The Immunoglobulin G Heavy Chain (IGHG) genes of the Atlantic bottlenose dolphin, Tursiops truncatus

The Immunoglobulin G Heavy Chain (IGHG) genes of the Atlanticbottlenose dolphin Tursiops truncatus

Annalaura Mancia a Tracy A Romano b Holly A Gefroh a Robert W Chapman cDarlene L Middleton a Gregory W Warr a Mats L Lundqvist d

a Marine Biomedicine and Environmental Science Center Medical University of South Carolina Hollings Marine Laboratories331 Ft Johnson Road Charleston SC 29412 USA

b Mystic Aquarium and Institute for Exploration Mystic CT 06355 USAc South Carolina Department of Natural Resources Charleston SC 29412 USA

d Center for Coastal Environmental Health and Biomolecular Research NOAA NOS Charleston SC 29412 USA

Received 13 September 2005 received in revised form 10 January 2006 accepted 11 January 2006Available online 7 March 2006

Abstract

Dolphin Immunoglobulin G Heavy Chain (IGHG) sequences were obtained by PCR amplification of cDNA from peripheral blood leukocytesusing degenerate primers Analysis of full-length sequences indicated the presence of two expressed isotypes IGHG1 and IGHG2 that differmainly in the hinge region of the molecule Genomic Southern blot analysis indicated that the IGHG1 and IGHG2 genes are most likely present insingle copies The inferred amino acid sequences show greatest similarity between the dolphin and other closely related artiodactyl species Thegenetic structure of the IGHG genes were deduced through genomic PCR and revealed that the hinge regions of both IGHG1 and IGHG2 areencoded by a single exon The transmembrane region of the dolphin IGHG chain shows similarity to the transmembrane region of othermammalian IGHG chains with a canonical CART motif This is in contrast to the unusual Ser to Gly substitution previously found in the dolphinIGHM transmembrane region and the functional significance of this variation for B cell antigen-receptor dimer activation remains unknowncopy 2006 Elsevier Inc All rights reserved

Keywords Bottlenose dolphin Antibody IGHG1 IGHG2 Immunoglobulin G IgG Isotype Subclass Allele Evolution

1 Introduction

The immune systems of terrestrial mammals (especiallyhuman and mouse) are well understood but those of marinemammals in particular those of the order Cetacea (whalesdolphins porpoises) which live in a totally aquatic environ-ment are not The ancestors of modern whales entered theoceans about 55ndash60 million years ago and the anatomy andphysiology of their descendants show major adaptations to lifein the aquatic environment These adaptations are morpholog-ical (the conversion of limbs to flippers and flukes)

physiological (deep and long diving) as well as behavioral(underwater communication navigation and echo-location)Life-long immersion in water brings with it exposure to acompletely different physical medium from that experienced bythe terrestrial mammals as well as exposure to a differentspectrum of microbes amongst them many pathogens Whilerelatively little is known about the cetacean immune system itcan be assumed that the unique challenges of the marineenvironment have led to adaptations in immune function Anumber of immune-related molecules have been cloned from arange of cetacean species (Murray et al 1995 King et al 1996Shoda et al 1998 Shirai et al 1998 Murray and White 1998Inoue et al 1999abc 2001 Romano et al 1999 St-Laurentet al 1999 St-Laurent and Archambault 2000 Shoji et al2001 Lundqvist et al 2002) and several serological andbiochemical studies on cetacean immunoglobulins (Ig) havebeen carried out (Nash and Mach 1971 Travis and Sanders

Comparative Biochemistry and Physiology Part B 144 (2006) 38ndash46wwwelseviercomlocatecbpb

A portion of this work was submitted in partial fulfillment of therequirements for the Master of Science Degree at the Medical University ofSouth Carolina by Holly Gefroh This work constitutes scientific contributionno 159 from the Sea Research Foundation Inc Corresponding author Tel +1 843 762 8962E-mail address manciamuscedu (A Mancia)

1096-4959$ - see front matter copy 2006 Elsevier Inc All rights reserveddoi101016jcbpb200601014

1972ab Cavagnolo 1979 Andreacutesdoacutettir et al 1987 Sweeneyet al 1987 Romano et al 1992) There are unique attributes ofthe cetacean immune system that suggest differences from othermammals It is clear that IGHM IGHG and IGHA are present incetaceans and subclasses of IGHG have been biochemicallyidentified in whales (Andreacutesdoacutettir et al 1987) A full-lengthIGHM cDNA of the Atlantic bottlenose dolphin Tursiopstruncatus has been sequenced and an initial analysis of VHgene usage in this species reported (Lundqvist et al 2002) Thetransmembrane region of the dolphin IGHM showed a uniquesubstitution in the highly conserved CART motif which isimportant for signaling in the B cell receptor (Lundqvist et al2002) In contrast to the single VH family usage described in thepig and cow two closely related artiodactyls the dolphin ex-presses at least 2 distinct VH families associated with the IGHM(Lundqvist et al 2002) Moreover the analysis and character-ization of CD4 in Delphinapterus leucas showed unique aminoacid (aa) substitutions in the cytoplasmic domain which ishighly conserved in human and mouse (Romano et al 1999)

The immune system of the Cetacea is of interest for reasonsadditional to the evolutionary radiation of the mammals Manycetacean species are endangered and threats to their healthinclude the immunosuppressive effects of anthropogenic stress-ors present in the marine environment (Lahvis et al 1995Moumlssner and Ballschmiter 1997 Van Bressem et al 1999Romano et al 2002) The characterization of immunoglobulinsis one important step in understanding the function of the im-mune system of the dolphin its evolutionary adaptations and itsresponse to environmental challenges Here we present the first

study to characterize the IGHG genes of the Atlantic bottlenosedolphin T truncatus

2 Material and methods

21 Sampling procedures

Blood samples were collected from dolphins maintained bythe US Navy Marine Mammal Program San Diego CA inaccordance with a protocol approved by the Institutional AnimalCare and Use Committee under the guidelines of the Associationfor the Accreditation of Laboratory Animal Care Peripheralblood leukocytes (PBL) were subsequently isolated from wholedolphin blood via centrifugation over Histopaque (Gibco-BRLRockville MD) Bottlenose dolphin liver and spleen sampleswere collected and immediately frozen on dry ice during nec-ropsies conducted by the US Navy Marine Mammal Programor the NOAA Center for Coastal Environmental Health andBiomolecular Research Charleston SC

22 RNA isolation and generation of cDNA

Total RNA was extracted from dolphin PBL or spleenusing the RNeasyreg kit (Qiagen Valencia CA) and integrityof the RNA verified on a 1 agarose gel Five g of totalRNA samples were subjected to first-strand synthesis andcDNA amplification was performed by RT-PCR using theSMARTtrade cDNA Library Construction Kit (Clontech PaloAlto CA)

Table 1Oligonucleotides used in this study

Name Description Position Sequence

G-414 3RACE anchor primer 5-TCTGAATTCTCGAGTCGACATCG-1857 IGHG_C1_forward a 351ndash387 5-GAGAATTCACMMRSCCASCARCRCCAARGTGGACAAG

b 357ndash393G-1858 IGHG_C3_reverse a 1039ndash1069 5-GAGGATCCTRTRGTGGTTGTGCARRSCCTCRTGYADCAC

b 1087ndash1117G-1910 IGHG_C3_forward a 852ndash870 5-CTGCATGGTCACCGACTTC

b 900ndash918G-1911 IGHG_C3 _ reverse a 905ndash921 5-TGGCTCCGGCTGCCCGT

b 953ndash969G-2143 IGHG_C3 _ forward a 896ndash914 5-GGCAGAGAAACGGGCAGCC

b 944ndash962G-2144 IGHG_3UTR_reverse a 1147ndash1165 5-TCGCTGGACCCTGAGAGCC

b 1195ndash1213G-2151 TM_ reverse d 342ndash362 5-CACCACYGAGGAGAAGATCCAG-2183 VH-FR3_ forward c 399ndash421 5-GACRCRGCCRTRTATTAYTGTGG-2322 IGHG_C1 _ forward a 329ndash346 5-CCGGCAAGACCTTCACCT

b 335ndash352G-2323 IGHG_C2 _ reverse a 451ndash468 5-GACGGACGGTCCTCCTGG

b 499ndash516G-2324 IGHG_C3 _ reverse a 851ndash868 5-AGTCGGTGACCATGCAGG

b 899ndash916

G-1857G1858 are degenerate primers used to amplify IGHG sequences (Aveskogh and Hellman 1998) G-414G-2143 was used in 3RACE and G-2183 to G-1911were used in 5RACE to obtain the full-length IGHG sequences G-2143 and degenerate G-2151 was used to obtain the TM sequence G-2322G-2323 was used toamplify the hinge exon region G-2322G-2323G-2324 was used to amplify the genomic sequence G-1910G-2144 was used to obtain a C3 exon fragment to use asa radioactively labeled probe in Southern blot hybridization C1 constant domain exon 1 C2 constant domain exon 2 C3 constant domain exon 3 a IGHG1Genbank accession no AY621036 b IGHG2 Genbank accession no AY621037 c IGHM Genbank accession no AF306861 d IGHG-TM Genbank accession noAY621030

39A Mancia et al Comparative Biochemistry and Physiology Part B 144 (2006) 38ndash46

23 PCR 3 and 5RACE

Dolphin spleen and PBL cDNA was used as template in aPCR with degenerate primers G-1857 and G-1858 for the C1exon (forward primer) and for the C3 exon (reverse primer)respectively (Table 1) (Aveskogh and Hellman 1998) toamplify IGHG sequences Full-length sequences were obtainedthrough overlapping 3 and 5 RACE PCR (Frohman et al

1988) conducted with the primers listed in Table 1 The PCRswere performed with the Advantagetrade cDNA PCR kit (Clon-tech Palo Alto CA) with primer concentrations of 10M and aMg2+ concentration of 35mM using a PCR profile with initialdenaturation at 94degC for 1min followed by 30 cycles of 94degC for20s 55degC for 30s 68degC for 15min and a final extension step of10min at 68degC Obtained fragments were agarose gel purifieddA-tailed with Taq DNA polymerase (Fisher Scientific

Fig 1 Nucleotide and inferred amino acid sequence of IGHG1 (A) and IGHG2 (B) Constant region domains the hinge regions and FR3 CDR3 and FR4 of the VHdomains are indicated by arrows The domain borders were identified from sequences of genomic PCR fragments or from (Lundqvist et al 2002) PotentialN-glycosylation sites following the NndashXndashS or T rule are underlined

40 A Mancia et al Comparative Biochemistry and Physiology Part B 144 (2006) 38ndash46

Pittsburg PA) and cloned into the pCRreg-21 TOPO-vector(Invitrogen Carlsbad CA) TOP-10 chemically competent cells(Invitrogen CarlsbadCA) were transformed and plasmids werepurified for sequencing The sequencing reactions were per-formed on both strands using the M13R and M13F primers(Invitrogen Carlsbad CA) as well as internal gene-specificprimers The primer concentration was 16pmolul and the se-quencing reactions were performed with CEQtrade Dye Termina-

tor Cycle Sequencing Quick Start Kit and analyzed in the CEQ8000 instrument (Beckman Coulter Fullerton CA) or at theBiotechnology Resource Laboratory of MUSC

24 Cloning of the IGHG transmembrane (TM) segment

Spleen cDNAwas used as template for PCR amplification ofthe dolphin IGHG-TM sequence using forward primer G-2143and degenerate reverse primer G-2151 (Table 1) designed fromsequence alignments of human and murine IgG transmembranesequences The PCR was performed using the Advantagetrade 2cDNA PCR kit (Clontech Palo Alto CA) in combination withthe Perfect Matchreg reagent (Stratagene La Jolla CA) using aPCR profile initial denaturation at 95degC for 5min an annealingstep at 58degC for 5min followed by 30 cycles of 95degC for 1min58degC for 2min 68degC for 2min and a final extension step of10min at 68degC Obtained fragments were agarose gel purifiedcloned and sequenced as described above

25 Genomic DNA Isolation and PCR

Genomic DNAwas phenol extracted (Sambrook et al 1989)from white blood cells and liver of 4 different animals In shortapproximately 025g of frozen sample was ground to a powderunder liquid nitrogen incubated overnight at 55degC in 20mL ofSTE (01M NaCl 10mM TrisndashCl pH 80 1mM EDTA pH 80)10 SDS supplemented with 002g of Proteinase K (Invitro-gen Carlsbad CA) After phenolndashchloroform extraction andethanol precipitation the genomic DNA was redissolved in25mL of TE (10mM TrisndashCl pH 80 1mM EDTA pH 80)The quality of the genomic DNAwas evaluated by agarose gelelectrophoresis and concentration determined by 260nm ab-sorbance measurements Genomic PCR was performed with50ng to 100ng of genomic DNA as template using a dolphinIGHG specific forward primer in the first constant exon C1(G-2322) in combination with a dolphin IGHG reverse primer

654321

231

94

65

44

2320

1311

09

Kb

Fig 2 Genomic representation of IGHG sequences by Southern blot analysisLane designations from left to right undigested DNA (Lane 1) DNA digestedwith BamHI (Lane 2) EcoRI (Lane 3) HindIII (Lane 4) PstI (Lane 5) or MboI(Lane 6) The spleen DNA from a single dolphin was separated on a 06agarose gel and transferred to a nylon filter The filters were hybridized with aprobe specific for the C3 exon Size markers are indicated in kilobases (Kb) tothe left of the figure

C1 exon intron C 2 exonhinge

F (G-2322) R (G-2323)

intron

13531078872603

310281

655606

bp 1 2

IGHG1 AAATCAACGGCCCAA------ACCCCTGTTCCAG------------------------------------GTGTCCCAAATGTCCAC 45IGHG2 AGTTCAAGGGCTCCACAACTCACCCATGCCCCAAGTGTCCCGAATGCCACAAGTGTCCCGAATGCCAACAGTGTCCCAAATGTCCAC 87

5rsquo 3rsquo

Fig 3 Hinge encoding exons of the dolphin IGHG genes Top left Ethidium bromide stained agarose gel (15) of PCR products amplified from white blood cell of asingle dolphin genomic DNA Lane 1 size marker Lane 2 PCR products Two major amplicons were generated 606 and 655bp in size respectively Top rightSchematic illustrating the PCR strategy The primers G-2322 (Forward priming within the C1 exon) and G-2323 (Reverse priming within the C2 exon) areindicated as arrows Bottom Genomic hinge region sequences obtained from the PCR amplified genomic fragments The 655bp long fragment was found tocorrespond to the IGHG2 cDNA sequence while the 606bp was found to correspond to the IGHG1 cDNA Conserved nucleotides in the hinge regions are indicated byasterisks

41A Mancia et al Comparative Biochemistry and Physiology Part B 144 (2006) 38ndash46

specific for either the second C2 (G-2323) or third C3 con-stant exons (listed in Table 1) The PCRs were performed withthe Advantagetrade-GC genomic PCR kit (Clontech Palo AltoCA) with primer concentrations of 10M using a PCR profilewith initial denaturation at 95degC for 1min 30 cycles of 95degCfor 30s 56degC for 30s 68degC for 4min followed by an incuba-tion for 10min at 68degC Obtained fragments were agarose gelpurified cloned and sequenced as described above

26 Southern blot analysis

Genomic DNA from the liver was digested to completionwith BamHI EcoRI HindIII PstI or MboI separated on 06agarose gels denatured and transferred to Nytranreg filters(Schleicher and Schuell Keene NH) by capillary blotting with05M NaOH overnight The damp filters were UV crosslinkedwith 150mJ and dried before pre-hybridization Blots were thenhybridized with a probe specific for the dolphin C3 exon in aformamide solution (50 formamide 5SSPE 2Denhardtssolution 1 SDS 50LmL yeast tRNA) overnight The probespecific to the C3 exon of dolphin IGHGwas obtained by PCRamplification using exon specific primers G-1910 and G-2144(Table 1) and spleen cDNA as template The PCR was per-formed using the Advantage2trade cDNA PCR Kit (ClontechPalo Alto CA) with primer concentrations of 10M and usinga PCR profile with initial denaturation at 94degC for 5min10 cycles of 94degC for 30s 58degC for 15s 68degC for 20s 20 cycles

of 94degC for 30s 58degC for 30s 68degC for 1min followed by7min at 68degC Authenticity of the fragment was verified bycloning and sequencing as described above The fragment wasthen released from the plasmid construct by EcoRI digest and gelpurified prior to radioactive labeling in PCR reaction containingprimers mentioned above a final concentration of 125mM ofdCTP dGTP and dTTP and 20Ci of [32P]dATP Afterhybridization the filters were washed for 330 min in 1SSPE01 SDS at 42degC and 230min in 05SSPE 01 SDS at65degC prior to exposure to X-Omat AR (Kodak Rochester NY)film for 72h at 80degC

27 Sequence alignments and phylogenetic tree

Amino acid sequences of immunoglobulin heavy chains wereobtained from GenBank and aligned using the software packageMEGA3 with default alignment parameters (Kumar et al 2004wwwmegasoftwarenet) Phylogenetic and molecular evolu-tionary analysis was conducted using the NeighbourndashJoining(NJ) method with 1000 bootstrap replications The sequenceswith the following accession numbers were obtained fromGenBank (T truncatus) dolphin IGHG1AAT65197IGHG2AAT65196 (Bos taurus) cattle IGHG1 AAB37381IGHG2a AAB37380 IGHG3 AAC48761 (Sus scrofa) pigIGHG1AAA52216 IGHG2aAAA52217 IGHG2bAAA52218IGHG3 AAA52219 IGHG4 AAA5220 (Mus musculus) mouseIGHG1 BAC44885 IGHG2a G2MSA IGHG2b P01866

IGHG1 AA ATC AAC GGC CCA AAC CCC TGT TCC AGG TGT CCC AAA TGT CCA C I N G P N P C S R C P K C P

IGHG201 AG TTC AAG TGCCCCAAGTGTCCCGAATGCCACAAGTGTCCCGAATGCCAACAGTGTCCCAAATGTCCACF K C P K C P E C H K C P E C Q Q C P K C P

IGHG203 AG TTC AAG TGCCCCAAGTGTCCCGAATGCCACAAGTGTCCCGAATGCCAACAGTGTCCCAAATGTCCACF K C P K C P E C H K C P E C Q Q C P K C P

GGC TCC ACA ACT CAC CCAG S T T H P

GTC TGC AAA GAT CCC AAAV C K D P K

Fig 4 Allelic variants for the hinge region of the 2 IGHG isotypes observed in a single dolphin Nucleotide and amino acids substitutions are shown in bold

Alleles IGHG1

IGHG101

IGHG102

IGHG103

Alleles IGHG2

IGHG201

IGHG202

IGHG203

Position

C 1 Intron Hinge Intron

Position

141 164 572

141 164

A A G

G A A

A G G

A G A G C C A C A C C

G G A G C C A C A C C

G A G T G A G A C A A

410 413 416 418 419 422 423 424

C 1 Intron Hinge Intron

61

Accession No

AY621031

AY621032

DQ173079

DQ173080

DQ173081

DQ173078

Fig 5 Allelic variants for the 2 IGHG isotypes in 4 different dolphins Variable positions are listed for each isotype with the numbering based on the genomic PCRfragments (GenBank Accession no AY621031 DQ173078 DQ173079 for IGHG101 02 03 and AY621032 DQ173080 DQ173081 for IGHG2010203)

42 A Mancia et al Comparative Biochemistry and Physiology Part B 144 (2006) 38ndash46

IGHG3 P03987 (Ovis aries) sheep IGHG1 X69797 IGHG2X70983 (Homo sapiens) human IGHG1 PO1857 IGHG2G2HU IGHG3 CAC10266 IGHG4 CAC20457

3 Results

31 Cloning of the IGHG cDNA

RT-PCR of dolphin PBL and spleen cDNA with degenerateprimers (G-1857 and G-1858 Table 1) for the constant domain1 exon (forward primer) and the constant domain 3 exon(reverse primer) of mammalian IGHG genes yielded two dif-ferent products of 718 and 760bp respectively The fragmentswere cloned and sequenced as described in Materials andMethods The BLASTbhttpwwwncbinlmnihgovBLASTNqueries of these sequences revealed that both sequences scoredhigh with IGHG genes of other mammals Using 3 and 5RACE techniques with the primers listed in Table 1 the fulllength IGHG dolphin genes were cloned as cDNA andsequenced The sequences termed IGHG1 and IGHG2 havebeen submitted to Genbank with accession AY621036 andAY621037 respectively (Fig 1A and B) The dolphin IGHG1and IGHG2 genes obtained show the characteristic structure of amammalian IGHG gene with a constant domain (C1) a hingeregion a second constant domain (C2) and a third constantdomain (C3) The hinge regions of the two IGHG sequencesdiffered considerably in lengthmdashthe shorter termed IGHG1

had a hinge region of 15 aa while the longer termed IGHG2had a hinge with 29 aa Comparisons of the inferred aasequences of the two genes showed an overall identity of 86While most of the differences outside the hinge regionsoccurred in the C1 and C2 domains with 17 and 13 aasubstitutions respectively the C3 domain had only 8 aasubstitutions Both IGHG1 and IGHG2 have 2 putative N-linked glycosylation sites one in the C2 domain which ishighly conserved within the mammals and has been shown inhuman studies to be important for the interaction between thetwo IGHG chains efficiency of Fc receptor binding andcomplement activation (Deisenhofer 1981 Rudd et al 19912001) and one in the C3 domain Although the glycosylationsite in C3 is not highly conserved among mammals it is alsopresent in mouse IGHG1 and similar sites are found in thehuman and cattle IGHG3 subclasses Overall the dolphinIGHG sequences were most closely related at both nucleotideand amino acid sequence level to those of artiodactyl speciesIn the amino acid sequence alignments the highest identityvalues were seen with cattle IGHG1 (705 identity to dolphinIGHG1 and 742 identity to dolphin IGHG2)

32 Genomic representation of IGHG sequences

Southern blot analysis was performed with a probe specificfor the C3 exon that would allow detection of both IGHGgenes Either 1 or 2 hybridizing bands ranging in size fromabout 23 to 23kb were obtained regardless of restriction en-zyme used (Fig 2) The single 23kb hybridization band ob-served in theMboI digested DNA suggests one of two things a)there are conserved sites for this restriction enzyme within theIGHG genes or b) the genes are located on the same MboIfragment The genomic structure of the hinge region of dolphinIGHG genes was investigated using PCR Genomic PCR using aforward primer (G-2322) in C1 and a reverse primer (G-2323)in C2 was conducted to amplify the hinge region with itsflanking introns This PCR generated 2 major amplicons as

Table 2Number of observed alleles for IGHG1 and IGHG2 in each one of 4 dolphins

Animal IGHG1 IGHG2

1 0203 02032 0102 01023 0101 01034 0103 0203

Data based on the sequence analysis of 227 recombinant clones derived from thegenomic PCR of the hinge region of dolphin IGHG genes

T L F

T S S

T S S

W T T I S I F I T L F L L S V C Y S A T V T L F

T I F

T S A

T S S

N M A T V G L F T

N M A T V L F T

A A T V L F T

A T V L F T

A A V F

IGHG12 dolphin

IGHG camel

IGHG2 human

IGHG3 human

IGHG1 mouse

IGHG2a mouse

IGHG2b mouse

IGHG3 mouse

IGHM dolphin

IGHM cow

IGHM human

IGHM mouse

IGHY duck

Fig 6 Alignment of immunoglobulin transmembrane regions The inferred amino acid sequence of the two dolphin IGHG TM regions is placed on top Residuesforming the highly conserved CARTmotif are boxed and shaded Amino acid identities are represented by dots Sequences were extracted from the following Genbankaccession numbers IGHG12 dolphin (Tursiops truncatus) AY621030 IGHG camel (Camelus dromedarius) CAB64865 IGHG2 human (Homo sapiens) BAA25141IGHG3 human (H sapiens) A45874 IGHG1 mouse (Mus musculus) P01869 IGHG2a mouse (M musculus) AAB59661 IGHG2b mouse (M musculus) AAB59659IGHG3 mouse (M musculus) P03987 IGHM dolphin (T truncatus) AJ320193 IGHM cow (Bos taurus) U63637 IGHM human (H sapiens) S14683 IGHM mouse(M musculus) P01873 IGHY duck (Anas platyrhynchos) X78357 and X78358

43A Mancia et al Comparative Biochemistry and Physiology Part B 144 (2006) 38ndash46

shown in Fig 3 Cloning and sequencing of these ampliconsidentified the longer fragment about 655bp in length as con-taining the hinge region of the IGHG2 gene The shorter frag-ment about 606bp in length contained the hinge region of theIGHG1 gene In both cases the hinge was found to be encodedby a single exon (Fig 3) Sequence analysis of additional PCRclones from the genomic DNA of lymphocytes and liver of 4different animals permitted the definition of allelic variations inthe sequence of the C1 exon the hinge exon and the twointrons flanking the hinge exon While IGHG2 showed allelicvariations in the hinge-encoding exon such allelic variants weremissing in IGHG1 as shown in Fig 4 The analysis of the C1exon as well as of the intron regions flanking the hinge exonpermitted the identification of 3 allelic variants for both IGHG1and IGHG2 (Fig 5) The independent observation of these allelicvariants of the IGHG genes in 4 different dolphins (Table 2)supported the conclusion that allelic variants had been correctlyidentified and diminished concerns that sequence variationscould be the result of PCR-introduced artifacts The distributionof the alleles in the 4 dolphins studied indicated that 3 animalswere heterozygous for IGHG1 and that all 4 were heterozygousfor IGHG2 (Table 2)

33 The transmembrane region of dolphin IgG

To characterize the transmembrane (TM) region of dolphinIGHG the membrane spanning region was amplified by RT-

PCR as described in Materials and Methods and the generatedsequence was deposited in GenBank under accession noAY621030 The sequence of the forward primer used was com-mon to both the IGHG1 and IGHG2 isotypes but still positionedso that the 2 isotypes could be distinguished All clones ex-amined yielded identical sequences for the both IGHG1 andIGHG2 TM regions The inferred amino acid sequence for theTM region of dolphin IGHG1IGHG2 was aligned with the TMsequences of other mammalian immunoglobulins (Fig 6) Thededuced sequence of the dolphin TM region showed a highdegree of similarity to the sequence of other mammalian speciesnot only in the TM region that contains the conserved CARTmotif (Campbell et al 1994) but also in the hydrophilic con-necting peptide that lies immediately N-terminal to the TMregion The highest overall sequence similarity was observedwith the camel sequence In contrast to the dolphin IGHM TMregion (Lundqvist et al 2002) the IGHG TM region contains aperfect consensus CART motif (Fig 6)

4 Discussion

The study reported here presents the characterization at thegenetic level of the bottlenose dolphin IGHG gene The majorconclusion is that two closely-related IGHG isotypes arepresent and expressed A detailed study of allelic variationsalong the whole length of the IGHG genes was not carried outin this study However while allelic polymorphisms were

HumanIGHG1

HumanIGHG3

HumanIGHG2

HumanIGHG4

PigIGHG1

PigIGHG3

PigIGHG2a

PigIGHG2b

PigIGHG4

DolphinIGHG1

DolphinIGHG2

SheepIGHG1

SheepIGHG2

CattleIGHG1

CattleIGHG2a

CattleIGHG3

MouseIGHG3

MouseIGHG2a

MouseIGHG2b

MouseIGHG1

99

55

85

100

73

67

100

100

100

100

94

100

90

61

99100

47

Fig 7 Phylogenetic tree of selected mammalian IGHG sequences A phylogenetic tree was constructed using Mega3 from the amino acid sequences (excluding thehinge regions) of the IGHG chains of various mammalian species (as described in Materials and Methods) Bootstrap values () in support of each node are indicated

44 A Mancia et al Comparative Biochemistry and Physiology Part B 144 (2006) 38ndash46

observed in both the IGHG1 and IGHG2 genes (Fig 4) thepositions showing allelic variation were relatively few withnone being observed in the hinge exon of IGHG1 or in theTM regions of either isotype (an identical TM sequence beingobserved for both the IGHG1 and IGHG2 isotypes) Anotherinteresting feature of the dolphin IGHG genes is that the hingeregions of both isotypes are encoded by a single exonmdashthehinge regions of other mammalian IGHG are encoded by asmany as 4 individual exons (Attanasio et al 2002) Thus astriking finding of this study is the close similarity betweenthe 2 IGHG isotypes their sequences are similar they haveonly a single hinge-region exon and their obtained TMsequences were identical These observations coupled withthe relatively low number of sequence positions showingallelic variation suggest that the gene duplication that gaverise to the dolphin IGHG genes may have been a relativelyrecent and lineage-specific event This is consistent with theview that the diversification of mammalian IGHG genes wasnot a basal event but that many lineage-specific IGHG geneduplication events have taken place during mammalianevolution (Takahashi et al 1982 Hayashida et al 1984Matsuda and Honjo 1996 Wagner et al 2002) Thishypothesis is also supported by the fact that in most casesphylogenetic trees cluster the IGHG subclass sequences in aspecies-specific manner as seen with all of the species(including the dolphin) in the tree of IGHG sequences shownin Fig 7 In this tree (Fig 7) the closest neighbors to thedolphin IGHG are the sequences of other artiodactyl species aresult that strengthens the suggestion of a close relationship ofthe cetacea to the artiodactyla it has even been suggested thatthese two orders should be combined into a single new orderthe Cetartiodactyla (Lum et al 2000 Gingerich et al 2001)However an interesting exception is observed with the pig oneof the artiodactyl species The swine sequences seem to showmore similarity to human IGHGs rather than to those of otherartiodactyl species as has been previously observed (Kacsko-vics et al 1994) and as occurs with the tree shown in Fig 7 TheIGHG sequences of human and pig clustered on the samebranch of the tree whether the hinge region was included in thesequence alignment (data not shown) or was excluded (Fig 7)

The membrane bound forms of the immunoglobulins areassociated with the CD79 molecules to form the B cell receptor(BCR) complex and together they span the lipid bilayer Theseinteractions are necessary to permit signal transduction over thecell membrane following antigen binding The TM region of theimmunoglobulins interacts with the CD79 molecules throughthe highly conserved CART motif (Campbell et al 1994)Previous reports of unusual variations in the CART motif in thedolphin IGHM chain (Lundqvist et al 2002) raised two ques-tions first does the dolphin IGHG possess a similar non-canonical CART motif and second does a non-canonicalCART motif affect the function of the BCR While thedescription of a canonical CARTmotif in the dolphin IGHG TMregion (Fig 5) answers the first question the issue of potentialfunctional impairments caused by the SerndashGly substitution inthe dolphin IGHM TM region will require direct experimentalinvestigation

Acknowledgements

We thank Wayne McFee of NOAACCEHBR and the USNavy Marine Mammal Program for the generous gift of dolphintissues and acknowledge the support of this research by awardsfrom the Office of Naval Research (N00014-02-1-0386 andN00014-00-1-0041 to TR) and from NOAANational MarineFisheries Service (to GW) This research was conducted underPermits 932-1489-03PRT009526 (NMFSUSFWS) and03US0209509 (CITES)

References

Andreacutesdoacutettir V Magnadoacutettir B Andreacutesson Oacute Peacutetursson G 1987Subclasses of IgG from whales Dev Comp Immunol 11 801ndash806

Attanasio R Jayashankar L Engleman CN Scinicariello F 2002 Baboonimmunoglobulin constant region heavy chains identification of four IGHGgenes Immunogenetics 54 556ndash561

Aveskogh M Hellman L 1998 Evidence for an early appearance of modernpost-switch isotypes in mammalian evolution cloning of IgE IgG and IgAfrom the marsupial Monodelphis domestica Eur J Immunol 282738ndash2750

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Cavagnolo RZ 1979 The immunology of marine mammals Dev CompImmunol 3 245ndash257

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Frohman MA Dush MK Martin GR 1988 Rapid production of full-length cDNAs from rare transcripts amplification using a single gene-specific oligonucleotide primer Proc Natl Acad Sci U S A 858998ndash9002

Gingerich PD Haq M-u Zalmout IS Khan IH Malkani AS 2001Origin of whales from early artiodactyls hand and feet of EoceneProtocetidae from Pakistan Science 293 2239ndash2242

Hayashida H Miyata T Yamawaki-Kataoka Y Honjo T Wels J BlattnerF 1984 Concerted evolution of the mouse immunoglobulin gamma chaingenes EMBO J 3 2047ndash2053

Kacskovics I Sun J Butler JE 1994 Five putative subclasses of swine IgGidentified from cDNA sequences of a single animal Immunology 1533565ndash3573

Inoue Y Itou T Ueda K Oike T Sakai T 1999a Cloning and sequencingof a bottle-nosed dolphin (Tursiops truncatus) interleukin-1 and -1$complementary DNAs J Vet Med Sci 61 1317ndash1321

Inoue Y Itou T Oike T Sakai T 1999b Cloning and sequencing of thebottle-nosed dolphin (Tursiops truncatus) interferon- gene J Vet MedSci 61 939ndash942

Inoue Y Itou T Sakai T Oike T 1999c Cloning and sequencing of abottle-nosed dolphin (Tursiops truncatus) interleukin-4 encoding cDNAJ Vet Med Sci 61 693ndash696

Inoue Y Itou T Jimbo T Syouji Y Ueda K Sakai T 2001 Molecularcloning and functional expression of bottle-nosed dolphin (Tursiopstruncatus) interleukin-1 receptor antagonist Vet Immunol Immunopathol78 131ndash141

King DP Schrenzel MD McKnight ML Reidarson TH Hanni KDStott JL Ferrick DA 1996 Molecular cloning and sequencing ofinterleukin 6 cDNA fragments from the harbor seal (Phoca vitulina) killerwhale (Orcinus orca) and southern sea otter (Enhydra lutris nereis)Immunogenetics 43 190ndash195

Kumar S Tamura K Nei M 2004 MEGA3 integrated software formolecular evolutionary genetics analysis and sequence alignment BriefBioinform 5 150ndash163

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Lahvis GP Wells RS Kuehl DW Stewart JL Rhinehart HL Via CS1995 Decreased lymphocyte responses in free-ranging bottlenose dolphins(Tursiops truncatus) are associated with increased concentrations of PCBsand DDT in peripheral blood Environ Health Perspect 103 67ndash72

Lum JK Nikaido M Shimamura M Shimodaira H Shedlock AMOkada N Hasegawa M 2000 Consistency of SINE insertion topologyand flanking sequence tree quantifying relationships among cetartiodactylsMol Biol Evol 17 1417ndash1424

Lundqvist ML Kohlberg KE Gefroh HA Arnaud P Middleton DLRomano TA Warr GW 2002 Cloning of the IgM heavy chain of thebottlenose dolphin (Tursiops truncatus) and initial analysis of VH geneanalysis of VH gene usage Dev Comp Immunol 26 551ndash562

Matsuda F Honjo T 1996 Organization of the human immunoglobulinheavy-chain locus Adv Immunol 62 1ndash29

Moumlssner S Ballschmiter K 1997 Marine mammals as global pollutionindicators for organochlorines Chemosphere 34 1285ndash1296

Murray BW White BN 1998 Sequence variation at the major histocom-patibility complex DRB loci in beluga (Delphinapterus leucas) and narwhal(Monodon monoceros) Immunogenetics 48 242ndash252

Murray BW Malik S White BN 1995 Sequence variation at the majorhistocompatibility complex locus DQB in beluga whales (Delphinapterusleucas) Mol Biol Evol 12 582ndash593

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Romano TA Olschowka JA Felten SY Quaranta Ridgway SH FeltenDL 2002 Immune response stress and environment implications forcetaceans In Pfeiffer CJ (Ed) Cell and Molecular Biology of MarineMammals Krieger Publishing Co Inc pp 253ndash279

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Rudd PM Elliot T Cresswell P Wilson IA Dwek RA 2001Glycosylation and the immune system Science 291 2370ndash2376

Sambrook J Fritsch EF Maniatis T 1989 Molecular Cloning ALaboratory Manual Cold Spring Harbor Laboratory Cold Spring Harbor

Shirai K Sakai T Oike T 1998 Molecular cloning of bottle-nosed dolphin(Tursiops truncatus) MHC class I cDNA J Vet Med Sci 60 1093ndash1096

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Travis JC Sanders BG 1972b Whale immunoglobulinsmdash II Heavy chainstructure Comp Biochem Physiol B 43 637ndash641

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46 A Mancia et al Comparative Biochemistry and Physiology Part B 144 (2006) 38ndash46