Immunogenetics (1995) 41:282-286 © Springer-Verlag 1995

Mayuri Patel • Douglas Selinger • George E. Mark Gerard J. Hickey • Gregory F. Hollis

Sequence of the dog immunoglobulin alpha and epsilon constant region genes

Received: 2 November 1994 / Revised: 10 January 1995

Abstract The immunoglobulin alpha (IGHAC) and epsi- lon (IGHEC) germline constant region genes were isolated from a dog liver genomic DNA library. Sequence analysis indicates that the dog IGHEC gene is encoded by four exons spread out over 1_7 kilobases (kb). The IGHAC sequence encompasses 1.5 kb and includes all three con- stant region coding exons. The complete exon/intron sequence of these genes is described.

Introduction

Immunoglobulin (Ig) proteins consist of two identical light (L) chains and two identical heavy (H) chains. Both Ig L and H chains contain an amino-terminal variable region of approximately 110 amino acids, which forms the antigen binding domain. The carboxy terminal constant (C) region domains of each chain is defined by two isotypes of IgL chain (kappa and lambda) and multiple isotypes of IgH chains (mu, delta, gamma, epsilon, and alpha, which define IgM, IgD, IgG, IgE, and IgA, respectively). The IgH chain C regions contain the effector functions common to anti- bodies of a given isotype (for review see Honjo 1983).

Substantial variations in the quantities of specific IgH chain isotypes are observed when different tissue fluids are

analyzed. For instance, IgA is the primary Ig isotype in mucosal fluids, but is found at low levels in serum. The preponderance of IgA found at mucosal sites reflects the critical role IgA provides as a first line of defense against pathogens invading epithelial surfaces (Delacroix et al. 1982; Underdown and Schiff 1986).

IgE antibodies are responsible for mediating allergic responses (Ishisizaka and Ishizaka 1978). IgE binds to mast cells and basophils through an Fce receptor and, when cross-linked by bound antigen, triggers a cascade of events that leads to the release of allergic mediators. Because of the central role that IgE plays in mediating allergic reactions, the region of the Iga constant region involved in Fce receptor binding is of great interest. Inhibition of binding of IgE to its receptor on mast cells may be a way to control allergic responses. Interestingly, of all five isotypes of immunoglobulin, the sequence of the IgE C region is the least well conserved across species. Conse- quently, studies of allergic reactions in a specific species are aided by having the primary amino acid sequence available for the IGHEC gene of that species.

We are interested in studying and modulating mucosal and allergic immune responses in the dog_ As a step toward that goal, we initiated studies designed to clone and sequence the dog IGHAC and IGHEC genes.

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers L 36871 (dog IGHAC) and L 36872 (dog IGHEC)

M. Patel • D. Selinger • G. E. Mark • G. E Hollis a (~) Department of Cellular and Molecular Biology, Merck Research Laboratories, R80Y-160, R O. Box 2000, Rahway, NJ 07065, USA

G. J. Hickey Department of Animal Drug Evaluation, Merck Research Laboratories, Rahway, NJ 07065, USA

Present address: i DuPont Merck Pharmaceutical Co., Experimental Station, E 400/5207, RO. Box 80400, Wilmington, DE 19880-0400, USA

Materials and methods

Genomic cloning

A dog liver genomic DNA bacteriophage library was purchased from ClonTech Inc. and 1 x 10e individual plaques were screened with a 4.3 kilobase (kb) XhoI-EcoRI fragment containing the entire human IGHAC1 gene (Max et al. 1982) as described previously (Hieter et al. 1981). Filters were hybridized overnight at 42 °C in a 10% dextran sulfate, 4 x standard sodium citrate (SSC), 50% formamide, and 0.8% Denhardt's Tris buffered solution. After hybridization, filters were washed with 2 x SSC, 0.1% sodium dodecyl sulfate (SDS) at room temperature for 30 min, 1 x SSC, 0.1% SDS at room temperature for 30 min and 1 x SSC, 0.1% SDS at 42 °C for 30 rain. Five positive bacteriophage were plaque purified, and large-scale lysates were prepared. Restriction digestions of positive bacteriophage clones

M. Patel et al.: Dog IGHAC and IGHEC sequences 283

10 30 50

1 AGT GAC C TAGC GT GTCAT T C T C4~C C CAGGT CTC GGCATAT GAAC TC~-ATGAC C TT GGGC T 60

610 630 650

601 GGC T GAC-C T CC CAGCAAGT GGC CAAGGTGGGGC C T C CA/GAAGGACC TGGAGGGT GGCAG 660

70 90 ii0

61 GTCACT GAC CATC TC TATGCAGTTTCC TCTAGT GCAAAGAAAAAATAGC CCTCAC CC T~C 120

670 690 710

661 ~GGCAC-GCAGAGGGTGCACACTGAC CTGTTCC~ATC T ~TCTCTC TC TC TC TCT C T 720

121

181

130 150 170

CTGTGAGGCCATGTAAGGGGTCCAGA~CTGC~CCAC~%GCTCACAGAGTGTCCT~T

190 210 230

GTUAC.AGAGTCC.~AAACCAGCCCCAGTGTGTTCCCGCTGAGCCTCTGCCA~CAGC-AGTCA XXSerLysThrSerProSerValPheProLeuSerLeuCysHiaGlnGluSer

180

240

250 270 290

241 G~AGGGTACGTGGTCATCGGCTGCCTGGTGCAGGGATTCTTCCCACCGGAGCCTGTGAAC 300 GluGlyTyrValValIleGlyCysLeuValGlnGlyPhePheProProGluProValAsn

730 750 770

721 CTCTCTCTGCTCCTGAA~TAACAGTCATCCGTGTCATCCATGTCCCTCGTGCAATGAGC spA~nSerHisPraCysHisSroCysProSerCysA~n~luP l i

790 810 830

781 CCCGCCTGTCACTACAGAAGCCAGCCCTCGAGGATCTGCTTTTAGGCTCCAATGCCAGCC r~krgLeuSerLeuGlnLysProAlaLeuGluAapLeuLeULeuGlySerAaDAlaSerL

850 870 890

841 TCACATGC_ACACTC~GTGGCCTGAAAGACCCCAAGGGTGCCACCTTCACCTGGAACCCCT euThrCysThrLeuSerGlyLeuLy~A~pProLy~GlyAlaThrPheThrTrpA~nProS

780

840

900

310 330 350

301 GTGACCTGGAATC-CCGGC-AAGGACAGCACATCTGTCAAGAACTTCCCCCCCATGAAGGCT 360 ValThrTrpAsnAlaSlyLysA~pSerThrSerValLysAsnPheProProMetLysAla 901

910 930 950

CCAAAGGGAAGGAACCCATCCAGAAGAATCCTGAGCGTGACTCCTGTGGCTGCTACAGTG erLysGlyLysG1uProIieGlnLy~A~nProGluArgl~pSerCysGlyCysTyrSerV

960

370 390 410

361 GCTACCG6AAGCCTATACACCATGAGCAGCCAGTTGACCCTGCCAGCCGCCCAGTGCCCT 420 Al~ThrG!yS~rLe~TyrThrM~Sar5erGlnL~uThrLeuProAlaAlaGlnCysPro

961

970 990 i010

TGTCCAGTGTCCTACCAGGCTGTGCTGATCCATGGAACCATGGGGACACCTTCTCCTGCA alSerSerValLeuProGlyCysAlaAspProTrp]ksn~i~GlyAspThrPheSerCysT

1020

421

430 450 470

GATGACTCGTCTGTGAAATGCCAAGTGCAGCATGCTTCCAGCCCCAGCAAGGCAGTGTCT AspAspSerSerValLysCysG!nValGl~HisAlaSerSerProSerLysAiaValSer

4~C 1021

1030 1050 1070

CAGCCACCCACCCTGAATCCAAGAGCCCGATCACTGTCAGCATCACCAAAACCACAGGTG hrAiaThrH!sProG/uSerLysSerProIleThrValSerIleThrLysThrThrG

1080

481

490 510 530

GTGCCCTGCAAAGGTCAGAGGGCAGGCTGGGGAGC~GGC-AGGGGCCCCACATCCTCACTCT ValProCymLysA

540 1081

1090 III0 1130

GC4: CCAGACC CTGC C C GT GAGGCACT GC TT GGCACACAAAAGT TT GT GAGGCAA~ T C ~TA 1140

541

550 570 590

GAC CCTCCACT TGGAGTTC TGGC CC ~CACTC CAC GGGGAGGACAGT GGC-C TGCT G 600

1150 1170 1190

1141 AGC CT GC TT C C TTCCTCTAGC C CC TGGGC TTGGGTGCTCCCAC CCACAT TTTACAAAGGG 1200

1270 1290 1310

1261 TGGCTCTCT GTCC TGCAGAGCACATC C CGC CCC.AGGTCCAC CTGC TGCC GCCGCCGTCGG lu~is~leProProGlnValHisLeULauProProProSerG

1330 1350 1370

1321 AAGAGC T GGC C C T CAATGAGC T GGT GACAE TGAC G T GeT T GGTGAGGGGCTT CAAAC CAK luGluLeuAlaLeua~nGluLeuValThrLeuThrCy~LeuValAxgGlyPheLysProL

1390 1410 1430

1381 AAGATGT GC TC GTA: OAT GGC T GCAAGGGAC C CAGGAGC TAC C C CAAGAGAAG TAC T TGA y~AspV al Le uValAr gTrpLeuGl nGl yThr G1 nGIUL euP roGlnGl uLys Ty r LeuT

145D 1470 1490

1441 C CT GGGAGC C C C T GAAGGASC C TGAC CAGA CCAACATGT TT C~ C G T GAC CAGCAT GC T GA hrTrpOluPr oLeuLysGluPr oAspGlnTh/AsnMe t PheAlaValThzs erMe tLeUA

1510 1530 1550

1501 GGGTGACAGC C C~kGACT ~ A A G T T CT CC TC~.ATGGT GGGC CAC G~mG r gValThrAlaGluAapTrpLys GlnGIyGIULy3 PheSerCysMetValG!yHi sGluA

1320

1330

1440

1500

1560

1210 1230 1250

1201 AAACTGT GGCATGGGGTGCTAT ~GAAGGCTC TTC CC CCAC CC CAGATC CC TGAC C 12_60

Fig. 1 Sequence of the dog IGHAC gene. The complete nucleotide and predicted amino acid sequence for dog IGHAC germline gene including exon and intron sequences. PolyA addition signal indicated by an underline. Hinge region at beginning of CH2 is marked by vertical lines

were performed according to the manufacturer's suggested conditions, with the restriction enzymes indicated. Regions of the clones contain- ing the dog IGHAC and IGHEC regions were identified using the human IGHAC1 region probe described above and a 2.8 kb BAMHI fragment encoding the human genomic IGHEC region (Max et al. 1982). The DNA fragments containing these dog Ig sequences were subcloned into pBluescript (Stratagene, La Jolla, CA) and are available upon request•

1561

1621

1681

1741

1570 1590 l&10

CTCTGCCCATGTCCTTCACCCAGAAGACCATCGACCGCCTGGCGGGTAAACCCACCCACG 1 lLeu9 rome t S e r pheThrGlr~Ly~Thr IleAspAxgLeuAl aGIyLys p roTbrHisV

1630 1650 1670

T ~KACGT GT ~TGT C~T CATGGC AE~kGGT GGAC GCF_AT ~T GCTAC T AAAc CGCC CAAT C T T alAsnValS erValValMe tAl aGluVa IA~pGI yI i eCy sTyr

1690 1710 1730

CCCTCCC TAAATAAA CT CCATGCTTGC C C ~ C CC GTGCTTC CA/CAGGCCGC CT

1750 1770

GTC T GT CCATAT TC GGGGT C T GT GGCATA C T ~ U , GGGTAGAGC TC 1789

1620

1680

1740

Table 1 Comparison of percent identity of nucleotides and amino acid sequences of dog IGHAC region with human and mouse Ig~ constant region genes and proteins

% Identity of dog IGHAC to IGHAC of other species

CH1 CH2 CH3 Total

59 73 78 71 52 67 73 65 72 74 83 76 57 70 82 70

Mouse Igh-2 (c~) DNA Mouse Igc~ Protein Human IGHACI DNA Human Igcd Protein

284 M. Patel et al.: Dog 1GHAC and IGHEC sequences

NucIeotide sequence analysis

The DNA sequences of the dog IGHAC and IGHEC genes were determined by the dideoxy chain termination method using the US Biochemicals (Cleveland, OH) Sequenase DNA sequencing kit. Syn- thetic oligonucleotides used as sequencing primers were synthesized on an ABI381A synthesizer or purchased from Stratagene. Nucleic acid alignments and translations were done using the University of Wisconsin Sequence analysis software package (Devereux et al. 1984).

Results

Initial genomic Southern blot analysis using probes that encoded the entire human or mouse IGHEC gene failed to detect dog IGHEC sequences under reduced-stringency blot washing conditions (data not shown). Previous work had shown that IGHAC genes are more closely conserved from species to species than IGHEC genes, but are closely linked to the IGHEC sequences (Auffray et al. 1981; Nishida et al. 1981; Flanagan and Rabbitts 1982a, Max et al. 1982; Shimizu et al. 1982). Therefore, a clone containing the dog IGHAC gene may also contain the IGHEC gene. A DNA fragment containing the human IGHAC1 gene was used as a probe to screen a dog genomic liver DNA bacteriophage library to isolate recombinant clones contain- ing the dog IGHAC gene. Five positive bacteriophage clones were identified and plaque purified. Each of these clones was probed with the human IGHACI and IGHEC gene fragments and one of the clones was shown to have sequences that hybridized to both probes. This clone was selected for further characterization.

Sequence analysis demonstrated that the dog IGHAC region gene is encoded in three exons spread over 1.5 kb of DNA (Fig. 1), consistent with the previously reported genomic structure of IGHAC genes from other species (Flanagan et al_ 1984; Osborne et al. 1988; Burnett et al. 1989; Kawamura et al. 1992). Sequence comparisons revealed that the predicted dog IGHAC gene product shares 65% and 70% amino acid identity with mouse and human, respectively, over the entire IGHAC coding region (Table 1)_ The CH3 domain is the most highly conserved, maintaining 78% (with mouse) and 83% (with human) amino acid identity. The hinge region of the dog IGHAC gene is fused to the 5' end of the CH2 domain and is 10 amino acids long. A consensus polyA addition signal is located 20 base pairs (bp) downstream of the translational termination codon.

Nucleotide sequence analysis indicates that the dog IGHEC gene is encoded by four exons, each of which corresponds to an Ig domain (Fig. 2). Overall, the predicted dog IGHEC gene product shares 49% and 57% amino acid identity with the mouse and human Ige constant region proteins, respectively [(Table 2) (Flannagan and Rabbitts 1982b; Ishida et al. 1982; Liu et al. 1982; Max et al. 1982)]. CH3 is the most conserved (62% in comparison with human) and CH2 is the most dissimilar (53% in compar- ison with human). Eighty-six bp downstream of the transla- tional termination codon is a consensus polyA addition signal.

Discussion

The effector functions of antibodies reside in the IgH chain C regions. The genes encoding the IgH chain isotypes are linked head-to-tail along the mammalian chromosome. In several species studied, the genes encoding the Ig~ and Iga heavy chain isotypes are closely linked (Flanagan and Rabbitts 1982a; Nishida et al. 1981; Max et al. 1982). Our analysis reveals that this organization is also present in the dog genome.

IgA is the principal immunoglobulin of mucosal sur- faces, where it is secreted as a polymeric antibody complex that contains J chain and secretory component (For review see Mestecky et al. 1991). To fully understand antibody- mediated immune responses at mucosal surfaces in a specific species, a knowledge of the IGHAC gene from that species is required.

Unlike other mammalian immunoglobulin genes, the hinge region of the IGHAC gene is not encoded by a separate exon, but is fused to the 5' end of the CH2 domain. This organization of hinge and CH2 is conserved in the dog IGHAC gene. The length of the Igc~ hinge region has been shown to vary. For instance, the human IGHAC1 gene has a hinge region of 18 amino acids, while the human IGHAC2 gene hinge region is only 5 amino acids long (Flanagan et al. 1984). The dog IGHAC gene hinge region is i0 amino acids long, identical in length to the mouse Igc~ hinge region (Auffray et al. 1981; Osborne et al. 1988).

Studies have suggested that the primary interactions between IgA and secretory component reside in the CH2 and CH3 domains of Igo~ constant region (Geneste et al. 1986). Secretory component is covalently linked to IgA through disulfide bonds. Cys 311 of the human Ig~ CH2 domain is responsible for this linkage (Fallgreen et al. 1993). Covalent linkage of dog Iga to secretory compo- nent may also occur at this position because this cysteine residue is conserved in the dog IGa chain.

The IGHEC gene has been cloned from many species including rat, mouse, and man as well as several non- human primates (Flanagan and Rabbitts 1982b; Max et al. 1982; Sakoyama et al. 1987; Kindsvogel et al. 1982). The C region of all functional IGHEC genes characterized to date is encoded in four exons, each containing one Ig domain (CHI-4) . Our analysis of the functional dog IGHEC gene reveals that it also is encoded by four exons, each representing an Ig domain.

The IgE antibody class plays a central role in type I immediate hypersensitivity. IgE binds to specific high- affinity receptors on mast cells and basophils and, when associated with antigen, triggers degranulation of vasoac- tire substances to produce allergic reactions (Ishisizaka and Ishizaka 1978). Because of its role in allergy, a substantial effort has been made to understand how the Ige constant region (which defines IgE) interacts with the Fca receptor on mast cells and basophils to trigger degranulation upon binding antigen (for review see Metzger 1992). These studies indicate that binding to the Fc~ receptor resides in the Ig~ CH3 and CH4 domains (Basu et al. 1993). Addi-

M. Patel et al.: Dog IGHAC and IGHEC sequences

I0 30 50

1 CAGAC~CAGATACCC-AC-GTC-A-ACAGCC~--~=CC%~--GCg~TA%~-'~q~=GC=GTC=~%CAG~CCC~-A~

61

70 90 110

CAGC=CACTGACACTC43CCCTGTCCCCACAGCCACCAGCCAC43ACCTGTCTG~T2CCCCT XA~hrSerGlnAspLeuSerValPheProL

121

130 150 170

TCCCCTCCTGCTGTAAAGACAACATCCCCAGTACCTCTGTTACAC~TCTC43TCA

euAlaSerCysCysLysAspAsnIleAlaSerThrSerValThrLeuGlyCysLeuValT

190 210 230

181 CCGGCTATCTCCCCATGTCGACAACTGTGACCTC~GACACGGGGTCTCTAAATAAGAATG hrGlyTyrLeuProMetSerThrThrValThrTrpAspThrGlySerLeuAsnLysAsnV

241

250 2 7 0 29O

TCACGACCTTCCCCACCACCTTCCACGAGACCTACGGCCTCCACAGCATCGTCAGCCAGG alThrTbrPheProThrThrPheHisGluThrTyrGlyLeuHisserIleValSerGlnV

301

310 330 350

TGACCGCCTCGGGCAAGTGGC42CAAACAGAGG~_ACCTC92AGCGTC~d2TCACC=CTC=AGT alThrAlaSerGlyLysTrpAlaLysGlnArgPheThrCysSerValAlaHisAlaGluS

361

370 390 410

CCACCGCCATCAACAAGACCS~/CAGTC~WAAC42CAGC43~CCACATGACACT erThrAlaIleAsnLysTnrPheSerA

43O 450 470

421 GGAGGGAGAAGGGACAGGCTGGGCGGGAGTGGTAGGAGAGGGGTC43TGGGCGGGCCCGAA

490 510 530

481 TGCCC42CAq~oCTGGTAACGCCCAC~_ACATG~TGGGGCTGACACATC=AGTCCCGT

550 57O 590

541 GGGC TCAGA;ACACCA~CACATGGCIC~CTCTACTrCTAGCATGTCCC TTAAAC T~

laCysAlaLeuAsnPhe

610 630 650

601 ATI~CCCCTACCGTGAAGCTCTTCCACTCCTCCIDCAACCCCGTCGGTGATACCCACACC IleProProThrValLysLeuPheHisSerSerCysAsnProValGlyAspThrHisThr

661

670 690 710

ACCATCCAGCTCCTGTGCCTCATCTCTGGCTACGTCCCAC~TGACATC~AGGTCATCTGG ThrIleGlnLeuLeuCysLeuIleSerGlyTyrValProGlyAspMetGluValIleTrp

721

730 750 770

CTCGTCGATC43GCkAAAC~CTACAACATATTCCCATACACTGCACCCC~CACAAAGGAG LeuValAspGlyGlnLysAlaThrAsnIlePheProTyrThrAlaProGlyThrLysGlu

781

790 810 830

GGCAACGTGACCTCTACCCACAGCGAGCTCAACATCACCCAQGGCC=AGTGC43TATCCCAA GlyAsnValThrSerThrNisSerGluLeuAsnIleThrGlnGlyGluTrpValSerGln

850 870 890

841 AAAACCTACACCTGCCAGGTCACCTATCAAGGCIqTACCTI~AAAGATGAGGCTCGCAAG LysYhrTyrTbrCysGlnValThrTyrGlnGlyPheThrPheLysAspGluAlaArgLys

901

910 93O 950

TCCTCAGGTATCGCCCCCCTGTCCCCCAGAAACCCAGATGCGCGAGGCTCAGAGATGAGG CysSerG

961

970 990 i010

CCCAAGGCACGCCCTCATGCACCCTCTCACACACTGCAGAGTCCGACCCCCGAGGCGTGA

luSerAspProArgGlyValT

60

120

180

240

300

360

420

480

54O

600

660

720

780

1021

840 1801

1030 1050 1070

CGAGCTACCTGAGCCCACCCAGCCCCC~fGACCTGTATGTCCACAAGGCCCCCAAGATCA hrSerTyrLeuSerProProSerProLeuAspLeuTyrValHisLysAlaProLysIleT

1080 iii0 1130

1081 c C TGCCTC43TAGTGGACCTCGCCACCATGGAAGGCATGAAC CTGACCTGGTACCGGGAGA hr CysLeuValValAspLeuAl aThrMe tGIuGIyMe tAsnLeuThrTrpTyrArgGluS

i150 1170 1190

ll41 GCAAAGAACCCGTGAACCCGGGCC~CAAGAAGGATCA~TGGGACGATCA erLy_sGluProValAsnProGlyProLe~AsnLysLysAspHisPheAsnGlyThrIleT

1210 1230 1250

1201 CAGTCACGTCTACCCTGCCAGTGAACACCAATGACTC~ATCGAQC~CGAGACCTACTA~

hrValThrserThrLeuProValAsnThrAsnAspTrpIleGluGl~GluTnrTyrTyrc

1270 1290 1310

1261 GCACC43TGACCCACCCC~&CCTC42CCAAGGACATCGTGCGCTCCATTC42CAAC~=CCCCTG ysArgValThrHisProHisLeuProLysAspIleValArgSerIleAlaLysAlaProG

1330 1350 1370

1321 GTGAGC CACCd~GCC CAC~C~=AC~TGGCCC~=CCTCCTC~AOCCGGAGC ~TGACC C C

285

108

114

120

12

13

1390 1410 1430

1381 ACACCTATCCACAGGCAAGCGTGCCCCCCCGGATGTGTACYTGTI~2CTGCCACCGGAGGA 14 lyLysArgAlaProProAspValTyrLeuPheLeuProProGluGl

1450 1470 1490

1441 C4~AGCAGGC43ACCAAGGACAGAGTCACCCTCACGTGCCTGATCCAG~CTTCTTCCCCGC 15 uGluGlnGlyThrLysAspArgValThrLeuThrCysLeuIleGlnAsnPhePheProAl

1510 1530 1550

1501 GGACATI~CAGTCCAATC4DCTC42C~AACGACAC42CCCATCCAGACAGACCAGTACACCAC 15 aAspIleSerValGlnTrpLeuArgAsnAspSerProIleGlnThrAspGlnTyrThxTh

1570 1590 1610

1551 CACCCGGCCCCACAAGGTCTCGQGCTCCAGGCCTGCCTTCTIC_ATCT2CAGCCGCCTGGA 16 rThrGlyProHisLysValSerGlySerArgProAlaPhePheIlePheSerArgLeuGl

1630 1650 1670

1621 CGT2AC4203Cd3TC~ACTGGGAC~-AGAAAAACAAATTCACCTGCCAAG~TGAGGC 16 uValSerArgValAspTrpGluGlnLysAsnLysPheThrCysGlnValValHisGluAl

1690 1710 1730

1681 C-CTGTCCGGCTCTAGGATCCTCCAGAAATGGGTGTCCAAAACCCCCGGTAAATGATGCCC aLeuSerGlySerArgIleLeuGlrLLysTrpValSerLysThrProGlyLys

1750 1770 1790

1741 ACCCTCCTC C CGC CCCCACC C CCCAGGC/2TCCAC CTGCTGGGAGGGA~TC~CAA

17

18

1861 192

900

1921

Fig. 2 predicted amino acid sequence for the dog IGHEC germline gene is

860 shown. PolyA addition signal is indicated by an underline

1810 1830 1850

GACCC TCCATCTGTCCTTGTCAATAAACACTCCAGTGTCTGCTTGGAGC C CTC~oC-CACAC 18

1870 1890 1710

C CATITC~TGGGCAC~GTTGCAGAGCAGGGATGTC ~CAGAAGGGTC C CC

CAGGGTGT 1928

Sequence of the dog IGHEC gene. The entire nucleotide and

1020 tional studies have used linear peptides to map the Ige binding site. In one of these studies, an octapeptide from the human Iga constant region (Pro345-Phe-Asp-Leu-Phe-Ile- Arg-Lys352) inhibited passive sensitization, presumably by

286

Table 2 Comparison of percent identity of nucleotides and amino acid sequences of dog IGHEC region with human and mouse Ige constant region genes and proteins

% Identity of dog IGHEC to IGHEC of other species

CH1 CH2 CH3 CH4 Total

Mouse Igh-7 (~) DNA 54 63 64 66 62 Mouse Iga Protein 42 42 55 56 49 Human IGHEC DNA 69 67 74 71 70 Human Ig8 protein 59 53 62 55 57

occupying the Fc~ receptor sites on cells (Nio et al_ 1993). The equivalent region of the dog Ig~ chain shares only 50% identity with this octapeptide (Dog sequence: Pro-Leu-Asp- Leu-Tyr-Val-His-Lys). Based on this observation, attempts to use Ige peptides involved in Fce receptor binding to modulate allergic reactions in dogs would require the use of peptides derived from the dog Ige sequence.

We have presented the complete nucleotide and pre- dicted amino acid sequence of the dog I G H A C and I G H E C

genes. These sequences should prove useful for studying immune responses in dogs.

Acknowledgments We thank Dr. Ronald Ellis for critically reviewing the manuscript, Ms. Susan Pols for help in preparing the manuscript, and Mr. Jeffrey Aaronson for his help with submission of the sequence data to GenBank.

References

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