Dev Comp Immunol
November 1, 2015;
Retention of duplicated ITAM-containing transmembrane signaling subunits in the tetraploid amphibian species Xenopus laevis.
The ITAM-bearing transmembrane signaling subunits (TSS) are indispensable components of activating leukocyte receptor complexes. The TSS-encoding genes map to paralogous chromosomal regions, which are thought to arise from ancient genome tetraploidization(s). To assess a possible role of tetraploidization in the TSS evolution, we studied TSS and other functionally linked genes in the amphibian species Xenopus laevis whose genome was duplicated about 40 MYR ago. We found that X. laevis has retained a duplicated set of sixteen TSS genes, all except one being transcribed. Furthermore, duplicated TCRα loci and genes encoding TSS-coupling protein kinases have also been retained. No clear evidence for functional divergence of the TSS paralogs was obtained from gene expression and sequence analyses. We suggest that the main factor of maintenance of duplicated TSS genes in X. laevis was a protein dosage effect and that this effect might have facilitated the TSS set expansion in early vertebrates.
Dev Comp Immunol
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Fig. 2. Multiple alignment of the deduced amino acid sequences of Xenopus laevis (Xl), Silurana tropicalis (St), chicken (Gg) and human (Hs) FcRγ and TcRζ subunits. Identical amino acid residues are denoted by white letters on black background, conserved residues – by black letters on gray background. Dashes designate gaps introduced for sequence alignments. Exclamation marks indicate exon-intron boundaries in human (upper line) and X. laevis (bottom line) genes. Asterisks designate ITAMs. Minus designates conserved negatively charged amino acid residue in the TM region.
Fig. 3. Multiple alignment of the deduced amino acid sequences of Xenopus laevis (Xl), Silurana tropicalis (St), and human (Hs) DAP10, DAP12, CD3 and CD79 subunits. Identical amino acid residues are denoted by white letters on black background, conserved residues – by black letters on gray background. Dashes designate gaps introduced for sequence alignments. Exclamation marks indicate exon-intron boundaries in human (upper line) and X. laevis (bottom line) genes. Asterisks designate tyrosine-based motifs. Minus designates conserved negatively charged amino acid residue in the TM region. St_DAP10 sequence was deduced using S. tropicalis genomic sequence along with EST cDNA sequences DN069735 and EL728268.
Fig. 4. RT-PCR analysis of mRNA coding for X. laevis TSS. Individual spleens of 6-month old adults were used for the analysis. The cDNA samples were normalized according to GAPDH or β-actin expression.
Fig. 5. RT-PCR analysis of FcRγ.a/b and TcRζ.a/b mRNA from X. laevis tissues of 6-month old adult (A), stage 56 tadpole (B) and stage 61–64 metamorphic (C) animals. Three individuals at each stage (a/t/m1-3) were used. Tissues analyzed were: Skin, Lung, Thymus, Spleen, Intestine, Kidney, Liver, Gills, Fat body and Muscles. All cDNA samples were normalized according to GAPDH expression. Either 0.1 pg of plasmid with corresponding cDNA insert (γa, γb, ζa, ζb) or clear water (0) were used as controls. “x” denotes reactions that were not carried out.
Fig. 6. Flow cytometry analysis of living 293T cells co-transfected with plasmids encoding HA-tagged X. laevis XFL2 receptor and c-myc-tagged X. laevis FcRγ.a/b transmembrane signaling subunits. The cells were stained with anti-HA antibodies and analyzed with a BD FACSCanto II cytometer and the BD FACSDiva software.
Fig. 7. Schematic representation of scaffolds fragments containing TCR genes. Headlines show the scaffold (Sc) number, the size of the scaffold (in parentheses) and the fragment coordinates on the scaffold. The bottom lines contain designations of the genes according to their counterparts in the human genome. The TCR genes and their neighbors are shown by open and filled rectangles, respectively. Pseudogenes are shown by gray rectangles. Arrows show transcription orientation. Double slash indicates a gap with a size shown in the corresponding bottom lines.