Immunoglobulin from Antarctic sh species of Rajidae family

Maria Rosaria Coscia , Ennio Cocca, Stefano Giacomelli, Fausta Cuccaro, Umberto Oreste
Institute of Protein Biochemistry, CNR, Via P. Castellino 111, Naples, Italy
a b s t r a c t a r t i c l e i n f o
Article history:
Received 27 July 2011
Received in revised form 6 September 2011
Accepted 7 September 2011
Keywords:
Antarctic skate
Cartilaginous sh immunoglobulin M
C gene diversity
Extra-cysteines
Immunoglobulins (Ig) of Chondroichthyes have been extensively studied in sharks; in contrast, in skates
investigations on Ig remain scarce and fragmentary despite the high occurrence of skates in all of the
major oceans of the world. To focus on Rajidae Ig, the most abundant heavy chain isotype, we have chosen
the Antarctic species Bathyraja eatonii, Bathyraja albomaculata, Bathyraja brachyurops, and Amblyraja
georgiana which live at high latitudes in the Southern Ocean, and at very low temperatures. We prepared
mRNA from the spleen of individuals of each species and performed RT-PCR experiments using two
oligonucleotides designed on the alignment of various elasmobranch Ig heavy chain sequences available
in GenBank. The PCR products, about 1400-nt long, were cloned and sequenced. Nucleotide sequence identi-
ties calculated for the constant region domains ranged from 88.5% to 97.5% between species, and from 91.1%
to 99.7% within species. In a distance tree, including also Raja erinacea sequences, two major branches were
obtained, one containing Arhynchobatinae sequences, the other one Rajinae sequences. Four presumptive D
gene segments were identied in the region of the VH/D/JH recombination; two different D segments were
often found in the same sequence. Moreover, 515 genomic fragments of different lengths, carrying the
gene locus encoding Ig chain were revealed by Southern blotting analysis. B. eatonii amino acid sequences
were analyzed for the positional diversity by Shannon entropy analysis, showing CH4 as the most conserved
domain, and CH3 as the most variable one. B. eatonii CDR3 region length varied between 11 and 15 amino
acid residues; the mean length (13.4 aa) was greater than that of Leucoraja eglanteria sequences (7.7 aa).
An alignment of representative sequences of Antarctic species and R. erinacea showed that more cysteine
residues not involved in the intradomain disulde bridges were present in Antarctic species.
2011 Elsevier B.V. All rights reserved.
1. Introduction
All extant cartilaginous sh fall into two major groups: the
holocephalans (chimeras and ratsh) and the elasmobranchs (sharks,
skates and rays). Sharks are not so common in Antarctic waters, and
only three species from 2 families, Squalidae and Lamnidae, were
recorded off South Georgia and in the sub-Antarctic. The only shark
that has really been more studied is the salmon shark (Lamna ditropis,
Hubbs & Follett, 1947). Indeed, skates (Rajiformes: Rajidae) are the
dominant component of the chondrichthyan fauna (Long, 1994)
despite not having been well studied due to the limited number of
specimens available.
It is not clear why there are so few cartilaginous sh in Antarctica.
The main reasons for this scarcity may be the atypical trophic or habitat
conditions in the Antarctic waters (Grande and Eastman, 1986).
Furthermore, the low diversity of teleosts in the Southern Ocean may
have limited the entry of sharks into the ecosystem(Verde et al., 2005).
Skates are a highly diverse sh group, comprising more species than
any other group of cartilaginous shes. The high degree of biodiversity
and endemism shown by skates is quite intriguing given their conserva-
tive morphology and apparent restrictive habitat, e.g. soft bottom
substrates. Skates are most diverse at higher latitudes; they are generally
found in shallower waters towards the poles, and occur deeper in warm
temperate to tropical equatorial waters (Ebert and Compagno, 2007).
Rajidae family includes two subfamilies, Arhynchobatinae (softnose
skates), and Rajinae (hardnose skates). The rst includes six genera, the
latter twelve genera.
Among Antarctic Rajidae, the major group is represented by six
species of Bathyraja: Bathyraja eatonii; Bathyraja maccaini; Bathyraja
meridionalis; Bathyraja murrayi; Bathyraja irrisa; and Bathyraja sp.
(dwarf) (Stehmann and Burkel, 1990). Three of these species have a
wide distribution, B. eatonii on the Kerguelen Plateau and in the
South Atlantic Ocean and Ross Sea, B. maccaini in the South Atlantic
and Pacic Oceans, and Bathyraja sp. (dwarf) in the Atlantic Ocean
and Ross Sea.
Cartilaginous sh are the oldest vertebrate class having an immune
system consisting of immunoglobulin (Ig), T cell receptors, the major
histocompatibility complex, besides somatic hypermutation, RAG-
mediated rearrangement, and the primary and secondary lymphoid
tissues (Flajnik, 2002). To date, three immunoglobulin heavy chain
Marine Genomics 5 (2012) 3541
Corresponding author at: Institute of Protein Biochemistry, CNR Via P. Castellino,
111 80131 Naples, Italy. Tel.: +39 081 6132556; fax: +39 081 6132629.
E-mail addresses: mr.coscia@ibp.cnr.it (M.R. Coscia), e.cocca@ibp.cnr.it (E. Cocca),
s.giacomelli@ibp.cnr.it (S. Giacomelli), f.cuccaro@ibp.cnr.it (F. Cuccaro),
u.oreste@ibp.cnr.it (U. Oreste).
1874-7787/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.margen.2011.09.001
Contents lists available at SciVerse ScienceDirect
Marine Genomics
j our nal homepage: www. el sevi er . com/ l ocat e/ mar gen
isotypes have been identied in cartilaginous sh. IgM which is
orthologous to mammalian IgM was identied in the serum of carti-
laginous sh approximately 45 years ago (Marchalonis and Edelman,
1966), and until recently was thought to be the only isotype present
in this class. The isotype IgMand the second isotype IgNAR, discovered
more recently, are detected in most species. IgNAR has been described
only in elasmobranchs, and it is a unique Ig composed of a heavy chain
dimer not associated with light chains. The third isotype, whose func-
tion is not known, is identied at the protein level in skates (so-called
IgX) (Harding et al., 1990a; Anderson et al., 1999; Kobayashi et al.,
1984), but only by immunoprecipitation or at the transcript level in
sharks (so-called IgW) (Bernstein et al., 1996; Greenberg et al., 1996).
The immunoglobulin genes of all elasmobranchs studied to date
showthe cluster-type organization, rather than the translocon organiza-
tion typical of teleosts (Wilson and Warr, 1992). Each of hundreds of in-
dependent clusters comprises one VHsegment, one or more Dsegments,
one JHsegment and a single constant gene (CH). This is considered to be
a gene arrangement ancestral to the complex IgH locus that exists in all
other vertebrates. Due to the limited number of elasmobranch species
investigated to date, the number of clusters for each isotype varies wide-
ly, both between species and between isotypes (Dooley and Flajnik,
2006). All these gene segments require somatic rearrangement to be
functional. Insome cases the gene segments within a cluster may be par-
tially (VHD-DJH, VHD-JH-CH and VHD-DJH-CH) or fully (VHDJHCH)
joined in the germline. The cluster type organization of immunoglobulin
heavy chain genes found in sharks has been conrmed in two represen-
tative species of Rajidae family, Raja erinacea (little skate) (Harding et al.,
1990a,b) and Leucoraja eglanteria (clearnose skate) (Anderson et al.,
1994). Among cartilaginous sh, L. eglanteria has been used as the
best-characterized model to study the ontogenetic development of the
Bcell repertoire andthe developmental-stage andtissue-specic expres-
sion patterns of a number of different genes that are involved in primary
antigen recognition (Miracle et al., 2001).
Studies we previously performed on secreted and membrane-bound
heavy chain of Immunoglobulin M from several Notothenioid teleosts
inhabiting the cold seawater of the Antarctic have revealed several pe-
culiar features to be considered as a hallmark of adaptation to the cool-
ing of the Antarctic environment (Coscia et al., 2010a,b). In order to
contribute to ll the knowledge gap relative to Antarctic Rajidae in
the eld of sh immunology, Immunoglobulin M heavy chain (Ig)
genes have been isolated and sequenced from the following species:
B. eatonii (Gnther, 1876), common on the Kerguelen Plateau, around
South Orkney and South Shetland islands. In the Antarctic Peninsula,
it occurs in the northeastern Weddell Sea, on Gunnerus Ridge, and in
the Ross Sea. Bathyraja brachyurops (Fowler, 1910) occurring in the
Strait of Magellan to the Falkland Islands. Bathyraja albomaculata
(Norman, 1937) occurring in Southwest Atlantic: Patagonia, and the
Falkland Islands. Amblyraja georgiana (Norman, 1938), occurring in
the Southern Ocean: around South Georgia Island and on the ridge
westward towards Black Rocks. Bathyraja belongs to the subfamily
Arhynchobatinae, while Amblyraja, Leucoraja, and Raja belong to the
subfamily Rajinae.
2. Materials and methods
2.1. Biological samples
Specimens of each skate species (listed in Table 1) were collected by
bottomtrawling near Lowand Brabant Islands in the Palmer Archipelago
(6325S, 6215W), or in the sub-Antarctic South Atlantic sector of the
Southern Ocean during the ICEFISH (International Collaborative Expedi-
tion to collect and study Fish Indigenous to Sub-Antarctic Habitats)
Cruise (2004). The animals were killed by truncation of spinal cord. The
spleens were dissected from adult specimens and frozen immediately.
Tissues were stored at 80 C until use.
2.2. PCR amplication of Ig heavy chain cDNA
Total RNA was isolated from the spleen of skate specimens using the
SV total RNA Isolation System kit (Promega), following the
manufacturer's instructions, and subjected to reverse transcription using
M-MLV Reverse Transcriptase (Ambion, Austin, TX, USA). To accomplish
PCR amplication of the double-stranded cDNA, the oligonucleotides
used as primers were designed on conserved regions of various elasmo-
branch heavy gene sequences available in GenBank, including sharks
since the number of skate sequences available to date is still limited.
The primers used are as follows: FR1Rj, 5-ggctccatcacactgaactg-3 or
FR3rj, 5-gctgtctattactgtgcgaga-3 (Forward primers); anti-secRj, 5-
ttctcattgacatgaattgacag-3 (Reverse primer). The sequences used for
primer design were under the following Accession Numbers: (Forward
primers) M29672, M29679, X15124, X15125, X16146 (R. erinacea). For
reverse primer design: M29679, M29676 (R. erinacea); X07782,
X07781, X07784, X07783 (Heterodontus francisci); M92851, AF327520
(Ginglymostoma cirratum); AAVX01635466.1 (Callorhinchus milii). The
amplication was performed as follows: 94 C for 2 min, 35 cycles of
94 C (30 s) 50 C (30 s), and 72 C (1 min) with a nal extension at
72 C for 10 min. The combination of FR1Rj and anti-secRj primers was
used only for B. albomaculata. The PCR products (about 14001600 bp)
were then analyzed on 1% agarose gel, puried by QIAquick PCR purica-
tion kit (Qiagen), and cloned into the StrataClone PCR Cloning kit
(Stratagene). The sequence of positive clones was determined with an
ABI PRISM 3100 automated sequencer at PRIMM (Naples, Italy).
2.3. Southern blotting
Genomic DNAs fromthe skates analyzed were prepared fromspleen
as described (Sambrook and Russell, 2001) and by using the DNA
Isolation Kit for Cells and Tissues (Roche). Aliquots of high molecular
weight genomic DNA (10 g) were digested with EcoR I or Hind III re-
striction endonucleases, separated by electrophoresis on 0.7% agarose
slab gels, running in 1 TAE buffer (40 mM Trisacetate, 1 mM EDTA)
at 34 V cm
1
, and transferred to nylon membranes (Hybond-N,
Amersham Biosciences). Southern replicas were probed for the Ig
genes by hybridization to the DIG-labeled Ig FR3Rj/anti-secRj cDNA
fragment from A. georgiana clone A5, cross-hybridizing with the other
species (labeled by PCR DIG Probe Synthesis Kit, Roche). Membrane
prehybridization and hybridization were carried out in DIG Easy Buffer
at 37 C for 4 h and 16 h, respectively. Membranes were washed to a
nal stringency of 0.1SSC, 0.1% SDS at 68 C, developed with immuno-
detection reagents, and nally autoradiographed.
2.4. Computational methods
The percentage of nucleotide identity between IgH sequence pairs
within each species and between different species was calculated
with the LALIGN program available at http://www.expasy.ch/. Se-
quence alignments were performed with the ClustalX 1.83 program
(Thompson et al., 1997).
Distance-based tree reconstruction was performed with the
Neighbor-Joining (NJ) method (Saitou and Nei, 1987) with the align-
ment generated by ClustalX, using the Gonnet series in the weight
matrix. Positions with gaps were excluded from the alignment used
to generate the tree.
Table 1
A list of Antarctic skate species investigated in the present work.
Authority Common name
Amblyraja georgiana Norman, 1938 Antarctic starry skate
Bathyraja brachyurops Fowler, 1910 Broadnose skate
Bathyraja eatonii Gnther, 1876 Eaton's skate
Bathyraja albomaculata Norman, 1937 White dotted skate
36 M.R. Coscia et al. / Marine Genomics 5 (2012) 3541
The observed distances were corrected for multiple substitutions
using Motoo Kimura's formula. 1000 bootstrap replicates were made.
The position variability from aligned amino acid sequences was
calculated as the Shannon entropy, at CVC Bioinformatics server
(http://bio.dfci.harvard.edu/).
2.5. Accession numbers
The sequences reported in this paper have been deposited in
EMBL/GenBank database under the following accession numbers:
GQ339110GQ339113, JF975618JF975624 (B. eatonii); JF975625
JF975627 (A. georgiana); JF975628JF975631 (B. brachyurops); and
JN127355 (B. albomaculata).
3. Results
3.1. Analysis of nucleotide sequences of the Rajidae Ig heavy chain
The PCR analysis of four species of Antarctic skates yielded several
secreted Ig heavy chain sequences. The primers designed on Ig se-
quences from various chondrichthyan species allowed us to isolate
three cDNA clones encoding immunoglobulin heavy chains from A.
georgiana, 11 from B. eatonii, four from B. brachyurops, one from B.
albomaculata. Fig. 1 shows B. eatonii clone c1 sequence encoding an
Ig heavy chain consisting of a partial VH domain, and four CH
domains. Domain boundaries were set based on comparison to Ig se-
quences from other elasmobranch sequences dened by IMGT
(Lefranc et al., 2005). CH1 consists of 101 residues, CH2, CH3, CH4,
and secretory region of 105, 102, 107, and 23 residues, respectively.
The number of negatively charged residues was found to be larger
than that of positively charged residues, serine being the most repre-
sented residue (Supplementary material Table S1), as observed for
Antarctic teleost immunoglobulins (Oreste and Coscia, 2002; Coscia
et al., 2008). One putative N-glycosylation site was found in the CH1
domain, two in CH2, one in CH3, and three in CH4. Four extra-
cysteines not involved in intradomain bonds were identied; one in
the CH1 domain responsible for linking to light chain, two in CH3,
and one in the secretory tailpiece (Fig. 1).
When the 11 B. eatonii cDNA clones were compared to each
other, the nucleotide sequence identities between constant region
domains were very high, ranging from 97.2% to 99.5% (mean value
98.3%). Sequences were then aligned and a distance tree was gen-
erated (Supplementary material Fig. S1). The tree shows two well-
supported major branches, the larger one of which containing
most sequences.
Sequence analysis was then extended to the other species.
Nucleotide sequence identities between the constant region domains
within individual species were even higher, ranging from 97.3% to
99.7% (mean value 98.1%) for B. brachyurops, and from 91.1% to
93.3% (mean value 92.4%) for A. georgiana. Moreover, the nucleotide
VH CH1
gctgtctattactgtgcgagactggtcagtgggtacaggatggttgactactgggcagaaggcaccatgctgacggtaaccaacgcggc
A V Y Y C A R L V S G Y R M V D Y W A E G T M L T V T N A A
tgcatccgcccccagcctcttcattctcttcacctgtgaggatgacggctccagtggttcgatcacctacggttgtctggtgttgggct
A S A P S L F I L F T C E D D G S S G S I T Y G C L V L G
attcccctgtgagcgccagtgtttcttggaagaaagatgatacagaactaaccaccggagtaatgacccacccggaagggttcaataag
Y S P V S A S V S W K K D D T E L T T G V M T H P E G F N K
aatggaacttacacccggagcagcgagttaaccatcaccgaggcggaggcagggggaggcgacatcttctgcgtggttcagcacggcca
N G T Y T R S S E L T I T E A E A G G G D I F C V V Q H G Q
CH2
aaatgagtacatcaagaaagtagcagatcccgtggtggttcatcatccaaacgtcgccattgccatcactgcccctgatgaaatcaagc
N E Y I K K V A D P V V V H H P N V A I A I T A P D E I K
gcagcagaaaagcgatcgccgtctgtttggtcaccgatttcaccccagcgcaatttactgtgaactggctgaagaatggaaagccattg
R S R K A I A V C L V T D F T P A Q F T V N W L K N G K P L
gattctggcatcgtcacctcccctgccttccaggtgaatggtgaaggtaactcctcggcgtccagccagttgacaatcccagccagcga
D S G I V T S P A F Q V N G E G N S S A S S Q L T I P A S E
CH3
gtggttcgtaggttccgtctatacctgccaagtcaaccatgaagaatatttgaaaagccaaaacatcagtaggtcgcaagattgtagtt
W F V G S V Y T C Q V N H E E Y L K S Q N I S R S Q D C S
cctgcggcgaaacaaaaatccttccaccacctgttgaacaagagttattcgagatgaccgtgacgttaacctgtctggtatccgatgca
S C G E T K I L P P P V E Q E L F E M T V T L T C L V S D A
ccgtttggtatcgaagtatcgtggaaccgcaaaaatatccctttggaatcagagatccagcccacagaacatgcggatatcgtggagag
P F G I E V S W N R K N I P L E S E I Q P T E H A D I V E S
caaagtaaacatttcgactcgtgactggctgagtggggataatttcgaatgcgtggtgaagcatgaagaaatacctactgcaaaaaccg
K V N I S T R D W L S G D N F E C V V K H E E I P T A K T
CH4
aacaaatcaactggaagaaaggtcaacatctgctgacaccgtccgcctcagtccttctgccgccgacagaagaaatctccgctcacaag
E Q I N W K K G Q H L L T P S A S V L L P P T E E I S A H K
actatcacccttacctgctttgtgagaggattctcccctcgcagagtcttcgtcacgtggacagttgatgacaagcgggtgcataggag
T I T L T C F V R G F S P R R V F V T W T V D D K R V H R S
caagtacaggaacaccgaggtggtggcggagaacggcaacaattccttcttcatgtacagcctattatccgttgggtcagagcagtggg
K Y R N T E V V A E N G N N S F F M Y S L L S V G S E Q W
caagcggagcctctttttcctgtctggttgggcacgaatcactccccatgaagaccatcgtcagaatggtcaataagtccagcggtaaa
A S G A S F S C L V G H E S L P M K T I V R M V N K S S G K
cccagttttgtgaacgtttcccttgtgttgatggatgctgtcaattcatgtcaatgagaa
P S F V N V S L V L M D A V N S C Q -
Fig. 1. Nucleotide and deduced amino acid sequence of B. eatonii Ig cDNA clone c1. The domain boundaries are based on comparison to Ig sequences from other skate species.
Extra-cysteines are in bold and underlined, N- putative glycosylation sites are shaded.
37 M.R. Coscia et al. / Marine Genomics 5 (2012) 3541
identities were calculated among representative sequences of each
species. They vary from 88.5% (between Arhynchobatinae and
Rajinae) and 97.5% (between Arhynchobatinae). CHregion nucleotide
sequences from the four Antarctic skates were aligned with the CH
sequence from R. erinacea and phylogenetic analysis was performed.
Fig. 2 shows sequences clustering by species with Arhynchobatinae
(Bathyraja) on one branch and Rajinae (Amblyraja genus, including
the Arctic species A. hyperborea, together with R. erinacea) on another
branch.
In order to characterize the multiplicity of clusters of Antarctic
skate Ig genes, genomic DNA, isolated from the four skate species,
was digested with EcoR I or Hind III and subjected to Southern blot-
ting using the CH specic probe generated by PCR and extended
from the end of FR3 to the end of CH4 of A. georgianus sequence
(Fig. 3); its ability to cross-hybridize with DNA from the other species
was veried in dot-blot experiments under medium stringency con-
ditions, before performing the Southern blotting genomic analyses.
The CH probe hybridized 5 to 15 fragments, depending on the en-
zyme used to digest the DNA and on the species analyzed. The num-
ber of CH-hybridizing bands is consistent with the presence of
multiple copies of the Ig gene. The signal given by each hybridizing
band was different in intensity, those more intense probably contain-
ing multiple fragments.
VH/D/JH junctions of cDNA clones of B. eatonii, B. brachyurops, A.
georgiana, and B. albomaculata were compared from the 3 end of
the VH sequence to the 3 of JH (Fig. 4). Nucleotides were tentatively
assigned to the VH, D, and JH segments, although there was an exten-
sive diversity generated by point mutations, deletion, and addition of
bases at the junctions during segmental rearrangement. Four distinct
putative D segments, with lengths from6 to 13 nucleotides, were ten-
tatively dened. Overall, 13 out of 18 clones showed the VHDDJH
conguration, at least one clone having this conguration in each spe-
cies. The sequences of the JH segments were identical to each other
with the exception of few positions. Based on single differences,
four different JH segments were identied in B. eatonii, two in B.
brachyurops, and two in A. georgiana.
3.2. Analysis of amino acid sequences of B. eatonii Ig heavy chain
The position variability for aligned amino acid sequences was calcu-
lated as the Shannon entropy, which takes into account the frequencies
of all amino acids and not only the most common one or two in each po-
sition (Supplementary material Fig. S2). As revealed by the Shannon
analysis, variable positions were distributed throughall four constant do-
mains, however the highest occurrence was observed at the CH1CH2
boundary. CH3 was the most variable domain, showing the highest en-
tropy value (15.59), expressed as sum of entropies for each position,
whereas CH4 exhibited the lowest sum entropy value of 7.95, being the
most conserved domain.
We determined the amino acid composition of B. eatonii CH region
in comparison with that of R. erinacea. We found in the Antarctic
skate that the content of serines was 11.2% vs 9.4%. Moreover, the ma-
jority of serine present (64.6%) was encoded by the TCN codon.
Among Antarctic skates, A. georgiana showed an overall composition
more similar to R. erinacea, both belonging to Rajinae subfamily,
than to the other Antarctic species (Supplementary Table S1).
3.3. Comparative analysis of Antarctic and temperate Rajidae Ig heavy
chain
The amino acid sequences of the four CHdomains, each representa-
tive of the four Antarctic skate species, were compared withthat fromR.
erinacea (Fig. 5). The CH boundaries were assigned by comparison to R.
erinacea CH exon sequences obtained from different genomic clones.
Fig. 2. Phylogenetic tree of CH regions from various skate species. The bootstrap values
reported are higher than 50%.
Fig. 3. Southern blotting analysis using a A. georgiana CH region probe. a, b, c, and d
refer to genomic DNA from A. georgiana, B. albomaculata, B. brachyurops, and B. eatonii,
respectively, digested with EcoR I or Hind III. Size markers (m) are reported on the left.
38 M.R. Coscia et al. / Marine Genomics 5 (2012) 3541
Conservation of canonical residues such as the two cysteines
forming intradomain disulde bonding was observed. The most
distinct differences between the Antarctic and non-Antarctic
skate species analyzed were the presence of one cysteine residue
other than canonical ones, observed at the beginning of CH3 exclu-
sively in the Antarctic skates, and a second one in two out of four
B. brachyurops sequences, and in B. albomaculata sequence (Sup-
plementary material Fig. S3). One extra-cysteine was observed at
the end of CH2 only in B. brachyurops clone B2, whereas the
extra-cysteine present in CH4 is conserved in both cold and tem-
perate skate species. The binding motif for C1q, HXDXXXP (Perkins
et al., 1991), was identied at the end of CH3 of A. georgiana and R.
erinacea whereas some differences in this motif were noted in CH3
from both B. eatonii and B. brachyurops, e.g. proline replaced by al-
anine (Fig. 5, and Supplementary material Fig. S3).
Putative N-glycosylation sites were almost all conserved in both
Antarctic and temperate skates: two in CH2, one in CH3 and three
in CH4 (except B. eatonii clone e15, see also Supplementary Fig. S3).
An exception is the CH1 domain which does not show any putative
N-glycosylation site in R. erinacea whereas shows one site in all
Antarctic species but in three B. eatonii clones, and in A. georgiana,
the latter showing sites at different positions according to the clone
analyzed (e.g. clone A4 lacks sites, whereas clone A6 shows one site
in a quite close position, and clone A5 shows two sites at the end of
the CH1 domain, Supplementary Fig. S3). The tailpiece of secretory
Ig chains is highly conserved throughout evolution: in particular, a
carboxy-terminal cysteine residue and a N-glycosylation site, found
in all species sequenced so far and involved in polymer formation
(de Lalla et al., 1998), are observed in all the sequences analyzed in
the present study.
We analyzed CDR3 length distribution in the Antarctic skate B.
eatonii in comparison with the temperate skate L. eglanteria (Fig. 6).
Antarctic skate CDR3 sequences are longer than L. eglanteria CDR3.
In accordance with the IMGT demarcations, the size of A. georgiana
CDR3 showed a mean length of 10.7 residues, B. brachyurops of 11.5
residues, and B. eatonii CDR3 a mean length of 13.4 residues, resulting
in overall greater length than that of L. eglanteria CDR3 that was of 7.7
residues (Table 2).
B.eatonii_clone_e22 GCTGTCTATTACTGTGCGAGATCTC-CAGTGGGTGCCC-----------ACTTCTATGACTACTGGGCAGAAGGCACCATGCTGACGGTAACCAAC
B.eatonii_clone_c4 GCTGTCTATTACTGTGCGAGAACGT-----CAACAGTGGGTGTTA----TACTTCTTGACTACTGGGGAGAAGGCACCATGCTGACGGTAACCAAC
B.eatonii_clone_e9 GCTGACTATTACTGTGCGAGAGGCCGAGTACAAC-GGGGGT-ATACGCGCG-ATCTTGACTACTGGGGAGAAGGCACCGTGCTGACGGTAACCAAC
B.eatonii_clone_c3 GCTGTCTATTACTGTGCGAGAGAGGGA-TACAACAGTGGGT-ACATCTGGG-TCTATGACTACTGGGCAGAAGGCACCATGCTGACGGTAACCAAC
B.eatonii_clone_e19 GCTGTCTATTACTGTGCGAGACAGATCCGT---------CATATCCG---CATTTATGACTACTGGGCAGAAGGCACCATGCTGACGGTAACCAAC
B.eatonii_clone_e15 GCTGTCTATTACTGTGCGAGACCCT---TACAACAGGGGGATATACTGGGGGGGTATGACTACTGGGCAGAAGGCACCATGCTGACGGTAACCAAC
B.eatonii_clone_e23 GCTGTCTATTACTGTGCGAGAGTA----TACAACAGG--CCTATAC-GGGG--CTATGACTACTGGGCAGAAGGCACCATGCTGACGGTAACCAAC
B.eatonii_clone_e1 GCTGTCTATTACTGTGCGAGAGGGGTA-TACAACAGG----TATACTGGGGGG-TATGACTACTGGGCAGAGGGCACCATGCTGACGGTAACCAAC
B.eatonii_clone_c1 GCTGTCTATTACTGTGCGAGACTGGTCAGTGGGTACA------------GGATGGTTGACTACTGGGCAGAAGGCACCATGCTGACGGTAACCAAC
B.eatonii_clone_e14 GCTGTCTATTACTGTGCGAGAC--------------ATACCCGTACTGGATGG-CTTGACTACTGGGGAGAAGGCACCGTGCTGACGGTAACCAAC
B.brachyurops_clone_B4 GCTGTCTATTACTGTGCGAGATGG-------AACAGTCGGAGTGG--------GCCTGACTACTGGGGAGAAGGCACCATGCTGACGGTAACCAAC
B.brachyurops_clone_B3 GCTGTCTATTACTGTGCGAGACGGC-CGG-GAGTCTC----ACTGGGTACGTATCTTGACTACTGGGGAGAAGGCACCATGCTGACGGTAACCAAC
B.brachyurops_clone_B2 GCTGTCTATTACTGTGCGAGATTCCT----------TAACCC-----------TCCCGACTACTGGGCAGAAGGCACCATGCTGACGGTAACCAAC
B.brachyurops_clone_B1 GCTGTCTATTACTGTGCGAGAAGGGGGGG-------TAACCCAC-----GTCGTCTTGACTACTGGGGAGAAGGCACCATGCTGACGGTAACCAAC
A.georgiana_clone_A4 GCTGTCTATTACTGTGCGAGAGGACG-----------------TACTGGGGA-TCTTGACTACTGGGGAGGAGGCACCATGGTGACGGTAACCAGC
A.georgiana_clone_A5 GCTGTCTATTACTGTGCGAGAGGGGTT-GAGGGTCTGG-------CAGGGGC-CCTTGACTACTGGGGAGGAGGCACCATGGTGACAGTAACCAGC
A.georgiana_clone_A6 GCTGTCTATTACTGTGCGAGAGA-------------TAACCC-----------CCCTGACTACTGGGGAGGAGGCACCATGGTGACGGTAACCAAC
B.albomaculata_clone_A1 GCTGTCTATTACTGTGCGAGGA----CAGTGAGCCGGGC---ATATTGGGG--ACTTGACTACTGGGGAGAAGGCACCATGCTGACGGTAACTAAC
**** *************** ********** ** ****** ** **** ***** * *
A Y Y C A D Y W G E G T T V T
Fig. 4. Alignment of the nucleotide sequence between the VH and JH regions of cDNA clones derived from four Antarctic skate species. Dashes indicate gaps. VH and JH gene seg-
ments are shaded in light gray; bases dening different JH are shaded in dark gray within each species. Nucleotide sequences of the four putative D segments are shaded in different
colors. Deduced amino acid residues shared by all the sequences are shown at the bottom.
CH1
_____A_______ ____B_____ __C___ ____D______ ______E______ ______F_____ ___G___
R.erinacea M29675 .APSAPSLFILFTCEDHGSSGSFTYGCLVLGYSPAGASVSWKKDDKQLTTGVKDYPAVFNKLGTYTRSSELTIAEAEA..GRGDIVCVVQHDQKEYETPLPE.
A.georgiana cloneA5 A------P---Y----------------A--------------------------------------I-----S----EA-----------N-T--HANVSDP
B.eatonii clone3 A-A-------------D-----I-----------VS---------TE-----MT-AE----N-----------T----..-G---F-----G-N--IEKVADP
B.albomaculata cloneA1 A-A----P--------D-----------------V----------TA-----MT--E----N-----------T----..-G---F-------N--IEKVADP
B.brachyurops cloneB3 A-A-------------D-----------------VS---------TE-----MT--E----N-----------T----..-G---F-----G-N--IEKVEDP
CH2
________A________ ____B_____ ___C___ ______D_____ _______E______ _____F_____ _____G____
R.erinacea M29675 LAVQHPTVTIAITAHDEIKRSKKAIAVCLVNDFTPAKFTVNWLKNGAPLDSDIVTSPAFQVNGNGNFSATSQLTFPAGEWFSGSVYTCQVDHGKYLKSQNISRPG
A.georgiana cloneA5 ..-V------T---R---T-----------------T--------------G-----------..----------------R--------H-K-D--------SE
B.eatonii clone3 VV-H--N-A-----P------R--------------Q---S--R--K----G-----------E--S--S----I--S---V--------N-EE---------SQ
B.albomaculata cloneA1 VV-H--N-A-----P------R--------------Q---------KL--FG-----------E--S--S----I--S---V--------N-EE---------SQ
B.brachyurops cloneB3 VV-Y--N-A-----P------R--------T-----Q---------K----G-----------E--S--S----I--S---V--------N-EEC--------SQ
CH3
_______A________ ____B_____ __C__ ____D_____ _____E______ ______F______ ____G_____
R.erinacea M29675 DGIPCVDLKITMLPPPVEQELMEMTVTLTCYRVSHAPYGITVSWSHMKKHLESEIQPTGHEDIVESKVNISTRDWLSGDYFECEVSHDEMPTPKTGRINWNK
A.georgiana cloneA5 -DCP-D-P---I-----------T------.---D-----I---Y-I-VH---------P--F--------------------V----D-----------K-
B.eatonii clone3 -CSS-G-T---I------------------L.--D--F--E---NRKNIP--------E-A------------------N---V-K-E-I--A--EQ---K-
B.albomaculata cloneA1 -CSS-C-T---I------------------L.--D--F--E---NRENIP--------E-A------------------N---V-K-E-I--A--EQ---K-
B.brachyurops cloneB3 -CSS-C-T---I---------F--------L.--D--F--E---NRENIP----------AY----------------EI---V-K-ED---A--E----K-
CH4
_______A________ ____B_____ ____C___ _____D______ _______E_______ ____F______ _____G______
R.erinacea M29675 GQHLLPPSVSILLPPTEEISAHKTITLTCFVRGFSPRRVFVTWTLDDKRVDESKYKNTEVVAENGNDTFFTYSLLSIGAEEWASGASFSCLVGHEALPMKTIVRTVNKSSGKPSFVNVSLVLMDTVNSCQ
A.georgiana cloneA5 -----T----V----------------------------------------D--------------Y---------V---Q----T--------------------------------------------
B.eatonii clone3 -----T----V---------------------------------------HR---R----------NS--M-----V---Q--------------S---------------------A------------
B.albomaculata cloneA1 -----T----V-----------------------A---------V-----HR---R----------NS--M-----V---Q-T------------S----------------------------------
B.brachyurops cloneB3 -----T----V---------------------------------------HR---R----------NS--M-----V---Q-A-----------------------------------------------
Fig. 5. Alignment of the deduced amino acid sequence of R. erinacea Ig constant domains (Accession number M29675) and representative amino acid sequences for the four Ant-
arctic skate species. Extra-cysteines and putative N-glycosylation sites are shaded in gray. The binding motif for C1q, HXDXXXP, is in bold.
39 M.R. Coscia et al. / Marine Genomics 5 (2012) 3541
4. Discussion
We investigated Ig heavy chain genes fromfour species of Antarctic
skates and compared them to temperate species. The species analyzed
comprise B. eatonii, B. brachyurops, B. albomaculata and A. georgiana,
and all four were sampled near Low and Brabant Islands in the Palmer
Archipelago (6325S, 6215W), or in the sub-Antarctic South Atlantic
sector of the Southern Ocean.
Isolation of the genes coding for Ig chain was achieved by PCR
using primers designed on alignment of several elasmobranch
heavy chain gene sequences available in GenBank. Due to the
limited number of skate sequences available to date, shark sequences
were also included in the alignment. Nucleotide sequence identities
between the constant region domains within individual species
range between 98.3% for Bathyraja and 92.4% for Amblyraja. The
nucleotide sequence identities among species representatives vary
from 88.5% between Arhynchobatinae and Rajinae, to 97.5% between
Arhynchobatinae, while R. erinacea and H. francisci constant domains
exhibit slightly lower identity values (89.4% and 93.3%, respectively).
The Antarctic skates investigated in the present study exhibit a
cluster type genomic organization similar to that described for other
cartilaginous sh, since multiple copies of CH genes have been
shown by Southern blotting.
Four distinct D gene segments were dened, two being present in
all the species analyzed. B. eatonii and B. brachyurops showed four D
segments, A. georgiana showed three D segments, B. albomaculata
two. In all the species analyzed the VH-D-D-JH conguration seemed
to be prevalent. However, in the absence of genomic data, we can
not exclude that it corresponds to the VHD
1
D
2
JH joined gene cong-
uration as that found in four R. erinacea genomic clones, representing
the third variation of germline fusion events, the other two being
VHDJH and VHDJH forms (Harding et al., 1990b). In this way, in-
creased diversity is assessed by N-region addition during DD joining.
The frequency of N addition in vertebrate heavy chain is higher than
70% (Gilllan et al., 1995), in 13 nurse shark heavy chain sequences
90% of junctions shows N regions (Fleurant et al., 2004). Therefore,
the Antarctic skate H chain gene seems to contain a similar extent
of N region addition. However, more clones have to be investigated
in each species to probably identify more D segments. Further, germ-
line information is necessary to clarify VH, D and JH sequences and to
assess contribution of the deletion and addition of bases at the junc-
tions during gene segment rearrangement in the diversication of an-
tibody VH regions.
Deduced amino acid sequences were analyzed for CDR3 diversity. Sig-
nicant differences in length were observed between the Antarctic skate
B. eatonii and the temperate skate L. eglanteria (Fig. 6). Specically, the
length of B. eatonii CDR3 ranges between 11 and 15 residues, that of L.
eglanteria between 6 and 10 residues. This greater diversity at Antarctic
skate CDR3, important for the mobility of the H3 loop, might allowhigher
exibility to cope with antigens in the cold environment. In accordance
with the IMGT demarcations, the size of A. georgiana CDR3 showed a
mean length of 10.7 residues, B. brachyurops of 11.5 residues, and B. eato-
nii CDR3 a mean length of 13.4 residues, resulting in overall higher length
variability than that of L. eglanteria CDR3 (7.7 residues). Moreover, nucle-
otide alignments showed evidence of contribution to variability of at least
four distinct D segments.
The amino acid sequences of the four CHdomains, each representa-
tive of the four Antarctic skate species, were compared withthat fromR.
erinacea. The presence of one more extra-cysteine residue observed at
the beginning of CH3 exclusively in the Antarctic skates, and a second
one in two out of four B. brachyurops sequences and in B. albomaculata
sequence (Supplementary material Fig. S3) is the most distinct differ-
ence between the Antarctic and non-Antarctic skate species analyzed.
Notably, in the CH3 domain, the two extra-cysteines fall within the
CXXC motif of thiol/disulde oxidoreductases, which is essential for
their catalysis of redox reactions (Chivers et al., 1996). The interconver-
sion between dithiol and disulde forms may allowexchanges inthe ar-
rangement of CysCys pairs for the formation of inter-heavy chain
disulde bonds, thus contributing to higher exibility of the IgM mole-
cule from Antarctic skates. One extra-cysteine was observed at the end
of CH2 only in B. brachyurops clone B2, whereas the extra-cysteine in
CH4 was present in both cold and temperate skate species; the latter
is one of the four conserved cysteines that are well known to be impor-
tant for assembly of polymeric IgM (Davis et al., 1989).
It does not seem that Antarctic skate heavy chains are particularly
rich in putative N-glycosylation sites, so are those fromAntarctic teleosts
(Coscia et al., 2010b). The number of putative N-glycosylation sites was
almost similar in both Antarctic and temperate skates, despite minor dif-
ferences in the position. The N-glycosylation site in the tailpiece of secre-
tory Ig chains was found in all the sequences analyzed in the present
study, conrming the conservation throughout evolution (de Lalla
et al., 1998).
The Shannon analysis of the position variability for aligned amino
acid sequences revealed that the CH3 domain is the most variable one
whereas CH4 exhibits the lowest sum entropy value, being the most
conserved domain. Overall, the sequence of the Antarctic skate CH3
tends to be the most diverse of the constant region in the middle of
the domain. Whether this could have some structural and/or func-
tional implications is difcult to say. There is little information on car-
tilaginous IgM effector functions. Differences in conserved residues
that are known to be important for complement activation have
been noted in Ig heavy chains from both teleost and cartilaginous
shes (Ota et al., 2003). The binding motif for C1q, HXDXXXP (Perkins
et al., 1991), was identied at the end of CH3 of Antarctic skates.
However, the motif was found to be conserved in B. eatonii and B.
brachyurops (Arhynchobatinae) while it showed some replacements
in A. georgiana and R. erinacea (Rajinae).
We determined the amino acid composition of Antarctic skate CHre-
gion in comparison with that of R. erinacea. We found Antarctic skates
possessing a higher content of serines, which are preferentially encoded
by the TCN codon. It appears that biased serine codon usage may be a
general feature of antibody molecule (Wagner and Neuberger, 1996). In
fact, regarding VH genes, it is well known that there is a clear preference
Fig. 6. CDR3 length distribution in B. eatonii (light gray) and L. eglanteria (dark gray).
B. eatonii CDR3 sequences are from this work, those from L. eglanteria are from Eason
et al. (2004). The limits of the CDR3 region were set according to the IMGT denition.
Table 2
Analysis of CDR3 lengths. Mean numbers of amino acid residues
determined for CDR3 sequences from different species. The
limits of the CDR3 region were set according to the IMGT
denition.
Amblyraja georgiana 10.7
Bathyraja eatonii 13.4
Bathyraja brachyurops 11.5
Raja eglanteria 7.7
40 M.R. Coscia et al. / Marine Genomics 5 (2012) 3541
for AGY serine codons in the CDRs where somatic mutation is more likely
to occur for afnity maturation, but a preference for TCNcodons inframe-
work regions. Similar observations have also been reported for the VH
genes of the Antarctic teleost Trematomus bernacchii (Oreste and Coscia,
2002), as well as in the amphibian Ambystoma mexicanum (Golub and
Charlemagne, 1998). It is shown that the codon bias for some amino
acids can be much higher at conserved rather than at non-conserved po-
sitions. In the particular case of serine, this led to the conclusion that
codon TCN probably appeared before codon AGY (Diaz-Lazcoz et al.,
1995). Based on this conclusion, we can suggest that the high occurrence
of TCN codons in the Antarctic skate constant region is to encode
conserved serine residues.
Taken together, these results have implications for the knowledge
of Ig from skates in the Southern Ocean. However, not as many pecu-
liar features as those identied in Antarctic teleost IgM have been
noted, probably because of the different evolutionary history of carti-
laginous and teleost shes of Antarctica. The most evolutionary prim-
itive vertebrates to possess the adaptive immune system are the
cartilaginous sh, which diverged fromthe teleost sh about 450 mil-
lion years ago. Furthermore, the evolutionary radiations of cartilagi-
nous and teleost shes were probably associated with a signicant
change in the organization, and regulation of immunoglobulin
genes. Similarly, other genes, e.g. globin genes (Marino et al., 2007)
and Otx genes (Kurokawa et al., 2006), have experienced different
evolutionary fates in cartilaginous and teleost sh.
At present, more studies are needed and the extension of these
preliminary data will be achieved only with the collection of addition-
al species from Antarctic and other sub-Antarctic areas.
Supplementary materials related to this article can be found
online at doi:10.1016/j.margen.2011.09.001.
Acknowledgments
This study was conducted in the framework of the Italian Antarctic
Research Program (PNRA), project 2005.1.2. We gratefully acknowl-
edge the logistic support provided to our research team at Palmer
Station, and on board RV LM Gould and RVIB NB Palmer (ICEFISH
Cruise) by the personnel of Raytheon Polar Services and by the cap-
tains and crews of RV LM Gould and RVIB NB Palmer. This work was
partly supported by the National Science Foundation Grant OPP-
0132032 to H. William Detrich (Northeastern University, Boston,
Mass.).
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