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Email: [email protected]. Received 29 October 2003. Accepted 28 ... BLAST program (http://www.ncbi.nlm.nih.gov). Expression of Japanese flounder MHC ...
Blackwell Science, LtdOxford, UK FISFisheries Science0919-92682004 Blackwell Science Asia Pty Ltd 702April 2004 800 Classes I and II MHC of Japanese flounder P Srisapoome et al. 10.1046/j.1444-2906.2003.00800.x Original Article264276BEES SGML

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2004; 70: 264–276

Cloning, characterization and expression of cDNA a containing major histocompatibility complex class I, IIa b genes of Japanese flounder Paralichthys olivaceus and IIb Prapansak SRISAPOOME, Tsuyoshi OHIRA, Ikuo HIRONO AND Takashi AOKI* Laboratory of Genome Science, Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Minato, Tokyo 108-8477, Japan ABSTRACT: The nucleotide sequences of Japanese flounder Paralichthys olivaceus, major histocompatibility complex (MHC) cDNA, classical MHC class Ia, non-classical MHC class Ib, MHC class IIa and IIb, were determined. The domain structures and antigen binding motifs of vertebrate MHC are conserved in the Japanese flounder MHC. A phylogenetic analysis supports the classification of these genes into class I and class II MHC. Classical MHC class Ia was ubiquitously expressed, whereas the non-classical MHC class Ib was expressed mainly in lymphoid organs, gills, intestine and stomach. The MHC classes IIa and IIb were also ubiquitously expressed. KEY WORDS: antigen presentation, evolution, fish immunity, Japanese flounder, major histocompatibility complex genes.

INTRODUCTION The major histocompatibility complex (MHC) is a region that has close association with immune function in vertebrates because it contains genes important for both innate and adaptive immune systems. In mammals, there are two classes of MHC gene. Class I genes can be subdivided into classical class I (Ia) and non-classical class I (Ib) based on structural and functional differences and expression pattern; class II MHC genes have two types, the a and b chains.1 Class I molecules are composed of an a chain and b2-microglobulin (b2m) and present the peptides derived from endogenously synthesized proteins to CD8+ T cells.2 In contrast, class II molecules are heterodimers that consists of an a and b chain. These molecules present exogenously derived peptides to CD4+ T cells.3 The genes encoding the MHC are highly polymorphic, with high variability in the peptide binding regions.1 Several MHC class I and II cDNA (and genes) have been cloned from a number of different fish species, mostly from modern bony fishes (teleostei),1,4,5 but also from cartilaginous fishes (Elasmobranchii)6 and a representative of the lobe-finned

*Corresponding author: Tel: 81-3-5463-0556. Fax: 81-3-5463-0690. Email: [email protected] Received 29 October 2003. Accepted 28 November 2003.

fish (sarcopterygii), the coelacanth.7 However, only a few reports have been published concerning the cloning of all types of MHC in a single fish species. This has been done only in rainbow trout,8,9 channel catfish,10–13 common carp and zebrafish.14 All of these species diverged early in the evolution of fish. Hence, in order to further elucidate the evolutionary relationships of MHC in the teleostean immune system, information of MHC from a species that diverged later in the evolutionary strata (such as the Japanese flounder) becomes necessary. In the present study, we cloned all two classes (classical class I, non-classical class I, class IIa and class IIb) of MHC from Japanese flounder Paralichthys olivaceus. MATERIALS AND METHODS Cloning and sequencing of Japanese flounder MHC cDNA We previously identified four different partial cDNA representing the two classes of MHC from an expressed sequence tag (EST) analysis of a Japanese flounder peripheral blood leukocyte cDNA library.15 The positive clones were used to screen 1 ¥ 105 recombinant phages from an amplified Japanese flounder leukocyte Lambda ZAP II library. Primers used in specific probes generated by polymerase chain reaction (PCR) for each gene

Classes I and II MHC of Japanese flounder

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are shown in Table 1. Positive clones were isolated and subcloned into pBluescript by in vivo excision (Stratagene, La Jolla, CA, USA). The cDNA clones were sequenced using ThermoSequenase (Amersham Pharmacia Biotech, Piscataway, NJ, USA) with M13 forward and M13 reverse primers and an automated DNA sequencer LC4200 (Li-Cor, Lincoln, NE, USA). Each determined sequence was compared with all sequences available in DNA Data Bank of Japan/European Molecular Biology Laboratory (DDBJ/EMBL)/GenBank using the BLAST program (http://www.ncbi.nlm.nih.gov). Expression of Japanese flounder MHC mRNA Total RNA was extracted from healthy Japanese flounder peripheral blood leukocytes (PBL), spleen, head kidney, trunk kidney, heart, liver, stomach, intestine, brain, gills, ovary, muscle, skin and eyes using Trizol (Life Technologies, Rockville, MD, USA). The purified total RNA (10 mg) was treated with DNase I (Promega, Madison, WI, USA) and then reverse transcribed into cDNA using an AMV Reverse Transcriptase First-Strand cDNA Synthesis Kit (Life Science, Arlington Heights, IL, USA) in a 20 mL synthesis reaction. One mL of the reversetranscribed sample was used in a 25 mL PCR reaction mixture using specific primers for each gene (Table 1). The PCR was performed with an initial denaturation step of 2 min at 95∞C, followed by 20 cycles of denaturation at 95∞C for 30 s, annealing at 55∞C for 30 s, and extension at 72∞C for 1 min. The PCR products were electrophoresed on 1.5% agarose gel. Phylogenetic analysis A phylogenetic tree of fish and vertebrate MHC genes was constructed with sequences available in the GenBank database. The ClustalX program was used for aligning the sequences.16 The phylogenetic analysis was based on genetic distance matrices and the neighbor-joining method.17 Phylogenetic trees were calculated using Phylip (version 3.57c, University of Washington) programs Protdist and Neighbor, and were calculated based on a bootstrap of 100 separate genetic distance matrices. RESULTS Characterization of class Ia and Ib cDNA Five different class Ia cDNA were cloned and sequenced from Japanese flounder. An alignment of amino acid sequences, their corresponding

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Table 1 Specific oligonucleotide primers used in PCR for probes generating and for genes expression analysis Name

Sequence (5¢-3¢)

Probe for cDNA screening MHC Ia MHC IaF TGCTTCTACTGGCAGGAACA MHC IaR AGAGCTCTTCCCATGCTCCA MHC Ib MHC IbF AATGACACAGACACCCACGT MHC IbR CCACACTGTTCCTTATCTGG MHC IIa MHC IIaF AGTCCGATGATCTACACCAG MHC IIaR CCATCAGCTGCAGCCAATCA MHC IIb MHC IIbF TGGAGTCGACGTAGAATCTG MHC IIbR TAGATCCAGGACCAAGTCCA Expression of MHC genes MHC Ia MHC IaF MHC IaR MHC Ib MHC IbF MHC IbR MHC IIa MHC IIaF¢ MHC IIaR¢ MHC IIb MHC IIbF¢ MHC IIbR¢ b-actin b-actin F b-actin R

TGCTTCTACTGGCAGGAACA AGAGCTCTTCCCATGCTCCA AATGACACAGACACCCACGT CCACACTGTTCCTTATCTGG ATAACCTGGATGGTGAAGAG CTCGTTCCCTTTGATGAGGA TACGTCAGGCTTCACTCTGT CAGGACCAGACTCAGTTAGT ACTACCTCAAGATCCTG TTGCTGATCCACATCTGCTG

MHC, major histocompatibility complex.

structure and characteristics are shown in Fig. 1. The cDNA are designated according to Klein et al. as MhcPaol-UA1 to -UA5.21 We also use these rules in the present study to designate other classes of MHC gene. The cDNA of these clones contained similar insert sizes of approximately 3 kb. The putative amino acid sequences of the cDNA exhibited features expected of MHC class Ia chains. An 18-aa hydrophobic leader peptide was predicted by using the signalP server.22 The leader peptides of these clones had a high similarity to each other except the amino acid at position -5. Clones PaolUA1 and -UA3 have a Gln residue at this position, whereas clones -UA2, -UA4 and -UA5 have His residue. Conserved cysteine residues among fish and human class I were also found in all of Japanese flounder MHC class Ia clones, at positions 98, 162, 198 and 257, which are capable of forming the characteristic immunoglobulin domain disulfide bonds. A potential N-linked glycosylation site at positions 84–86 appeared in the same position at the edge of the a1 domain. Important residues for b2-microglobulin binding23 (Thr10, Gln93, Gln112, Asp233) and salt bridges (His3, Asp28, His90, Asp116) are found to be highly conserved among these clones and vertebrates. Inspection of the peptide-binding motif indicated that many structural residues in channel catfish and Atlantic salmon MHC class I molecules are conserved in

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Leader peptide -10

-1

Paol-UA1 MHTLLFLLLLAGTQSATA Paol-UA2 -------------H---Paol-UA3 -----------------Paol-UA4 -------------H---Paol-UA5 -------------H---Auha-517 M-F-G-AAuha-705 MEKKMKCV-I--LCCHA-SS Auha-706 MLKVWMFMVMKVMKVFIFF--L-MQG-AFuru-I1 MKSLDF-L--A-LSLPDSSFuru-I2 MKSLDF-L--A-MSLPDSSFuru-I3 MKSLDF-L--A-LSVPDSSGamr-UA*C28 -KA-TG----VFGHGVSS Gamr-UF*6 -KL-TG----VFGHGVSS Gamr-UF*8 -KL-TG----VFGHGGSS Onmy-UBA*0201 MKS-II--L-INA-SOnmy-UBA*502 MKGII-LV-GIGLLHT-SOnmy-UBA*301 MKGII-LV-GIGLLHT-SStvi-C1-1 MKTLVVAV---GLMV-D-SStvi-C1-3 MKTLVVAV---GLMV-D-SStvi-C1a2 MKTLVVAV---GLMV-D-SStvi-C1a49 MKTLVVAV---GLMV-D-SHLA-A2 MAVMAPRTLV-L-SGALALTQTW-

Paol-UA1 Paol-UA2 Paol-UA3 Paol-UA4 Paol-UA5 Auha-517 Auha-705 Auha-706 Furu-I1 Furu-I2 Furu-I3 Gamr-UA*C28 Gamr-UF*6 Gamr-UF*8 Onmy-UBA*0201 Onmy-UBA*502 Onmy-UBA*301 Stvi-C1-1 Stvi-C1-3 Stvi-C1a2 Stvi-C1a49 HLA-A2

Paol-UA1 Paol-UA2 Paol-UA3 Paol-UA4 Paol-UA5 Auha-517 Auha-705 Auha-706 Furu-I1 Furu-I2 Furu-I3 Gamr-UA*C28 Gamr-UF*6 Gamr-UF*8 Onmy-UBA*0201 Onmy-UBA*502 Onmy-UBA*301 Stvi-C1-1 Stvi-C1-3 Stvi-C1a2 Stvi-C1a49 HLA-A2

1

10 20 30 40 50 60 70 80 ^ v + ^ v v RTHSLQYFYTASSHVPNFPEFVYVGLVDEVPISHYDSNTMKTEPKQDWMLKAT**DSQYWESETQKSLGEQQWFKVNSETLKERFNQTE* -----K--F-G--Q----------------QMI--------A-S---------**----------IG--H-------I-IA--------* -----H------------------------T----------------------**----------------------S-----------* -----K--F-G--Q----------------QMI--------A-S---------**----------IG--H-------I-IA--------* -----K----G--Q--------G--Y----Q-I--------V-L---------**--H---R--RSF--K--T---DI-IA--------* V----K--F-G--Q--------V--M--D-QVV----D-E-AGP----FARN-**-Q-----Q-GNL--S--T--A-I--A-Q-----G* VK---K--C-ETPG-QSI----A-AF----Q-GDFNNVRG*AEP-K--IKFFADHPEHLEWYSSISKQ*SH-V--A-I--FRQ-L---D* V----K-------Q--------T--M--D-QVDY---D-E-AEP----IARN-**-Q----RN-DIYR-S--S--A-I-IV-Q-----G* V--T-K----G--G--------A--M--D-QMVR-----RRAQP--E--KEV-AD-P--LDRD-GNLM-T--T--A-I-IA-Q-----G* V--T-K-------G--------I--M--D-QMVR-----RRMQP--E--KEF-AD-P--LDTQS-NAF-A--IY-A-I-IA-------G* V--T-K-------G--------A--M--D-QMVR-----RRTQP--E--KDV-ED-P--LDRN-GNF--S--TY-G-I-I--P-----G* VL---H-------GLTT-----A--M--G-QML----VSKRAVA-----ERYARE-RD-L-GQ-GGLQ-Q--T--A-IGIA-Q-----G* VL---QF------GLTA-----A--M--G-QFYY-----QRAVL-----EQV-SG-AD-LKRN-EN-Q-S--A--A-IGIA-Q-----G* VL---QF------GLTA-----A--M--G-QFYY-----QRAVL-----EQV-SAH*D-LKRN-EN-Q-S--A--A-IGIA-Q-----G* V----K------TGIEG--Q--A--I-NGMQ-DYF-GVSEKNVL--S--EGVR**-*E**KMI-NTRK-H--T--ASV-IVMQ-----T* V----K-------E--------V-AM--G-QMV-----SQRAVP----VN---**-P----RN-GIFK-S--T--A-IDIA-Q----SG* V----K----G--E--------V-AM--GAQMV-----SQRAVP-----NR-AETLP-----Q-GIFK-D--T--A-ID-A-Q----SG* V---MK-------G--------G-----D-EMV------KRTEP-----SRV-ED-P----RN-GNFV-S--V--AGI-I--P-----G* V----K-------G--------T-----D-E--------KREEL-----SRV-ED-P--LQRN-EIFV-T--A--A-I-VA-Q-----G* V---MK----G--G------Y-S-----D-EMV------KRTEP-----SRV-ED-P--LQRN-EI-E-D--T--A-I-VA-Q-----G* A----K-------G--------T-----G-EMV------KRAEP-----SRV-ED-P---QRN-DGLV-H--A--A-I-VA-Q-----G* GS--MR--F-SV-RPGRGE-RFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPE--DG--R-VKAHS-THRVDLG--RGYY--S-A o o o o oo

90 100 110 120 130 140 150 160 170 180 ^ + + ^ v v vv v v GVHIIQRMYGCEWDDETGEVK*GYDQHGYDGEDFISLDLETETWIAPTPQAVITKLKWDNNKALLANQKNYYTQVCPDWLKKYLEHGKSSLLRT ------W--------------*--R-F---------------------------------D--D--HR---L--E---------------------------------------*-----------------------------------------------------------------------------W--------------*--R-D---------------------------------D--D--HR---L--E------------------------W--S-----------*--E-F------L-----------TQR---------------Q--HL---LIQE------IFV-------------V-W----------NV-NS-FH-----------F--K-L-----KQ-G----H---K-TV-NDYWN--HN-L--E-----VSY-R---Q-----M---N----------IN*-FN-F-------L----K-L---T-K--------R--AY--R-EGNR-FLGHR--EF--Q--QY-R-F-QTA -F-LY-W----------N---*-FE-D-------L-F-MK---F-T-V---IV--H-LES-RGFI-QKA-----I--E-----VNY-R---M-K ---MV--------------*-N-FN-F-F--A-----K-K-GI-V-AKRE--K--W---QDE-WIEQETD----I-IK--Q-FVNY-----M-----Y-N----------P-*-N-FF-D----A----FK-KEGI-V-AKRE-E---H---QDV-GIEQLKS----I-IQ--Q--VNY-----M-----A--------------*-N-FR-D----A----FK-KEGIFV-AKRE-E---H---QDV-GIEQLKS----I-IQ--Q--VNY-----M--A-MF-W--------DDDS*TD--E-F-------------HL--V--VR--FA--R---ED--I-QYK---H-KE-V-------AY---T-Q--A-MA-L-C-------D-T*TD--N-QA-----------K-M--V-AVR--FS--QR--GL--FNEQ--H---VE-V-------AY---T-Q--A-MA-L-C-------D-T*TD--N-QA------L----K-M--V-AVR--FS--QR--GL--FNEQ--H---VE-V-------AY---T-Q----TF-L-----LG-*D-IPR*-DF-L----A--L---KS-L--T-ANQK---------ATG-EANF----LENT-IE-----VNY--DT-E-K ----N--------N----*ATG--S-D--------AF--K-K-----K-------F---SDI-MTERD-H-L--T-IE------DY---T-MT----V-M-C---------*-TE-FN----------AF--K-TK------------H---S-T-NNEYW---I--E-I------VDY--NT-M-----V---------------*N-FS-W---------F--K--SYV--KQ------H----D--EISQR-H-L--E--E-V----NY-R---------L---------------*N----D--------VF--K-ESYV--KQ------H-------QI-QR-H-L--I--E------NY-R---------L-I-------------*N-FS-D--------VF--K-ESYV--KQ------H-L-----QI-QR-H-F--I--E------NY-R---------F---------------*N-FM-Y---------F--K-------KQ------H-------GI-QY-H-L--E--E-V----NY-R------S-TV------*DVGSDWRFLR--H-YA---K-Y-A-KEDLRS-TAADMA-QT--H--EAAHV*AEQLRA-LEGT-VE--RR---N--ET-Q-o o o o o o o oo o oo o o o

CP/TM Paol-UA1 Paol-UA2 Paol-UA3 Paol-UA4 Paol-UA5 Auha-517 Auha-705 Auha-706 Furu-I1 Furu-I2 Furu-I3 Gamr-UA-C28 Gamr-UF*6 Gamr-UF*8 Onmy-UBA*0201 Onmy-UBA*502 Onmy-UBA*301 Stvi-C1-1 Stvi-C1-3 Stvi-C1a2 Stvi-C1a49 HLA-A2

a2

190 200 210 220 230 240 250 260 270 280 + ELPSMSLLQKSPS***SPVSCHATGFYPDSAMLFWRKDGEEHHEDVYVGEVLPNHDGTFQMSADLQVSSIPPEDWRRYDCVFQLSGVKEDIVKRLDKDSVESNREK -------------***-------------------------L----------------------------------------------------------------------------***------------------------------------------------------------------------------------------------------***-------------------------L----------------------------------------------------------------------------***-------------------------L--------------------T--------------------I---------------------V---V-----T--***----------H-NR-E-V-----V-L--G-EK--I-T-N--N----V--DF--VTS---K----------LN----TK---NAIKT-WGV---V------*-***------------NR-EMF-------L--G-DP--I-R-N-------V--KL--DT--E-E------H---*V----TK-NQALIRT-P-G V---V-V---TS-***-QFH--------NR-EMF-----V----G-VK--I---N-E-----V--DL--VT----DK---------VN----T----VTKKEF*** ----V-----T--***-------------R-T-S---GE--L----DH--M-L-P-------V--K---V-----SS-K-------*AKE-TTT---NQIRT-W** ----V-----T--***-------------R-T-S---GE--L----DH--M-L-P-------V--K---V-----SS-K-------*AKE-TTT---NQIRT-W** ----V-----T--***-------------R-T-S---GE--L----DH--M-L-P-------V--N---V-----SS-K-------*AKE-TTT---TQIRT-W** DR-RV----R---***---V---------RVVVF--R--Q-L--Q-DP-------N----V-V--NLKAV-Q---G--E--V--KG*I---STP--PALIRT-G** -R-RV----R---***---V---------RVVVF--R--Q-L--L-DP------------V-V--NLKAV-Q---G--E--V--RG*I---STP--PALIRT-G** -R-RV----R---***---V---------RVVVF--R--Q-L--L-DP------------V-V--NLKAV-Q---G--E--V--RG*I---STP--PALIRT-NG* VR--V-----T--***---T--------SGV-VF-Q---Q-Q-G--EH--I-Q-D-----K-TH-T-TP*EEWKNNK-Q--V--AGIED--T-V-IESEIQT-F** VP--V-----T--***---T--------SGV-VF-Q---QDQ-G--EH--T---D-----K-TH-T-TP*EEWKKKQ-Q--V-VTGI---DFIKVLTE-EIKTNW* VP--V-----T--***---T--------SDV-VS-Q---QD-----EY--T---D-----K-SH-T-TP*EDRKNSK-Q--V-VKGI---FIEV-PVPDAANVV** DR--V--------***-------------R--MF------------D---T---N--S----V--DL--V-A--------------M-D---TK---AKIRT---DR--V--------***-------------R--MF------------D---T---N--S----V--DL--V-A--------------M-D--ITK--IAKIRT---DR--V--------***-------------R--MF------------D---T------S----V--DL--V-A--------------M-D---TK--IAKIRT---DR--V--------***------------DR--MF------------D---T--S---S----V--DL--V-A--------------M-D---TK--IAKIRT---DA-KTHMTHHAV-DHEATLR-W-LS---AEIT-T-Q----DQTQ-TELV-TR-AG-----KWAAVVVP-G**QE*Q--T-HV-HE-*LPKPLT*-RWE*P*-**** o o o o o o o o oo o o

290

300

a1

a3

CT 310

PTDVTT**IIIVAAVVVLALVLAVTGFIVY ------**--------A--V---------------**---------------------------**--------A--V----I----------II--------A--V----V-----SIRSDGGSSDSTGTIIGVV-VMLLLL-IGLGIF NIRKE-PSGMLISIIASVVVA-ILVLVA-GVAVY -VG-VVGV-TGLLLLA-SIAGVY GKPGVR*GDG*AEDPSNTAVIA-VAVVVLALVLIAVVGFLLY GKPGVR*GDG**EDPSNTAVIA-VAVVVLALVLIAVVGFLLY GKPGVR*GDG*AEDPSNTAVIA-VAVVVLALVLIAVVGFLLY GKS****GHT-PIIIGL-V-L--AAAAV-GVLLY GRTRLRVAFT-PIIIGFVV-LP-AAAAV-GVLLY GRTRLRVAFT-PIIIGFVV-LP-AAAAV-GVLLY GKTNRGSNDP-TIGLIIGGVIALLVIIV-GVVIW NDPAPNIVL--GVV-*A-L--IVA*VVG-VIW -IIGGVV*ALLLVV-A-VVG-VIW ---M--PI-AAVVVLALVL-AVIGVLVY ---MRERERERERELREREREREREREREPRAESARGMMVAPLLSRTACL ---M--PI-AA-VVLALVL-AVIGVLVY ---M--PI-AA-VVLALVL-AVIGVLVY SQPTIPI*VGII-G*L-*LFGAVIT-AVVAAVMW

320

330

340

RKRNAKCPPSPGKDPNVLKPLNPDGQVSNSPSETSS -----E------N----------Q ----------------------------------------E------N----------Q -----E-L----N---A-N-----*K--I-S----FWKRRTNGQDKKGLNVPTLLRLHLH-LKIQTLRKRAVKSRR K-KKERNTEPSPG-VSEIEE-RS-YLRN MWQKKYKTFK-TNTSDTSST --KKVAGKCTKPEGHSEVQEPLKPNPN --KK**GKCTKPKGHSEVKEPLKPNPN --KK**GKCTKTEGHSEVKVPLKPNPN K-----DKRHKP*-GSDTSSENTE--NPA-EAQPLTKV K--KPSDQRHKPVATSDTSSEDA K--KPSDQRHKPVATSDTSSEDA K-K-K-GFVPASTSDTDSENSGKGI-KI K-KSK-GFIQAQSSDTDSDNSGRAA-QI K--SK-GFVPASTSDTDSENSGKAAPQI --KK------TDSSEVNEKLNQET WRSWC K-KKK-LRLVLPRAEFGTRV-L-HYGAHR-CALQNSAGQAGYLSFSAPLTCAAL --K*KADKTQNKPANYRTVP-NGSDTS-G-SPNGSNEVVNSSTPLS RRKSSDRKGGSYSQAASSDSAQGSDVSLTACKV

Fig. 1 Alignment of amino acid sequences encoded by Paol-UA1 to -UA5 clones with classical major histocompatibility complex (MHC) class I. The number is based on the Paol-UA1 sequence. Asterisks indicate gaps. Conserved residues are indicated as follows: (v) antigen binding residues; (^) salt bridges; (+) b2-microglobulin contact residues; boxes: CD8 binding loop. Circles indicate conserved cysteine residues involved in disulfide bond and other conserved residues. Underline: potential N-linked glycosylation sites. CP/TM, connecting peptide/transmembrane; CT, cytoplasmic domain. Bold: TM region of each sequence. The amino acid sequences were taken from the following references: Furu, pufferfish (Timon et al.);18 Auha, cichlid fish (Sato et al.);19 Stvi, walleye (Fujiki et al.).20 Human, HLA-A2; OnmyUBA*201, -UBA*502 and -UBA*301 of rainbow trout and Gamr-UA*C28, -UF*6, -UF*8 of Atlantic cod were taken from GenBank accession numbers K02883, AF287485, AF287488, AF287486, AF414203, AF414205 and AF414207, respectively.

Classes I and II MHC of Japanese flounder

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Leader peptide -30

-20

-10

-1

Paol-UB1 MKNTMKNKVDTCPVFLFVSVLGLLSMFPVLSA Bain-ZE*0201 MRTSVTTI-ALFCMFL-YGTL-S-QBain-ZE*0401 MMMTIRKYCTSLPKYEVESTGIRPNHDGTFQLKRSVEIQEDEKAEYDCFVSHRNFKPIIIKLGQATESL-AETPAAGRERAGATSDSSVTG-LEK-IQN Bain-ZE*0501 MMMTIRKYCTSLPEYEVKSTGIRPNHDGTFQLRKSVEIQGNEKAEYDCFVHHRNFKSIIIKLGQTTESLSAETPAAGRERAGESSDSSVTG-LEK-IQN Cyca-ZE*0101 MGTSVVTAS-ALLCVFILCE-S-QT Dare-ZE*0101 MA--AVLFSAVM-LAIVPAWT Dare-ZE*0201 MA--AVLFSAVM-LSVVPAWT HLA-E MVDGTLLLLSSEALALTQTW-

Paol-UB1 Bain-ZE*0201 Bain-ZE*0401 Bain-ZE*0501 Cyca-ZE*0101 Dare-ZE*0101 Dare-ZE*0201 HLA-E

Paol-UB1 Bain-ZE*0201 Bain-ZE*0401 Bain-ZE*0501 Cyca-ZE*0101 Dare-ZE*0101 Dare-ZE*0201 HLA-E

Paol-UB1 Bain-ZE*0201 Bain-ZE*0401 Bain-ZE*0501 Cyca-ZE*0101 Dare-ZE*0101 Dare-ZE*0201 HLA-E

10 20 30 40 50 60 70 80 90 ^ v + ^ v v ERHSLTYIYTAFSKPVQSPGLHEFTAMGMVDQKMIDYFDSDLNLKVPKEPWM*EQNMGKDYWKMGTESRQSK**QKWFNVNIDILMKRLRQNDT -K---Y-----L----DL--IYQ-----LL-DRP---YN-KDQK-I--QT--K-*K-QE---EK--Q--**-SKE------V------M-H-ES -K---Y-----L----DL--IYQ-----LL-DIQ---YN-REQR-I--QT--K-*KLQE---EK--Q--**-SKE------V------M-H-ES -K---Y-----L----DL--IYQ-----LL-DRQ---YN-EEQK-I--QT--K-*K-HE---EK--Q--**-SKE------V------M-H-ES -K---Y-----L----DQ--IYQ-----LL-DRE---YN-KEQR-I-RQH--K-*K-QE---EK--Q--**-SKE------V------M-H-ES -K---Y-----L-R--NL--IH------LL-DRQ---YN-QEQK-I--Q---K-*K-QE---EK--Q--**-SKE------VN---E-M-H-KS -K---F-----L-R--DL--IY------LL-DRQ---YN-IDQK-IH-Q---K-*K-QE---EK--Q--**-SKE------VN---D-M-H-KS GS---K-FH-SV-R-GRGEPRFISVGYVDDT-FVRFDN-AASPRM--RA---*--EGSE*--DRE-R-ARDTA**-I-R--LRT-RGYYN-SEA ooo o o o o o oo o o o oo o

100 110 120 130 140 150 160 170 180 ^ + + v ^ v vv v v DTHVLQWLHGCETSRSTASMKFYRGVDTYSYDGNDFLHFDDGHGVWVSSGAAADDTKRKWDGVQTLKDYTKGYLENECLKWLEKFVTYRQKQLQAAP -L-----R----VEQQGDEV--SK-ISE-G---DN--S---KESQ--APVE--LP------N-PI-NQ-------K--VD--N--RE-GDKG-RHG* -L-----R----VEQKGDEV--SK-ISE-G---E---T---KESQ--A-V---LP-----GN-PI-NQ-A-----K--VD--N--RE-ADEEIRHG* -L-----R----VEQQGDEV--SK-ISE-G---DN--S---KESQ--APVD--LP------N-PI-NQ-------K--VD--N--RE-GD-E-RHG* -V--F--R----IEKNGDEV--SK-IDE-----AN--S---KEFQ--APV---VP------N-TI-NQ-------K--VD--N--RE-GDEVIRKG* -V-----R----IESQDNNVR-SK-IDE-----EN--S---ADSQ--APVD--LP------N-PI-NQ-------K--VD--N--RE-GDEE-KQG* -V-----R----IDSQGNDVR-SK-IDE-----EN--A---AESR--APVEE-LP------N-PI-NQ-------K--VD--K--RE-GDQE-RKV* GS-T---M----LGPDRRFLRG-EQFAYDGK-**LYTLNE-LRS*-TAVDT--QISEQ-S*NDASEAE-QRA---DT-VE--H-YLEKGKET-LHL* o oo oooo o o o o o o ooo o oo o

190 200 210 220 230 240 250 260 270 280 + SPSMFM*FAKKSHTETSVI*LTCLATGFLQRQVELEMRRDGRLLTAQDGVRTSNVRPNDADSYQIRNSVEILKTDKSLFTCEVLHRESSLSVATTW --PAVHL---R-IKDR-KLK---M----YP-D-T-GI-KSRTF-PEDETES-*GI---HDG---M-K---TQEDE-TNYD-F-S--TLKETIIV--SPTVHV---R-IKDK-KLK---M----YP-D-T-LI-KSHTS-P*D-*E*-*GT---HDGTF-VKK---IQEDE-AEYD-F-S--TLKETIIV---LAVHV---R-IKDR-KLK---M----YP-D-T-GI-KSRTF-SEDETES-*GI---HDG---M-K---TQEDE-TNYD-F-S--TLKETIIV---PEVHV---RCTRDKTKLE---F----YPKD-I-SI-KYRSP-PEKEIESS*G----HDGT--LKKT-IIQEDE-ADYD-V-S-STLNETII-K-APKVHV---RYVNGKAKLK---L----YPKD-Y-TI-KYRTA-SDSEVESS*G----HDGTF-L-K-TYI-EEE-AEYD-Y-A--TLNAP-V----RDVHV---RYINGKDKLK---L----YPKD-Y-TI-KYRTA-SDN-LESS*G----HDGTF-L-K-TYINEDE-AEYD-Y-N--TLKEP-IIRE-PKTHVTHHP*ISDHEAT*-R-W-L--YPAEIT-TWQQD-EGH-QDTELVE**T--AGDGTF-KWAA-VVPSGEEQRYT-H-Q-EGLPEP-TLRo o o oo o oo o o o o o

CP/TM 290 Paol-UB1 Bain-ZE*0201 Bain-ZE*0401 Bain-ZE*0501 Cyca-ZE*0101 Dare-ZE*0101 Dare-ZE*0201 HLA-E

300

DRKLPNAA*SGLTYVGVGVGLVVLVVLG -G-YSPVTI# -GE# -G-YSPVTI# -GTC-DCGS-LVIGIVI-AV--IV--AAVCLYLF -G-CSDCSKESAIGLI--*A*I*IGAVVVAAIVVVAFI NGECLSEPPIAMIAGII-*V-IL-GAI-VTV LRWKPASQPTIPIVGIIAGLVLLGS-VSGAVVAA

a1

a2

a3

CT 310 VQPL

MTKRLTCRQQSATNGQVQGAYEVNENLLPGYNSNGHA LKKNNKFCFRTTQEPSEENGRVLMKDPVFKE WILKKKNIIGNGDEKHFVVSTVSGNKDENGFVVSTVSGNKD VIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL

Fig. 2 Alignment of the deduced amino acid sequences of Paol-UB1 with non-classical major histocompatibility complex (MHC) class I molecules. Conserved residues are indicated as follows: (v) antigen binding residues; (^) salt bridges; (+) b2-microglobulin contact residues; boxes: CD8 binding loop. Asterisks indicate gaps. Circles: conserved cysteines involved in disulfide bond and other conserved residues. Underline: potential N-linked glycosylation sites. CP/TM, connecting peptide/transmembrane; CT, cytoplasmic domain. Bold: TM region of each sequence. (#) Premature termination codons. Class I ZE amino acid sequences identified in common carp (Cyca), large barbus (Bain), zebrafish (Dare) were taken from Kruiswijk et al.14 and human HLA-E accession number M20022.

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Leader peptide Paol-D(01)A Paol-D(02)A Auha-DAA1 Cyca-DXA101 Dare-19446 Icpu-A-1 Mosa-(A-R2) Onmy-DAA-02 Gici-pSa5-1 Bola AQ16-2 Cafa-L37332 Feca-DRA0101 Ovar-DQA 2.2 Scab-DRA Susc-SLA-DRA1-c HLA-DMA HLA-DRA

-10 -1 MNMNV*LVLCFVLS*V ---*--------*MKHA*PLLILILNNFCVF MYG-L-M-ALIVSTE MDLFGFL-TFTVI--NMRLFLLCFT-VC-KDTE MKM-KIMMV-VLWF-CMKTSMIV-I--CQVYAE MEARNYFSVL-LVLIQGGWAGK MNRALILGALALTTMMS-SG MTISGVPVLGFFIMAFLMGPQE AVSRVPVLGFCIMALLMGPQE *VLNRALILGALALTTMMSPSG MARSEV-VLGFFFMAVLMNPQE MTILGVPVLG*FVITIL*NLQK M GHEQNQGAALLQMLPLLWLLPHSWAVPEAPTPMWPD MAISGVPVLGFFIIAVLMSAQE-WA

10 Paol-D(01)A Paol-D(02)A Auha-DAA1 Cyca DXA101 Dare-L19446 Icpu-A-1 Mosa-(A-R2) Onmy-DAA-02 Gici-pSa5-1 Bola AQ16-2 Cafa-L37332 Feca-DRA0101 Ovar-DQA 2.2 Scab-DRA Susc-SLA-DRA1-c HLA-DMA HLA-DRA

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a1

180

PPSSPMIYTRDAVELGGENTLICHVTGFYPAPVHVYWTKNGVDVTEGTSLNVPYPNTDGSFRQTARLKFIAQQGDVYSCTVSH*LALDQSLTKIW -------------------------------------------------E---------------------------------*---E------D-PEIVLFSS-K----V--S---F-NH---PSIN-T-----HP-ST-V--SRYF--K-QT-H-FST-T-TPSE--F-----E-*S--ETPK-R-A-Q-S*--ARTG-Q--S--L----ASR-F-P--R-R----NL---DKS--SQY---D-ET-N-FSH-P-TP-E----T---Q-*E--QTPD-RT----PV--P--D----EK---------------K-------KN------I----L-K-NT-T--S--E--P-L--M---S-K-*-S-KDP--RF--H-S*--P--D---EVK-------S--F-P--R-R--R-NQN----GRIST------VT-N-FSS-S-TPEE--I-G---E-*KG-TEP--RIA-PDASV-SEGD-V-GVQ---------LF-P--N-S----NQI---DV--SQYRRKN--T-NIFSS---TPAE--I-----Y-KALESRFI--TD-PVTS--SE-E-V-DER----------F-P--N-S----NDI---EI-FSQYRR-S--T-NMFSA---TPAE--I-----N-RSIQG-PN--TVRPEVSV-SE-L--WGQL-----FAD----PHITMK-RR-NEPM-D-DNITEF-IKE-FT-RRFSY-SIVPSP--M---H-E-*SS-QDPV-VFV-*EVTVFSKSP-M-GQP-------DNIF-PVINIT-LK--HA----V-ETSFL-KD-H--LKIGY-T-LPSDN-I-D-K-E-WGLDEPL-*-H--*EVTVLSNTP---GEP-I---FIDK-S-PVIN-T-LR--NP--T-V-ETIFL-RE-HL--KFHY-P-LPSAE---D-K-E-WGLDEPL-*-H--*EVTVLSNSP---GEP-I---FIDK-S-PVIN-T-LR--KP--T-V-ET-FL-RE-HL--KFHY-P-LPSTE---D-K-E-WGLDEPL-*-HV-*EVTVFSKSP-M-GQP-------DNIF-PVINIT-L---HA----V-ETSFL-KD-H--LKIGY-T-LPSDD---Y-K-E-WGLEEPL-*-H--*EVTVLSSGP---GEP-V---FIDK-S-PVVN-R-L---EP--T-V-ET-FL-RK-QL--KFHY-L-LPSNE-F-D-E-D-LGLDRPL-*-H--*EVTVLSDKP---GEP-I---FIDK-S-PVVN-T-LR--SP--R-V-ET-FL-RE-HL--KFHY-P-MPSTE---D-Q-E-WGLDKPL-*-HGFPIAEVF-LKPL-FGKP--LV-F-SNLF-PMLT-N--HDHSVPV--FGPTFVSAVDGL--QAFSY-N-TPEPS-IF--I-T-EIDRYTAIA*Y--*EVTVL-NSP---REP-V---FIDK-T-PV-N-T-LR--KP--T-V-ET-FL-RE-HL--KFHY-P-LPSTE---D-R-E-WGLDEPL-*-Ho o o o o o o o o o

CP/TM 190 Paol-D(01)A Paol-D(02)A Auha-DAA1 Cyca DXA101 Dare-L9446 Icpu-A-1 Mosa-(A-R2) Onmy-DAA-02 Gici-pSa5-1 Bola AQ16-2 Cafa-L37332 Feca-DRA0101 Ovar-DQA 2.2 Scab-DRA Susc SLA-DRA1-c HLA-DMA HLA-DRA

50

SAEGLHVDLHVSGCSDHEGEDVYNLDGEELWFAD*FIKKEGVEPQP*PFIDHITYRDGTYQRAEANQHVCKTNLDVIRTAMKDFKIEHE** ----------------------------------*--------E--*-------F------N---------------------------** -QIPHEMTYV-*--FVQGLTE-QFEFDG-EFLYAN-P---I-YTV-NFIFPDPSHVLVGLSIL-DALDNGIWC-LITKI-EMEE-YVP-EK TQVVNR*-VQFV----T-R-FLIGF------H--*--R----VTV-*D-A-*PIGFP-F-ETGV-LME---Q--ALNIKVY*KPTD-QL** AQAEHR*-VDFF----T-K-YLQGF-----YHS-*--R-V--VTA-*D-A-*PMSYP-F-ENSV-QME---QD-ATDIKAY*NSPE-QL** AQNKF-*HLEL-A--EKDK-YMVGS----MFY--*-E--DI-NAL-*P-A-PGEF*T-GFAF--SKTAN-QA--Q-LSVEF--KPLPQD** --DD--E-FRIA----SD--EM-G-----V-Y--*--N-K------*S-I--TS-VE---EQ-VA--QI-RQ--GLALK-V--*PQKFD** *DKV--T-IYI-----SD-V-M-G-------Y--*-N-----VAL-*P-A-Q-SF*P-F-EQ-VGDLEIL-G--AKCIK-Y-NPPETID** YLYDFTQVYFVQQR-PEKHF-*VME--D-IFYM-*-NL-KE-ARI-*E-A*-LYMQG-EAAISANIAI*V-N--K-DMNLSAGTPEPK*** GEDIVADHVGSY-TEIYQSHGPSGQYTQ-FDGDEM-YVDL-KKETVWRLPMFSQFAGFDP-A-LSEIATA-H----LTKRSNFTPVIN-** -WAVKEEHVIIQA*EFYLTPDPSGEFMFDFDGDEI-HVDMEKKETVWRLEEFGRFASFEA-G-LA-IA-D-A---TMIKRSNHTPNTNV** -LAIKEEHVIIQA*EFYLKPDSSGEFMFDFDGDEI-HVDMEKKETVWRLEEFGRFASFEA-G-LA-IA-D-A---ILIKRSNNTPNTN-** GEDIVADHVGIY-ADFYQSHGPSGQYTH-FDGDEL-YVDL-KKETVWRLPMFGEFTSFDP-G-LSEIATA-H---IMIKRSNFTPVIN-** -WAIKEEHSIIQA*EFYLSPDQSGEFMFDFDGDEI-HVDVDKKETIWRLKEFGQFASFEA-G-LA-IA-D-A---IMIKRSNHTPDSNV** -WAIVENHVIIQA*EFYLSPDKSGEFMFDFDGDEI-HVDMEKRETVWRLEEFGHFASFEA-G-LA-IA-D-A--EILIKRSNNTPNTNV** DLQNHTFLHT-YCQDGSPSVGLSEAYD-DQLFFFD-SQNTRVPRL-EFA-WAQEQG-APA-LFDKEFCEWMIQQ*IGPKLDGKIPVSR*** IK-E*--IIQ**AEFYLN*PDQSGEFMFDFDGDEI-HVDMAKKETVWRLEEFGRFASFEA-G-LA-IA-D-A--EIMTKRSNYTP-TNV** o

90 Paol-D(01)A Paol-D(02)A Auha-DAA1 Icpu-A-1 Mosa-(A-R2) Onmy-DAA-02 Cyca-DXA101 Dare-19446 Gici-pSa5-1 Bola-AQ16-2 Cafa-L37332 Feca-DRA0101 Ovar-DQA 2.2 Scab-DRA Susc-SLA-DRA1-c HLA-DMA HLA-DRA

20

200

CT 210

DVDVQQPSVGPAVFCGVGLSVGLLGVAAGTFFLI ---------------------------------EAE-MNSDQSLGPAIFC-VGLS-GLLGVTVGVFF E---AV-G------------L------------E---EL-------------VL------------E-N-DL------------VA-------T-----V D-EKPE--I-------LG-T-------------EPE-S---VA---------TL------T------QG-PEEKS-HG*TIICALGLT-GIIS-VVGIIL GMYEFH-FRYSLF-YPIQNTLSFGPSISQSKACF EFEPPT-LP*ET*TEN-VCAL--*I-GLVGIITGTIF EF-APT-LP*ET*TEN-VCAL--*I-GLVGIIVGTIF EP*EI*-*A*-M**SELTET-***VC-L-LTMGLVGIVVGTIF EFEE-T-LP*ET*KEN-VCAL--*A-GLVGIVVGTIF EFEA-T-LP*ET*TENTVCAL--*I--LVGIIVGTVL VPRNAL--DLLENVLCGVAFGLGVLGIIVGIV--IY EF-APS-LP*ET*TEN-VCALG-*T-GLVGIIIGTIF

220 KGNECS -----FVKGHQRQ -L-N-N ---N-N ---Q-N --------Q-N LIK-RQRLQAQQHGI PHFKVSKIILHSLPKPGALSFCRINTTPPA IIKGMRKVKAGERRGPL IIKGMRKVNAGERQGPL IIQGLRSGGASRHQGPL IIKGTRKNPTAERRGPL IIKGVRKGNATEHRGPL FRKPCSGD IIKGVRKSNAAERRGPL

a2

Classes I and II MHC of Japanese flounder

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the Japanese flounder MHC class I molecules. However, certain unique aspects within the antigenic cleft are also displayed by different Japanese flounder cDNA. The conserved peptide-binding motif in mammals, Tyr7-Tyr57-Tyr82-Tyr120Thr140-Lys143-Trp144-Tyr157-Tyr169 (YYYYTKWYY),8 was also found in the Paol-UA1 to -UA4 clones, except for two substitutions of Arg82 and Phe120 (YYRFTKWYY). However, clone Paol-UA5 has two different substitutions of Leu120 and Phe169 (YYRLTKWYF) (numbering based on PaolUA1). The region of the a3 domain, the residues of which bind to CD8,24 is well conserved with respect to other fish but not in human MHC class I regions. It does possess an acidic character in having four of eight acidic residues in Japanese flounder. The five sequences obtained from these clones and all other teleosts share three amino acids deletion between amino acids 193 and 194 in the a3 domain when compared with the human MHC class I sequence. Among the five Japanese flounder MHC class Ia cDNA, three different cytoplasmic domain sequences were found as predicted by the Strictest Cutoff DAS Transmembrane Prediction Program.25 Besides classical MHC class I cDNA, we also cloned a cDNA (Paol-UB1), which was suspected to be of the non-classical MHC class I (class Ib) and which can be distinguished from classical genes by several criteria. First, there is a high degree of sequence dissimilarity; second, non-classical genes are expressed in an erratic or tissue-specific manner; and third, the polymorphism of non-classical genes is much lower than that of classical genes.26 This clone contained a 2.8 kb insert encoding 345 amino acid residues, and had low levels of sequence similarity (27–30%) when compared with the other five Japanese flounder classical MHC class I cDNA. The complete deduced amino acid sequence of this clone contained a 32-residue leader peptide, 282 residues of the extracellular domain (a1–a3), 31 residues (positions 283–313) of connecting peptide, transmembrane and cytoplasmic domain (Fig. 2). The deduced amino acid sequence of Paol-UB1 was aligned with common carp, large barbus, zebrafish, and human nonclassical MHC class I molecules (Fig. 2). The a1 domain of this clone had 56–58% identity to the a1

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domain of non-classical MHC class I molecules of three species of fish (common carp, large barbus and zebrafish).14 The a2 and a3 domains of this clone were 42–45% identical with that of the a2 and a3 domains of non-classical MHC class I clones of common carp, large barbus and zebrafish.14 Most of the features known to be conserved in classical and non-classical MHC class I molecules23,27 are present in all non-classical MHC sequences. The Japanese flounder non-classical MHC sequences possessed four cysteine residues (Cys102, Cys169, Cys209 and Cys267) in the a2 and a3 domains to form disulfide bonds within these domains, as was previously reported to be the case for non-classical MHC. It also contains conserved residues of His3, Asp31, His94, Asp132 in the a1 and a2 domains, forming two salt bridges within these domains. However, another important motif, Phe-Tyr-Pro, for the formation of salt bridges in other vertebrates was replaced with Phe-Leu-Gln (positions 214–216) in Japanese flounder. The acidic residues in the exposed loop of the a3 domain form a major CD8 binding site in Japanese flounder non-classical MHC class I sequences, possessing only one strongly acidic residue within the same region of other species. Four residues, Thr10, Gln97, Asp123 and Gln250, which are known to be involved in b2microglobulin binding of human MHC class I molecules,23 are found to be conserved in Paol-UB1. The Japanese flounder non-classical MHC class I molecule, like all other MHC class I molecules, possesses a putative N-linked glycosylation site at the end of the a1 domain with amino acid sequence NDT (Fig. 2). The putative peptide binding motif Tyr7-Tyr60-Arg85-Phe127-Thr147Lys150-Trp151-Tyr160-Phe176 (YYRFTKWYF) of Paol-UB1 is found to be slightly different from Japanese flounder classical class Ia molecules. a cDNA Characterization of class IIa Two clones were isolated that had 39–64% homology to previously reported MHC class IIa molecules.9,28–30 The Japanese flounder MHC class IIa cDNA were named Paol-D(01)A and Paol-D(02)A. They contained 235 and 233 amino acid residues,

 Fig. 3 Alignment of amino acid sequences of Paol-D(01)A and Paol-D(02)A with major histocompatibility complex (MHC) class IIa molecules. Circles: conserved cysteine involved in disulfide bond and other conserved residues. Asterisks indicate gaps. Underline: potential N-linked glycosylation sites. CP/TM, connecting peptide/transmembrane; CT, cytoplasmic domain. Bold: TM region of each sequence. The sequences used for this alignment were taken from GenBank are as follows: Auha (cichlid fish; AF091557); Cyca (common carp; X95432); Dare (zebrafish; L19446); Icpu (channel catfish; AF103006); Mosa (striped bass; L35065); Onmy (rainbow trout; AJ251432); Gici (nurse shark; M89951); Bola (cow; D50049); Cafa (dog; L37332); Feca (cat; U51576); Ovar (sheep; I47092); Scab (squirrel; M97615); Susc (pig; M92445); HLA-DMA and HLA-DRA (human; X62744 and K01171, respectively).

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Leader peptide -10 Paol-D(01)B Paol-D(02)B Mosa-(C1) Mosa-(C2) Mosa-(C22) Mosa-(S-1) Mosa-R41 Onmy-DAB 01 Onmy-DBB Onmy-DBB01 Orla-DAB Pore-DXB-01 Sasa-clone c157 Sasa-DAB-0101 HLA-DRB-0101

-1

MAS*FILSFSL*FFIT**VCTANG ---*-------*----**----D---S-*-----*L---S*LY-G----S-*-----*L---S*LY-A----S-*-----*L---S*LY-A----S-*-----*L---S*LY-A----S-*-----*L---S*LY-A--SMPIAFYIC-*TLLWSIFSGT--SKSIRFYIC-*AVALSTLYET--SKSIRFYIC-*AVALSTLYET--DSSSLC***-**LFL*TL-SA-A -AQAQGC-V--*VLFL*VFSPGGA -S**I*FCV--*TLVLSIFSGT--S**I*FCV--*TLVLSIFSGT-MVCLKLPGGSC-TALTVTLMV-SSRLALAGD-RPR o o

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Paol-D(01)B Paol-D(02)B Mosa-(C1) Mosa-(C2) Mosa-(C22) Mosa-(S-1) Mosa-R41 Onmy-DAB 01 OnmyDBB OnmyDBB01 Orla-DAB Pore-DXB-01 Sasa-clone c157 Sasa-DAB-0101 HLA-DRB-0101

FRYYVVNSCEFNSSKLNDIEFTESYYYNKLEYIRFSSSVGKFVGYTEHGIKNAERWNNGPEVI*SSRGEKERYCLNNVGVDVESALTNT -LH-M-D-------------YIY-H----------------------V-------L-------*NR-A*-*P---T-IN-**V*RL*** -LN-A--R-V---TDPKN--YIY-H------IA-------E------F-V-Q-KY--SD-SELARRSAQ--TV-QH-INI-YQVV-DKS -LN-A-GR-V---TDPKN--YIY-E------IA-------E------Y-L-Q-KY--SD-SELAQM-AA-----QH-INI-YQA--DKS FLN-A-GR-V---TDPKN--YIY-E------IA-------E------Y-L-Q-KY--SD-SELAQM-AA-----QH-INI-YQA--DKS ---FWTDR-V---TDPRN--YIN--------YA-----E-E------L-V-----F-KD-SYLAQR-A-------T-INI-YQN--DKS ---FETDR-V---TD-K--KYIR-E------IA-------E------L-LRW-KY---N-SYLAQM--------QH-I-NWYSNI-DKS YFEQ--RQ-RYS-KD-HG---ID--VF--A-HV--N-T--RY-------L----A--SDAGILGQEQA-L----KPSAAI-YSAI-DKYASD--TR-LYS-IDMHGA--IQ--TF--V-HL--N-T--E------L-L---K-L-R-Q--VQM*---L--L-KP-ADIHYRAI-DKYASD--TR-LYS-IDMHGA--IQ--TF--V-HL--N-T--E------L-L---K-L-R-Q--VQM*---L--L-KP-ADIHYRAI-DK-ME-A--R------D-T---YIY-M---RK-YA----SL-KY-----F-V-----F-KDTSELSVR-AQ--T--KH-IDI-YQT--SKS -YLS-LER-QSS-SDGH-AVLLDQV-F--ILEVEYN-T--KMI----*KTEALAIIL-NNPEFITHEIW-TNL-KR-TPLA*QKL--*P YFEQ--RQ-RYS-KD-QG---ID--VF--A-YI--N-T--K------L-V----A--SDAA-LAVE---L----KH-ADLHYSTI-DKYFEQ--RQ-RYS-KD-QG---ID--VF--A-YI--N-T--K------L-V----A--SDAA-LAVE---L----KH-ADLHYSTI-DK-LWQLKFE-H-FNGTER*VRLL-RCI--QE-SV--D-D--EYRAVE-L-RPD--Y--SQKDLLEQK--QVDN--RH-Y--GESFTVQRR o o o o o o

Paol-D(01)B Paol-D(02)B Mosa-(C1) Mosa-(C2) Mosa-(C22) Mosa-(S-1) Mosa-R41 Onmy-DAB 01 Onmy-DBB Onmy-DBB01 Orla-DAB Pore-DXB-01 Sasa-clone c157 Sasa-DAB-0101 HLA-DRB-0101

QTLRQASL*CGAP**SWQTCMLVCSVFDFYPKRIKVSWQRDGQEVTSDVTSTDELADGDWYYQIHSHLEYMPKSGEKISCVVEHASLSKPLITDW *HSV*APP***-G***K*HA--------------------------------------------------------------------------VEPSVVLHS*V--PAGKHPS------Y--F--H-R---L------------------A--F---------T-R--------------KE--V--VQPSVVLHS*V--PAGKHPS------Y--F--H-R---L-----------P------A--F---------T-R--------------KE--V--VQPSVVLHS*V--PAGKHPS------Y--F--H-R---L-----------P------A--F---------T-R--------------KE--V--VKPSVVLHS*V--PAGKHPS------Y--F--H-R---L------------------A--F---------T-R--------------KE--V--VQSYVRLHS*V--PAGKHPS------C--F--H-R---L------------------A--F---------T-R--------------KE--V--VEPHVRLSS*VT-PSGRHPA--M--AY-----Q-R-T-L---R--K------E---N-------------T---------M---I--TE-MMYHVEPHVRLSS*VT-PSGRHPA--M--AY-----P-R-T-L---R--K------E---N-------------T---------M---I--TE-MMYHVEPHVRLSS*VT-PSGRHPA--M--AY-----P-R-T-L---R--K------E---N-------------T---------M---I--TE-MMYHVQPRVRVQSLAPSG*GHHPA--V---Y-----T-R-S-L-GKE--S------A-ME--------------T-R-------K------KD--V--VEPYVQLR*LEKAEY-QHQQ--I--AY-----Q-R-T-L---K------------PN-N-L----TY--FT--P----T-ML-----K--NLY-VEPHVRLSS*V--PSGRHPA--M--AY-----P-R-T-L---R--K------E---N-------------T-R-------M---I--TE-MVYHVEPHVRLSS*V--PSGRHPA--M--AY-----P-R-T-L---R--K------E---N-------------T-R-------M---I--TE-MVYHVEPKVTVYPS*KTQPLQHHNL-V---SG---GS-E-R-F-N---EKAG-V--GLIQN---TF-TLVM--IV-R---VYT-Q---P-VTS--TVEo oo o o o o o o o o o o o oo oo o oo o o o

90

100

110

120

CP/TM 190 Paol-D(01)B Paol-D(02)B Mosa-(C1) Mosa-(C2) Mosa-(C22) Mosa-(S-1) Mosa-R41 Onmy-DAB 01 Onmy-DBB Onmy-DBB01 Orla-DAB Pore-DXB-01 Sasa clone c157 Sasa-DAB-0101 HLA-DRB-0101

200

130

140

CT 210

DPSMPESERNKIAIGTSGLILGLTLSLAGFIYYKRKAQ ----------------------------------------------------A-------I-------------R ---------------A-------I-------------R ---------------A-------I-------------R ---------------A-------I-------------R ---------------A-------I-------------R ---L--A--------A---V--TI-A---L----K-SS ---L--A--------A---V--TI-A---L----K-SS ---L--A--------A---V--TI-A---L----K-SS -----------V---A-------V-------------R ELEP*D-KWS--VV-SA--L---VF-I-------TTSN ---L--A--------A---V--AI-A---L----K-SS ---L--A--------A---V--AI-A---L----K-SS *RARS--AQS-MLS-V--FV---LFLG--LFIY o o o oo oo

220 GRILVPTN -------------S------S------S---P--S------S-VL***** -VL***** -VL***** ------SS --VV-PTTEDVCPEETL -VL***** -VL***** FRNQKGHSGVQPTGFLS

150

160

170

b1

180

b2

Classes I and II MHC of Japanese flounder

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respectively, and have a high amino acid sequence identity of 96%, differing from each other in the length of the leader peptide sequence and amino acid positions 43, 53 and 60 in the a1 domain, and positions 137 and 174 in the a2 domain (Fig. 3). The amino acid sequences of Japanese flounder MHC class IIa have two cysteines in the a1 domain, which are unique to teleosts and all human MHC class IIa human leukocyte antigen (HLA)-DMA chains (positions 14 and 68). However, neither of the Japanese flounder MHC class IIa clones possesses a potential N-linked glycosylation site, in contrast to most previously reported MHC class IIa molecules wherein the N-linked glycosylation site is present in the a1 or a2 domain. The transmembrane region of the Japanese flounder MHC class IIa molecule contains the GXGXXXGXXGXXXG motif (where X is any hydrophobic residue other than Gly), which is believed to be important for correct interaction with the MHC class IIb chain. Finally, a relatively short cytoplasmic tail of six amino acid residues is present, similar in length to the cytoplasmic domains of channel catfish, common carp, rainbow trout, striped bass and zebrafish (Fig. 3).

271

Expression of MHC mRNAs in Japanese flounder tissues The Japanese flounder MHC classical class Ia (Paol-UA) gene was expressed in all tissues. The mRNA of class Ib (Paol-UB1) was detected mainly in gills, head kidney, intestine, PBL, spleen, stomach and trunk kidney, and relatively small quantities were detected in brain, eye, heart, liver, muscle, ovary, and skin. Both class IIa and class IIb chain mRNAs were expressed in all tissues (Fig. 5). Phylogenetic analysis The phylogenetic tree of Japanese flounder MHC class I (classical and non-classical), class IIa and class IIb among MHC of other vertebrates is shown in Fig. 6. Paol-UA1 to Paol-UA5, Paol-UB1, PaolD(01)A and Paol-D(02)A and, Paol-D(01)B and Paol-D(02)B of Japanese flounder MHC were found to cluster with the classical MHC class I, nonclassical MHC class I, MHC class IIa and MHC class IIb, respectively. DISCUSSION

b cDNA Characterization of class IIb Two distinct MHC class IIb molecules of Japanese flounder Paol-D(01)B and Paol-D(02)B were cloned and characterized. These two clones contained 246 and 232 amino acid residues and had an amino acid identity of 78%. The derived amino acid sequences of MHC class IIb molecules of Japanese flounder, teleost fish and human are aligned in Fig. 4. Both Japanese flounder MHC class IIb chains have conserved cysteine residues at positions Cys9, Cys73, Cys109 and Cys165. In the b2 domain of Japanese flounder MHC class IIb, a Phe-Tyr-Pro motif (positions 114–116) and two tryptophan residues (positions 123 and 145), which are important for intrasalt bridge formation,1 are mostly conserved. The connecting peptide, transmembrane and cytoplasmic domains of teleosts1 have high identity. The overall sequence similarity revealed that Japanese flounder MHC class IIb domain 2 is more highly conserved than domain 1.

Five different MHC class Ia chain cDNA (Paol-UA1 to Paol-UA5) were isolated from a PBL cDNA library of Japanese flounder. Each exhibited characteristic class I features, strongly suggesting that these genes encode functional proteins that bind peptides, that associate with b2-microglobulin,23 and that have contact with the CD8 molecule.24 The a1, a2 and a3 domains of the five Japanese flounder MHC class Ia were 50–67% identical to the corresponding domains in teleost fish5,8,13 and 33% identical to corresponding domains in the human.32 There is a general lack of sequence conservation in the cytoplasmic domains of all fish MHC class I sequences and the Japanese flounder is no exception, having three completely different cytoplasmic domain sequences in the five sequences obtained. In addition, the carboxy terminus of Japanese flounder MHC class Ia contains some Ser residues that might act as phosphorylation sites, as is the case in human MHC class Ia molecules.32 Interestingly, we found the Ser resi-

 Fig. 4 Alignment of amino acid sequences of Paol-D(01)B and Paol-D(02)B with major histocompatibility complext (MHC) class IIb molecules. CP/TM, connecting peptide/transmembrane; CT, cytoplasmic domain. Bold: TM region of each sequence. Circles: conserved cysteine involved in disulfide bond and other conserved residues. Asterisks indicate gaps. Underline: potential N-linked glycosylation sites. The sequences used for this alignment were taken from GenBank are as follows: Mosa (striped bass clones C1, C2, C22, S-1 and R41 accession numbers L33962–L33966); Onmy (rainbow trout; from Ristow et al.);31 Orla (Japanese medaka; AF080585); Sasa (Atlantic salmon clone c157 and -DAB*0101; X70166 and X70167, respectively); HLA-DRB (human; M11161).

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dues in clones Paol-UA1, Paol-UA3 (Ser339, 341, 343) and Paol-UA5 (Ser339, 341–343) but not in clones Paol-UA2 and Paol-UA4. The presence of nine evolutionarily conserved putative peptide-anchoring residues in the amino acid sequence of classical class I genes has been found to be a useful criterion in the discrimination of classical class I genes and non-classical class I genes in many vertebrates.33–36 In the classical class I molecules of mammals this motif of nine amino acids is YYYYTKWYY,14 while in classical class I molecules of non-mammalian vertebrates it is slightly changed in some residues.14 Alignment of Paol-UA1 to Paol-UA5 sequences with human classical class I molecules reveals the presence of the conserved non-mammalian motif YYRFTKWYY or YYRLTKWYF for classical class I (Fig. 1).8,11,14 However, the non-classical class I molecules of both Japanese flounder and human (HLA-E) have some different residues of the motif (YYRFTKWYF and YYYYSKSYY, respectively; Fig. 2). The expression pattern of Japanese flounder non-classical MHC class I agrees with those in mammals.37 Consequently, in common carp and large barbus, non-classical MHC class I expression was observed in the lymphoid organs, intestine or liver.14 Based on sequence and phylogenetic analyses as well as the expression pattern, we conclude that Paol-UB1 indeed belongs to the non-classical MHC class I (Fig. 6). This is in agreement with the fact that nonclassical MHC class I molecules are not limited to common carp and gold fish38 as previously suggested,39 but are also found in large barbus and zebrafish,14 and now in Japanese flounder. Also, non-classical MHC class I molecules are suspected to be present in channel catfish,13 coelacanth,7 salmonid fish,8 and shark.6,34 All of these

P Srisapoome et al.

findings indicate that the non-classical MHC class I gene is common in fish. The deduced amino acid sequences of Japanese flounder Paol-D(01)A and Paol-D(02)A were 46–49% identical with MHC class IIa molecules of other teleosts and 29–31% identical with those of other vertebrates. Based on these identities and their structure, we classified Paol-D(01)A and Paol-D(02)A as belonging to MHC class IIa. Japanese flounder MHC class IIa has two cysteine residues (Cys14 and Cys68) in the a1 domain. This is also the case in fish and human HLADMA40 but not in other vertebrates. In contrast, an N-linked glycosylation site in the terminal region of the a1 domain was not observed in fish MHC class IIa molecules and human HLA-DMA, but it is present in MHC class IIa molecules of other vertebrates.41–44 It is unclear from a structural point of view whether these cysteines are essential for proper folding of the teleostean class II a1 domain. The lack of the cysteine residues in class II a1 domains of shark and all warmblooded species, argues perhaps in favor of the hypothesis that there is no structural need for these residues to be present.30 Two clones of Japanese flounder MHC class IIb were also found and characterized. Alignment of the putative amino acid sequences (Fig. 4) shows that the general structure of MHC class IIb molecules is similar to that of higher vertebrates.1 They are all composed of two extracellular domains, a connecting peptide, a transmembrane domain, and a cytoplasmic domain. The Paol-D(01)B and Paol-D(02)B have 70% amino acid similarity in domain 1 and 90% homology in domain 2. Similar results were found in other teleost fish such as cichlids,45 striped bass46 and Atlantic salmon.47 A number of MHC class IIb molecules from these

A 1

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563 bp

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502 bp 524 bp

b-actin

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14 590 bp

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422 bp

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Fig. 5 Expression of major histocompatibility complex (MHC) genes in tissues and organs of Japanese flounder. (A) Expression of classical MHC Ia and non-classical MHC class Ib; (B) expression of MHC class IIa and IIb. Lane 1, brain; lane 2, eye; lane 3, gills; lane 4, head kidney; lane 5, heart; lane 6, intestine; lane 7, peripheral blood leukocytes (PBLs); lane 8, liver; lane 9, muscle; lane 10, ovary; lane 11, skin; lane 12, spleen; lane 13, stomach; lane 14, trunk kidney. b-actin was included as a positive control.

Classes I and II MHC of Japanese flounder

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Class Ib

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Class Ia

Bain-ZE*0201 Cyca-ZE0101 Dare-ZE0101 Onmy-UBA0201 Paol-UB1 Gamo-UA*C28 Auha-517 Furu-I1 Stvi-C11 HLA-E Paol-UA1 HLA-A2 PaolUA3 Paol-UA1 H2-Kd Paol-UA2 Paol-UA4 H2-M3 Hefr-19 Hefr-20

Mr1 Xela-XNC1.1

Gici-UAA01

Xela-UAA1f

Trsc-UAA*101

Sasa-C22 Sasa-C157 Onmy-DAB*01

MICA

Onmy-DBB*01 Auha-DAA*1

Paol-D(02)B

Auha-DBA*S01 Paol-D(01)B Mosa-AS8

Mosa-C1

Paol-D(02)A Paol-D(01)A Pore-DXB*01

Onmy-DAA*01

HLA-DRB Cyca-DXA*101 HLA-DRA

Class IIb 0.1

Dare-L19446 Icpu-DBA*01 Icpu-DAA1*01

Gici-pSa51 HLA-DMA

Class IIa Fig. 6 Phylogenetic tree of two classes of major histocompatibility complex (MHC) genes in Japanese flounder and other vertebrates. The accession numbers of sequences used in phylogenetic tree construction are as follows. Human: HAL-A*0201 (K02883), HLA-E (M20022), MICA (A55739), Mr1 (U22963), HLA-DRA (K01171), HLA-DRB (M11161), HLADMA (X62744); mouse: H-2 Kd (J00402), H-2 M3 (U18797); frog: Xela-UAA1f (L20733), Xela-XNC 1.1; leopard shark: Trsc-UAA*101 (AF034316); horned shark: Hefr-19 (AF028558), Hefr-20 (AF028559); shark: Gici-UAA01 (AF220063), Gici-pSa5–1 (M89951); rainbow trout: Onmy-UBA*201 (AF287485), Onmy-DAA*01 (AJ251431), Onmy-DAB*01 (U20943), Onmy-DBB*01 (AF115529); Atlantic cod: Gamr-UA*C28 (AJ132511); cichlid fish: Auha-517 (AF038550), Auha-DAA*01 (AF091557), Auha-DBA*S01 (AF212849); walleye: Stvi-C1-1 (AY057455); striped bass: Mosa-AS8 (L35063), Mosa-C1 (L33962); common carp: Cyca-DXA1*01 (X95432), Cyca-ZE*0101 (AJ420951); zebrafish: Dare (L19446), Dare-ZE*0101 (AJ420953); channel catfish: Icpu-DAA1*01 (AF103002), Icpu-DBA1*01 (AF103055); guppy: Pore-DXB*01 (AF080585); large barbus: Bain-ZE*0201 (AJ420277); Atlantic salmon: Sasa-C157 (X70166), Sasa-C22 (X70167).

species was sequenced. An extensive variability was detected in the b1 domain, which corresponds with the functional peptide-binding region of known MHC class II molecules, whereas the b2 domains were highly conserved among the determined sequences. At the amino acid level, the b1 and 2 domains of Paol-D(01)B and Paol-D(02)B have high identification with the MHC class II molecules of other fish: 63–72% in striped bass (Mosa),46 61% in Japanese medaka (Orla) (AF080585) and 44% in Atlantic salmon (Sasa),47 and low identification with human HLA-DRB48 (34%) (Fig. 4). As in Japanese flounder MHC class IIa, we could not find the N-linked glycosylation

site in the domain 1 of Japanese flounder MHC class IIb. This feature was also found in MHC class IIb molecules of striped bass (Mosa)46 except clone R41, Japanese medaka (Orla; AB033212) and human HLA-DRB.48 However, this N-linked glycosylation site was found in the b1 domains of MHC class IIb of rainbow trout (Onmy),49 guppy (Pore; AF080585) and Atlantic salmon (Sasa).50 Investigations of MHC gene expression by reverse transcription–polymerase chain reaction (RT-PCR) revealed that the Japanese flounder MHC class Ia expression pattern is consistent with both that of other teleost species13,20,51 and with mammalian MHC class Ia gene expression,1

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which are constitutively expressed on virtually all nucleated cells (Fig. 5). However, the expression of MHC class II genes, which are normally expressed on only dendritic cells, B cells and macrophages in mammals,1 was found in all investigated tissues of Japanese founder. The presence of MHC class II mRNA in all tissues could be a result of contamination from leukocytes because it can be difficult to eliminate contaminated leukocytes of the blood in tissues. The similar result of expression of MHC class II gene has been reported in Atlantic salmon.52 This suggests that MHC class II gene expression in all investigated fish tissues is possible as a result of the lymphoid/myeloid cell content of the tissues. Remarkably, highly expressed levels of both classes of MHC genes in Japanese flounder were observed in gills, intestine and stomach. This may be a reflection of the fact that these organs are the portals through which antigens such as microbes enter into the fish body, and where the fish immune response is initiated. In conclusion, all four types of MHC (classical MHC class I, non-classical MHC class I, MHC class IIa and MHC class IIb) cDNA in a Japanese flounder were determined. This indicates that the MHC gene is polygenic because there are several MHC classes, which encoded protein with different ranges of peptide binding specificity.1 In the case of MHC class Ia and IIb, two alleles were found. These results reflect the characteristic of the MHC gene, which is highly polymorphic.1 The number of MHC genes in the genome of teleost fish is not accurately known. The extensive studies were focused on MHC class Ia, in which three or four genes per genome seems to be a common number for teleosts, and this is the optimal number required to provide enough alleles to protect against a range of pathogens.45 Recently, the gene organization of MHC genes of teleosts has been described differently from all other jawed vertebrates because the MHC genes of teleosts are located in at least three linkage groups on separate chromosomes.14 In the future, identification of polymorphisms in Japanese flounder MHC may provide a tool for analyzing the fish immune system. The nucleotide sequence data reported in the present paper have been submitted to the GenBank database and have been assigned the accession numbers as follows: Paol-UA1–UA5 accession numbers AB126917–AB126921, Paol-UB1 accession number AB126916, Paol-D(01)A and PaolD(02)A accession numbers AB126914 and AB126915, respectively. Paol-D(01)B and PaolD(02)B, accession numbers AB126922 and AB126923, respectively.

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ACKNOWLEDGMENT The present study was supported in part by a Grant-in-Aid for Scientific Research (S) (No.15108003) of the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

REFERENCES 1. Dixon B, van Erp SH, Rodrigues PN, Egberts E, Stet RJ. Fish major histocompatibility complex genes: an expansion. Dev. Comp. Immunol. 1995; 19: 109–133. 2. Rothbard JB, Gefter ML. Interactions between immunogenic peptides and MHC proteins. Annu. Rev. Immunol. 1991; 9: 527–565. 3. Konig R, Huang LY, Germain RN. MHC class II interaction with CD4 mediated by a region analogous to the MHC class I binding site for CD8. Nature 1992; 30: 796–798. 4. Takeuchi H, Figueroa F, O’hUigin C, Klein J. Cloning and characterization of class I Mhc genes of the zebrafish, Brachydanio rerio. Immunogenetics 1995; 42: 77–84. 5. van Erp SH, Dixon B, Figueroa F, Egberts E, Stet RJ. Identification and characterization of a new major histocompatibility complex class I gene in carp (Cyprinus carpio L.). Immunogenetics 1996; 44: 49–61. 6. Ohta Y, Okamura K, McKinney EC, Bartl S, Hashimoto K, Flajnik MF. Primitive synteny of vertebrate major histocompatibility complex class I and class II genes. Proc. Natl Acad. Sci. USA 2000; 97: 4712–4717. 7. Betz UA, Mayer WE, Klein J. Major histocompatibility complex class I genes of the coelacanth Latimeria chalumnae. Proc. Natl Acad. Sci. USA 1994; 91: 11 065–11 069. 8. Shum BP, Rajalingam R, Magor KE, Azumi K, Carr WH, Dixon B, Stet RJ, Adkison MA, Hedrick RP, Parham P. A divergent non-classical class I gene conserved in salmonids. Immunogenetics 1999; 49: 479–490. 9. Shum BP, Guethlein L, Flodin LR, Adkison MA, Hedrick RP, Nehring RB, Stet RJ, Secombes C, Parham P. Modes of salmonid MHC class I and II evolution differ from the primate paradigm. J. Immunol. 2001; 166: 3297–3308. 10. Godwin UB, Antao A, Wilson MR, Chinchar VG, Miller NW, Clem LW, McConnell TJ. MHC class II B genes in the channel catfish (Ictalurus punctatus). Dev. Comp. Immunol. 1997; 21: 13–23. 11. Antao AB, Chinchar VG, McConnell TJ, Miller NW, Clem LW, Wilson MR. MHC class I genes of the channel catfish. sequence analysis and expression. Immunogenetics 1999; 49: 303–311. 12. Godwin UB, Flores M, Quiniou S, Wilson MR, Miller NW, Clem LW, McConnell TJ. MHC class II A genes in the channel catfish (Ictalurus punctatus). Dev. Comp. Immunol. 2000; 24: 609–622. 13. Antao AB, Wilson M, Wang J, Bengten E, Miller NW, Clem LW, Chinchar VG. Genomic organization and differential expression of channel catfish MHC class I genes. Dev. Comp. Immunol. 2001; 25: 579–595. 14. Kruiswijk CP, Hermsen TT, Westphal AH, Savelkoul HF, Stet RJ. A novel functional class I lineage in zebrafish (Danio rerio), carp (Cyprinus carpio), and large barbus (Barbus intermedius) showing an unusual conservation of

Classes I and II MHC of Japanese flounder

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

FISHERIES SCIENCE

the peptide binding domains. J. Immunol. 2002; 15: 1936– 1947. Nam BH, Yamamoto E, Hirono I, Aoki T. A survey of expressed genes in the leukocytes of Japanese flounder, Paralichthys olivaceus, infected with Hirame rhabdovirus. Dev. Comp. Immunol. 2000; 24: 13–24. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997; 25: 4876–4882. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1987; 4: 406–425. Timon M, Elgar G, Habu S, Okumura K, Beverley PC. Molecular cloning of major histocompatibility complex class I cDNAs from the pufferfish Fugu rubripes. Immunogenetics 1998; 47: 170–173. Sato A, Klein D, Sultmann H, Figueroa F, O’hUigin C, Klein J. Class I Mhc genes of cichlid fishes: identification, expression, and polymorphism. Immunogenetics 1997; 46: 63–72. Fujiki K, Booman M, Chin-Dixon E, Dixon B. Cloning and characterization of cDNA clones encoding membranebound and potentially secreted major histocompatibility class I receptors from walleye (Stizostedion vitreum). Immunogenetics 2001; 53: 760–769. Klein J, Bontrop RE, Dawkins RL, Erlich HA, Gyllensten UB, Heise ER, Jones PP, Parham P, Wakeland EK, Watkins DI. Nomenclature for the major histocompatibility complexes of different species: a proposal. Immunogenetics 1990; 31: 217–219. Nielsen H, Engelbrecht J, Brunak S, von Heijne G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 1997; 10: 1–6. Kaufman J, Salomonsen J, Flajnik M. Evolutionary conservation of MHC class I and class II molecules: different yet the same. Semin. Immunol. 1994; 6: 411–424. Salter RD, Norment AM, Chen BP, Clayberger C, Krensky AM, Littman DR, Parham P. Polymorphism in the alpha 3 domain of HLA-A molecules affects binding to CD8. Nature 1989; 338: 345–347. Cserzo M, Wallin E, Simon I, von Heijne G, Elofsson A. Prediction of transmembrane alpha-helices in prokaryotic membrane proteins: the dense alignment surface method. Protein Eng. 1997; 10: 673–676. Klein J, O’hUigin C. The conundrum of nonclassical major histocompatibility complex genes. Proc. Natl Acad. Sci. USA 1994; 91: 6251–6252. Saper MA, Bjorkman PJ, Wiley DC. Refined structure of the human histocompatibility antigen HLA-A2 at 2.6 Å resolution. J. Mol. Biol. 1991; 20: 277–319. van Erp SH, Egberts E, Stet RJ. Characterization of class II A and B genes in a gynogenetic carp clone. Immunogenetics 1996; 44: 192–202. Murray BW, Sultmann H, Klein J. New family of Mhc class II A genes identified from cDNA sequences in the cichlid fish Aulonocara hansbaenschi. Immunogenetics 1999; 49: 544–548. Grimholt U, Getahun A, Hermsen T, Stet RJ. The major histocompatibility class II alpha chain in salmonid fishes. Dev. Comp. Immunol. 2000; 24: 751–763. Ristow SS, Grabowski LD, Thompson SM, Warr GW, Kaattari SL, de Avila JM, Thorgaard GH. Coding sequences of the

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35.

36.

37. 38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

275

MHC II b chain of homozygous rainbow trout (Oncorhynchus mykiss). Dev. Comp. Immunol. 1999; 23: 51–60. Guild BC, Strominger JL. Human and murine class I MHC antigens share conserved serine 335, the site of HLA phosphorylation in vivo. J. Biol. Chem. 1984; 259: 9235–9240. Shum BP, Avila D, Du Pasquier L, Kasahara M, Flajnik MF. Isolation of a classical MHC class I cDNA from an amphibian. Evidence for only one class I locus in the Xenopus MHC. J. Immunol. 1993; 15: 5376–5386. Bartl S, Baish MA, Flajnik MF, Ohta Y. Identification of class I genes in cartilaginous fish, the most ancient group of vertebrates displaying an adaptive immune response. J. Immunol. 1997; 159: 6097–6104. Kaufman J, Andersen R, Avila D, Engberg J, Lambris J, Salomonsen J, Welinder K, Skjodt K. Different features of the MHC class I heterodimer have evolved at different rates. Chicken B-F and beta 2-microglobulin sequences reveal invariant surface residues. J. Immunol. 1992; 148: 1532– 1546. Flajnik MF, Kasahara M, Shum BP, Salter-Cid L, Taylor E, Du Pasquier L. A novel type of class I gene organization in vertebrates: a large family of non-MHC-linked class I genes is expressed at the RNA level in the amphibian Xenopus. EMBO J. 1993; 12: 4385–4396. Abbas AK, Lichtman AH, Pober JS. Cellular and Molecular Immunology. WB Saunders, Philadelphia. 2000. Stet RJ, Kruiswijk CP, Saeij JP, Wiegertjes GF. Major histocompatibility genes in cyprinid fishes: theory and practice. Immunol. Rev. 1998; 166: 301–316. Figueroa F, Mayer WE, Sato A, Zaleska-Rutczynska Z, Hess B, Tichy H, Klein J. Mhc class I genes of swordtail fishes, Xiphophorus: variation in the number of loci and existence of ancient gene families. Immunogenetics 2001; 53: 695–708. Kelly AP, Monaco JJ, Cho SG, Trowsdale J. A new human HLA class II-related locus, DM. Nature 1991; 353: 571– 573. Kasahara M, Vazquez M, Sato K, McKinney EC, Flajnik MF. Evolution of the major histocompatibility complex: isolation of class II A cDNA clones from the cartilaginous fish. Proc. Natl Acad. Sci. USA 1992; 89: 6688–6692. Hirsch F, Germana S, Gustafsson K, Pratt K, Sachs DH, Leguern C. Structure and expression of class II alpha genes in miniature swine. J. Immunol. 1992; 149: 841–846. Wagner JL, DeRose SA, Burnett RC, Storb R. Nucleotide sequence and polymorphism analysis of canine DRA cDNA clones. Tissue Antigens 1995; 45: 284–287. Yuhki N, O’Brien SJ. Nature and origin of polymorphism in feline MHC class II DRA and DRB genes. J. Immunol. 1997; 158: 2822–2833. Figueroa F, Mayer WE, Sultmann H, O’hUigin C, Tichy H, Satta Y, Takezaki N, Takahata N, Klein J. Mhc class II B gene evolution in East African cichlid fishes. Immunogenetics 2000; 51: 556–575. Walker RA, McConnell TJ. Variability in an MHCMosa class IIb chain-encoding gene in striped bass (Morone saxatilis). Dev. Comp. Immunol. 1994; 18: 325–342. Langefors A, Lohm J, von Schantz T. Allelic polymorphism in MHC class II B in four populations of Atlantic salmon (Salmo salar). Immunogenetics 2001; 53: 329–336. Bell JI, Estess P, St. John T, Saiki R., Watling DL, Erlich HA, McDevitt HO. DNA sequence and characterization of human class II major histocompatibility complex beta

276

FISHERIES SCIENCE

chains from the DR1 haplotype. Proc. Natl Acad. Sci. USA 1985; 82: 3405–3409. 49. Glamann J. Complete coding sequence of rainbow trout Mhc II b chain. Scand. J. Immunol. 1995; 41: 365–372. 50. Hordvik I, Grimholt U, Fosse VM, Lie O, Endresen C. Cloning and sequence analysis of cDNAs encoding the MHC class II b chain in Atlantic salmon (Salmo salar). Immunogenetics 1993; 37: 437–441.

P Srisapoome et al.

51. Koppang EO, Lundin M, Press CMcL, Rønningen K, Lie Ø. Expression of Mhc class I mRNA in tissues from vaccinated and non-vaccinated Atlantic salmon (Salmo salar L.). Fish Shellfish Immunol. 1998; 8: 577–587. 52. Koppang EO, Press CMcL, Rønningen K, Lie Ø. Differing levels of Mhc class IIb chain expression in a range of tissues from vaccinated and non-vaccinated Atlantic salmon (Salmo salar L.). Fish Shellfish Immunol. 1998; 8: 183–196.