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* Center for Biotechnology and Center for Oral Biology, Department of Bioscience at Novum, Karolinska Institute, Huddinge, Stockholm, Sweden;
Department of Physiology and Biochemistry, University of Veterinary Science, Budapest, Hungary;
Department of Biochemistry and Biophysics and Stockholm Bioinformatics Center, Stockholm University, Stockholm, Sweden;
Department of Animal Science, Texas A&M University, Houston, TX 77843; and
¶ Immune and Gene Therapy Laboratory, Cancer Center Karolinska, Karolinska Hospital, Stockholm, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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genes have been identified and shown to be
transcriptionally active. The deduced amino acid sequences from their
cDNAs show that artiodactyl IgD H chains are structurally similar to
human IgD, where the cow, sheep, and pig IgD H chain constant regions
all contain three domains and a hinge region, sharing homologies of
43.6, 44, and 46.8% with their human counterpart, respectively.
According to a phylogenetic analysis, the C gene appears to have
been duplicated from the Cµ gene >300 million yr ago. The ruminant
µCH1 exon and its upstream region was again duplicated before the
speciation of the cow and sheep, 
20 million yr ago, inserted
upstream of the gene hinge regions, and later modified by gene
conversion. A short S (switch ) sequence resulting from the
second duplication, is located immediately upstream of the bovine C
gene and directs regular µ- class switch recombination in the cow.
The presence of C genes in artiodactyls, possibly in most mammals,
suggests that IgD may have some as yet unknown biological properties,
distinct from those of IgM, conferring a survival
advantage. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene, however, were recently found in teleosts, including channel
catfish (Ictalurus punctatus), Atlantic cod (Gadus
morhua), and Atlantic salmon (Salmo salar)
(6, 7, 8), suggesting a more ancient origin. In these species
the µCH1 exon is spliced onto gene-like sequences, yielding
chimeric H chains containing a large number of constant domains, but
devoid of a hinge segment (6, 7, 8).
A comparison of the genes in different species suggests that,
unlike the µ gene, considerable structural alterations have occurred
during evolution, where exon duplications in fish (7, 8)
and exon deletions, resulting in a lack of CH2, in rodents (9, 10) have played key roles in their phylogeny.
The currently held view is that the C genes have been evolutionarily
deleted in all other mammalian species except primates and rodents
(11, 12). This conclusion is based on experiments that
have failed to detect either the IgD protein or the C gene in
additional species (11, 12, 13). In recent years we have
witnessed a rapid progress of research on large scale analysis of
animal genomes, where databases for expressed sequence tags
(ESTs),4 facilitate
the identification of new genes. In this paper we have identified and
characterized the cow, sheep, and pig C genes using an EST-based
approach.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Total RNA was extracted from animal spleen or blood using TRIzol
(Life Technologies, Gaithersburg, MD) following the manufacturer’s
instructions. About 5 µg total RNA was used to synthesize
first-strand cDNA with a First-Strand cDNA Synthesis Kit (Amersham
Pharmacia Biotech, Uppsala, Sweden). The primers used for the
bovine IgD RACE PCR were: bIgD-5'RACE1, 5'-TCA TCG CTG CTC
TCG TCC TG-3'; bIgD-5'RACE2, 5'-GCC GTG GCA AAC TGG GAA GG-3';
bIgD-3'RACE1, CAA TCT GAC CCT CCG CAC TG-3'; and bIgD-3'RACE2, GTG GAG
CCA GGA CGA GAG CA-3'; they were designed based on a bovine EST
sequence (accession no. AW653692). The primers for the cloning of the
sheep Ig cDNA were: bIg-JH, 5'-GCC AAG GAC TCC TGG TCA CCG TCT
C-3'; bIgD-5'RACE2, 5'-GCC GTG GCA AAC TGG GAA GG-3'; and bIgD-3'S,
5'-GAC ATC CTC CTC ACG TGG CTG-3'. The primers for cloning of the pig
IgD cDNA were: swine-JH, 5'-CCA GGC GTT GAA GTC GTC GTG T-3'; and a
degenerated primer, IgD-CH3-conas, 5'-CRG AYA CYT CRC ACA GGA GCC A-3'.
The RACE PCR amplifications were conducted according to the
instructions of the 5'RACE System for Rapid Amplification of cDNA Ends
(Life Technologies).
Cloning of cow, sheep, and pig full-length IgD H chain-encoding sequences
All the constant region primers were designed based on the
sequences derived in this study, while the variable region primers, all
located in the leader sequence of the V exon, were designed based on
VH sequences available in the National Center for
Biotechnology Information GenBank. Whereas the sheep IgD H chain
cDNA was amplified using primers sheep-IgVHs (5'-ACC CAC TGT GGA CCC
TCC TCT T-3') and sheep-IgDas (5'-GGG AGC AGC AGG CAG CGT GGA G-3'),
the cow and pig IgD H chain cDNAs were obtained by employing nested
PCR. The primers used for amplification of cow IgD were bovine-IgVHs1
(5'-GCT CCA AGA TGA ACC CAC TGT G-3'), bovine-IgDas1 (5'-CAT GAT GCC
CTC CTC TTG GTC T-3'), bovine-IgVHs2 (5'-ACC CTC CTC TTT GTG CTC
TCA-3'), and bovine-IgDas2 (5'-GGG CTG GGC TCT GTG ATG GAC-3'). The
primers to amplify the pig IgD H chain were pig-IgVHs1 (5'-TTC GGC TGA
ACT GGG TGG TCT T-3'), pig-IgDas1 (5'-CGG GGC TAC TTC ACC TTG AG-3'),
pig-IgVHs2 (5'-CGG CTG AAC TGG GTG GTC TTG T-3'), and pig-IgDas2
(5'-GGG GCT ACT TCA CCT TGA GG-3'). The resulting PCR products were all
cloned into a T-vector, and after transformation into Escherichia
coli, randomly picked recombinants were screened using
CH3-based degenerated primers.
Bacterial artificial chromosome (BAC) clones
A bovine µ, 
3, and 1 gene-positive BAC clone,
BAC66R4C11, was isolated from a previously constructed bovine genomic
BAC library (14).
Long PCR amplifications of the bovine and ovine genomic fragments
A long PCR kit (Expand Long Template PCR System Kit, Roche
Diagnostics Scandinavia, Bromma, Sweden) was employed to amplify the
genomic DNA fragment containing the bovine gene. Primers bIgMS
(5'-GAC TCC TGT GCG ACC CGA TAG-3') and bIgD-ESTas (5'-CAG CCA CGT GAG
GAG GAT GTC-3') were used to produce clone bMD, and primers bIgD-3'S
(5'-GAC ATC CTC CTC ACG TGG CTG-3') and bIgD-3'As (5'-ATG ATG CCC TCC
TCT TGG TCT-3') were used to produce clone bDE. The exon-intron
boundaries were identified by comparison of the genomic sequences with
the cDNA sequence of the bovine C gene. The primers used for cloning
of the bovine JH-Cµ intron were bIg-JH (as mentioned above) and
bIgM-CH2as (5'-GCG GGA CAA AGA CAC TCA CGA CTG G-3'). To amplify the
sheep genomic fragment containing the ovine µTM exon and part of the
CH1 exon, the primers sheep IgM-TMs (5'-ACC TTC ATT GTG CTC TTC CTC
CTG-3') and sheep IgD-CH1as (5'-CGC TGC TGA CCG TGC TGT TGT TGA G-3')
were used.
PCR amplifications of the recombined Sµ-S DNA fragments in cow
A nested PCR was used to amplify the recombined DNA fragments
created by class switching using Sµ- and S-specific primers Sµ1
(5'-TCT GAG GGT GGC AAG CGT GTC-3'), Sµ2 (5'-AGG GAA GCT AAA GTC GTC
AC-3'), S1 (5'-CCT GAG GTC AGC CCA GTG TTG-3'), and S2 (5'-GTC
AAG CCC AGG CAG TTC AT-3'). The DNA samples were purified from the
peripheral blood of cows in a herd located in a research facility of
the Swedish Agricultural University (Uppsala, Sweden). The cows are of
the Swedish red and white breed.
Cloning of PCR products, preparation of plasmids, and DNA sequencing
PCR products were recovered from the agarose gel using the
QIAquick Gel Extraction kit (Qiagen, Valencia, CA) and were
subsequently cloned into the pGEM-T vector (pGEM-T Vector System I,
Promega, Madison, WI) with E. coli DH5
as a bacterial
host. The plasmids and BAC DNA were prepared using QIAprep Spin
Miniprep kits and Qiagen plasmid Maxi kits (Qiagen), respectively. The
ABI PRISM BigDye Terminator Ready Reaction kit (PerkinElmer, Foster,
CA) was used for sequencing.
Northern and Southern blots and restriction enzyme digestion analysis
All the restriction enzymes were purchased from Promega. The
Northern and Southern blots were performed using ExpressHyb
hybridization solution (Clontech, Palo Alto, CA) according to the
manufacturer’s instructions. A bovine CH3-derived DNA fragment
(probe A) was used for both Northern and Southern blottings. Another
DNA fragment, spanning the bovine CH3 and membrane-bound form
encoding cDNA 3' end (probe B), was used to visualize both cow and
sheep genes. The probes were labeled with an oligolabeling kit
(Amersham Pharmacia Biotech, Uppsala, Sweden). To identify the bands
obtained in Northern blotting, a DNA fragment from the 3' untranslated
region (UTR) of the bovine IgD membrane-bound form cDNA (probe C) was
amplified and used in a second Northern blot.
Computational analysis of DNA sequences and construction of phylogenetic trees
A DNA sequence homology search was conducted using the NCBI
BLAST program. Sequence alignment and comparison, was conducted using
the MegAlign program (DNASTAR, Madison, WI). The dot plot comparison
was performed using the same program with the following parameters:
percentage, 80; window, 30; min quality, 1. Construction of the
phylogenetic trees of IgM and IgD were made using both
dnapars and dnaml (DNA maximum likelihood method)
programs from the PHYLIP package (15). A consensus tree
was taken from 1000 bootstrapped phylogenetic trees. To estimate the
divergence time for different gene sequences, pairwise neutral
evolutionary distance (NED) (16) was calculated. NED
(NED = 0.5e-kt + 0.5; where k is the
assumed first order rate constant, and t is the number of years) is an
evolutionary distance based upon the rate of 2-fold degenerate
synonymous transition and basically represents the proportion of
conserved 2-fold degenerate codons (Asp, Cys, Glu, Phe, His, Lys, Asn,
Gln, and Tyr) between pairs of nucleic acid sequences, where the
differences between each of these codons are represented solely by
transitions (16). Except for the bovine, ovine, and
porcine gene sequences that were generated in this study, all other
sequences were obtained from the NCBI GenBank with the following
accession numbers: cow IgM, U63637; human IgM, X14940; mouse IgM,
J00443; rat IgM, X78895; horse IgM, L49414; pig IgM, U50149; sheep IgM,
L04260; rabbit IgM, J00666; hamster IgM, X02804; chicken IgM, X01613;
cod IgM, X58870; axolotl IgM, X68700; catfish IgM, M27230; char IgM,
X83373; clawed frog IgM, M20484; duck IgM, U27213; trout IgM, X65262;
turtle IgM, U53567; human IgD, X57331; mouse IgD, J00447; rat IgD,
J00741; catfish IgD, U67437; and salmon IgD, AF141605.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cDNAs
A homology search of the human C gene sequence using the NCBI
BLAST program yielded a bovine EST clone (accession no. AW653692),
where the deduced peptide showed a sequence similarity of 53% to the
corresponding region of the human CH2 and CH3 domains (Fig. 1
a). This EST clone was
derived from a cDNA library made from pooled tissue from lymph node,
ovary, fat, hypothalamus, and pituitary (17). The RACE
technique was subsequently employed to clone a full-length cDNA from
bovine spleen total RNA. Three overlapping clones, bD5E, bDM, and bD3E,
containing the 5' part, the middle part, and the 3' end of the
gene, respectively, were obtained. Sequencing of these three clones
yielded a typical Ig H chain mRNA transcript containing a rearranged
variable region segment and a constant region. The deduced peptide
sequence included a typical Ig transmembrane region, indicating that it
represented a membrane-bound form of an Ig H chain (accession no.
AF411240).
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A Northern blot, using mRNA isolated from bovine spleen, was conducted
to analyze the transcriptional pattern of the bovine C gene. Two
bands were detected using a C gene-specific probe (Fig. 2a, probe A), where the 2-kb
mRNA transcripts, corresponding to the membrane-bound form of IgD, gave
a strong signal. The 1.5-kb band, corresponding to the secreted form of
IgD, was weaker, suggesting that the bovine C gene is mainly
transcribed as a membrane-bound encoding form in the spleen. The
identities of the two bands were confirmed by a second hybridization
using a DNA fragment derived from the 3' UTR of the membrane-bound form
encoding cDNA (Fig. 2b, probe C).
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gene in other ruminants, we performed a Southern
blot using the bovine C gene as a probe on sheep and pig genomic
DNA. Under stringent hybridizing conditions, positive bands were
visualized in restriction enzyme-digested sheep (Fig. 2
c,
probe B) and pig DNA (Fig. 2d, probe A), suggesting that
both genomes contain a gene homologous to the bovine C. The multiple
bands obtained in the bovine NcoI-digested sample were
probably due to the presence of at least two NcoI sites in
the probe-spanning genomic region. To clone the sheep cDNA, a
primer, bIg-JH, designed based on the bovine JH sequence that is known
to be highly homologous to the sheep JH, and primer bIgD5'RACE2 were
used to amplify the 5' portion of the sheep C gene from sheep blood
total RNA. The 3' end of a membrane-bound encoding form of cDNA was
obtained using 3'RACE PCR with the primer bIgD3's and an anchored
primer.
The deduced sheep C amino acid sequence from the cDNA (accession
no. AF411238) shows 87.5 and 44% homology to cow and human IgD,
respectively. As in the cow, the first domain is also highly homologous
to its µCH1 (24), showing a similarity of 96.6% (Fig. 6b) and 93.5% at the DNA and protein levels, respectively,
whereas the CH2 and CH3 domains only show an overall amino acid
homology of 27.6% to the last two Cµ domains.
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b), was designed
based on the conserved sequence of the human, sheep, and cow CH3
domain-encoding sequences to clone the porcine C cDNA. We first
cloned the 5' portion from blood lymphocyte total RNA using the primers
swine-JH and IgD-CH3-conas, the former being based on the published
porcine JH sequence (11). Furthermore, the 3' end of a
secreted form of -encoding cDNA was amplified using RACE PCR,
employing the primers, pig-IgD-3'RACE, and an anchored primer. The porcine IgD (accession no. AF411239) is more similar to cow (60.6%) and sheep (60.9%) than human (46.8%) and mouse (38.4%) when comparing the amino acid sequences of the whole IgD H chain constant region. Sequence data from four independent cDNA clones suggest that, like teleost fish, the pig may use the µCH1, which is spliced onto a short hinge segment and unique CH2- and CH3-encoding exons to produce IgD, since all the sequenced cDNA contained sequences that were identical with the µCH1 (25).
A sequence comparison of the deduced peptides of cow, sheep, and pig
-chains with those of human, mouse, and channel catfish is shown in
Fig. 3.
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All the above IgD constant region sequences were obtained and
compiled using RACE PCR. To prove functionality and integrity of the
IgD H chains in these three species, we directly amplified and cloned
the IgD H chain cDNAs encompassing both the variable region and the
constant region sequences (accession no. AF515672–AF515674). Sequence
analysis showed that these cDNAs were functional and encoded normal Ig
H chains, as no stop codon resulting in premature termination or other
sequence abnormalities were found. Compared with our previously
sequenced clones, a single nucleotide polymorphism was found in both
cow and sheep CH1 exons (Fig. 4 and
Fig. 6, a and b). The CH1 domain-encoding
sequence of the pig IgD clone showed a 3-bp difference from the first
sequenced pig cDNA (Fig. 6d), again suggesting allotypic
polymorphism.
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gene
We have previously isolated a cow Cµ and C3 gene-positive BAC
clone, termed BAC66R4C11, from a bovine library constructed using the
pBeloBAC11 vector (14). Based on the gene cDNA and the
sequences downstream of the Cµ gene, two overlapping genomic clones,
bMD and bDE (Fig. 5), were obtained by
cloning the long PCR products, amplified using BAC66R4C11 DNA as a
template. The insert of the clone bMD spans 4 kb DNA in size,
containing a 1-kb sequence upstream of the C gene, CH1, H1,
H2, CH2, and part of CH3, while the clone bDE contains part of
CH3, TM1, TM2, and part of the 3' UTR (Fig. 5). Since the
sense primer generating the clone bMD was based on the sequence 4 kb
downstream of the bovine Cµ gene, it can be deduced that the 7.4
kb long bovine C gene is located 5.1 kb downstream of the bovine
Cµ gene. Bovine genomic DNA was used as a control to ensure that the
BAC clone used had not been rearranged during the cloning process and
yielded the same results (data not shown).
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gene excluded the possibility that the first
domain-encoding sequence of the cDNA was spliced from the Cµ gene.
However, there is a striking similarity between the two, and comparing
the 324-bp DNA sequences of the CH1 and µCH1 exons, only a 10-bp
difference was observed (Fig. 6a).
The genomic organization of the bovine C gene was constructed based
on a comparison of the cDNA and genomic sequences (Fig. 5). The bovine
C gene resembles the human C gene in the number of exons, but
differs slightly in the length of the introns. The boundaries for
the exon-intron junctions are shown in Table I. It is worth noting that a noncanonical
splicing site, GC-AG, is used in the intron between the TM1- and
TM2-encoding exons.
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and JH-Cµ introns
To look for the origin of the CH1, we determined the complete
sequence of the 5.1-kb intron between the bovine Cµ and C
genes. A detailed sequence analysis of the Cµ-C intron
(accession no. AF411241) shows the presence of a bovine non-long
terminal repeat retro-element, Bov B-long interspersed nuclear element
(B-LINE) (26), with a truncated 5' end, in the Cµ-C
intron (Fig. 5). This retro-element contains a reverse
transcriptase-encoding region that is thought to be responsible for DNA
transposition (26).
A BLAST search using the whole intron sequence showed that, except for
the Bov B-LINE, the bovine Cµ-C intron was highly homologous to
the sheep JH-Cµ intronic DNA, indicating that the bovine Cµ-C
intron may have been duplicated from its JH-Cµ intron. To address
this question, we cloned and sequenced the bovine JH-Cµ intron
(accession no. AF411242 and AF211243). The 7-kb region was
PCR-amplified from the BAC66R4C11 DNA using primers bIg-JH-S and
bIgM-As and cloned into pGEM-T for sequencing. The sequence data
revealed that the fragment contained two functional JH segments, JH1
and JH2, the bovine intronic enhancer region, and the Sµ (switch µ)
region.
A dot plot analysis of the Cµ-C and JH-Cµ introns showed long
homologous DNA stretches in the two introns (Fig. 7), strongly suggesting that the CH1
exon together with close to 4 kb upstream DNA originated from the
3'-flanking region of the bovine intronic enhancer down to the µCH1
exon (Fig. 5). The duplicated sequence was later interrupted by
introduction of the retro-element, Bov B-LINE.
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genes in sheep and pig, where the
results show that the sheep C gene is located 6.5 kb downstream
of the Cµ gene, while the pig C gene is located roughly 4 kb
downstream of the Cµ gene (data not shown).
A short S region mediates IgD class switching in cow
Consistent with the Southern blot results reported by Knight et
al. (27), a short, 280-bp Sµ-like region, abundant in
switch µ motifs, CTGGG (15 repeats) and CTGAG (12 repeats), was
identified immediately upstream of the C gene (Fig. 5). The S may
theoretically be used to mediate class switch recombination. To test
this hypothesis, a nested PCR, shown in Fig. 8a, was conducted to amplify
recombined DNA fragments. While the Sµ-specific primers Sµ1 and
Sµ2 are located in the 5'-flanking region of the switch µ region,
the S-specific primers S1 and S2 are located in the
3'-flanking region of the switch . Several fragments ranging from
400–600 bp in size were generated using genomic DNA from different
cows and were subsequently cloned and sequenced. Although there is a
very high sequence homology between the very 5' part of the Sµ and
S regions, we could identify the recombination breakpoints using the
mismatches between the two regions as markers (Fig. 8b). Our
data clearly show that switch recombination, involving the Sµ and
S regions, occurs in the cow.
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and Cµ genes in vertebrates
One striking feature of the IgD in cows and sheep is that their
CH1 domains share almost the same sequence as their IgM CH1. An
examination of the bovine JH-Cµ and Cµ-C intron sequences
supports the idea that the bovine CH1 exon, together with its
5'-flanking sequence were recently duplicated from the DNA-spanning,
3'-flanking region of the intronic enhancer to the µCH1 exon. Since
the sheep C gene also has a µCH1-like CH1, the duplication
would be expected to have occurred before the speciation of cows and
sheep. To estimate when the bovine and ovine CH1 exons were
duplicated from their respective Cµ genes, unrooted phylogenetic
trees were constructed using the first domain of both IgD and IgM from
a number of species (Fig. 9, a
and b). Unexpectedly, the results suggested that the
duplication event creating the present CH1 exon occurred
independently in cows and sheep after the evolutionary divergence of
these two species 20 million yr ago (28). However, it
is widely appreciated that gene conversion and other nonrandom
processes act on immune system genes, which, in turn, are well
documented to misrepresent phylogenetic relationships
(29). To analyze the pairwise distances in more detail,
NED values were therefore calculated (16). The results
indicated that all bovidae sequence pairs were approximately equally
related (NEDsheep IgD:cow IgD = 0.11;
NEDsheep IgD:sheep IgM = 0; NEDcow
IgD:cow IgM = 0.10), with the exception of those involving
cow IgM and sheep IgM/IgD (NED = 0.22). This provides support for
a gene conversion event occurring after duplication in the common
ancestor of cows and sheep. Even more convincing data, supporting a
gene conversion model, can be deduced from a comparison of the µ and
CH1 exon sequences in both cows and sheep, where alignments of
sheep and cow CH1 with their respective µCH1 sequences show that
the differences are clustered in their 3' ends (Fig. 6, a
and b). However the most 3' 16-bp DNA in both sheep and cow
CH1 are identical (Fig. 6c), indicating that a gene
conversion event between the µCH1 and CH1, following the second
duplication event that either replaced a pre-existing CH1 or
introduced a missing exon, may have occurred after speciation in both
sheep and cows.
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genes suggest that
the latter was initially duplicated from the former. The recent
identification of a C like gene in teleost fish suggests that the
C gene appeared in primitive vertebrates, ancestral to birds,
reptiles, and mammals, since the lineage leading to teleost fish
diverged from other vertebrates 450 million yr ago (28, 30). This is consistent with the phylogenetic tree constructed
using the IgD CH3 and IgM CH4 domains (Fig. 9, c and
d), where the calculated NED values (NEDmammalian
IgM:bird IgM = 0.45, NEDmammalian IgM:mammalian
IgD = 0.91, NEDmammalian IgD:bird IgM
= 1.43) suggest that the first duplication of the Cµ gene, generating
the C gene, is much more ancient than the divergence between birds
and mammals, 310 million years ago (28). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene
in mammals other than primates and rodents and sheds significant light
on the evolution of the mammalian IgD H chain constant region gene.
Apparently, the mammalian IgD constant regions have developed
structural diversity with regard to both the hinge region and the CH2
domain (9, 10, 31). Structurally, the ruminant IgD H chain
constant region is more similar to that of humans than rodents, since
they all share three CH domains and a relatively long hinge segment,
which is encoded by two separate exons. However, the hinge regions of
these molecules differ from each other not only in length, but also
with regard to their peptide sequences (Fig. 3).
Unlike their counterparts in human and rodents, the CH1 domain of IgD
constant regions in cows and sheep share an extremely high homology
with their respective µCH1 domains (Fig. 6, a and
b). In the cow we have demonstrated that the CH1 exon is
used to produce the IgD H chain mRNA transcripts. In pigs, however,
sequence data derived from two animals yielded inconsistent results,
necessitating further research. One possibility is that the genomic
CH1-encoding sequence is indeed present in pigs, but in some animals
these exon sequences are not used and, as in teleost fish, the µCH1
is spliced onto the CH2 and CH3 sequences. The deduced pig IgD H
chain is characterized by a shorter hinge region compared with those of
humans, cows, and sheep, and although the hinge segment is similar to
that of mice and rats in length, the homology between them is quite low
(Fig. 3).
The finding of the C gene in artiodactyls raises the question of
whether the gene is widely distributed in vertebrates and not present
only in some selected mammals and teleost fish. The results derived
from the phylogenetic analysis indicate that the C gene might be
present in birds. However, in the recently sequenced duck Ig H chain
constant region locus (32), the 4-kb intron between the
Cµ and C genes does not contain any Ig-like sequences, questioning
the existence of a C gene in birds. We have previously mapped the
chicken Ig H chain constant region gene locus (33) and
shown that, as in the duck, an inverted C gene is located between
the Cµ and C
genes. Our own unpublished observations also show
that there is no C gene in the intron between the µTM- and
TM-encoding exons, indicating that the insertion of the C gene
may have deleted or displaced the avian C gene.
In human and mouse B cells, coexpression of IgM and IgD depends on
alternative splicing of a long primary transcript. In teleost fish,
post-transcriptional RNA splicing is involved, resulting in a chimeric
form of IgD H chain with the µCH1 domain being fused to unique C
sequences (6, 7, 8). Due to the absence of an authentic S
region, B cells expressing exclusively IgD
(IgM-IgD+) are extremely
rare in humans and are almost absent in mice (34).
Homologous recombination mediated by two 442-bp repeats localized
upstream of the Sµ and within the Cµ-C intron, or nonhomologous
recombination between Sµ and 
regions has been suggested as the
molecular basis for generating these rare
IgM-IgD+ cells (35, 36). To date, the cow is the only species in which a true S
region has been demonstrated. The identification of Sµ-S
recombination junctions, suggests that the expression of bovine IgD
might depend on a deletional event, class switch recombination, which
is a dominant mechanism for IgG, IgA, and IgE switching in most
species. The bovine S region is, however, rather short compared with
other switch regions (typically 2–10 kb), which may reflect a gradual
deletion of the S sequences during evolution. This process might
also, assuming that these sequences were involved in the first
duplication event, have led to a complete loss of S sequences in
humans and mice.
It has previously been shown that IgD may replace IgM in B cell
ontogeny (37), and the presence of C genes in
artiodactyls, possibly in most mammals, suggests that IgD may have some
as yet unknown biological properties distinct from those of IgM,
conferring a survival advantage.
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Footnotes |
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2 All the sequences reported in this paper have been submitted to National Center for Biotechnology Information GenBank with accession numbers AF411238–AF411247.
3 Address correspondence and reprint requests to Dr. Yaofeng Zhao, Center for Biotechnology, Novum, SE-14157 Huddinge, Sweden. E-mail address: yafe{at}csb.ki.se
4 Abbreviations used in this paper: EST, expressed sequence tag; BAC, bacterial artificial chromosome; LINE, long interspersed nuclear element; NED, neutral evolutionary distance; S, switch region; UTR, untranslated region.
Received for publication February 19, 2002. Accepted for publication August 21, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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heavy chain of human immunoglobulin D. Proc. Natl. Acad. Sci. USA 79:2850. gene. Immunology 91:1.[Medline]
1 and 2 genes. Mol. Immunol. 26:841.[Medline]
heavy chain. Science 228:733. gene upstream of a condensed upsilon gene. Immunology 101:348.[Medline]
locus accompanies Cµ deletion and switch to immunoglobulin D secretion. Mol. Cell. Biol. 11:5660.This article has been cited by other articles:
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  Z. Wei, Q. Wu, L. Ren, X. Hu, Y. Guo, G. W. Warr, L. Hammarstrom, N. Li, and Y. Zhao Expression of IgM, IgD, and IgY in a Reptile, Anolis carolinensis J. Immunol., September 15, 2009; 183(6): 3858 - 3864. [Abstract] [Full Text] [PDF]
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Y. Zhao, H. Cui, C. M. Whittington, Z. Wei, X. Zhang, Z. Zhang, L. Yu, L. Ren, X. Hu, Y. Zhang, et al. Ornithorhynchus anatinus (Platypus) Links the Evolution of Immunoglobulin Genes in Eutherian Mammals and Nonmammalian Tetrapods J. Immunol., September 1, 2009; 183(5): 3285 - 3293. [Abstract] [Full Text] [PDF]
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I. Achour, P. Cavelier, M. Tichit, C. Bouchier, P. Lafaye, and F. Rougeon Tetrameric and Homodimeric Camelid IgGs Originate from the Same IgH Locus J. Immunol., August 1, 2008; 181(3): 2001 - 2009. [Abstract] [Full Text] [PDF]
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 Y. Zhao, Q. Pan-Hammarstrom, S. Yu, N. Wertz, X. Zhang, N. Li, J. E. Butler, and L. Hammarstrom Identification of IgF, a hinge-region-containing Ig class, and IgD in Xenopus tropicalis PNAS, August 8, 2006; 103(32): 12087 - 12092. [Abstract] [Full Text] [PDF]
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Y. Ohta and M. Flajnik IgD, like IgM, is a primordial immunoglobulin class perpetuated in most jawed vertebrates PNAS, July 11, 2006; 103(28): 10723 - 10728. [Abstract] [Full Text] [PDF]
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B. Wagner, D. C. Miller, T. L. Lear, and D. F. Antczak The Complete Map of the Ig Heavy Chain Constant Gene Region Reveals Evidence for Seven IgG Isotypes and for IgD in the Horse J. Immunol., September 1, 2004; 173(5): 3230 - 3242. [Abstract] [Full Text] [PDF]
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 H. M. Farrell Jr., R. Jimenez-Flores, G. T. Bleck, E. M. Brown, J. E. Butler, L. K. Creamer, C. L. Hicks, C. M. Hollar, K. F. Ng-Kwai-Hang, and H. E. Swaisgood Nomenclature of the Proteins of Cows' Milk--Sixth Revision J Dairy Sci, June 1, 2004; 87(6): 1641 - 1674. [Abstract] [Full Text] [PDF]
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 A. Hosseini, G. Campbell, M. Prorocic, and R. Aitken Duplicated copies of the bovine JH locus contribute to the Ig repertoire Int. Immunol., June 1, 2004; 16(6): 843 - 852. [Abstract] [Full Text] [PDF]
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G. A. Loset, K. H. Roux, P. Zhu, T. E. Michaelsen, and I. Sandlie Differential Segmental Flexibility and Reach Dictate the Antigen Binding Mode of Chimeric IgD and IgM: Implications for the Function of the B Cell Receptor J. Immunol., March 1, 2004; 172(5): 2925 - 2934. [Abstract] [Full Text] [PDF]
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C. D. Lemke, J. S. Haynes, R. Spaete, D. Adolphson, A. Vorwald, K. Lager, and J. E. Butler Lymphoid Hyperplasia Resulting in Immune Dysregulation Is Caused by Porcine Reproductive and Respiratory Syndrome Virus Infection in Neonatal Pigs J. Immunol., February 1, 2004; 172(3): 1916 - 1925. [Abstract] [Full Text]
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 Y. Zhao, I. Kacskovics, H. Rabbani, and L. Hammarstrom Physical Mapping of the Bovine Immunoglobulin Heavy Chain Constant Region Gene Locus J. Biol. Chem., September 12, 2003; 278(37): 35024 - 35032. [Abstract] [Full Text] [PDF]
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Y. Zhao, Q. Pan-Hammarstrom, I. Kacskovics, and L. Hammarstrom The Porcine Ig {delta} Gene: Unique Chimeric Splicing of the First Constant Region Domain in its Heavy Chain Transcripts J. Immunol., August 1, 2003; 171(3): 1312 - 1318. [Abstract] [Full Text] [PDF]
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Q. Pan-Hammarstrom, S. Dai, Y. Zhao, I. F. van Dijk-Hard, R. A. Gatti, A.-L. Borresen-Dale, and L. Hammarstrom ATM Is Not Required in Somatic Hypermutation of VH, but Is Involved in the Introduction of Mutations in the Switch {micro} Region J. Immunol., April 1, 2003; 170(7): 3707 - 3716. [Abstract] [Full Text] [PDF]
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, Nucleotides that are
polymorphic in different cows and sheep. The indicated T in the bovine
, Polymorphism found in different clones where C is replaced by
T.
, Coding exons; lower thick lines, DNA fragments
involved in the putative duplication; Eµ, 5' intronic enhancer; Sµ,
switch µ; S
