Previous studies on the immunoglobulin (Ig) genes in avian species are limited (mainly to galliformes and anseriformes) but have revealed several interesting features, including the absence of the IgD and Igκ encoding genes, inversion of the IgA encoding gene and the use of gene conversion as the primary mechanism to generate an antibody repertoire. To better understand the Ig genes and their evolutionary development in birds, we analyzed the Ig genes in the ostrich (Struthio camelus), which is one of the most primitive birds. Similar to the chicken and duck, the ostrich expressed only three IgH chain isotypes (IgM, IgA and IgY) and λ light chains. The IgM and IgY constant domains are similar to their counterparts described in other vertebrates. Although conventional IgM, IgA and IgY cDNAs were identified in the ostrich, we also detected a transcript encoding a short membrane-bound form of IgA (lacking the last two CH exons) that was undetectable at the protein level. No IgD or κ encoding genes were identified. The presence of a single leader peptide in the expressed heavy chain and light chain V regions indicates that gene conversion also plays a major role in the generation of antibody diversity in the ostrich. Because the ostrich is one of the most primitive living aves, this study suggests that the distinct features of the bird Ig genes appeared very early during the divergence of the avian species and are thus shared by most, if not all, avian species.
Citation: Huang T, Zhang M, Wei Z, Wang P, Sun Y, Hu X, et al. (2012) Analysis of Immunoglobulin Transcripts in the Ostrich Struthio camelus, a Primitive Avian Species. PLoS ONE 7(3): e34346. https://doi.org/10.1371/journal.pone.0034346
Editor: Sebastian D. Fugmann, National Institute on Aging, United States of America
Received: November 21, 2011; Accepted: February 26, 2012; Published: March 29, 2012
Copyright: © 2012 Huang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Science Fund for Distinguished Young Scholars (30725029), the Taishan Scholar Foundation of Shandong Province, and the National Basic Research Program of China (973 Program-2010CB945300). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The adaptive immune system of jawed vertebrates is characterized by the production of immunoglobulins (Igs) in response to antigens . The B cell antigen receptor Ig is a heterodimeric protein that is usually composed of two identical heavy (H) chains and two identical light (L) chains. A disulfide bond formed by cysteine residues between the CL and CH1 domains covalently joins the L chain to the H chain, and the two V domains associate non-covalently to form the antigen-binding site .
The Ig classes (in mammals, IgM, IgA, IgD, IgG and IgE) are defined by the isotypes of the heavy chain constant genes (μ, α, δ, γ and ε). Additional Ig isotypes have been identified in lower jawed vertebrates, including birds, reptiles, amphibians, bony fish and cartilaginous fish . IgM is structurally conserved throughout evolution and is expressed in all jawed vertebrates. IgD is as ancient as IgM and has been described in elasmobranchs (in which it was previously known as IgW), bony fish, amphibians, reptiles and mammals , , , , . IgD is, however, absent in birds and several mammals such as rabbits, opossum and elephants , , , . Compared with IgM, IgD shows a high degree of structural plasticity because of variance in the copy number and number of CH encoding exons as well as alternative RNA splicing . In addition to these ancient Ig classes, some additional distinct Ig classes have been found in different vertebrates, such as IgY in lower tetrapods , , , IgNAR in cartilaginous fish , IgT/IgZ in the trout and zebrafish , , IgX and IgF in amphibians , , and IgO in the platypus .
The L chains contribute considerably to combinatorial antibody diversity by their association with H chains . It is known that cartilaginous fish, teleost fish and amphibians express three IgL isotypes: κ, λ and σ , , . A fourth IgL isotype, σ-cart, is only found in sharks . Evolutionarily, fewer types of Ig light chains are present in mammals and reptiles, which express only λ and κ. The two light-chain loci differ significantly in their genomic organization. At the λ locus, multiple Vλ segments are followed by Jλ-Cλ repeats. By contrast, the κ chain–encoding locus contains only a single Cκ gene with a small cluster of Jκ and multiple Vκ genes located upstream , , . Surprisingly, birds exclusively express λ light chains , . The chicken and zebra finch IgL loci include only one functional IGVL gene and one IGJL gene, but multiple IGVL pseudogenes are located upstream of this functional IGVL gene , . Light chain diversity is generated by intrachromosomal gene conversion using the upstream pseudo-Vλ gene segments as donor sequences .
The avian species described to date express only three immunoglobulin classes: IgM, IgA and IgY, which are encoded by Cμ, Cα and Cυ respectively , , and no IgD encoding gene has been identified. The Cυ and Cα genes in the chicken and duck IgH loci are positioned in reverse orientation , , which raises questions regarding the mechanism of class switch recombination in birds and the evolution of the IGHC gene locus. IgY is a monomeric antibody of low molecular weight found in amphibians, reptiles, and birds and is thought to be the ancestor of mammalian IgG and IgE . In addition to the full-length IgY, ducks can also generate a truncated IgY termed IgY(ΔFc), which is expressed by the alternative transcriptional termination of the single υ gene , .
Birds represent an enormously diverse group of vertebrates comprising nearly 9000 species. Our knowledge of the avian Ig genes is currently restricted to a few galliform (chicken, turkey, pheasant and quail) and anseriform birds (duck) . According to phylogenetic studies, these two groups of birds diverged only recently (approximately 100 million years ago [MYA]) . The ostrich (Struthio camelus) belongs to the ratitae order, which represents the most primitive living aves, i.e., birds that diverged from other avian lineages approximately as early as 140 MYA . In the present study, we analyzed the Ig genes in this species to investigate whether it expresses other Ig isotypes in addition to the IgM, IgA, IgY and λ light chains. Our objective was to provide additional clues to understand the evolution of Ig genes in birds.
IgH classes expressed in the ostrich
To analyze the IgH classes expressed in the ostrich, we generated two Ig-specific mini-libraries using the total RNA isolated from the spleen and intestine. In total, 234 clones derived from the spleen library were analyzed. Most clones (199) were found to contain IgA cDNA, whereas only 16 IgM- and 5 IgY-containing clones were identified. The remaining 14 clones were shown to contain non-Ig sequences. It is surprising that the IgA clones comprised such a large portion of the library. This finding is likely the result of PCR bias in the construction of the library. We only identified IgA clones (319 clones) from the intestine library (total of 327 clones analyzed). These data suggest that the ostrich also expresses IgM, IgY and IgA, similar to chickens and ducks. The presence of these encoding genes in the ostrich genome was subsequently confirmed by Southern blotting using Cμ-, Cα- and Cυ-specific full-length probes (Fig. 1A).
A. Southern blot detection of the ostrich Ig heavy chain constant region genes using Cμ-, Cα-, and Cυ-specific full-length probes. EI, EcoR I; EV, EcoR V; B, BamH I; X, Xba I; D, Dra I; H, Hind III; P, Pst I. B. Southern blot detection of the ostrich Ig heavy chain constant region genes using Cμ4, Cα3, Cυ4 single-exon probes. EI, EcoR I; EV, EcoR V; B, BamH I; H, Hind III; PI, Pst I; PII, Pvu II.
To investigate whether the ostrich expresses IgD, we designed several pairs of degenerate primers based on the conserved Cδ regions of other species. However, we did not to amplify any putative IgD sequence regardless of whether cDNA or genomic DNA was used.
Analysis of the ostrich Cμ gene
Analysis of the obtained IgM heavy chain constant-region cDNA clones revealed only a unique sequence, which suggests the expression of a single μ gene. However, four bands were detected when the μCH4 sequence (containing no Hind III site) was used as a probe in the Southern detection of Hind III-digested genomic DNA (Fig. 1B), which indicates the presence of more than one μ genes in the ostrich genome.
The obtained ostrich secretory IgM heavy chain constant-region cDNA encodes 447 amino acids, in which 12 cysteines are positionally conserved compared with Cμ in other species (Fig. S1). All of the aligned Cμ sequences (secreted form) exhibit an identical three-amino-acid motif (TCY) in their carboxy terminals (Fig. S1), which is where the cysteine is assumed to bind the J chain to form polymeric IgM . The entire ostrich IgM constant region contains four potential N-linked glycosylation sites (N–X–S/T): N-46, N-127, N-199 and N-434. Only N-46 and N-434 are conserved among reptiles, birds and mammals , . The N-127 site is conserved in birds and reptiles. The N-199 site is found exclusively in birds (Fig. S1). Alignment of the ostrich IgM constant region with those of other species demonstrated that the Cμ3 and Cμ4 domains to be less divergent than the Cμ1 and Cμ2 domains (Fig. S1). The ostrich IgM constant region shares an overall identity of 53.1% and 63.1% with the chicken and duck Cμ, respectively, at the protein level. The identity of the ostrich IgM is supported by a phylogenetic analysis (Fig. 2).
The amino-acid sequences of all CH domains were used for the tree construction. For those species that express IgD (and IgW) longer than four CH domains, only the first four CH domains were used in the tree construction. The scale bar represents the genetic distance. The credibility value for each node is shown.
Northern blotting to detect IgM gene expression further showed that the ostrich μ gene was primarily expressed in the spleen and large intestine and only weakly expressed in the liver and small intestine (Fig. 3) although RT-PCR showed IgM transcripts to be present in all tissues examined (Fig. S2).
Analysis of the ostrich α gene
Southern blotting with either the full-length or Cα3 exon as probes suggested that a single α gene was present in the ostrich genome (Fig. 1). IgA is the principal antibody class in mucosal secretions and acts as an important first line of defense . It is usually highly expressed in mucosal tissues but only weakly expressed in the spleen. However, most clones in our spleen-derived Ig-specific mini-library were found to be IgA. This finding could be the result of a PCR bias during the process of 3′ RACE. Indeed, our RT-PCR and Northern blotting data showed that the ostrich IgA was primarily expressed in the large and small intestines (Fig. S2, Fig. 3).
When comparing the ostrich IgA heavy chain constant region with those of other species, 10 conserved cysteines were observed. There are three N-linked glycosylation sites in Cα2, Cα3 and the canonical secretory tail: N-165, N-221 and N-419, all of which are conserved in birds (Fig. S3). The ostrich Cα gene shares 44% sequence identity with chicken and 66% with duck Cα.
When performing 3′RACE PCR using the spleen RNA and JH-derived primers, we observed an 850-bp band in addition to the major 1.6-kb products (all 4-Cα containing transcripts). Sequencing of this band showed that it encoded a short, membrane-bound IgA lacking the last two Cα domains (i.e., VDJ-Cα1-Cα2-TM) (Fig. S4). To further confirm the presence of this short transcript, we used primers derived from the Cα1 to perform IgA-specific 3′RACE. In addition the full-length of 1.4-kb IgA transcript, we again detected the short IgA transcript, which contained only the first two Cα domains (Fig. 4A). To determine whether the short IgA is only expressed in the spleen, we then performed RT-PCR using the primers derived from the Cα1 and TM regions. The short form was detected in multiple tissues (Fig. 4B). Northern blotting with the first two Cα exons as a probe showed the short form to be mainly expressed in the intestine, albeit at a much lower level than the full-length form (Fig. 4C). To confirm that the short IgA transcript was derived from alternative splicing, we amplified and sequenced the exon-intron boundaries of Cα2-intron-Cα3, and Cα4-intron-TM, which clearly demonstrated that the short form to be derived from splicing of the Cα2 onto the TM exon.
A. PCR product of IgA 3′RACE. M, 100-bp DNA marker; 1, full-length IgA form; 2, non-specific band; 3, short form of IgA. B. RT-PCR detection of the short form of transmembrane IgA gene expression in different tissues. C. Northern blot analysis of ostrich full length and short-form of IgA gene expression in different tissues.
The presence of the short IgA transmembrane transcript raises a question as to whether the ostrich is able to express a secreted IgA form lacking the last two Cα domains (i.e., IgA(ΔFc), similar to IgY(ΔFc) in ducks), although we did not observe such transcripts in the RACE experiments. We thoroughly analyzed the intron sequence between Cα2 and Cα3, and did not find any potential transcriptional termination signal or polyadenylation signal sequence (i.e., AATAAA). A polyclonal rabbit antiserum against the ostrich Cα1 and Cα2 were used in Western blotting. Only the intact form of IgA (approximately 65 KD under reducing conditions) was detected in the intestine membrane and cytoplasmic proteins (Fig. 5A). No short form of IgA could be identified at the protein level, probably because of an extremely low level of expression. The IgA in secretions of the large intestine appeared to be dimeric (approximately 350 KD under non-reducing conditions), as under reducing conditions, the molecular weight of the IgA heavy chain (without light chains) is approximately 65 KD (Fig. 5B).
A. IgA expression in tissues and secretions. 1, Large-intestine cytoplasmic proteins; 2, Small-intestine cytoplasmic proteins; 3, Cell membrane proteins of the large intestine; 4, Cell membrane proteins of the small intestine; 5, Large-intestine secretions. B. Dimerized IgA detected in large-intestine secretions.
Analysis of the ostrich υ gene
The full-length IgY heavy chain constant region cDNA (secreted form) was obtained by screening the spleen Ig-specific mini-library. A phylogenetic analysis indicated that it was the ostrich υ gene (Fig. 2). Similar to ostrich μ, we only obtained a single IgY heavy chain constant-region cDNA, although Southern blotting indicated that more than one υ gene was present in the ostrich genome (Fig. 1). Alignment of the ostrich IgY heavy chain constant region with those of other species revealed two cysteines in the Cυ1, which suggests that these molecules can associate with light chains. Seven additional cysteines were distributed in Cυ2-Cυ4, all of which are conserved across all species examined (Fig. S5). Cυ contains two N-linked glycosylation sites: N-166 in the Cυ2 conserved in birds and lizards and N-265 in the Cυ3 conserved in Xenopus and humans (Fig. S5). A domain-by-domain comparison of the Cυ regions indicated that the Cυ1 displayed the lowest amino acid identity in birds (Fig. S5).
The expression pattern of the ostrich IgY transcript was examined using RT-PCR and Northern blotting suggested that the υ gene was primarily expressed in the spleen and large intestine (Fig. S2, Fig. 3).
Analysis of rearranged VDJ fragments
To analyze the expressed VDJ sequences, 5′RACE was performed using the primers derived from the μ, α and υ chain constant regions. The inferred amino acid sequences were aligned and showed relatively low sequence diversity. The amino acid sequence variabilities of the VH region were mostly confined to the CDR regions, in particularly the CDR3 region . We sequenced 83 cDNA fragments, which provided 54 unique CDR3 (Fig. S6). The length of CDR3 varies from 9 to 24 residues to create considerable variability with an average of 14.33±2.18 codons, which is longer than the CDR3 of Xenopus (8.6 codons) and mice (8.7 codons) . Analysis of the FR4 sequences suggests that there are two distinct JH gene segments in the ostrich: JH1 and JH2, which differ by seven nucleotides but have only one amino-acid substitution (Fig. S7). Among the obtained VH clones, more than 10 contained leader peptide-encoding sequences that were identical in sequence (MGPRLPGFVLLLLLLAALPGLRA). It is highly likely that only a single VH gene segment was available for VDJ recombination events in the ostrich.
Analysis of the ostrich light chain genes
To analyze the light chain genes in the ostrich, we designed several pairs of degenerate primers for the λ and κ genes based on the conserved Cλ and Cκ sequences of other species. These primers were used in PCR amplifications with the spleen cDNA as templates. We were only able to amplify the λ gene in the ostrich, as a phylogenetic analysis clearly showed that the identified gene belonged to the λ lineage (Fig. 6). We further performed 5′ RACE amplifications based on the Cλ sequence that we obtained. In total, 57 clones were sequenced and shown to encode the full-length Vλ domain and the same leader peptide (MAWAPLLLAVLAHGSGSLV). Overall, the inferred Vλ region amino acid homology among these clones ranged from 87.2 to 99.2%. The average length of the CDR3 was 9.81±2.38 codons, with a range of 4 to 14 codons. The tetrapod IGVL sequences generally have a fairly well-conserved DEAD (Asp–Glu–Ala–Asp) motif in the FR3 region . However, as in the chicken IGVL sequence (DEAV), the Asp residue is also substituted by a Val in the ostrich. An analysis of these Vλ sequences revealed only a single Jλ in the ostrich.
The amino acid sequences were used for the tree construction.
We subsequently performed 3′RACE using the leader peptide specific primers and which identified a single Cλ in the ostrich. The Cλ sequence shows a 67.0%, 55.1% and 64.5% amino acid sequence homology to the chicken, lizard and human Cλ1, respectively. A protein sequence alignment of Cλ in amphibians, reptiles, birds and mammals revealed an identical pattern with regard to the cysteine distribution (Fig. S8).
In the present study, three Ig isotypes (IgM, IgA and IgY, but not IgD nor Igκ), were identified in the ostrich, which is a primitive avian species belonging to the order struthioniformes. Although Southern blotting indicated that there was more than one copy of the μ and υ genes in the ostrich genome, we were only able to obtain one copy of the μ and υ expressed at the cDNA level. We amplified and sequenced the intron between Cμ1 and Cμ2 and obtained only a single sequence. We also performed PCR using two pairs of primers derived from the conserved Cυ4 sequences using genomic DNA. All sequenced clones were shown to contain the same sequence. The additional μ and υ genes detected by Southern blotting were likely pseudogenized and have diverged. We also performed Southern blotting using probes for the IgD and Igκ constant regions of crocodiles and could not detect any bands (data not shown). It is likely that, as in chickens and ducks, the δ and κ genes are both absent in the ostrich; however a definite conclusion cannot currently be reached. All of the expressed ostrich heavy chain and light chain V regions harbored the same signal peptide, which indicates that there is only one functional VH and Vλ involved in V(D)J recombination in the ostrich. It is reasonable to assume that the ostrich also uses gene conversion as a major mechanism for generating antibody diversity. The ostrich Ig genes essentially exhibit the same distinct features that have been previously observed in chickens and ducks. This similarity demonstrates that the typical bird Ig system was likely already present in the common ancestor of carinatae and ratitae bird species and has remained unchanged over a long period of evolution (Fig. 7).
Reptiles are the closest relatives of the aves, and they are believed to have diverged approximately 250 MYA . Recent studies have shown that reptiles, such as lizards and turtles, express IgD and κ light chains , , ; this finding suggests that the evolutionary loss of these two genes must have occurred in birds after their divergence from reptiles (Fig. 7). Another interesting issue regarding IgA evolution also arises when considering findings present in both reptiles and birds. The IgA-encoding gene in ducks and chickens shows a transcriptional orientation opposite to that of IgM and IgY , . We also recently showed that the IgA encoding gene was absent in lizards and some other reptiles ( and our unpublished data), which suggests that the IgA gene in the lineages leading to reptiles and birds has undergone some gene rearrangements that either deleted or inverted this gene. These germ-line DNA rearrangements in the IgH locus might also account for the evolutionary loss of the IgD gene in birds. A future investigation on the Ig genes in more primitive living birds or reptiles may help to clarify this issue.
When analyzing the ostrich IgA transcripts, we identified a shorter membrane-bound IgA encoding form with the last two Cα exons removed, as the Cα2 exon was directly spliced onto the TM exon. However, this short form of IgA could not be detected at the protein level, which suggests limited to no functional significance. Indeed, this short form of the IgA transcript was present at a very low level even at the mRNA level, and its presence may simply be to the result of accidental RNA splicing caused by non-critical mutations around the splice sites.
In summary, we characterized three Ig heavy chain classes (IgM, IgA and IgY) and the λ light chain in the ostrich in this study. This study enriches the current knowledge of ratitae Igs, provides support for the continuous evolution of immunoglobulins in birds and highlights the importance of comparative studies in understanding the evolutionary history of the immune system.
Materials and Methods
Animals, RNA and DNA isolation
Ostriches (Struthio camelus) were purchased from a local Beijing farm. The animals were treated in accordance with the guidelines of China Agricultural University regarding the protection of animals used for experimental and other scientific purposes. The study was approved by the ethics committee of China Agricultural University (ID number 20110302). The total RNA was extracted from different tissues using the TRNzol kit (TianGen Biotech), following the manufacturer's instructions. Genomic DNA was extracted from the liver following routine protocols.
Construction and screening of spleen and intestinal Ig-cDNA libraries
Approximately 2 µg of spleen and intestine total RNA was used to synthesize first-strand cDNA with a First-Strand cDNA synthesis kit (Promega, USA). One pair of degenerate primers, VHs (5′-CCH RGV AAG GGG CTS GAG TGG GT-3′) and IgAas (5′-TTG ACW TKG GTG GGT TTA CC-3′), was designed based on the conserved VH and IgA CH regions. The products were gel-extracted, ligated into the pMD19-T vector (Takara) and sequenced. JH-specific primers were designed according to the analysis results. First-strand cDNA synthesis was performed with Not I-d (T) 18 primers (5′-AAC TGG AAG AAT TCG CGG CCG CAG GAA TTT TTT TTT TTT TTT TTT-3′). JH1s (5′-GCC GGG GCA CCT CGG TCA CCG TCT CCT CA-3′) and JH2s (5′-GCC GCG GGA CGG CCG TCA CCG TGT CCT CA-3′) were mixed and used as sense primers for one round of 3′RACE PCR for the ostrich heavy chain constant regions. The resulting 1.6-kb PCR products were cloned into a T vector to generate an Ig cDNA mini-library. The white clones (after blue-white screening) were subjected to PCR screening using the universal primers M13F (5′-GCT TCG AAT GTA AAA CGA CGG CCA GT -3′) and M13R (5′-GCT TCG AAC AGG AAA CAG CTA TGA C -3′) for positively recombined clones containing the correct insert size; IgM1 (5′-CGT CAA CGA CAG CAT CTC CA -3′) and IgM2 (5′-GCG AGC ACC AGG GAC ACA TT-3′) for IgM-positive clones; IgA1 (5′-GCG CTC TGG ACG TGA CCT CCG A -3′) and IgA2 (5′-GAC GCT CAG CTT GCT GTA GAC -3′) for IgA-positive clones; and IgY1 (5′-CTG CCT CAT CTC CCA CTT CTA C-3′) and IgY2 (5′-TCT CGG TGC AGT GGC TCA AGA AC-3′) for IgY-positive clones. The clones that contained correct insert size but were negative for IgM, IgA and IgY were sequenced to identify the inserts. The primers used for cloning the ostrich VH region were MGSP1 (5′-CTG GAG ATG CTG TCG TTG ACG TAG T-3′), MGSP2 (5′-TGA CGT AGT TGG TCC AGG AGA A-3′), AGSP1 (5′-CCG GGT AGG TCA AGA CCT CTG A-3′), AGSP2 (5′-TCG CTG GAA ACC CAG GTG AC-3′), YGSP1 (5′-TGG CGG CCA TCG CAA ACT GGC T-3′), and YGSP2 (5′-TAG AGG CCG GAG CGG AAG AG-3′); all of the primers were designed from the CH regions that we obtained. The PCR experiments were performed according to the instructions of the 5′-RACE System for Rapid Amplification of cDNA Ends (Invitrogen, USA). The 650-bp PCR products were cloned into the pMD19-T vector and sequenced directly. Similarly, the ostrich Ig light-chain spleen and intestinal cDNA min-libraries were constructed and screened. The primers used to amplify the Igλ constant region were CLs (5′-AAH AAG GCC ACM CTG GTG TG-3′) and CLas (5′-CAG GTA RCT GCT GDC CAT RTA-3′). Primers LGSP1 (5′-GTA CTG GTT GTT GCT CTG-3′) and LGSP2 (5′-CCA CAC CGT TGG AGA TGG G-3′) were used to perform the 5′RACE. Primers L1 (5′-TCG CGG TGC TCG CCC ACG G-3′) and L2 (5′-CAC GGC TCA GGT TCC CTG GTC-3′) were used to perform the 3′RACE.
Fourteen micrograms of liver genomic DNA digested with EcoR I, EcoR V, BamH I, Xba I, Pst I, Hind III, Dra I and Pvu II were fractionated in 0.9% agarose and transferred to Hybond N+ nylon membranes. Cμ-, Cα-, and Cυ-specific full-length as well as single-exon probes were labeled using a PCR digoxigenin probe synthesis kit (Roche, Germany). The primers used to amplify the full-length Cμ and Cμ4 exon probes were Cμs (5′- CGT CAA CGA CAG CAT CTC CA-3′), Cμas (5′-CAT TGA CCG AGG TGG GTT TA-3′), Cμ4s (5′-GCC AGA GCC CCG ACC ATC TAC-3′), and Cμ4as (5′- GAG GAC TTG TCC ACC GAC TTC-3′). The primers used to amplify the full-length Cα and Cα3 exon probes were Cαs (5′- CTG CGA CGA GGG AAA CGT CAC-3′), Cαas (5′-GAC GCT CAG CTT GCT GTA GAC-3′), Cα3s (5′-CTG CCC GTG GTC TCC ATC CTC-3′), and Cα3as (5′-AGT TCC TTC TGT GCA GAT TTG-3′). The primers used to amplify the full-length Cυ and Cυ4 exon probes were Cυs (5′- CTG CCT CAT CTC CCA CTT CTA C-3′), Cυas (5′- TTT ACC GGG GCT CCT GCT GAT-3′), Cυ4s (5′- CTG GCG CCC AGC GTC TAC CT-3′), and Cυ4as (5′- TGC GTT GCA CGA ACT TCA TG-3′). The hybridization and detection were performed following the manufacturer's hybridization instructions.
PCR and Northern blotting detection of ostrich Ig gene expression in different tissues
The synthesized cDNA samples derived from RNA isolated from different organs (heart, liver, spleen, lung, kidney, large intestine, small intestine, and stomach) were used in RT-PCR to detect the expression of IgM, IgA, and IgY. The ostrich EIF1A1 gene was used as an internal control. The PCR primers were IgM-detections (5′-GGC CCC GTT GAT GTG GTG CCC A-3′); IgM-detectionAs (5′-GCT CGA AGC CGC ACT CCA G-3′); IgA-detections (5′-AAG AAC ATC GGG GAC TTA TG-3′); IgA-detectionAs (5′-GAC GCT CAG CTT GCT GTA GAC-3′); IgY-detections (5′-CTG CCT CAT CTC CCA CTT CTA C-3′); and IgY-detectionAs (5′-GAT GGT GCG TTG CAC GAA CTT-3′). Total RNA was used (7 µg/lane) for Northern analysis (the same kit as for the Southern blots). The primers used for probe amplification were the following: IgMs (5′-CCT GGA CCA ACT ACG TCA AC-3′); IgMas (5′-TTG CTC GTG TTC CTC ATC TC-3′); IgA1s (5′-CTG CGA CGA GGG AAA CGT CAC-3′); IgA1as (5′-GGA CAC TCG GCA AGC GAA CTC-3′); IgA2s (5′-GGA CCT CTA CCT CAG CCA GAA-3′); IgA2as (5′-GTG GAC GAA GGT GAT GGG GAT-3′); IgYs (5′-GAT CCA CGT CTT CGC CTT GC-3′); IgYas (5′-GAT GGT GCG TTG CAC GAA CTT-3′); IgLs (5′-GTG CAC CTC TTC CCT CCA TC-3′); IgLas (5′-TAG GAG GAG CAC TCG GAT CT-3′); EIF1A1s (5′-AAG GAG AAG ACC CAC ATC AAC-3′); and EIF1A1as (5′-GAG GAT CAT TCT TGC TGT CAC-3′).
Membrane and cytosol proteins derived from different ostrich tissues were prepared using extraction kits (Beyotime, Beijing). Large-intestine secretions were diluted with 3% PBS. The oligopeptide that encodes ostrich IgA CH1–CH2 exons was synthesized, modified and coupled with KLH, and then intravenously injected into New Zealand rabbits. A polyclonal rabbit antiserum against the ostrich IgA CH1–CH2 domains was obtained and purified (Cwbiotech, Beijing). Samples were thermally denatured, separated by 12% SDS-PAGE and transferred to nitrocellulose membranes (Millipore, USA). The blot was blocked in Tris-buffered saline (TBS) containing 5% skim milk (w/v) for 1 h. Rabbit pAb and HRP-conjugated goat anti-rabbit IgG secondary antibodies (Cwbiotech, Beijing) were diluted using TBS+5% milk to 1∶600 and 1∶5000, respectively. The membranes were washed six times in TBS+0.05% Tween20 (TBST) between each step, and all incubations were performed at room temperature for 1 h. The bands were detected by incubation with Pierce ECL plus western blotting substrate (Thermo Fisher Scientific, USA) following the manufacturer's instructions.
Sequence alignment and phylogenetic analysis
DNA and protein sequence editing, alignments, and comparisons were performed with the MegAlign software (DNASTAR). Phylogenetic trees were generated using MrBayes3.1.2  and viewed in TreeView . Multiple protein sequence alignments for the tree construction were performed using ClustalW. The accession numbers for the sequences (http://www.ncbi.nlm.nih.gov/sites/entrez) used for phylogenetic analysis are as follows: α or χ gene: chicken, S40610; cow, AF109167; duck, AJ314754; human, J00220; mouse, J00475; platypus, AY055778; X. laevis, BC072981; axolotl, AM774592; gecko, DQ523197; X. tropicalis, BC157650. δ gene: catfish, U67437; cow, AF411245; human, BC021276; mouse, J00449; X. tropicalis, DQ350886; lizard, EF690359. γ gene: human, J00228; mouse, J00453; platypus, AY055781; horse, AJ302055. ε gene: cow, AY221098; human, J00222; mouse, X01857; platypus, AY055780. μ gene: nurse shark, M92851; skate, M29679; catfish, X52617; lungfish, AF437724; zebrafish, AF281480; X. laevis, BC084123; axolotl, AM419754; lizard,EF690357; gecko, EU287911; chicken, X01613; duck, AJ314754; human, X14940; mouse, V00818; platypus, AY168639. υ gene: chicken, X07175; duck, X78273; X. laevis, X15114; lizard, EF690360; gecko, EU827594; X. tropicalis, BC089679; axolotl, X69492; Chinese soft-shell turtle, FJ605148. The accession numbers of sequences used for IgL constant regions are as follows; λ genes: chicken, X04768; duck, X82069; platypus, AF525122; human, J00252; mouse, AC14021; X. laevis type III, BC082898; X. tropicalis type III, BC121563; zebra finch, ACH44209; lizard IGIC1, IGIC2 (Ref.25); skate type II, L25566; sandbar shark type II, M81314; horn shark type III, L25561. κ genes: mouse, EF392842; human, AC210709; cow, BC122795; lizard (Ref.25); X. laevis BC068859; zebrafish IGIC1, AF246185; zebrafish IGIC3, AF246193; nurse shark NS4, L16765; carp IGIC1, AB015902; carp IGIC3, AB035730.σ genes: X. laevis, NM_001094414; X. tropicalis, AAH87749; zebrafish IGIC2, AF246162; nurse shark, EF114766; horn shark, EF114760; carp IGIC2 (AB091120). σ-cart genes: nurse shark NS5, AY720857; skate type, L25568; horn shark type I, X15316.
Sequence alignment of the ostrich IgM CH region with that of other species. Dots are used to denote identical amino acids, and dashes are used to adjust the sequence alignment. Canonical cysteines are shaded and conserved N-linked glycosylation sites across species are in red. The alignment was performed using ClustalW with some manual adjustments.
RT-PCR detection of the ostrich IgH gene expression in different tissues.
Sequence alignment of the ostrich IgA CH region compared with that of other species. The alignment was performed using the ClustalW method in MegAlign. Canonical cysteines are shaded, and conserved N-linked glycosylation sites across species are in red.
Sequence of the short IgA membrane-bound form (VDJ-Cα1-Cα2-TM).
Sequence alignment of the ostrich IgY CH region compared with that of other species. The alignment was performed by using the ClustalW method in MegAlign. Canonical cysteines are shaded and conserved N-linked glycosylation sites across species are in red.
Sequence alignment of the 54 CDR3.
Sequence alignment of the ostrich JH gene segments.
We are indebted to Drs. Tao Wang, Gang Cheng, Qinghe Li, Beilei Xu and Lingxiao Li for their inspiring suggestions. The authors also wish to thank Dr. Xueqian Cheng for helping with the sample collection. The sequences reported in this study have been deposited in the NCBI GenBank (http://www.ncbi.nlm.nih.gov/geo) under the following accession numbers: JN709443–JN709460.
Conceived and designed the experiments: TH LH NL YZ. Performed the experiments: TH MZ. Analyzed the data: TH MZ ZW PW YS XH LR YZ. Contributed reagents/materials/analysis tools: QM RZ YG. Wrote the paper: TH LH YZ.
- 1. Flajnik MF (2002) Comparative analyses of immunoglobulin genes: surprises and portents. Nat Rev Immunol 2: 688–698.MF Flajnik2002Comparative analyses of immunoglobulin genes: surprises and portents.Nat Rev Immunol2688698
- 2. Bengten E, Wilson M, Miller N, Clem LW, Pilstrom L, et al. (2000) Immunoglobulin isotypes: structure, function, and genetics. Curr Top Microbiol Immunol 248: 189–219.E. BengtenM. WilsonN. MillerLW ClemL. Pilstrom2000Immunoglobulin isotypes: structure, function, and genetics.Curr Top Microbiol Immunol248189219
- 3. Litman GW, Anderson MK, Rast JP (1999) Evolution of antigen binding receptors. Annu Rev Immunol 17: 109–147.GW LitmanMK AndersonJP Rast1999Evolution of antigen binding receptors.Annu Rev Immunol17109147
- 4. Ohta Y, Flajnik M (2006) IgD, like IgM, is a primordial immunoglobulin class perpetuated in most jawed vertebrates. Proc Natl Acad Sci U S A 103: 10723–10728.Y. OhtaM. Flajnik2006IgD, like IgM, is a primordial immunoglobulin class perpetuated in most jawed vertebrates.Proc Natl Acad Sci U S A1031072310728
- 5. Wilson M, Bengten E, Miller NW, Clem LW, Du Pasquier L, et al. (1997) A novel chimeric Ig heavy chain from a teleost fish shares similarities to IgD. Proc Natl Acad Sci U S A 94: 4593–4597.M. WilsonE. BengtenNW MillerLW ClemL. Du Pasquier1997A novel chimeric Ig heavy chain from a teleost fish shares similarities to IgD.Proc Natl Acad Sci U S A9445934597
- 6. Zhao Y, Pan-Hammarstrom Q, Yu S, Wertz N, Zhang X, et al. (2006) Identification of IgF, a hinge-region-containing Ig class, and IgD in Xenopus tropicalis. Proc Natl Acad Sci U S A 103: 12087–12092.Y. ZhaoQ. Pan-HammarstromS. YuN. WertzX. Zhang2006Identification of IgF, a hinge-region-containing Ig class, and IgD in Xenopus tropicalis.Proc Natl Acad Sci U S A1031208712092
- 7. Wei Z, Wu Q, Ren L, Hu X, Guo Y, et al. (2009) Expression of IgM, IgD, and IgY in a reptile, Anolis carolinensis. J Immunol 183: 3858–3864.Z. WeiQ. WuL. RenX. HuY. Guo2009Expression of IgM, IgD, and IgY in a reptile, Anolis carolinensis.J Immunol18338583864
- 8. Zhao Y, Kacskovics I, Pan Q, Liberles DA, Geli J, et al. (2002) Artiodactyl IgD: the missing link. J Immunol 169: 4408–4416.Y. ZhaoI. KacskovicsQ. PanDA LiberlesJ. Geli2002Artiodactyl IgD: the missing link.J Immunol16944084416
- 9. Lundqvist ML, Middleton DL, Hazard S, Warr GW (2001) The immunoglobulin heavy chain locus of the duck. Genomic organization and expression of D, J, and C region genes. J Biol Chem 276: 46729–46736.ML LundqvistDL MiddletonS. HazardGW Warr2001The immunoglobulin heavy chain locus of the duck. Genomic organization and expression of D, J, and C region genes.J Biol Chem2764672946736
- 10. Ros F, Puels J, Reichenberger N, van Schooten W, Buelow R, et al. (2004) Sequence analysis of 0.5 Mb of the rabbit germline immunoglobulin heavy chain locus. Gene 330: 49–59.F. RosJ. PuelsN. ReichenbergerW. van SchootenR. Buelow2004Sequence analysis of 0.5 Mb of the rabbit germline immunoglobulin heavy chain locus.Gene3304959
- 11. Wang X, Olp JJ, Miller RD (2009) On the genomics of immunoglobulins in the gray, short-tailed opossum Monodelphis domestica. Immunogenetics 61: 581–596.X. WangJJ OlpRD Miller2009On the genomics of immunoglobulins in the gray, short-tailed opossum Monodelphis domestica.Immunogenetics61581596
- 12. Guo Y, Bao Y, Wang H, Hu X, Zhao Z, et al. (2011) A preliminary analysis of the immunoglobulin genes in the African elephant (Loxodonta africana). PLoS One 6: e16889.Y. GuoY. BaoH. WangX. HuZ. Zhao2011A preliminary analysis of the immunoglobulin genes in the African elephant (Loxodonta africana).PLoS One6e16889
- 13. Sun Y, Wei Z, Hammarstrom L, Zhao Y (2011) The immunoglobulin delta gene in jawed vertebrates: A comparative overview. Dev Comp Immunol 35: 975–981.Y. SunZ. WeiL. HammarstromY. Zhao2011The immunoglobulin delta gene in jawed vertebrates: A comparative overview.Dev Comp Immunol35975981
- 14. Zhao Y, Rabbani H, Shimizu A, Hammarstrom L (2000) Mapping of the chicken immunoglobulin heavy-chain constant region gene locus reveals an inverted alpha gene upstream of a condensed upsilon gene. Immunology 101: 348–353.Y. ZhaoH. RabbaniA. ShimizuL. Hammarstrom2000Mapping of the chicken immunoglobulin heavy-chain constant region gene locus reveals an inverted alpha gene upstream of a condensed upsilon gene.Immunology101348353
- 15. Greenberg AS, Avila D, Hughes M, Hughes A, McKinney EC, et al. (1995) A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks. Nature 374: 168–173.AS GreenbergD. AvilaM. HughesA. HughesEC McKinney1995A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks.Nature374168173
- 16. Hansen JD, Landis ED, Phillips RB (2005) Discovery of a unique Ig heavy-chain isotype (IgT) in rainbow trout: Implications for a distinctive B cell developmental pathway in teleost fish. Proc Natl Acad Sci U S A 102: 6919–6924.JD HansenED LandisRB Phillips2005Discovery of a unique Ig heavy-chain isotype (IgT) in rainbow trout: Implications for a distinctive B cell developmental pathway in teleost fish.Proc Natl Acad Sci U S A10269196924
- 17. Danilova N, Bussmann J, Jekosch K, Steiner LA (2005) The immunoglobulin heavy-chain locus in zebrafish: identification and expression of a previously unknown isotype, immunoglobulin Z. Nat Immunol 6: 295–302.N. DanilovaJ. BussmannK. JekoschLA Steiner2005The immunoglobulin heavy-chain locus in zebrafish: identification and expression of a previously unknown isotype, immunoglobulin Z.Nat Immunol6295302
- 18. Hsu E, Flajnik MF, Du Pasquier L (1985) A third immunoglobulin class in amphibians. J Immunol 135: 1998–2004.E. HsuMF FlajnikL. Du Pasquier1985A third immunoglobulin class in amphibians.J Immunol13519982004
- 19. Zhao Y, Cui H, Whittington CM, Wei Z, Zhang X, et al. (2009) Ornithorhynchus anatinus (platypus) links the evolution of immunoglobulin genes in eutherian mammals and nonmammalian tetrapods. J Immunol 183: 3285–3293.Y. ZhaoH. CuiCM WhittingtonZ. WeiX. Zhang2009Ornithorhynchus anatinus (platypus) links the evolution of immunoglobulin genes in eutherian mammals and nonmammalian tetrapods.J Immunol18332853293
- 20. Nemazee D (2006) Receptor editing in lymphocyte development and central tolerance. Nat Rev Immunol 6: 728–740.D. Nemazee2006Receptor editing in lymphocyte development and central tolerance.Nat Rev Immunol6728740
- 21. Criscitiello MF, Flajnik MF (2007) Four primordial immunoglobulin light chain isotypes, including lambda and kappa, identified in the most primitive living jawed vertebrates. Eur J Immunol 37: 2683–2694.MF CriscitielloMF Flajnik2007Four primordial immunoglobulin light chain isotypes, including lambda and kappa, identified in the most primitive living jawed vertebrates.Eur J Immunol3726832694
- 22. Haire RN, Rast JP, Litman RT, Litman GW (2000) Characterization of three isotypes of immunoglobulin light chains and T-cell antigen receptor alpha in zebrafish. Immunogenetics 51: 915–923.RN HaireJP RastRT LitmanGW Litman2000Characterization of three isotypes of immunoglobulin light chains and T-cell antigen receptor alpha in zebrafish.Immunogenetics51915923
- 23. Qin T, Ren L, Hu X, Guo Y, Fei J, et al. (2008) Genomic organization of the immunoglobulin light chain gene loci in Xenopus tropicalis: evolutionary implications. Dev Comp Immunol 32: 156–165.T. QinL. RenX. HuY. GuoJ. Fei2008Genomic organization of the immunoglobulin light chain gene loci in Xenopus tropicalis: evolutionary implications.Dev Comp Immunol32156165
- 24. Fleurant M, Changchien L, Chen CT, Flajnik MF, Hsu E (2004) Shark Ig light chain junctions are as diverse as in heavy chains. J Immunol 173: 5574–5582.M. FleurantL. ChangchienCT ChenMF FlajnikE. Hsu2004Shark Ig light chain junctions are as diverse as in heavy chains.J Immunol17355745582
- 25. Gerdes T, Wabl M (2002) Physical map of the mouse lambda light chain and related loci. Immunogenetics 54: 62–65.T. GerdesM. Wabl2002Physical map of the mouse lambda light chain and related loci.Immunogenetics546265
- 26. Kawasaki K, Minoshima S, Nakato E, Shibuya K, Shintani A, et al. (2001) Evolutionary dynamics of the human immunoglobulin kappa locus and the germline repertoire of the Vkappa genes. Eur J Immunol 31: 1017–1028.K. KawasakiS. MinoshimaE. NakatoK. ShibuyaA. Shintani2001Evolutionary dynamics of the human immunoglobulin kappa locus and the germline repertoire of the Vkappa genes.Eur J Immunol3110171028
- 27. Wu Q, Wei Z, Yang Z, Wang T, Ren L, et al. (2010) Phylogeny, genomic organization and expression of lambda and kappa immunoglobulin light chain genes in a reptile, Anolis carolinensis. Dev Comp Immunol 34: 579–589.Q. WuZ. WeiZ. YangT. WangL. Ren2010Phylogeny, genomic organization and expression of lambda and kappa immunoglobulin light chain genes in a reptile, Anolis carolinensis.Dev Comp Immunol34579589
- 28. Sanders BG, Travis JC (1975) Evidence for one immunoglobulin light-chain type in chickens: absence of a blocked N-terminal light-chain type. Biochem Genet 13: 779–782.BG SandersJC Travis1975Evidence for one immunoglobulin light-chain type in chickens: absence of a blocked N-terminal light-chain type.Biochem Genet13779782
- 29. Magor KE, Higgins DA, Middleton DL, Warr GW (1994) cDNA sequence and organization of the immunoglobulin light chain gene of the duck, Anas platyrhynchos. Dev Comp Immunol 18: 523–531.KE MagorDA HigginsDL MiddletonGW Warr1994cDNA sequence and organization of the immunoglobulin light chain gene of the duck, Anas platyrhynchos.Dev Comp Immunol18523531
- 30. Parvari R, Ziv E, Lentner F, Tel-Or S, Burstein Y, et al. (1987) Analyses of chicken immunoglobulin light chain cDNA clones indicate a few germline V lambda genes and allotypes of the C lambda locus. EMBO J 6: 97–102.R. ParvariE. ZivF. LentnerS. Tel-OrY. Burstein1987Analyses of chicken immunoglobulin light chain cDNA clones indicate a few germline V lambda genes and allotypes of the C lambda locus.EMBO J697102
- 31. Das S, Mohamedy U, Hirano M, Nei M, Nikolaidis N (2010) Analysis of the immunoglobulin light chain genes in zebra finch: evolutionary implications. Mol Biol Evol 27: 113–120.S. DasU. MohamedyM. HiranoM. NeiN. Nikolaidis2010Analysis of the immunoglobulin light chain genes in zebra finch: evolutionary implications.Mol Biol Evol27113120
- 32. McCormack WT, Thompson CB (1990) Chicken IgL variable region gene conversions display pseudogene donor preference and 5′ to 3′ polarity. Genes Dev 4: 548–558.WT McCormackCB Thompson1990Chicken IgL variable region gene conversions display pseudogene donor preference and 5′ to 3′ polarity.Genes Dev4548558
- 33. Lundqvist ML, Middleton DL, Radford C, Warr GW, Magor KE (2006) Immunoglobulins of the non-galliform birds: antibody expression and repertoire in the duck. Dev Comp Immunol 30: 93–100.ML LundqvistDL MiddletonC. RadfordGW WarrKE Magor2006Immunoglobulins of the non-galliform birds: antibody expression and repertoire in the duck.Dev Comp Immunol3093100
- 34. Choi JW, Kim JK, Seo HW, Cho BW, Song G, et al. (2010) Molecular cloning and comparative analysis of immunoglobulin heavy chain genes from Phasianus colchicus, Meleagris gallopavo, and Coturnix japonica. Vet Immunol Immunopathol 136: 248–256.JW ChoiJK KimHW SeoBW ChoG. Song2010Molecular cloning and comparative analysis of immunoglobulin heavy chain genes from Phasianus colchicus, Meleagris gallopavo, and Coturnix japonica.Vet Immunol Immunopathol136248256
- 35. Magor KE, Higgins DA, Middleton DL, Warr GW (1999) Opposite orientation of the alpha- and upsilon-chain constant region genes in the immunoglobulin heavy chain locus of the duck. Immunogenetics 49: 692–695.KE MagorDA HigginsDL MiddletonGW Warr1999Opposite orientation of the alpha- and upsilon-chain constant region genes in the immunoglobulin heavy chain locus of the duck.Immunogenetics49692695
- 36. Warr GW, Magor KE, Higgins DA (1995) IgY: clues to the origins of modern antibodies. Immunol Today 16: 392–398.GW WarrKE MagorDA Higgins1995IgY: clues to the origins of modern antibodies.Immunol Today16392398
- 37. Magor KE, Higgins DA, Middleton DL, Warr GW (1994) One gene encodes the heavy chains for three different forms of IgY in the duck. J Immunol 153: 5549–5555.KE MagorDA HigginsDL MiddletonGW Warr1994One gene encodes the heavy chains for three different forms of IgY in the duck.J Immunol15355495555
- 38. Magor KE, Warr GW, Middleton D, Wilson MR, Higgins DA (1992) Structural relationship between the two IgY of the duck, Anas platyrhynchos: molecular genetic evidence. J Immunol 149: 2627–2633.KE MagorGW WarrD. MiddletonMR WilsonDA Higgins1992Structural relationship between the two IgY of the duck, Anas platyrhynchos: molecular genetic evidence.J Immunol14926272633
- 39. van Tuinen M, Hedges SB (2001) Calibration of avian molecular clocks. Mol Biol Evol 18: 206–213.M. van TuinenSB Hedges2001Calibration of avian molecular clocks.Mol Biol Evol18206213
- 40. Pereira SL, Baker AJ (2006) A mitogenomic timescale for birds detects variable phylogenetic rates of molecular evolution and refutes the standard molecular clock. Mol Biol Evol 23: 1731–1740.SL PereiraAJ Baker2006A mitogenomic timescale for birds detects variable phylogenetic rates of molecular evolution and refutes the standard molecular clock.Mol Biol Evol2317311740
- 41. Sorensen V, Rasmussen IB, Sundvold V, Michaelsen TE, Sandlie I (2000) Structural requirements for incorporation of J chain into human IgM and IgA. Int Immunol 12: 19–27.V. SorensenIB RasmussenV. SundvoldTE MichaelsenI. Sandlie2000Structural requirements for incorporation of J chain into human IgM and IgA.Int Immunol121927
- 42. Zhao Z, Zhao Y, Pan-Hammarstrom Q, Liu W, Liu Z, et al. (2007) Physical mapping of the giant panda immunoglobulin heavy chain constant region genes. Dev Comp Immunol 31: 1034–1049.Z. ZhaoY. ZhaoQ. Pan-HammarstromW. LiuZ. Liu2007Physical mapping of the giant panda immunoglobulin heavy chain constant region genes.Dev Comp Immunol3110341049
- 43. de Lalla C, Fagioli C, Cessi FS, Smilovich D, Sitia R (1998) Biogenesis and function of IgM: the role of the conserved mu-chain tailpiece glycans. Molecular Immunology 35: 837–845.C. de LallaC. FagioliFS CessiD. SmilovichR. Sitia1998Biogenesis and function of IgM: the role of the conserved mu-chain tailpiece glycans.Molecular Immunology35837845
- 44. Woof JM, Kerr MA (2006) The function of immunoglobulin A in immunity. J Pathol 208: 270–282.JM WoofMA Kerr2006The function of immunoglobulin A in immunity.J Pathol208270282
- 45. Roman T, De Guerra A, Charlemagne J (1995) Evolution of specific antigen recognition: size reduction and restricted length distribution of the CDRH3 regions in the rainbow trout. Eur J Immunol 25: 269–273.T. RomanA. De GuerraJ. Charlemagne1995Evolution of specific antigen recognition: size reduction and restricted length distribution of the CDRH3 regions in the rainbow trout.Eur J Immunol25269273
- 46. Patel HM, Hsu E (1997) Abbreviated junctional sequences impoverish antibody diversity in urodele amphibians. J Immunol 159: 3391–3399.HM PatelE. Hsu1997Abbreviated junctional sequences impoverish antibody diversity in urodele amphibians.J Immunol15933913399
- 47. Das S, Nikolaidis N, Klein J, Nei M (2008) Evolutionary redefinition of immunoglobulin light chain isotypes in tetrapods using molecular markers. Proc Natl Acad Sci U S A 105: 16647–16652.S. DasN. NikolaidisJ. KleinM. Nei2008Evolutionary redefinition of immunoglobulin light chain isotypes in tetrapods using molecular markers.Proc Natl Acad Sci U S A1051664716652
- 48. Janke A, Arnason U (1997) The complete mitochondrial genome of Alligator mississippiensis and the separation between recent archosauria (birds and crocodiles). Mol Biol Evol 14: 1266–1272.A. JankeU. Arnason1997The complete mitochondrial genome of Alligator mississippiensis and the separation between recent archosauria (birds and crocodiles).Mol Biol Evol1412661272
- 49. Xu Z, Wang GL, Nie P (2009) IgM, IgD and IgY and their expression pattern in the Chinese soft-shelled turtle Pelodiscus sinensis. Molecular Immunology 46: 2124–2132.Z. XuGL WangP. Nie2009IgM, IgD and IgY and their expression pattern in the Chinese soft-shelled turtle Pelodiscus sinensis.Molecular Immunology4621242132
- 50. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574.F. RonquistJP Huelsenbeck2003MrBayes 3: Bayesian phylogenetic inference under mixed models.Bioinformatics1915721574
- 51. Page RD (1996) TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12: 357–358.RD Page1996TreeView: an application to display phylogenetic trees on personal computers.Comput Appl Biosci12357358
- 52. Hackett SJ, Kimball RT, Reddy S, Bowie RC, Braun EL, et al. (2008) A phylogenomic study of birds reveals their evolutionary history. Science 320: 1763–1768.SJ HackettRT KimballS. ReddyRC BowieEL Braun2008A phylogenomic study of birds reveals their evolutionary history.Science32017631768