Genomic organisation analysis of novel immunoglobulin-like transcripts in Atlantic salmon (Salmo salar) reveals a tightly clustered and multigene family

Abstract

Background: Several novel immunoglobulin-like transcripts (NILTs) which have previously been identified in the salmonid species rainbow trout (Oncorhynchus mykiss) contain either one or two extracellular Ig domains of the V-type. NILTs also possess either an immunoreceptor tyrosine-based activating motif (ITAM) or immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic region resulting in different signalling abilities. Here we report for the first time the genomic organisation and structure of the multigene family of NILTs in Atlantic salmon (Salmo salar) using a BAC sequencing approach.

Results: We have identified six novel Atlantic salmon NILT genes (Ssa-NILT1-6), two pseudogenes (Ssa-NILTp1 and Ssa-NILTp2) and seven genes encoding putative transposable elements in one BAC covering more than 200 kbp. Ssa-NILT1, 2, 4, 5 and 6 contain one Ig domain, all having a CX₃C motif, whereas Ssa-NILT3 contains two Ig domains, having a CX₆C motif in Ig1 and a CX₇C motif in Ig2. Atlantic salmon NILTs possess several ITIMs in the cytoplasmic region and the ITIM-bearing exons are in phase 0. A comparison of identity between the amino acid sequences of the CX₃C Ig domains from NILTs varies from 77% to 96%. Ssa-NILT1, 2, 3 and 4 were all confirmed to be expressed either by their presence in EST databases (Ssa-NILT1) or RT-PCR (Ssa-NILT2, 3, and 4) using cDNA as template. A survey of the repertoire of putative NILT genes in a single individual revealed three novel genes (Ssa-NILT7-9) represented by the Ig domain, which together with Ig domains from Ssa-NILT1-6 could be divided into different groups based on specific motifs.

Conclusions: This report reveals a tightly clustered, multigene NILT family in Atlantic salmon. By screening a highly redundant Atlantic salmon BAC library we have identified and characterised the genomic organisation of six genes encoding NILT receptors. The genes show similar characteristics to NILTs previously identified in rainbow trout, having highly conserved cysteines in the Ig domain and several inhibitory signalling motifs in the cytoplasmic region. In a single individual three unique NILT Ig domain sequences were discovered at the genomic DNA level, which were divided into two different groups based on a four residue motif after the third cysteine. Our results from the BAC screening and analysis on the repertoire of NILT genes in a single individual indicates that many genes of this expanding Ig containing NILT family are still to be discovered in fish.

Figure 1

Screenshot of FPC contig 341 adapted from http://www.asalbase.org. BAC clone S0024B13 was chosen for shotgun sequencing (indicated in red). BAC clones positive for NILT hybridization probe are indicated by a red square. The position of PCR markers are highlighted with dark and light green bars. All BAC clones have been end sequenced (indicated in light red).

Figure 2

Assembly of NILT-containing BAC clone. a. Graphic illustration of the orientation and the number of contigs (ctg) after assembly. b. Genomic organization of the Atlantic salmon (Ssa) NILT genes identified in BAC clone S0024B13. Contig 3 contains a partial and a full-length predicted NILT. Contig 4 contains four full-length NILT genes, one partial NILT gene and two NILT pseudogenes.

Figure 3

Exon-intron structure of NILT genes from the Atlantic salmon (Ssa), zebrafish (Dre), and carp (Cca). The exons are indicated by boxes and introns by horizontal lines. Phase of the introns are shown below each intron.

Figure 4

Comparison and variability analysis of NILTs from Atlantic salmon. a. Multiple sequence alignment of the deduced amino acid sequences of Ssa-NILT1-6 using Clustal W. Conserved cysteines are indicated in black. The transmembrane region is highlighted in gray. Putative inhibitory signalling motifs (ITIMs) in the cytoplasmic region are underlined. The predicted eight beta-strands are denoted by asterisks. LP, leader peptide; CP, connecting peptide; TM, transmembrane region; Cyt, cytoplasmic region. Dots (.) indicate identity to Ssa-NILT1 and dashes (-) denote gaps introduced for optimal alignment. b. Analysis of the variability of the entire Ssa-NILT protein sequences using Shannon entropy calculations. Positions with a Shannon entropy higher than 1.3 are considered significantly variable (indicated by black line).

Figure 5

Phylogenetic tree showing the relationship between NILTs from Atlantic salmon, rainbow trout, carp and zebrafish and NKp44 and TREM molecules from human using the amino acid sequences of the Ig domains. The unrooted tree was built by the neighbour-joining method using Clustal W and the MEGA 4 packages and bootstrapped 10,000 times. The scale bar corresponds to 0.1 estimated amino acid substitutions per site. [GenBank: Omy-NILT1 [23]; Omy-NILT2, FM180056; Omy-NILT3, FM180057; Omy-NILT4, FM180058; Cca-NILT1, CAH19212; Cca-NILT2, CAH19213; Dre-NILT1, BN001234; Dre-NILT2, BN001235; Human-TREM1, AAL74018; Human-TREM2, AAH32362; Human-NKp44, AJ225109].

Figure 6

Multiple alignment of the amino acid sequences of NILT Ig domains from the Atlantic salmon. Characteristic residues are highlighted in black and gray. Dots (.) indicate identity to Ssa-NILT1 Ig1 and dashes (-) denote gaps introduced for optimal alignment. Below is a rooted phylogenetic tree showing the division into different groups of NILT Ig domains from the Atlantic salmon. The unrooted tree was built by the neighbour-joining method using Clustal W [41] and the MEGA 4 [42] packages and bootstrapped 10,000 times.