Evidence for a transposition event in a second NITR gene cluster in zebrafish

Abstract

Novel immune-type receptors (NITRs) are immunoglobulin-variable (V) domain-containing cell surface proteins that possess characteristic activating/inhibitory signaling motifs and are expressed in hematopoietic cells. NITRs are encoded by multigene families and have been identified in bony fish species. A single gene cluster, which encodes 36 NITRs that can be classified into 12 families, has been mapped to zebrafish chromosome 7. We report herein the presence of a second NITR gene cluster on zebrafish chromosome 14, which is comprised of three genes (nitr13, nitr14a, and nitr14b) representing two additional NITR gene families. Phylogenetic analyses indicate that the V domains encoded by the nitr13 and nitr14 genes are more similar to each other than any other zebrafish NITR suggesting that these genes arose from a tandem gene duplication event. Similar analyses comparing zebrafish Nitr13 and Nitr14 to NITRs from other fish species indicate that the nitr13 and nitr14 genes are phylogenetically related to the catfish IpNITR13 and IpNITR15 genes. Sequence features of the chromosomal region encoding nitr13 suggest that this gene arose via retrotransposition.

Figure 1. Nitr13 and Nitr14 sequences

(a) The peptide sequences encoded by the nitr13 (Nitr13), nitr14a-Long (Nitr14a-L) and Nitr14a-Short (Nitr14a-S) cDNAs are aligned with the Nitr14b peptide sequence predicted from genomic sequence. Black shading indicates identical residues whereas gray shading indicates functionally similar residues. The locations of the predicted leader peptide sequences, transmembrane domains (TM), and joining (J) domains (FGXGTXLX(L/I)) are indicated with gray bars above the alignment. Residues, which are highly conserved in Ig domains (Litman et al. 2001), are indicated above the alignment using the IMGT numbering system (Giudicelli et al. 2006). Black circles indicate the locations of two tyrosines present in the cytoplasmic tail of Nitr14aL. (b) Predicted protein structures for Nitr13 and members of the Nitr14 family. Variable (V), transmembrane (TM), joining (J) and carboxy-terminal tyrosines (Y) are indicated. Only Nitr14a-L includes a transmembrane domain. The asterisk (*) next to Nitr14b indicates that this sequence is predicted from genomic sequence.

Figure 2. Allelic complexity of the NITR gene cluster on chromosome 14

A percent identity plot (PIP) was generated using two alleles of the NITR gene cluster on chromosome 14. Sequence from BAC DKEY-149K8 was used as the reference for comparison to sequence from BAC RP71-24B12. Note that only 2 of 5 exons for nitr14a are identifiable from BAC RP71-24B12.

Figure 3. Cytogenetic localization of NITR gene clusters to chromosomes 7 and 14

BACs corresponding to the NITR gene cluster on chromosome 7 (DKEY-7N10) and the newly identified NITR gene cluster (DKEY-149K8) were localized to specific zebrafish chromosomes using 2-color fluorescence in situ hybridization (Freeman et al. 2007). (a) DKEY-7N10 hybridized uniquely to the subtelomeric region of chromosome 7q and is shown (orange) in relation to the near-telomeric marker for the q-arm of chromosome 7, CH211-128L16 (green). (b) DKEY-149K8 hybridized uniquely to the subtelomeric region of chromosome 14q and is shown (orange) in relation to the near-telomeric marker for the p-arm of chromosome 14, CH211-117N19 (green).

Figure 4. Phylogenetic analyses of zebrafish NITR V domains

Phylogenetic analyses of the V domains from zebrafish Nitr13, Nitr14a and Nitr14b with V domains from the zebrafish NITR families described previously (Yoder et al. 2004). Protein symbols are abbreviated (e.g. Nitr1a is represented by “1a”). Bootstrap values less than 70% are not shown. Branch lengths are measured in terms of amino acid substitutions, with the scale indicated below the tree.

Figure 5. Phylogenetic analyses of NITR V domains from multiple species

Neighbor-joining tree of V domains encoded by NITR genes in zebrafish (Danio rerio, “Dr”); channel catfish (Ictalurus punctatus, “Ip”); rainbow trout (Oncorhynchus mykiss, “Om”); Southern pufferfish (Sphoeroides nephelus, “Sn”); and Japanese flounder (Paralichthys olivaceus, “Po”). Protein symbols are abbreviated to the NITR number (e.g. zebrafish Nitr14a is shown as “Dr 14a”). In the instances of NITR gene families, only one member was included in the analysis. For example Sn 16 represents Sn 16 and 22; Sn 13 represents Sn 13 and 18; Sn 20 represents Sn 14, 15, 17, 19, 20, 21, 23-26; Ip 1 represents Ip 1 and 3; and Ip 5 represents Ip 5-11. The number assigned to each interior branch is the bootstrap value; bootstrap values less than 50 are not shown. The branch lengths are measured in terms of the number of amino acid substitutions estimated by Poisson correction, with the scale given below the tree. Note that zebrafish Nitr13 and Nitr14 V domains group with catfish IpNITR13 and 15 V domains (gray shading): these 4 V domains also group together when using maximal parsimony and UPGMA methods (not shown).

Figure 6. The nitr13 gene originated via transposition

(a) The nitr13 gene encodes 2 exons (top). The protein coding sequence of the nitr13 gene is comprised of 2 segments (black rectangles) in exons 1 and 2. The nitr13 mRNA possesses coding sequences for a leader (L), variable (V), intermediate (I) and transmembrane (TM) domains (middle). However a stop codon (TAA) and a reading frame shift exist between the V and I domains: a frame shift also exists between the I and the TM domains. Three non-coding sequences (gray rectangles) indicate that nitr13 arose via retrotransposition of a mature NITR mRNA: 1) a leader (L) sequence is found at the 3' end of the single intron, which is in frame with the variable (V) domain in exon 2, and 2) an Ig domain of the intermediate (I) type and 3) a transmembrane domain in the 3' untranslated region. One possible model for the nitr13 precursor mRNA is shown (bottom). (b) The protein structure of the predicted precursor to Nitr13 is compared to Nitr13.

Figure 7. Phylogenetic analyses of the untranslated I domain within the nitr13 gene

(a) The untranslated I domain encoded in the 3' untranslated region of nitr13 (termed Nitr13^P for precursor of Nitr13) is aligned with I domains from other zebrafish NITRs. Black shading indicates identical residues whereas gray shading indicates functionally similar residues. The J-related domain and residues highly conserved in Ig domains (Litman et al. 2001) are indicated above the alignment using the IMGT numbering system (Giudicelli et al. 2006). Six highly conserved cysteines, representative of NITR I domains, are indicated by asterisks below the alignment (Litman et al. 2001). (b) Phylogenetic analyses of the I domain from zebrafish Nitr13^P and I domains from the zebrafish NITR families described previously (Yoder et al. 2004). Analyses and presentation are as in Fig. 4.