Xenopus in the Amphibian Ancestral Organization of the MHC Revealed

Ancestral Organization of the MHC Revealed in the Amphibian Xenopus Yuko Ohta, Wilfried Goetz, M. Zulfiquer Hossain, Masaru Nonaka and Martin F. Flajnik This information is current as of September 29, 2021. J Immunol 2006; 176:3674-3685; ; doi: 10.4049/jimmunol.176.6.3674 http://www.jimmunol.org/content/176/6/3674 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Ancestral Organization of the MHC Revealed in the Amphibian Xenopus1

Yuko Ohta,2* Wilfried Goetz,* M. Zulﬁquer Hossain,* Masaru Nonaka,† and Martin F. Flajnik*

With the advent of the Xenopus tropicalis genome project, we analyzed scaffolds containing MHC genes. On eight scaffolds encompassing 3.65 Mbp, 122 MHC genes were found of which 110 genes were annotated. Expressed sequence tag database screening showed that most of these genes are expressed. In the extended class II and class III regions the genomic organization, excluding several block inversions, is remarkably similar to that of the human MHC. Genes in the human extended class I region are also well conserved in Xenopus, excluding the class I genes themselves. As expected from previous work on the Xenopus MHC, the single classical class I gene is tightly linked to immunoproteasome and transporter genes, defining the true class I region, present in all nonmammalian jawed vertebrates studied to date. Surprisingly, the immunoproteasome gene PSMB10 is found in Downloaded from the class III region rather than in the class I region, likely reflecting the ancestral condition. Xenopus DM␣, DM␤, and C2 genes were identified, which are not present or not clearly identifiable in the genomes of any teleosts. Of great interest are novel V-type Ig superfamily (Igsf) genes in the class III region, some of which have inhibitory motifs (ITIM) in their cytoplasmic domains. Our analysis indicates that the vertebrate MHC experienced a vigorous rearrangement in the bony fish and bird lineages, and a translocation and expansion of the class I genes in the mammalian lineage. Thus, the amphibian MHC is the most evolutionary conserved MHC so far analyzed. The Journal of Immunology, 2006, 176: 3674–3685. http://www.jimmunol.org/

he MHC is the most gene-dense region in the human ge- gesting that this class I region is the primordial organization (5–7). nome and plays an indispensable role in the adaptive im- In some nonmammalian species, there is only a single or few clas- mune system (1). Class I and class II Ag-presenting mol- sical class I genes, perhaps due to a selection for coevolution with T ϩ ecules present small peptides derived from pathogens to CD8 and the Ag-processing genes. Thus, plasticity of class I genes in mam- CD4ϩ T cells, respectively. In the class I system, endogenous pep- malian species is an evolutionarily derived characteristic (5, 7). tides derived from intracellular pathogens are enzymatically Xenopus (especially Xenopus laevis and more recently Xenopus cleaved into small peptides by the immunoproteasome containing tropicalis) has been used historically for developmental studies by guest on September 29, 2021 the specialized ␤-subunits PSMB8, PSMB9, and PSMB10, which (8). Regarding the MHC, this animal is the most comprehensively upon infection replace the constitutive subunits, PSMB5, PSMB6, studied amphibian for characteristics of the adaptive immune sys- and PSMB7, respectively (2). Short peptides of 8–11 aas are trans- tem. Xenopus is a unique model because there are several ported into endoplasmic reticulum by the TAP (TAP1 and TAP2) polyploid species (2n–12n) within the genus that arose by recent and then loaded onto class I molecules associated with tapasin genome-wide duplication (from 2 to 30 million years ago) (9). (TAPBP). The resulting class I-peptide complexes move to the cell Because of its important phylogenetic position, and because it is a surface, where they are recognized by Ag-speciﬁc TCRs expressed true diploid (genome size approximately half that of human), X. by CD8ϩ T cells (3). Interestingly, in most mammals, the genes tropicalis has been selected as a model organism for a whole ge- responsible for class I Ag processing are embedded in the class II nome sequencing project (͗www.jgi.doe.gov/xenopus͘). BAC li- region (e.g. PSMB8, PSMB9, TAP1, and TAP2) or in the extended braries have been constructed and available to the public for anal- class II region (e.g., TAPBP, class I transcription regulator, RXRB), ysis and genetic manipulation. In addition, different sources of whereas class I genes themselves are found in another region (4). expressed sequences have been deposited into the expressed se- In contrast, studies of nonmammalian vertebrates have shown that quence tag (EST)3 databases for X. tropicalis and X. laevis, which class I genes are tightly linked to class I-processing genes, sug- facilitates gene annotation. In our previous studies of the Xenopus MHC in which we te- diously cloned the genes orthologous to those of humans one by *University of Maryland, Department of Microbiology and Immunology, 655 West one, it was shown that synteny seemed to be stable between the Baltimore Street, BRB13-009, Baltimore, MD 21201; and †Department of Biological two species separated by 350 million years (6, 10). This is in Sciences, Graduate School of Science, University ofTokyo, Hongo, Bunkyo-ku, To- kyo, Japan contrast to some other nonmammalian vertebrates in which the MHC genes are scattered over the genome, especially for class II Received for publication November 1, 2005. Accepted for publication January 9, 2005. and class III region genes (5, 7, 11–20). In this study, we took The costs of publication of this article were defrayed in part by the payment of page advantage of the genome project and the various EST databases charges. This article must therefore be hereby marked advertisement in accordance and mined them for MHC genes. Our results reveal that the entire with 18 U.S.C. Section 1734 solely to indicate this fact. architecture of the Xenopus MHC is remarkably conserved when 1 This work was supported by National Institutes of Health Grant AI27877 (to Y.O., compared with human, and further show that the teleost and, to a W.G., and M.F.F.) and Grant 15207019 from The Ministry of Education, Culture, Sports, Science, and Technology (to M.N.). 2 Address correspondence and reprint requests to Dr. Yuko Ohta, University of Mary- 3 Abbreviations used in this paper: EST, expressed sequence tag; Igsf, Ig superfamily; land, Department of Microbiology and Immunology, 655 West Baltimore Street, BLAST, Basic local alignment search tool; ORF, open reading frame; TM, trans- BRB13-009, Baltimore, MD 21201. E-mail address: [email protected] membrane; XMIV, Xenopus MHC-linked Ig superfamily.

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 3675 lesser extent, bird MHCs are highly derived. In addition, analysis with HindIII or SacI, and fragmented DNA was separated on an agarose gel of the Xenopus MHC has revealed that some major immune genes and blotted onto membranes. The DNA amount was increased proportion- seem to have emerged at the level of amphibians and has uncov- ally to the ploidy level. The gene-specific Ig-domain probe (EST entry CN328971; nt 300–587) was made by using PCR from cDNA library made ered some new Ig superfamily (Igsf) genes that are activating or from X. laevis spleen and intestine, and the sequence was confirmed. Prim- inhibitory receptor candidates, similar to those first discovered on ers used for amplification were as follows: 5Ј-AAA GTG GAA CAG CCT NK cells (21–23). GAG CG-3Ј and 5Ј-CAT CAC ATG CAC AAT GGT TCC-3Ј. Hybrid- ization was performed under low stringency conditions (30% formamide; Materials and Methods 6 ϫ SSC) at 42°C for overnight, and washed in 2 ϫ SSC, 1% SDS at room temperature, followed by 2 ϫ SSC, 0.1% SDS at 55°C (38). The same blot cDNA sequence database searches for MHC genes was later washed under high stringency conditions (0.2 ϫ SSC, 0.1% SDS We obtained accession numbers for genes listed in the human MHC, ex- at 65°C) to eliminate low-homology signals. cluding pseudogenes, from the Wellcome Trust Sanger Institute web site (͗www.sanger.ac.uk͘). Basic local alignment search tool (BLAST)p and Results tBLASTn were performed on the National Center for Bioinformatics In- stitute (NCBI) web site (͗www.ncbi.nlm.nih.gov͘) with either full-length Database mining amino acid sequences or domain-by-domain in the X. laevis, X. tropicalis, The chicken DM␣1 and ␤1-encoding exons (obtained from and/or EST_Others databases using the BLOSUM 45 matrix. Genes with AL023516) were used to search databases for the Xenopus DM E-values of Ͻ0.05 were further confirmed by BLASTp or BLASTx searches in the vertebrate databases using the BLOSUM 45 matrix. When genes; the deduced amino acid sequences of these regions of the no positive result was obtained, we further searched Xenopus EST data- bird sequences were found to be more specific for DM compared bases in the Wellcome Trust Sanger Institute using the BLOSUM 50 ␣ ␤

with their 2or 2 Igsf domains, which more readily selected Downloaded from matrix. classical class II sequences in BLAST searches. We and others (S. Data-mining the X. tropicalis genome project Beck, personal communication) have done exhaustive searches in the EST and genomic databases for teleost DM genes and could We began this study with BLASTn searches of X. tropicalis version 3.0 (estimated genomic coverage of 7.4ϫ) at the Department of Energy Joint not identify them, suggesting either that teleosts have lost the DM Genome Institute (JGI; ͗www.jgi.doe.gov/xenopus͘) with MHC genes that genes or they arose in the tetrapod lineage after its divergence from were isolated over the past 10 years (class I (24), class II (25, 26), TAP1 bony fish. The Xenopus sequences were used in a phylogenetic http://www.jimmunol.org/ (10), TAP2 (27), PSMB8 (28), PSMB9 (29), Ring3 (30), C4 (31), Factor B analysis, and the trees solidify the hypothesis that the DM class II (32), HSP70 (33), and RXRB (34, 35)). In most cases, X. laevis genes were genes are as old as classical class II␣ and class II␤ (Ref. 42 and used for the searches because most genes were cloned from this species, and we were fortunate that usually there is enough sequence similarity in Fig. 1). Thus, we think it is more likely that these genes have been coding regions between X. laevis and X. tropicalis to permit isolation of the lost in teleosts and will be found in the cartilaginous fish. orthologues across species. Most scaffolds were large enough to contain From the initial EST searches with human MHC genes (full- multiple genes, and thus we used various MHC candidate genes found in length amino acid sequences), we found most of the Xenopus the EST databases to screen other scaffolds containing the X. tropicalis orthologues of the human MHC genes. Individual scaffolds were then re- housekeeping genes with significant E-values. Each gene was fur- trieved from the JGI browser window, and all “fgenesh” entries and EST ther confirmed by BLASTx for their orthology. Using these genes

hits were examined manually. To confirm the gene annotation, we searched found in the EST databases, we then BLAST-searched the X. tropi- by guest on September 29, 2021 all predicted genes by BLASTx in the NCBI vertebrate database, using the calis scaffolds version 3.0 (͘www.jgi.doe.gov/xenopus͗). All scaf- BLOSUM 45 matrix. In cases when we did not find Xenopus genes in the folds so-identified were then inspected for open reading frames EST databases, we searched EST databases using reconstructed nucleotide sequences from the scaffolds. We tried to follow the nomenclature used in (ORF), which were manually verified and then used to rescreen the the map to the HUGO Gene Nomenclature Committee (36) and its GenBank database (see percentage of identities in Table I). During database (37). this process, we identified other genes on the scaffolds that were X. laevis cDNA library screening then used to screen the EST databases. A total of 122 ORFs were found on the eight genomic scaffolds We isolated two genes that have important roles in the mammalian immune (Tables I and II) encompassing 3.65 Mbp of which 110 genes were system. Probes were made from an EST entry for the partial DM␤ gene (BX845472) by PCR at nucleotide positions 63–331, from a X. laevis annotated that showed significant similarity to genes in the data- cDNA library made from mixture of spleen and intestine mRNA. The C2 bases. Twelve genes had no database match (denoted as ORF). At probe was made by PCR using primers taken from EST entry (BX853282) least one gene on each X. tropicalis scaffold shown in Fig. 2 has corresponding to nucleotide positions 42–462, from a X. laevis cDNA been rigorously analyzed for MHC linkage previously (24–33) library made from mixture of liver, spleen, and thymus mRNA (10). The FABGL DAXX PCR amplicons were cloned into the TA cloning vector (Invitrogen Life (Y. Ohta and M. F. Flajnik, unpublished data for , , Technologies) and sequenced. Both library screenings and washings were and FLOT1), and thus we are certain that all of these scaffolds are conducted under high stringency conditions (38). Positive clones were iso- in the Xenopus MHC. lated and sequenced in their entirety. The sequences are deposited to Gen- We could not decide by phylogenetic analyses whether DDAH ␤ Bank, and accession numbers are given as DQ268506 for X. laevis DM and NOTCH were orthologues of the human MHC-encoded and DQ268507 for X. laevis C2. DDAH2 and NOTCH4 or their paralogues found on human chro- Phylogenetic trees mosomes 1, 9, and 19 (5). Their location within the MHC makes The deduced DM␣ (EST clone, AAH61681) and DM␤ amino acid se- it likely that they are the orthologues of the MHC-encoded human quences were aligned using Clustal X, and Neighbor-Joining bootstrapping genes. Conversely, several genes were found in the Xenopus MHC trees (1000 trial runs) were made and viewed in the TreeView 1.6.6 pro- that are present on different chromosomes in the human, some- gram (39). The deduced X. laevis and X. tropicalis (reconstructed from times in paralogous regions (Table I and red numbered loci in Fig. scaffold) C2 amino acid sequences were also aligned with factor B and C2 of tetrapod species, bony and cartilaginous fish Bf/C2, whose assignment 2). This is likely due to differential silencing of genes after diver- to Bf or C2 is not clear, and lamprey and invertebrate Bf/C2 are considered gence from the common ancestor. These subjects are further de- to represent the preduplication Bf/C2 state (40, 41). For both trees gaps scribed and discussed in more detail below. were included, and multiple substitutions were not taken into account. Southern blotting Extended class II region Genomic DNA from different Xenopus species (2n–12n), or from siblings All 15 functional genes in the human extended class II region and in a family ( f/g ϫ f/r) with known MHC haplotypes (27), was digested 3 of 4 genes flanking this region were found in ϳ415- and 200-kb 3676 SYNTENIC INTEGRITY OF MHC THROUGH EVOLUTION

regions of two X. tropicalis scaffolds, respectively; the gene den- sity is ϳ24 kb/gene and ϳ40 kb/gene, respectively. The genes between RXRB and PHF1 are inverted but in the same order compared with the human MHC (4) (Fig. 2), implying an en bloc inversion. Because all genes in this inverted region are found on a single scaffold, it is unlikely to be an assembly artifact. By BLAST searching the end of scaffold 917 for contiguous scaffolds, we were able to connect scaffolds 917 and 726, covering over 1 Mbp genomic region linking the extended class II region to the class II␣ gene. In a later version of the genome assembly (version 4.0 and 4.1), these scaffolds are indeed connected (scaffold 396; see Table III). In summary, this region is remarkably well conserved between Xenopus and human.

Class II region and the specialized nonmammalian class I region We previously mapped class II␣, class II␤, Ring3 (BRD2), proteasome PSMB8 and PSMB9, and transporter TAP1 (ABCB2) and TAP2 (ABCB3) genes to the MHC by segregation analyses in X. Downloaded from laevis families (24–33). We now report the order of these genes in the class II region and the primordial class I region (Fig. 2). In addition, the nonclassical class II molecules, DM␣ and DM␤, were mapped into the class II region. The class II region (five genes, from classical class II␣ to DM␤) encompasses ϳ217 kb on scaffold 1109, whereas the class I region (five genes, from class Ia to TAP2)isϳ274 kb on scaffold 1316. From Southern blotting anal- http://www.jimmunol.org/ ysis, two class II␣ and class II␤ genes were found in X. tropicalis (L. Du Pasquier, personal communication). Class II␣ genes are split onto two scaffolds (exons 1 and 2 on 917 and 3 and 4 on 1109); however, it is likely that the presence of the two tandemly duplicated highly homologous genes obstructed a correct sequence assembly. So far, only one class II␤ gene was found on the scaffolds. However, the distance between class II␤ and class II␣ on the ϳ scaffold is 244 kb, seemingly too large compared with intergenic by guest on September 29, 2021 distances in other MHC regions. There are many repetitive elements and fragments of retrotransposons in this area, including 2 contigs that match perfectly to Magnetococcus sp. MC-1 sequences (AAAN03000014). Thus, this region seems to have been contaminated with sequences from other species (even in the version 4.0 scaffold), and thus we must wait to clarify the sequence and distance between the class II loci. PSMB9 is also split between two scaffolds (1109 and 1316); however, because there is only a single locus from Southern blotting analysis (29), these scaffolds FIGURE 1. Phylogenetic analysis of vertebrate MHC molecules dem- are within an intron length of each other. onstrates an ancient origin of DM. The extracellular domains for the full- A BTNL-II gene (butyrophilin-like MHC class II-associated), length EST for DM␣ (GenBank accession no. AAH61681; A) and our located at the border of the mammalian class II and class III re- full-length clone for DM␤ (ABB85336; B) were used in the construction of gions (43), was found neither in the EST databases nor the the trees. Genetic distance is shown as a bar on the bottom. Accession nos. genomic scaffolds. However, other BTN genes are found in the for each sequences are as follows: human DM␣ (AAH11447), mouse DM␣ human class I region. The BTN genes are Igsf members (44) that ␣ ␣ (NP_034516), rat DM (CAA89831), cow DM (BAA11171), chicken display notable sequence similarity to other MHC genes, particu- ␣ ␣ ␣ DM (CAA18966), quail DM (BAC82512), Xenopus DM larly to human MOG and bird B-G (13) (see below). (AAH61681), human DP␣ (NP_291032), human DQ␣ (NP_002113), human DR␣ (NP_061984), mouse IA␣ (AAB81529), rat II␣ (AAB35454), Xenopus DBAf2 (AAH57744), Xenopus DAAf1 (AAL58430), Caiman II␣ Class III region (AAF99282), catfish II␣ (AAD39871), zebrafish II␣ (NP_001007049), Forty-five human genes listed in the human class III region were ␤ ␤ nurse shark DAA (AAA49310), human DM (NP_002109), mouse DM 2 found on five scaffolds spanning ϳ2 Mbp (Fig. 2), suggesting that (A55242), rat DM␤ (CAA89832), cow DM␤ (BAA11172), rabbit DM␤ like the extended class II region, the class III region is old and (AAB53264), chicken DM␤1 (CAA18968), chicken DM␤2 (CAA18967), quail DM␤1 (BAC82514), quail DM␤2 (BAC82513), human DO␤ extremely well conserved. Because the NOTCH gene is in scaffold (AAA59717), mouse A␤2 (AAA51637), chicken II␤ (AAS00716), quail 1316, where the majority of genes are in the class I region, the II␤ (BAC82510), Xenopus II␤ (BAA02845), medaka II␤ (BAA94279), class III region is contiguous with the class I region. Like the catfish II␤ (AAB67871), carp II␤ (CAA64709), trout II␤ (AAD53026), extended class II region, there seems to have been at least three en shark II␤ (L20274), human A1 (NP_002107), mouse H2-K (AAA80451), bloc inversions between C4 and PPT2, STK19 and C6orf29, and rat RT1␣ (XP_579224), chicken B-F (CAA18972), Xenopus I␣ HSP70 and CSNK2B. There are also potential translocations (e.g., (AAA16064), nurse shark UAA (AAC60347). ATP6V1G2, BAT1, and NEU1). PCR was used to identify the gap between scaffolds 895 and 1207. A 0.8-kb fragment was sequenced The Journal of Immunology 3677

Table I. List of genes, scaffold numbers, and database accession no. found in the databases

Percentage of Gene Scaffold X. tropicalisa X. laevisa Identities/aab

Region ﬂanking the extended class II subregion ZBTB9 726 No hit BC087446 (F) 54/196 (Full) C6orf82 726 No hit BC078516 (F) 73/109 (Full) PHF1 726 No hit AF130453 (F) 54/571 (Full) Extended class II subregion KIFC 726 CR848323 (F) U82809 (F) AAB40402 DAXX 726 BX759743 BC079997 48/229 (Gen) ZNF297 726 No hit BJ042776 51/538 (Gen) TAPBP 726 No hit CF283998 31/193 (Part) RGL2 726 AL594530 No hit 41/734 (Gen) HKE2 726 AL633707 BC084766 (F) 75/156 (Full) C6org11 726 BX737105 BC077337 (F) 60/551 (Full) B3GALT4 726 No hit BP688393 38/186 (Gen) RPS18 726 AL960564 BC068873 (F) 98/131 (Full) VPS52 726 BX758938 BJ057872 72/621 (Gen) RING1 726 AL628421 BC081039 (F) Q66J69 HSD17B8 726 CR760242 (F) No Hit 67/251 (Full) SLC39A7 726 BX721639 BQ733936 73/161 (Gen) Downloaded from AL968958 RXRB 726 AL868786 BC073179 AAH73179 917 Col11A2 917 No hit CB942366 67/1476 (Gen) BF845697 Classical class II subregion

DO No hit No hit No hit NA http://www.jimmunol.org/ BRD2 1109 BX750317 U51449 AAB18943 DM␣ 1109 No hit BC061681 (F) 38/229 (Full) DM␤ 1109 No hit DQ268506 (F) 31/241 (Full) Class II␣ 917 CR760040 AF454378 (F) AAL58434 1109 CF524557 Class II␤ 917 BC087775 D13684 (F) BAA02841 PSMB9 1109 NM_001003660 D87687 (F) BAA19759 1316 TAP1 1316 No hit AY204552 (F) AAP36718 PSMB8 1316 AB033151 D44540 (F) BAA07945 TAP2 1316 No hit AY204554 (F) AAP36720 by guest on September 29, 2021 C6orf10 No hit No hit No hit NA Class III subregion NOTCH4 1316 No hit No hit 38/1426 (Gen) GPSM3 1026 No hit BP703429 53/62 (Gen) PBX2 1026 AL679273 BC071048 (F) 84/357 (Full) BX733561 RNF5 1026 AL848378 CA793548 (F) 80/120 (Full) AGPAT1 1026 AL959082 BC081085 (F) 71/265 (Full) BX758119 EGFL8 1026 CR761323 (F) No hit 42/281 (Full) PPT2 1207 No Hit BC059297 (F) 66/283 (Full) C6orf31 1026 BX730761 BC074389 64/225 (Gen) KFBPL 1026 BX741700 No hit 40/259 (Gen) CREBL1 1026 AL878690 No hit 53/402 (Gen) AL872993 TNXB 1026 No hit BX846582 40/1103 (Gen) CYP21A2 1026 No hit BC079793 (F) 45/460 (Full) C4 1026 No hit D78003 (F) BAA11188 STK19 895 AL866392 BC087397 (F) 45/266 (Full) AL680203 DOM3Z 895 CR761194 BC088900 (F) 53/392 (Full) SKIV2L 1207 AL648835 CV077949 56/341 (Gen) RDBP 1207 BX705721 No hit 78/135 (Gen) BF 1207 AL658338 D49373 (F) BAA08371 BQ519832 895 BX711550 C2 1207 BX739487 DQ268507 39/752 (Full) CV811291 ZBTB12 1207 BX772914 BC072114 (F) 58/468 (Full) BAT8 1207 AL882026 BX854837 69/558 (Gen) C6orf29 1207 AL656289 BC073678 (F) 67/703 (Full) AL867952 NEU1 895 BX730249 BC074166 (F) P12890 BX736306 HSP70 547 CX414897 X01102 (F) P02827 (Table continues) 3678 SYNTENIC INTEGRITY OF MHC THROUGH EVOLUTION

Table I. Continued

Percentage of Gene Scaffold X. tropicalisa X. laevisa Identities/aab

LSM2 547 BX753796 (F) BP708188 (F) AAH90606 VARS2 547 AL967562 BC084762 (F) 66/1211 (Full) BX730148 C6orf26 547 CF224182 AW644535 36/149 (Part) CF224183 MSH5 547 No hit BP691385 57/512 (Gen) CLIC1 547 BC059765 (F) AY277695 (F) AAH59765 DDAH2 547 BC075381 (F) BC078574 (F) 55/278 (Full) LY6G6C 547 BX687777 (F) No hit 31/100 (Full) LY6 547 AL958403 (F) No hit 30/123 (Full) BAT5 547 BC075571 (F) BC077872 (F) AAH75571 CSNK2B 547 BC077003 (F) BC083993 (F) 93/234 (Full) BAT4 895 BG487159 BJ045925 39/361 (Gen) APOM 895 No hit BC078609 45/188 (Gen) BAT3 895 BX764202 BC060479 (F) 51/1185 (Full) BG348638 BG486156 AL955051 Downloaded from AL966216 BAT2 895 BX698872 BC051009 (F) 37/2232 (Full) AL633000 AL792183 BX732414 BX716126

LTB 895 BX771103 BQ398178 34/152 (Gen) http://www.jimmunol.org/ TNF 895 No hit BU905532 35/207 (Gen) LTA 895 No hit No hit 38/159 (Gen) NFKBIL1 895 AL661130 BJ614008 34/392 (Gen) ATP6V1G2 895 No Hit CD254193 68/117 (Gen) BAT1 895 BC061280 (F) BC084004 (F) AAH61280 Classical and extended class I subregion POU5F1 No hit No hit No hit NA TCF19 547 BX769629 AW782810 40/332 (Gen) C6orf18 547 AL646254 BF613095 32/712 (Gen) DDR1 547 No hit BC060755 (F) 60/930 (Full) FLOT1 547 AL866886 AF545659 (F) AAN86277 by guest on September 29, 2021 CR760749 TUBB 547 BC074549 (F) BC049004 (F) AAH75549 MDC1 547 AL969933 BP681667 44/246 (Gen) BX752964 BX779269 NRM 547 No hit BC073486 (F) 53/253 (Full) KIAA1949 547 BX738291 BJ636557 48/145 (Gen) DHX16 547 AL632457 BP709014 89/529 (Gen) BX696867 C6orf136 547 BC087624 (F) No hit 45/204 (Full) C6orf134 547 No hit BC089285 (F) 55/429 (Full) MRPS18B 547 BX740904 BP705701 55/201 (Gen) PPP1R10 547 AL633731 BC074405 (F) Q6GLQ4 AL871337 AL629506 AL628261 ABCF1 547 BX44344 BC081034 (F) 87/600 (Full) GNL1 547 No hit BJ614782 61/618 (Gen) GABBR1 726 BX727166 No hit 78/629 (Gen) BX731212 Olfactory receptor 726 No hit No hit 49/291 (Gen) Olfactory receptor 726 No hit No hit 48/309 (Gen)

Human chromosome Percentage of Gene Scaffold no. X. tropicalisa X. laevisa Identities/aab

Genes not found in the human MHC, but in the Xenopus scaffoldsa KIAA1720 726 1 AL774574 BC084800 (F) 62/398 (Full) 408 ribosomal protein S5 726 X BX718023 BC054263 (F) 96/203 (Full) CR848217 RASA1 726 5 No Hit CD253980 70/708 (Gen) HGMA1L1 726 X BX741519 BX849417 43/91 (Gen) Thymopoietin 917 12 No hit No hit 31/107 (Gen) MAP1 1109 14 CX40492 BC073201 26/361 (Gen) Carnitine O-acetyltransferase 1026 9 BC063356 (F) BC072849 (F) 50/587 (Full) (Table continues) The Journal of Immunology 3679

Table I. Continued

Human chromosome Percentage of Gene Scaffold no. X. tropicalisa X. laevisa Identities/aab

CENPA 1026 2 AL863713 BC092389 (F) 67/90 (Full) PSMB10 1026 16 No hit BC056039 (F) 62/276 (Full) PDE4DIP 1207 1 AL633679 No Hit 22/350 (Gen) BX700846 Similar to CTGF 547 6 (not MHC) No hit BG234029 40/132 (Gen) NPDC1-like 547 9 No hit No hit 30/120 (Gen) RGS3 895 9 No hit CA788699 36/176 (Gen)

Scaffold X. tropicalis X. laevis

Unknown genes (ORF) found in the Xenopus scaffolds 726 AL635951 No hit 726 No hit No hit 726 BX751117 No hit 1109 No hit CD099547 BP700625 1109 BX688173 No hit Downloaded from 1316 AL963961 No hit 547 BX781893 No hit 547 BX717682 No hit 547 No hit No hit 895 AL868311 No hit 895 No hit CB942034

895 No hit No hit http://www.jimmunol.org/

a Percentage of Full-length DNA sequences are noted as (F). b Percentage of amino acid identities are shown in this Table over the matching region. We used the longest and more reliable sequences, either partial cDNA, or genomic sequences retrieved from scaffolds, or full-length cDNA sequences when available. The query sequences used for this column were as follows: full-length cDNA sequences (Full), partial-length cDNA sequences (Part), retrieved genomic sequences (Gen). Accession no. are shown when the sequences were annotated. NA, Not applicable. from both ends, confirming that the gap between scaffolds 895 and characteristic. We await studies of the elasmobranch class I region 1207 is short and contains a single intron of a factor B gene. to elucidate the original location of PSMB10. In the MHC of all teleosts so far studied, the three (or more) We found a gene similar to the complement C2 gene in the by guest on September 29, 2021 immunoproteasome genes are tightly linked to class I genes (Refs. scaffold 1207 near the factor B gene. Phylogenetic analysis 18, 20, 45–47; also see Fig. 5), and thus we were surprised to find strongly supports that the gene is more similar to C2 than to factor the third immunoproteasome gene PSMB10 in the class III region. B (Fig. 3). In teleost fish, the Bf/C2 genes are often duplicated, and Because teleost MHC genes are found in many linkage groups and one of these genes encodes a protein that, like C2, functions in the spread onto different chromosomes, PSMB10 consequently may classical complement pathway (48, 52–54). However, upon phy- have remained in the class I region in bony fish as a result of logenetic tree analysis, most of the teleost genes form a monophy- translocation of ancestral class III region genes out of the MHC (12, 15, 48) and coevolution via “functional clustering” of immu- letic cluster independent of tetrapod Bf and C2 clusters. Thus, noproteasome and class I genes (49–51). In contrast, because most whereas it is still not clear whether the Bf/C2 gene duplication and genes have maintained their ancient synteny in the Xenopus MHC, functional differentiation predated the emergence of teleosts, our it is likely that early in evolution PSMB10 indeed was located in data clearly demonstrate that the Bf/C2 duplication predated the the class III region. Alternatively, PSMB10 translocated out of the appearance of amphibians. The three TNF members encoded in class I region in Xenopus, and its present location is a derived the human class III region (LTA, LTB, and TNF) are found in the

Table II. Genes found in the Xenopus MHC

Number Category of Genes Genes

Ag processing/presentation 11 Class I, class II␣, class II␤,DM␣,DM␤, PSMB8, PSMB9, PSMB10, TAP1, TAP2, TAPBP Inﬂammation 5 ABCF1, DAXX, LTA, LTB, TNF␣ Leukocyte maturation 3 DDAH1/2, LY6, LY6G6C Complement 3 BF, C2, C4 Immune regulation 3 NFKBIL1, RXRB, FKBPL Stress response 1 HSP70 Ig superfamily 6 XMIV Olfactory receptors 2 Nonimmune genes 64 Genes not found in the human MHC 13 PSMB10 and others Unknown genes 12 ORF Total number of genes 122 3680 SYNTENIC INTEGRITY OF MHC THROUGH EVOLUTION

Table III. Update on scaffold assembly

Scaffolds

Version 3.0 Version 4.0 726 (656,544 bp) 396 (1,113,890 bp) 917 (462,912 bp) 396 (1,113,890 bp) 1109 (310,645 bp) 895 (310,644 bp) 1316 (205,920 bp) 1038 (205,919 bp) 1026 (374,626 bp) 744 (484,856 bp) 1207 (255,515 bp) 744 (484,856 bp) 1207 (255,515 bp) 1175 (141,455 bp) 895 (482,803 bp) 752 (471,867 bp) 547 (899,709 bp) 488 (901,819 bp)

Xenopus MHC, suggesting that the synteny is old, and that non- MHC linkage of the teleost TNF family members is a derived feature (55, 56), like most of the other class III genes in this phylogenetic group.

Of greatest interest to us, there is a cluster of related genes Downloaded from residing on the edge of the two scaffolds 547 and 895 (Fig. 2 and Table II). From the order, positions, and similarity of these genes, we predict that scaffolds 547 and 895 are tightly linked; however, BLAST searches using end sequences failed to unite them. The conserved cysteines (C), tryptophan (W), and spacing between the

deduced amino acid sequences mark these genes as Igsf members, http://www.jimmunol.org/ and the GXG motif in the G-strand suggests that these sequences could be V domains in the Ag receptor family (Fig. 4A), thus we named them XMIV (Xenopus MHC-linked Ig superfamily V genes). In fact, BLAST searches usually selected TCR or IgNAR V domains (58) as the most similar sequences, although with low sequence identity (data not shown). The putative expressed sequences were pieced together from the genomic exons. Some of the XMIV genes seem to contain cytoplasmic tails followed by one or two ITIMs (shaded in Fig. 4A) (22, 23), suggesting these XMIV by guest on September 29, 2021 molecules are inhibitory receptors. Other genes have positively charged amino acids in their transmembrane (TM) regions, suggesting that they could interact with ITAM-containing adaptors (21); however, these lysine (K) and/or arginine (R) residues are usually located centrally in the TM in activating receptors. One to four additional cysteine residues found in the IgSF domains in the XMIV may form intra- and/or inter-chain disulfide bridges (boxed in Fig. 4A). Unfortunately, our EST database searches resulted in only one full-length entry (CN328971) for these XMIV genes, in X. laevis (Fig. 4A). To confirm that CN328971 is indeed the X. laevis counterpart of the X. tropicalis genes, we performed Southern blotting on a family with known MHC haplotypes (27). In all 20 siblings, restriction fragment length polymorphism for CN328971 matched perfectly to the known MHC haplotypes in the family (Fig. 4B). In the human MHC, NKp30 is found in the location where XMIV genes are present in the Xenopus MHC (see NCR3 in Fig. 2). However, we found multiple genes that showed significant similarity to human NKp30 in other scaffolds, and these XMIV genes are not similar to NKp30 (amino acid identity Ͻ22–26%), as

scaffolds on both sides. Subregions of the human MHC are color-coded: area ﬂanking to the extended class II region, gray; extended class II region, pink; class II region, blue; class III region, peach; class I region, green; extended class I region, yellow. Gene symbols were followed as assigned by the HUGO Gene Nomenclature Committee and ImMunoGeneTics/HLA Sequence Databases. Xenopus genes found outside the human MHC are marked as green boxes, with the human chromosome numbers listed as red FIGURE 2. Comparison of the X. tropicalis MHC to the human MHC superscripts. Igsf genes unique to the Xenopus MHC on scaffolds 547 and (modiﬁed from Ref. 4) reveals extraordinary conservation. Partial human 895 are shown in red boxes. Transcriptional orientations are indicated as MHC genes are listed as the template in the center and the Xenopus MHC gradients and arrows on the right bottom. The Journal of Immunology 3681

EST databases (BC073304, BC074259) when screening with the human gene (E-value of ϽeϪ39). Xenopus C9orf58 gene is on scaffold 191 and linked to other genes encoded on human chromosome 9. Thus, owing to differential silencing after the en bloc duplications early in vertebrate evolution (59), Xenopus AIF1 was shut down in the MHC, whereas functional human AIF1 and C9orf58 genes are on chromosomes 6 and 9, respectively.

Genes in the mammalian class I region The human class I region designation cannot be applied to nonmammalian species, because class I genes are embedded within the class II region closely linked to the immunoproteasome and transporter genes (Figs. 2 and 5). Despite the absence of class I genes in this region, we found 15 Xenopus genes orthologous to the human genes, including TUBB and FLOT1, which are also located in the teleost MHC (linked to the teleost class I region) (Fig. 5). Thus, the architecture/framework of the extant mammalian class I region pre-existed 450 million years ago and appears stable over Downloaded from evolutionary time; class I genes were translocated from the true class I region and expanded in the modern class I region in the mammalian lineage, as previously proposed (6). GABBR1 and two olfactory genes on scaffold 726 are found outside of the extended class II region, suggesting a reorganization of the genes either in an ancestor of Xenopus or human. http://www.jimmunol.org/ No MOG-containing Igsf domains were found in scaffolds 726 and 547, consistent with the fact that we did not ﬁnd any other Igsf-containing human homologues such as AGER, C6orf25,or BTN in any region of the MHC. However, when we extended our analysis to the Xenopus nonclassical class I (XNC) genes (60), which are located at the telomere of the same arm of the chromosome as MHC (which is near the centromere), a cluster of BTN FIGURE 3. Phylogenetic tree of Factor B and C2 sequences identiﬁes

genes was indeed identified, near to the XNC genes (data not by guest on September 29, 2021 Xenopus C2. The tree was constructed by the Neighbor-Joining Method based on an alignment of amino acid sequences using Clustal X version shown). These data demonstrate that the class I-BTN association 1.81. Numbers indicate the bootstrap values, supporting the depicted par- is old. titioning from 1000 trials. Genetic distance is shown as a bar on the bottom. Abbreviations and accession nos. for each sequence are as follows: Hosa Bf, Homo sapiens Bf (P00751); Hosa C2 (AAB97607); Mumu Bf, Mus Categories of genes in the Xenopus MHC musculus Bf (NM_008198); Mumu C2 (NM_013484); Xela BfA, X. laevis Next, we classified genes found in the Xenopus MHC by their BfA (BAA06179); Xela BfB (BAA08371); Xela C2, X. laevis C2 functions (Table II). We found genes belonging to each category as (ABB85337); Omny Bf-1, Oncorhynchus mykiss Bf-1 (AAC83699); Omny detailed in the human MHC such as those involved in the follow- Bf-2 (AAC83698); Orla Bf/C2, Oryzias latipes Bf/C2 (BAA12207); Dare Bf, Danio rerio Bf (NP_571413); Cyca Bf/C2B, Cyprinus carpio Bf/C2B ing: Ag processing for class I and class II molecules, inflammation, (BAA34707); Cyca Bf/C2-A3 (BAB32650); Trsc Bf, Triakis scyllium Bf leukocyte maturation, complement, immune regulation, Igsf, and (BAB63203); Leja Bf, Lethenteron japonicum Bf (I50807); and Stpu Bf, heat shock protein (HSP). Again, the overall MHC architecture is Strongylocentrotus purpuratus Bf (AAC79682). well conserved. In the human MHC, ϳ28% of the expressed tran- scripts are potentially associated with immunity (4). In the Xeno- pus MHC, 32 genes (26.2%) fall into this category, also quite mentioned above. It is possible that the scaffold containing Xeno- similar to that of human. Thirteen genes were found in Xenopus pus NKp30 (data not shown) could be between scaffolds 547 and that are not in the human MHC, five of which are encoded on 895, although it seems unlikely because this would be too large of MHC paralogous regions: KIAA1720 and ODE4DIP on human a disruption (ϳ2 Mb) in the midst of the other densely packed chromosome 1, and RGS3, Carnitine acetyltransferase, and class III genes. Furthermore, searches of the X. tropicalis genome NPDC1-like on human chromosome 9. As described above for with Igsf domains only selected these MHC scaffolds, suggesting AIF1, the likely explanation for this finding is differential silencing that all XMIV members are in the MHC. All of the Xenopus species (in these cases) on the MHC paralogous regions in Xenopus. Fur- (2n–12n) have multiple copies of XMIV genes, with no obvious thermore, as mentioned above PSMB10 is found in the Xenopus increase in gene numbers in the higher-order polyploids (Fig. 4C), MHC, whereas it is located in humans on chromosome 16. Previ- like for many other immune genes (Ref. 58 and unpublished data). ous work in teleosts suggested that PSMB10 was originally located The following class III genes were not detected on the scaffolds in the MHC class I region, and subsequently translocated onto a or in the EST databases: AGER, C6orf48, C6orf27, C6orf25, separate chromosome in the mammalian genome (51). Interest- LY6G6E, LY6G6D, LY6G5C, LY6G5B, C6orf47, and LST1. The ingly, we found carnitine acetyltransferase in the vicinity of the LY6 family members in human seem to be derived from recent constitutive proteasome subunit and direct homologue of PSMB10 duplications and thus would not be expected to be found in Xe- and PSMB7, further supporting the idea that PSMB8,-9, and -10 nopus. The AIF1 paralogue, C9orf58, was found in the X. laevis arose from duplication of PSMB5,-6, and -7, with subsequent 3682 SYNTENIC INTEGRITY OF MHC THROUGH EVOLUTION

FIGURE 4. A, Deduced amino acid alignment of XMIV genes found in the X. tropicalis scaffolds 547 and 895, and an entry found in the X. laevis EST database Downloaded from CN328971. Sequence numbers correlate with the scaffold and location. ORFs for XMIV1 and -4 contain two Igsf domains and are designated as Ϫ1 and Ϫ2. Evo- lutionary conserved amino acids, ITIM, and positively charged residues in the

TM and cytoplasmic (CYT) regions are http://www.jimmunol.org/ highlighted in gray, and extra cycteins are boxed. The X. tropicalis sequences were pieced together from exons on the two scaffolds. B, Linkage of the X. laevis gene (CN328971) to the Xenopus MHC. A X. laevis family (f/g ϫ f/r) with known MHC haplotypes (f, g, r) was used for the linkage analysis. The father is indicated as P, and siblings are indicated with by guest on September 29, 2021 numbers. The previously determined MHC haplotypes are shown above the blot (27). Haplotype-speciﬁc bands are shown as arrows on the left. C, Novel MHC-linked XMIV members are present in other polyploid Xenopus species. Southern blotting with the Igsf domain of CN328971 (X. laevis) probe was performed under low stringency conditions. Ploidy levels are noted underneath the blot. The Journal of Immunology 3683

can be explained as differential subfunctionalization or neofunc- tionalization of the genes between human and Xenopus over evolutionary time. For these reasons, each scaffold was carefully examined by eye, and the final decision was made in most cases when the linkage was conserved in the vicinity of the gene in question. For example, TNXB is in MHC, and TNC is in paralogous regions of chromosome 9; searching the genomic database with TNC resulted in selection of a scaffold that does not contain genes orthologous to MHC, whereas we found C4 and PSMB10 genes closely linked to TNXB. Because of the highly conserved synteny and relatively large scaffolds, we were able to distinguish MHC scaffolds from paralogous scaffolds. An additional point to be emphasized is that the scaffolds have been assembled automatically, and although the standard of the assembly is high, the assembly is incomplete and perhaps incorrect in some places. From our previous family analyses, we identified two class II␣ and two class II␤ genes in X. tropicalis (personal Downloaded from observation), but our searches did not select other scaffolds. In addition, this region contains highly repetitive and transposable elements (and, unfortunately, an artifact), making assembly difficult. Previously, we found multiple MHC-linked HSP70 genes (unpublished data), but we only found one in these scaffolds, sug-

gesting that the sequence may not be entirely accurate in these http://www.jimmunol.org/ regions. It is possible, or even likely, that regions containing some repeats are biased or difficult to sequence and/or assemble. During the writing of this manuscript new versions of the ge- FIGURE 5. Genomic organization in the Xenopus class I region compared nome assembly were released (version 4.0 and 4.1, coverage 7.65 with the medaka (47) and the chicken (13) MHCs. Transcriptional orientations genome equivalents). The sizes and general organization are al- are indicated on each side of the center bars (arrows at bottom right). Boxes noting class Ia genes and pseudogenes are p and f, respectively. most identical with the previous version except that two scaffolds were connected (Table III). From our previous work using recombinant X. laevis,weor- translocation of PSMB5 and -6 (51). Synteny of PSMB10 and car- dered the nonmammalian class I region as class II, TAP/LMP, class by guest on September 29, 2021 nitine acetyltransferase in the Xenopus MHC suggests that differ- I/C4 (6, 10). However, as shown in Fig. 2, the order of the genes ential silencing resulted from the presence of PSMB7-carnitine in the X. tropicalis scaffolds is class II, class I, TAP/LMP, C4. acetyltransferase in the primordial MHC. Two olfactory genes Again, this could either be due to an assembly error in X. tropicalis were found, but it is not clear whether these genes are orthologous or alternatively to genomic re-organization that happened during to those in the human MHC. There are 12 unknown genes of which tetraploidization. The frog used for the scaffold assembly was het- nine genes are found in the EST database, suggesting functional erozygous for the two class I region lineages that are found in all genes. of the Xenopus species (10, 64). We have found that the lineages of class I/PSMB8/TAP1/TAP2 are always found within a set in Discussion wild-caught animals (10), suggesting that there is a block in re- Genes involved in immune responses evolve rapidly, likely to combination between genes in these lineages, perhaps because of combat rapidly evolving or emerging pathogens (61, 62). This major sequence modifications in noncoding regions, recently rapid evolution of immune genes can be obstructive when pursuing shown to be true in medaka (65). Unfortunately, the assembly was orthologous genes in divergent species; BLAST searches of the complete for only one of the lineages (lineage A) in this particular genome using amino acid sequences from other species often re- region, so we will have to wait to test this hypothesis. sulted in “no hit.” Sometimes, even using the relatively closely There are two large clusters of histone and tRNA genes in the related X. laevis did not result in identification of the X. tropicalis human extended class I subregion. It is proposed that the MHC sequences. By contrast, searches using genes in large families such as TAP1 and TAP2 resulted in too many hits because the conserved may have hitchhiked with these clusters (or vice versa) to maxi- ATP-binding domain selects other ABC transporter genes as well. mize transcriptional activity (4). Unfortunately, our analysis of Xe- Similar problems were observed with the paralogous genes. Ver- nopus MHC did not include this extended class I region to examine tebrate genome analysis had revealed that there are three other whether the cluster of histone and tRNA genes is an evolutionary gene complexes similar to the MHC (63). We were usually suc- conserved feature of MHC. We await future versions of the ge- cessful in identifying which of the paralogues was orthologous to nome project to examine this question. the human MHC counterpart, but sometimes it was not clear. For In the vicinity of the human MHC, there are 34 olfactory-re- example, we could not identify Xenopus DDAH as either DDAH2 ceptor loci, 14 of which are potentially functional. Sperm-ex- (human MHC) or DDAH1 (chromosome 9), and thus named the pressed olfactory-receptor genes may be functionally involved in Xenopus clone DDAH1/2 in Fig. 2. This was true of NOTCH as the selection of spermatozoa by the female (sperm receptor selec- well, where different exons seemed to be orthologous to the dif- tion hypothesis), as well as many other functions (66). Thus far, we ferent NOTCH paralogues; therefore, we could not confidently have found two olfactory receptors in the MHC and XNC scaffolds identify the MHC-encoded gene as NOTCH4. These phenomena (data not shown). Therefore, it will be interesting to determine 3684 SYNTENIC INTEGRITY OF MHC THROUGH EVOLUTION whether there are larger clusters of olfactory genes near the Xe- 6. Nonaka, M., C. Namikawa, Y. Kato, M. Sasaki, L. Salter-Cid, and M. F. Flajnik. nopus MHC genes and determine their tissue and ontogenic 1997. Major histocompatibility complex gene mapping in the amphibian Xenopus implies a primordial organization. Proc. Natl. Acad. Sci. USA 94: 5789–5791. distribution. 7. Kelley, J., L. Walter, and J. Trowsdale. 2005. 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somal duplication of major histocompatibility complex class I regions in rainbow by guest on September 29, 2021 pensed altogether with immunoproteasome genes and perhaps trout (Oncorhynchus mykiss), a species with a presumably recent tetraploid another housekeeping genes. In contrast, this current study (and our cestry. Immunogenetics 56: 878–893. previous predictions) has shown that Xenopus is much similar to 21. Reth, M. 1989. Antigen receptor tail clue. Nature 338: 383–384. 22. Long, E. O., D. N. Burshtyn, W. P. Clark, M. Peruzzi, S. Rajagopalan, S. Rojo, human and allows for an understanding of the common MHC an- N. Wagtmann, and C. C. Winter. 1997. Killer cell inhibitory receptors: diversity, cestor, at least at the level of amphibian emergence. By contrast, specificity, and function. Immunol. Rev. 155: 135–144. the mammalian MHC has a peculiar organization in which the 23. Vivier, E., and M. Daeron. 1997. Immunoreceptor tyrosine-based inhibition motifs. Immunol. Today 18: 286–291. class I genes are not closely linked to TAP and PSMB genes. 24. Shum, B. P., D. Avila, L. Du Pasquier, M. Kasahara, and M. F. Flajnik. 1993. The loss of this linkage seems to be accompanied by the loss of the Isolation of a classical MHC class I cDNA from an amphibian: evidence for only genetic dimorphism of the linked class I/PSMB/TAP genes observed one class I locus in the Xenopus MHC. J. Immunol. 151: 5376–5386. 25. Sato, K., M. F. Flajnik, L. Du Pasquier, M. Katagiri, and M. Kasahara. 1993. in amphibian, teleosts, and cartilaginous fish (10, 64, 65, 73). Thus, Evolution of the MHC: isolation of class II ␤-chain cDNA clones from the am- the amphibian seems to be the best model to study evolution of the phibian Xenopus laevis. J. Immunol. 150: 2831–2843. vertebrate MHC. 26. Liu, Y., M. Kasahara, L. L. Rumfelt, and M. F. Flajnik. 2002. Xenopus class II A genes: studies of genetics, polymorphism, and expression. Dev. Comp. Immu- nol. 26: 735–750. 27. Ohta, Y., S. J. Powis, W. J. Coadwell, D. E. Haliniewski, Y. Liu, H. Li, and Acknowledgments M. F. Flajnik. 1999. Identification and genetic mapping of Xenopus TAP2 genes. We thank Louis Du Pasquier for discussions about the data. Immunogenetics 49: 171–182. 28. Namikawa, C., L. Salter-Cid, M. F. Flajnik, Y. Kato, M. Nonaka, and M. Sasaki. 1995. Isolation of Xenopus LMP-7 homologues: striking allelic diversity and Disclosures linkage to MHC. J. Immunol. 155: 1964–1971. The authors have no financial conflict of interest. 29. Nonaka, M., C. Namikawa-Yamada, M. Sasaki, L. Salter-Cid, and M. F. Flajnik. 1997. Evolution of proteasome subunits delta and LMP2: complementary DNA cloning and linkage analysis with MHC in lower vertebrates. J. Immunol. 159: 734–740. References 30. Salter-Cid, L., L. Du Pasquier, and M. Flajnik. 1996. RING3 is linked to the 1. Kumanovics, A., T. Takada, and K. F. Lindahl. 2003. Genomic organization of Xenopus major histocompatibility complex. Immunogenetics 44: 397–399. the mammalian MHC. Annu. Rev. Immunol. 21: 629–657. 31. Mo, R., Y. Kato, M. Nonaka, K. Nakayama, and M. Takahashi. 1996. Fourth 2. Driscoll, J., M. G. Brown, D. Finley, and J. J. Monaco. 1993. MHC-linked LMP component of Xenopus laevis complement: cDNA cloning and linkage analysis of gene products specifically alter peptidase activities of the proteasome. Nature the frog MHC. Immunogenetics 43: 360–369. 365: 262–264. 32. Kato, Y., L. Salter-Cid, M. F. Flajnik, M. Kasahara, C. Namikawa, M. Sasaki, 3. Antoniou, A. N., S. J. Powis, and T. Elliott. 2003. Assembly and export of MHC and M. Nonaka. 1994. Isolation of the Xenopus complement factor B comple- class I peptide ligands. Curr. Opin. Immunol. 15: 75–81. mentary DNA and linkage of the gene to the frog MHC. J. Immunol. 153: 4. Horton, R., L. Wilming, V. Rand, R. C. Lovering, E. A. Bruford, V. K. Khodiyar, 4546–4554. M. J. Lush, S. Povey, C. C. Talbot, Jr., M. W. Wright, et al. 2004. Gene map of 33. Salter-Cid, L., M. Kasahara, and M. F. Flajnik. 1994. Hsp70 genes are linked to the extended human MHC. Nat. Rev. Genet. 5: 889–899. the Xenopus major histocompatibility complex. Immunogenetics 39: 1–7. 5. Flajnik, M. F., and M. Kasahara. 2001. Comparative genomics of the MHC: 34. Marklew, S., D. P. Smith, C. S. Mason, and R. W. Old. 1994. Isolation of a novel glimpses into the evolution of the adaptive immune system. Immunity 15: RXR from Xenopus that most closely resembles mammalian RXR ␤ and is ex- 351–362. pressed throughout early development Biochim. Biophys. Acta 1218: 267–272. The Journal of Immunology 3685

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