadus morhua)

Abstract

NR-13, Mcl-1, and BCL-X(L), are conserved anti-apoptotic proteins that belong to the anti-apoptotic Bcl-2 sub-family, which inhibits cell death by preventing mitochondrial membrane permeabilization (MMP). Given the anti-apoptotic functions of these proteins in vertebrates (e.g. human, mouse, and zebrafish) and the involvement of apoptotic regulation in immune responses, we studied the sequences of these genes and their transcript expression in Atlantic cod (Gadus morhua) during innate immune responses to viral and bacterial stimuli. Based on previously generated Atlantic cod expressed sequence tags (ESTs), we identified partial cDNA sequences of putative orthologues of Atlantic cod NR-13, Mcl-1, and Bcl-X, and obtained the full-length cDNA, genomic, and promoter region sequences for these genes. The analyses of Atlantic cod cDNA sequences, and comparisons of the cod deduced amino acid sequences to putative orthologues in other species, revealed the presence of highly conserved Bcl-2 homology (BH) and transmembrane (TM) domains in the Atlantic cod sequences. Analysis of gene structure revealed conserved intron/exon boundaries within the coding regions of human and Atlantic cod putative orthologues. We found that an intron/exon boundary immediately following the codon for the 8th residue (tryptophan) of the BH2 domain exists in all anti-apoptotic Bcl-2 sub-family genes regardless of vast evolutionary distance. We also identified a non-coding exon in the Atlantic cod NR-13-like gene, which appears to be absent in its putative mammalian orthologues. Quantitative reverse transcription-polymerase chain reaction (QPCR) was used to study constitutive gene expression in six tissues (blood, brain, gill, head kidney, pyloric caecum, and spleen) of non-stressed juvenile cod; NR-13 and Bcl-X2 were most highly expressed in gill, whereas Mcl-1 and Bcl-X1 were most highly expressed in blood. In cod challenged with intraperitoneal (IP) injections of the viral mimic polyriboinosinic polyribocytidylic acid (pIC), (1) NR-13 mRNA expression was significantly up-regulated (compared to both 0h pre-injection and timed saline injected controls) in spleen at 6h post-injection and in head kidney at both 6 and 24h post-injection (HPI), and (2) both Mcl-1 and Bcl-X2 were significantly up-regulated (compared to both 0h pre-injection and timed saline injected controls) in spleen at 6 HPI. QPCR was used to show that, in cod challenged with IP injections of formalin-killed, atypical Aeromonas salmonicida (ASAL), only NR-13 appeared to be responsive (significantly up-regulated in spleen at 6 HPI compared to 0h pre-injection controls). Interestingly, QPCR showed that saline injection had a mild (less than 3-fold) but significant inductive effect (compared to 0h pre-injection controls) on both NR-13 and Mcl-1 transcript expression in spleen at 2 HPI. Although we only obtained partial cDNA and genomic sequences for Bcl-X2, sufficient evidence was accumulated to show that two Bcl-X paralogues exist in Atlantic cod, possibly due to the teleost-specific genome duplication event. Promoter regions for NR-13, Mcl-1, and Bcl-X1 were obtained and analyzed for the first time in fish, and potential regulatory sites (e.g. putative NF-kappaB binding sites) that were found in the promoter regions of NR-13 and Mcl-1 may account for their transcriptional activation by pIC.