Zhao Y, Pan-Hammarström Q, Yu S, Wertz N, Zhang X, Li N, Butler JE, Hammarström L.

	Fig. 1. Assembly of the X. tropicalis IgH gene locus. VH, heavy-chain variable genes; DH, heavy-chain diversity gene segments; JH, heavy-chain joining gene segments; Cμ, IgM encoding gene; Cδ, IgD encoding gene. The filled boxes indicate exons encoding structurally conserved IgC domains: Cχ, IgX encoding gene; Cυ, IgY encoding gene; Cφ, IgF encoding gene; M, membrane exon. The domains encoding exons of each constant region gene are indicated with Arabic numbers. The position of the χCH1 exon is uncertain because it is missing in Scaffold_928 due to a small sequence gap.
	Fig. 2. Structure of IgD heavy chains in different vertebrates. Catfish IgD (GenBank accession no. U67437); human IgD (GenBank accession no. AAB21246); H, hinge region.
	Fig. 3. An unrooted phylogenetic tree of Igs in vertebrates. The tree was constructed by using protein sequences of the first and last CH domains (fish δCH6 and Xenopus δCH7 were used as the last domain) of all heavy-chain classes. Except for the Ig sequences obtained in this study, all other sequences were taken from the GenBank database, with the following accession numbers: Cδ gene: catfish (AF363450), fugu (AB159481), human (BC021276), mouse (J00449), and zebrafish (BX510335); Cμ gene: catfish (M27230), chicken (X01613), duck (AJ314750), human (X14940), mouse (V00818), nurse shark (M92851), little skate (M29679), X. laevis (BC084123), and zebrafish (AY643751); Cα gene: chicken (S40610), cow (AF109617), duck (U27222), human (P01877), and mouse (BC010324); Cε gene: cow (BTU63640), human (AK130825), and mouse (X01857); Cγ gene: cow (S82407), human (BX640623), llama (AF305955), and mouse (AY498569); Cυ gene: chicken (X07174), duck (AJ314754), and X. laevis (X15114); and Cχ gene: X. laevis (BC072981), nurse shark NAR (U18701), sandbar shark IgW (U40560), and lungfish IgW (AF437727).
	Fig. 4. Structure of Igs in X. tropicalis. (a) A ribbon representation of the predicted structural model of the X. tropicalis IgF heavy chain. The CH1 and CH2 domains are colored green and blue, respectively. The putative hinge region between the two domains is colored red. Note that the hinge between CH1 and CH2 contains a gap (Ser-248 to Gly-252), which is due to the absence of corresponding residues in the template structure. The figure was prepared with PyMOL software. (b) Domain structure of IgF as compared with IgM, IgX, and IgY. There is only one cysteine in the C terminus of the CH2 domain of IgM for potential inter-heavy-chain disulfide bonding. CH, heavy-chain constant region domain; CL, light-chain constant region domain; VH, heavy-chain variable region; VL, light-chain variable region.
	Fig. 5. Expression of X. tropicalis IgD, IgF, IgX, IgY, and IgM in different organs as detected by RT-PCR. BA, β actin; 1, kidney; 2, thymus; 3, intestine; 4, spleen; 5, stomach; 6, liver; 7, caecum; 8, negative control.
	Fig. 6. Sequence alignment of the IgM heavy-chain constant region in Xenopus tropicalis and Xenopus laevis. The alignment was performed by using the ClustalW method in MegAlign (DNASTAR, Madison, WI). Trop, X. tropicalis; Laev, X. laevis.
	Fig. 7. Sequence alignment of the IgX heavy-chain constant region in X. tropicalis and X. laevis. The alignment was performed by using the ClustalW method in MegAlign (DNASTAR). Trop, X. tropicalis; Laev, X. laevis.
	Fig. 8. Sequence alignment of the IgY heavy-chain constant region in X. tropicalis and X. laevis. The alignment was performed by using the ClustalW method in MegAlign (DNASTAR). Trop, X. tropicalis; Laev, X. laevis.
	Fig. 9. Sequence alignment of IgD transmembrane regions from different species. The alignment was performed by using the ClustalW method in MegAlign (DNASTAR). The percentage value after each sequence indicates its percentage identity to the X. tropicalis IgD transmembrane region. Except for the X. tropicalis IgD sequence, which was identified in this study, all other sequences were taken from the GenBank database, with the following accession numbers: catfish IgD, U67437; cod IgD, AF155200; cow IgD, AF515672; human IgD, BC063384; mouse IgD, AJ851868; pig IgD, AF515674; rat IgD, AY148495; salmon IgD, AF141606; and sheep IgD, AF515673.
	Fig. 10. Deduced amino acid sequence of the X. tropicalis IgD heavy-chain constant region.
	Fig. 11. Nucleotide and the deduced amino acid sequences of an IgF cDNA (GenBank accession no. BC087793). Leader, leader peptide; VDJ, rearranged variable region. The hinge, including segments encoded within CH1 and CH2 exons, is underlined. The cysteine and prolines are in bold.
	Fig. 12. Sequence of the membrane-bound form of IgF. Stop codons are indicated by inverted filled triangles; the poly(A) addition signal is underlined. CH2, second constant region domain; M1–4, exons encoding the transmembrane region and the cytoplasmic tail.
	Fig. 13. Long PCR amplification of the intron DNA between the u and f genes. (a) Strategy for the long PCR. The primers IgYTMs and IgFCH1as were used. M, region encoding transmembrane region; CH, region encoding the constant domain exon; h, hinge-encoding exon. (b) Long PCR amplification result. Lane 1, 1-kb DNA ladder; lane 2, amplified PCR product; lane 3, 5-kb DNA ladder.
	Fig. 14. DH (a) and JH (b) gene segments in X. tropicalis. Recombination sequence signals, including nonamers and heptamers, are in bold.
	Fig. 15. Dotplot analysis of the JH–Cm intron sequence. Sequence comparison was carried out by using the one-pair dotplot of MegAlign (DNASTAR). The JH–Cm intron was analyzed by using the following parameters: percentage, 80%; window, 30; minimum quality, 1. The parameters used for all of the other sequences were: percentage, 65%; window, 30; minimum quality, 1.
	Fig. 16. Sequence comparison of the IgF hinge–CH2 intron and the CH2 exon.
	Fig. 17. Sequence alignment of the IgF and IgY heavy-chain constant regions in X. tropicalis. The alignment was performed by using the ClustalW method in MegAlign (DNASTAR). Trop, X. tropicalis.

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