Analysis of Immunoglobulin Transcripts in the Ostrich Struthio camelus, a Primitive Avian Species
a Primitive Avian
Species. PLoS ONE 7(3): e34346. doi:10.1371/journal.pone.0034346
Analysis of Immunoglobulin Transcripts in the Ostrich Struthio camelus , a Primitive Avian Species
Tian Huang 0
Min Zhang 0
Zhiguo Wei 0
Ping Wang 0
Yi Sun 0
Xiaoxiang Hu 0
Liming Ren 0
Qingyong Meng 0
Ran Zhang 0
Ying Guo 0
Lennart Hammarstrom 0
Ning Li 0
Yaofeng Zhao 0
Sebastian D. Fugmann, National Institute on Aging, United States of America
0 1 State Key Laboratory of Agrobiotechnology, College of Biological Sciences, National Engineering Laboratory for Animal Breeding, China Agricultural University , Beijing , People's Republic of China, 2 College of Animal Science and Technology, Henan University of Science and Technology , Henan , People's Republic of China, 3 Division of Clinical Immunology, Department of Laboratory Medicine, Karolinska University Hospital Huddinge, Stockholm, Sweden, 4 Key Laboratory of Animal Reproduction and Germplasm Enhancement in Universities of Shandong, College of Animal Science and Technology, Qingdao Agricultural University , Qingdao , People's Republic of China
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 Igk 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 l 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 k 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.
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 (m, a, d, c
and e). 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 [4,5,6,7,8]. IgD is, however, absent in birds and
several mammals such as rabbits, opossum and elephants
[9,10,11,12]. 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 [7,9,14], IgNAR in cartilaginous fish ,
IgT/IgZ in the trout and zebrafish [16,17], IgX and IgF in
amphibians [6,18], 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: k, l and s [21,22,23]. A fourth IgL isotype, s-cart, is
only found in sharks . Evolutionarily, fewer types of Ig light
chains are present in mammals and reptiles, which express only l
and k. The two light-chain loci differ significantly in their genomic
organization. At the l locus, multiple Vl segments are followed by
Jl-Cl repeats. By contrast, the k chainencoding locus contains
only a single Ck gene with a small cluster of Jk and multiple Vk
genes located upstream [25,26,27]. Surprisingly, birds exclusively
express l light chains [28,29]. 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 [30,31]. Light chain diversity is generated
by intrachromosomal gene conversion using the upstream
Vl gene segments as donor sequences .
The avian species described to date express only three
immunoglobulin classes: IgM, IgA and IgY, which are encoded
by Cm, Ca and Cu respectively [33,34], and no IgD encoding gene
has been identified. The Cu and Ca genes in the chicken and duck
IgH loci are positioned in reverse orientation [14,35], 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(DFc), which is
expressed by the alternative transcriptional termination of the
single u gene [37,38].
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 l 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
IgYcontaining 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 Cm-, Ca- and
Cu-specific full-length probes (Fig. 1A).
To investigate whether the ostrich expresses IgD, we designed
several pairs of degenerate primers based on the conserved Cd
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 Cm gene
Analysis of the obtained IgM heavy chain constant-region
cDNA clones revealed only a unique sequence, which suggests the
expression of a single m gene. However, four bands were detected
when the mCH4 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 m
genes in the ostrich genome.
The obtained ostrich secretory IgM heavy chain
constantregion cDNA encodes 447 amino acids, in which 12 cysteines are
positionally conserved compared with Cm in other species (Fig.
S1). All of the aligned Cm 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 (NX
S/T): N-46, N-127, N-199 and N-434. Only N-46 and N-434 are
conserved among reptiles, birds and mammals [42,43]. 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
Cm3 and Cm4 domains to be less divergent than the Cm1 and Cm2
domains (Fig. S1). The ostrich IgM constant region shares an
overall identity of 53.1% and 63.1% with the chicken and duck
Cm, respectively, at the protein level. The identity of the ostrich
IgM is supported by a phylogenetic analysis (Fig. 2).
Northern blotting to detect IgM gene expression further showed
that the ostrich m 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 a gene
Southern blotting with either the full-length or Ca3 exon as
probes suggested that a single a 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
spleenderived Ig-specific mini-library were found to be IgA. This finding
could be the result of a PCR bias during the process of 39 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 Ca2, Ca3 and the
canonical secretory tail: N-165, N-221 and N-419, all of which are
conserved in birds (Fig. S3). The ostrich Ca gene shares 44%
sequence identity with chicken and 66% with duck Ca.
When performing 39RACE PCR using the spleen RNA and
JHderived primers, we observed an 850-bp band in addition to the
major 1.6-kb products (all 4-Ca containing transcripts).
Sequencing of this band showed that it encoded a short, membrane-bound
IgA lacking the last two Ca domains (i.e., VDJ-Ca1-Ca2-TM)
(Fig. S4). To further confirm the presence of this short transcript,
we used primers derived from the Ca1 to perform IgA-specific
39RACE. In addition the full-length of 1.4-kb IgA transcript, we
again detected the short IgA transcript, which contained only the
first two Ca 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 Ca1 and TM regions. The
short form was detected in multiple tissues (Fig. 4B). Northern
blotting with the first two Ca 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
Ca2intron-Ca3, and Ca4-intron-TM, which clearly demonstrated that
the short form to be derived from splicing of the Ca2 onto the TM
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 Ca domains (i.e., IgA(DFc), similar to
IgY(DFc) in ducks), although we did not observe such transcripts in
the RACE experiments. We thoroughly analyzed the intron
sequence between Ca2 and Ca3, and did not find any potential
Figure 1. Southern blot detection of the ostrich Ig heavy chain constant-region genes. A. Southern blot detection of the ostrich Ig heavy
chain constant region genes using Cm-, Ca-, and Cu-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 Cm4, Ca3, Cu4 single-exon probes. EI, EcoR I; EV, EcoR V; B, BamH I;
H, Hind III; PI, Pst I; PII, Pvu II.
transcriptional termination signal or polyadenylation signal
sequence (i.e., AATAAA). A polyclonal rabbit antiserum against
the ostrich Ca1 and Ca2 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
Analysis of the ostrich u 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 u gene (Fig. 2). Similar to ostrich m, we only obtained a
single IgY heavy chain constant-region cDNA, although Southern
blotting indicated that more than one u 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 Cu1, which suggests that these molecules can associate with
light chains. Seven additional cysteines were distributed in
Cu2Cu4, all of which are conserved across all species examined (Fig.
S5). Cu contains two N-linked glycosylation sites: N-166 in the
Cu2 conserved in birds and lizards and N-265 in the Cu3
conserved in Xenopus and humans (Fig. S5). A domain-by-domain
comparison of the Cu regions indicated that the Cu1 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 u gene was primarily expressed in the spleen and large intestine
(Fig. S2, Fig. 3).
Figure 2. Phylogenetic analysis of the ostrich immunoglobulin genes. 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.
Analysis of rearranged VDJ fragments
To analyze the expressed VDJ sequences, 59RACE was
performed using the primers derived from the m, a and u 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 14.3362.18
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
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 l and k genes based on
the conserved Cl and Ck sequences of other species. These
primers were used in PCR amplifications with the spleen cDNA as
templates. We were only able to amplify the l gene in the ostrich,
as a phylogenetic analysis clearly showed that the identified gene
belonged to the l lineage (Fig. 6). We further performed 59 RACE
amplifications based on the Cl sequence that we obtained. In
total, 57 clones were sequenced and shown to encode the
fulllength Vl domain and the same leader peptide
(MAWAPLLLAVLAHGSGSLV). Overall, the inferred Vl region amino acid
homology among these clones ranged from 87.2 to 99.2%. The
average length of the CDR3 was 9.8162.38 codons, with a range
of 4 to 14 codons. The tetrapod IGVL sequences generally have a
fairly well-conserved DEAD (AspGluAlaAsp) 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 Vl sequences revealed only a single Jl in the
We subsequently performed 39RACE using the leader peptide
specific primers and which identified a single Cl in the ostrich.
The Cl sequence shows a 67.0%, 55.1% and 64.5% amino acid
sequence homology to the chicken, lizard and human Cl1,
respectively. A protein sequence alignment of Cl 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 Igk), 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 m and u genes in the ostrich genome, we were only
able to obtain one copy of the m and u expressed at the cDNA
level. We amplified and sequenced the intron between Cm1 and
Cm2 and obtained only a single sequence. We also performed PCR
using two pairs of primers derived from the conserved Cu4
sequences using genomic DNA. All sequenced clones were shown
to contain the same sequence. The additional m and u genes
detected by Southern blotting were likely pseudogenized and have
diverged. We also performed Southern blotting using probes for
the IgD and Igk constant regions of crocodiles and could not
detect any bands (data not shown). It is likely that, as in chickens
and ducks, the d and k 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
Figure 4. PCR and Northern blot analysis of IgA gene expression. A. PCR product of IgA 39RACE. 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.
Figure 5. Western blot detection of ostrich IgA. A. IgA expression in tissues and secretions. 1, Large-intestine cytoplasmic proteins; 2,
Smallintestine 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.
only one functional VH and Vl 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 k light chains [7,27,49]; 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 [9,14]. 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 Ca
exons removed, as the Ca2 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 l 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 manufacturers instructions. Genomic DNA was
extracted from the liver following routine protocols.
Construction and screening of spleen and intestinal
Approximately 2 mg 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 (59-CCH RGV AAG GGG CTS GAG TGG GT-39) and
IgAas (59-TTG ACW TKG GTG GGT TTA CC-39), 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 (59-AAC TGG AAG
AAT TCG CGG CCG CAG GAA TTT TTT TTT TTT TTT
TTT-39). JH1s (59-GCC GGG GCA CCT CGG TCA CCG
TCT CCT CA-39) and JH2s (59-GCC GCG GGA CGG CCG
TCA CCG TGT CCT CA-39) were mixed and used as sense
primers for one round of 39RACE 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 (59-GCT TCG AAT
GTA AAA CGA CGG CCA GT -39) and M13R (59-GCT TCG
AAC AGG AAA CAG CTA TGA C -39) for positively
recombined clones containing the correct insert size; IgM1
(59CGT CAA CGA CAG CAT CTC CA -39) and IgM2 (59-GCG
AGC ACC AGG GAC ACA TT-39) for IgM-positive clones;
IgA1 (59-GCG CTC TGG ACG TGA CCT CCG A -39) and
IgA2 (59-GAC GCT CAG CTT GCT GTA GAC -39) for
IgApositive clones; and IgY1 (59-CTG CCT CAT CTC CCA CTT
CTA C-39) and IgY2 (59-TCT CGG TGC AGT GGC TCA
AGA AC-39) 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 (59-CTG GAG ATG CTG
TCG TTG ACG TAG T-39), MGSP2 (59-TGA CGT AGT
TGG TCC AGG AGA A-39), AGSP1 (59-CCG GGT AGG TCA
AGA CCT CTG A-39), AGSP2 (59-TCG CTG GAA ACC CAG
GTG AC-39), YGSP1 (59-TGG CGG CCA TCG CAA ACT
GGC T-39), and YGSP2 (59-TAG AGG CCG GAG CGG AAG
AG-39); all of the primers were designed from the CH regions that
we obtained. The PCR experiments were performed according to
the instructions of the 59-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 Igl constant region were CLs (59-AAH AAG GCC
ACM CTG GTG TG-39) and CLas (59-CAG GTA RCT GCT
GDC CAT RTA-39). Primers LGSP1 (59-GTA CTG GTT GTT
GCT CTG-39) and LGSP2 (59-CCA CAC CGT TGG AGA
TGG G-39) were used to perform the 59RACE. Primers L1
(59TCG CGG TGC TCG CCC ACG G-39) and L2 (59-CAC GGC
TCA GGT TCC CTG GTC-39) were used to perform the
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. Cm-, Ca-, and Cu-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 Cm and Cm4 exon probes were Cms (59- CGT CAA
CGA CAG CAT CTC CA-39), Cmas (59-CAT TGA CCG AGG
TGG GTT TA-39), Cm4s (59-GCC AGA GCC CCG ACC ATC
TAC-39), and Cm4as (59- GAG GAC TTG TCC ACC GAC
TTC-39). The primers used to amplify the full-length Ca and
Ca3 exon probes were Cas (59- CTG CGA CGA GGG AAA
CGT CAC-39), Caas (59-GAC GCT CAG CTT GCT GTA
GAC-39), Ca3s (59-CTG CCC GTG GTC TCC ATC CTC-39),
and Ca3as (59-AGT TCC TTC TGT GCA GAT TTG-39). The
primers used to amplify the full-length Cu and Cu4 exon probes
were Cus (59- CTG CCT CAT CTC CCA CTT CTA C-39),
Cuas (59- TTT ACC GGG GCT CCT GCT GAT-39), Cu4s
(59CTG GCG CCC AGC GTC TAC CT-39), and Cu4as (59- TGC
GTT GCA CGA ACT TCA TG-39). The hybridization and
detection were performed following the manufacturers
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
IgMdetections (59-GGC CCC GTT GAT GTG GTG CCC A-39);
IgM-detectionAs (59-GCT CGA AGC CGC ACT CCA G-39);
IgA-detections (59-AAG AAC ATC GGG GAC TTA TG-39);
IgA-detectionAs (59-GAC GCT CAG CTT GCT GTA GAC-39);
IgY-detections (59-CTG CCT CAT CTC CCA CTT CTA C-39);
and IgY-detectionAs (59-GAT GGT GCG TTG CAC GAA
CTT-39). Total RNA was used (7 mg/lane) for Northern analysis
(the same kit as for the Southern blots). The primers used for probe
amplification were the following: IgMs (59-CCT GGA CCA ACT
ACG TCA AC-39); IgMas (59-TTG CTC GTG TTC CTC ATC
TC-39); IgA1s (59-CTG CGA CGA GGG AAA CGT CAC-39);
IgA1as (59-GGA CAC TCG GCA AGC GAA CTC-39); IgA2s
(59-GGA CCT CTA CCT CAG CCA GAA-39); IgA2as (59-GTG
GAC GAA GGT GAT GGG GAT-39); IgYs (59-GAT CCA CGT
CTT CGC CTT GC-39); IgYas (59-GAT GGT GCG TTG CAC
GAA CTT-39); IgLs (59-GTG CAC CTC TTC CCT CCA
TC39); IgLas (59-TAG GAG GAG CAC TCG GAT CT-39); EIF1A1s
(59-AAG GAG AAG ACC CAC ATC AAC-39); and EIF1A1as
(59-GAG GAT CAT TCT TGC TGT CAC-39).
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 CH1CH2 exons was
synthesized, modified and coupled with KLH, and then
intravenously injected into New Zealand rabbits. A polyclonal
rabbit antiserum against the ostrich IgA CH1CH2 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 manufacturers
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: a or
x gene: chicken, S40610; cow, AF109167; duck, AJ314754;
human, J00220; mouse, J00475; platypus, AY055778; X. laevis,
BC072981; axolotl, AM774592; gecko, DQ523197; X. tropicalis,
BC157650. d gene: catfish, U67437; cow, AF411245; human,
BC021276; mouse, J00449; X. tropicalis, DQ350886; lizard,
EF690359. c gene: human, J00228; mouse, J00453; platypus,
AY055781; horse, AJ302055. e gene: cow, AY221098; human,
J00222; mouse, X01857; platypus, AY055780. m 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. u 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; l 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. k 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.s
genes: X. laevis, NM_001094414; X. tropicalis, AAH87749;
zebrafish IGIC2, AF246162; nurse shark, EF114766; horn shark,
EF114760; carp IGIC2 (AB091120). s-cart genes: nurse shark
NS5, AY720857; skate type, L25568; horn shark type I, X15316.
Figure S1 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.
Figure S3 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.
Figure S5 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
Figure S8 Sequence alignment of the ostrich IgL
constant region compared with that of other species.
The alignment was performed using the ClustalW method in
MegAlign. Canonical cysteines are shaded.
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: JN709443JN709460.
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.
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