Kerry J. Laing

Identification and analysis of an interleukin 8-like molecule in rainbow trout Oncorhynchus mykiss.

An interleukin 8 (IL-8) homologue has been identified in the rainbow trout Oncorhynchus mykiss. The transcript contains an open reading frame of 294 nucleotides that translates into a 97 amino acid putative peptide, with 5 and 3 untranslated regions (UTR) of 171 and 453 nucleotides, respectively. As with previously sequenced lamprey and flounder genes, the trout amino acid sequence lacks the typical ELR motif upstream of the first pair of cysteines, where DLR is present. The trout IL-8 gene contains four exons divided by three short introns of 341, 247 and 292bp, and occupies 1824bp of genomic DNA. RT-PCR reveals a low level constitutive expression of the IL-8 homologue in many tissues, including spleen, heart, liver, head kidney and gill. Expression was not detectable in the brain. Whilst no apparent affect of lipopolysaccharide (LPS) on IL-8 expression was observed in vivo, stimulation of a trout macrophage cell line (RTS-11) with either LPS or poly I:C did result in clear up-regulation of IL-8 expression, detectable by RT-PCR and Northern blot analysis.

Expression of an inducible nitric oxide synthase gene in rainbow trout Oncorhynchus mykiss

Using oligonucleotide primers based on mammalian nitric oxide synthases (NOS), expression of an inducible NOS (iNOS) gene was detected in head kidney and gill tissue of bacterially-challenged rainbow trout. Three overlapping fragments were amplified by RT-PCR and used to construct a contiguous sequence of 1410bp, with high nucleotide homology to iNOS in birds (61%) and mammals (57-59%). The nucleotide sequence translated in one reading frame to produce a partial peptide containing 470 amino acids, with 69-71% amino acid homology with mammalian iNOS, 81% homology with chicken iNOS and 85% homology with a partial (492bp) goldfish iNOS sequence. In vitro stimulation of head kidney macrophages with LPS also induced expression of the trout iNOS RNA, with optimal expression seen using 20-50 microg/ml LPS at 2h to 6h post-stimulation. The evolutionary and functional significance of the trout iNOS sequence are discussed.

A partial sequence for nitric oxide synthase from a goldfish (Carassius auratus) macrophage cell line

Expression of inducible nitric oxide synthase (iNOS) mRNA was detected in a recently developed goldfish macrophage cell line by RT‐PCR. using degenerate primers designed against conserved nucleotide motifs within the different mammalian isoforms of NOS. Increased expression of iNOS poststimulation with LPS was found, and suggests that it is a functional enzyme in goldfish macrophages. supporting the view that iNOS regulation is pretranslational. The nucleotide sequence translated in one reading frame with no stop codons to produce a partial peptide containing 164 amino acids, with highest homology (85%) to a recently identified rainbow trout iNOS sequence. The peptide translation also gave an insight into the conservation of binding motifs, since two cofactor binding sites were present in the amplified PCR product (FMN and calmodulin). In addition, a 42 aa motif present in the region just upstream of the FMN binding motif of mammalian endothelial and neuronal NOS isoforms was absent in the translation, in agreement with every published sequence for iNOS. Finally, the translation was used to construct an unrooted phylogenetic tree.

Cloning and sequencing of caspase 6 in rainbow trout, Oncorhynchus mykiss, and analysis of its expression under conditions known to induce apoptosis.

The rainbow trout caspase 6 gene has been cloned and sequenced. The open reading frame consisted of 906bp, which translated into a protein of 302 amino acids, containing the caspase active site pentapeptide (QACRG) and the caspase family signature (HADADCFVCVFLSHG). Amino acids involved in catalysis and those known to form the P1 carbohydrate binding pocket were conserved. Phylogenetic tree analysis showed a tight grouping with other known caspase 6 genes. Conserved aspartic acid residues at positions 33, 191 and 202 suggested that this molecule is produced as a proenzyme that is subsequently cleaved to release active subunits, with the region between Asp-191 and Ala-203 acting as a linker that is cleaved out. RT-PCR analysis revealed that the trout caspase 6 gene was expressed in brain, blood, gill, liver, head kidney and spleen. Addition of LPS or cortisol to head kidney leucocyte cultures had no effect upon caspase 6 expression. However, addition of LPS after preincubation with cortisol increased expression relative to control cultures. Incubation with RU486 abrogated this effect, confirming it was mediated via glucocorticoid receptors. Lastly, a confinement stress in vivo increased caspase 6 expression. The data are discussed with respect to the immunoregulatory role of apoptosis in fish immune responses.

TGF-β3 exists in bony fish

Abstract A partial nucleotide sequence of transforming growth factor-β3 (TGF-β3) has been isolated from the Siberian sturgeon (Acipenser baeri), rainbow trout (Oncorhynchus mykiss) and European eel (Anguilla anguilla), confirming a ubiquitous presence in the ray-finned (Actinopterygian) bony fish. The bony fish TGF-β3 is highly conserved, with some 83–84% nucleotide identity (coding region) and 90–95% predicted amino acid identity to known homeotherm TGF-β3’s. Far lower homologies are apparent with other known TGF-β isoforms in fish (e.g. 64–66% and 81–82% amino acid identity to trout TGF-β1/5 and carp TGF-β2 respectively). Phylogenetic tree analysis showed that the fish TGF-β3’s clustered with the known homeotherm TGF-β3’s. The relatively tight clustering of TGF-β1, TGF-β2 and TGF-β3 was in contrast to the TGF-β5’s, which are clearly a more heterogenous group.

Phylogeny of vertebrate cytokines.

Considerable progress has been made in recent years in the sequencing of cytokine genes in non-mammalian vertebrates. This review will focus primarily on the recent advances made in isolating interleukin-1β (IL-1β), transforming growth factor-β (TGF-β) genes and chemokine genes, since they have been found in several vertebrate groups (typically fish, amphibians and birds) allowing some discussion of cytokine gene evolution. For recent advances on cytokine genes that have been isolated to date in only birds (outwith mammals), namely interferons and IL-2, the reader is referred to Schat & Kaiser (1997), Kaiser et al. 1998a,b), Sick et al. (1998), Michalski et al. (1999) and Kaiser & Mariani (1999).