Download Xenopus in the Amphibian Ancestral Organization of the MHC

Ancestral Organization of the MHC Revealed
in the Amphibian Xenopus
This information is current as
of June 16, 2017.
Subscription
Permissions
Email Alerts
J Immunol 2006; 176:3674-3685; ;
doi: 10.4049/jimmunol.176.6.3674
http://www.jimmunol.org/content/176/6/3674
This article cites 70 articles, 23 of which you can access for free at:
http://www.jimmunol.org/content/176/6/3674.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
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.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
References
Yuko Ohta, Wilfried Goetz, M. Zulfiquer Hossain, Masaru
Nonaka and Martin F. Flajnik
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*
T
he MHC is the most gene-dense region in the human genome and plays an indispensable role in the adaptive immune system (1). Class I and class II Ag-presenting molecules present small peptides derived from pathogens to CD8⫹ and
CD4⫹ T cells, respectively. In the class I system, endogenous peptides derived from intracellular pathogens are enzymatically
cleaved into small peptides by the immunoproteasome containing
the specialized ␤-subunits PSMB8, PSMB9, and PSMB10, which
upon infection replace the constitutive subunits, PSMB5, PSMB6,
and PSMB7, respectively (2). Short peptides of 8 –11 aas are transported into endoplasmic reticulum by the TAP (TAP1 and TAP2)
and then loaded onto class I molecules associated with tapasin
(TAPBP). The resulting class I-peptide complexes move to the cell
surface, where they are recognized by Ag-specific TCRs expressed
by CD8⫹ T cells (3). Interestingly, in most mammals, the genes
responsible for class I Ag processing are embedded in the class II
region (e.g. PSMB8, PSMB9, TAP1, and TAP2) or in the extended
class II region (e.g., TAPBP, class I transcription regulator, RXRB),
whereas class I genes themselves are found in another region (4).
In contrast, studies of nonmammalian vertebrates have shown that
class I genes are tightly linked to class I-processing genes, sug-
*University of Maryland, Department of Microbiology and Immunology, 655 West
Baltimore Street, BRB13-009, Baltimore, MD 21201; and †Department of Biological
Sciences, Graduate School of Science, University ofTokyo, Hongo, Bunkyo-ku, Tokyo, Japan
Received for publication November 1, 2005. Accepted for publication January
9, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grant AI27877 (to Y.O.,
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 Maryland, Department of Microbiology and Immunology, 655 West Baltimore Street,
BRB13-009, Baltimore, MD 21201. E-mail address: [email protected]
Copyright © 2006 by The American Association of Immunologists, Inc.
gesting that this class I region is the primordial organization (5–7).
In some nonmammalian species, there is only a single or few classical class I genes, perhaps due to a selection for coevolution with
the Ag-processing genes. Thus, plasticity of class I genes in mammalian species is an evolutionarily derived characteristic (5, 7).
Xenopus (especially Xenopus laevis and more recently Xenopus
tropicalis) has been used historically for developmental studies
(8). Regarding the MHC, this animal is the most comprehensively
studied amphibian for characteristics of the adaptive immune system. Xenopus is a unique model because there are several
polyploid species (2n–12n) within the genus that arose by recent
genome-wide duplication (from 2 to 30 million years ago) (9).
Because of its important phylogenetic position, and because it is a
true diploid (genome size approximately half that of human), X.
tropicalis has been selected as a model organism for a whole genome sequencing project (具www.jgi.doe.gov/xenopus典). BAC libraries have been constructed and available to the public for analysis and genetic manipulation. In addition, different sources of
expressed sequences have been deposited into the expressed sequence tag (EST)3 databases for X. tropicalis and X. laevis, which
facilitates gene annotation.
In our previous studies of the Xenopus MHC in which we tediously cloned the genes orthologous to those of humans one by
one, it was shown that synteny seemed to be stable between the
two species separated by 350 million years (6, 10). This is in
contrast to some other nonmammalian vertebrates in which the
MHC genes are scattered over the genome, especially for class II
and class III region genes (5, 7, 11–20). In this study, we took
advantage of the genome project and the various EST databases
and mined them for MHC genes. Our results reveal that the entire
architecture of the Xenopus MHC is remarkably conserved when
compared with human, and further show that the teleost and, to a
3
Abbreviations used in this paper: EST, expressed sequence tag; Igsf, Ig superfamily;
BLAST, Basic local alignment search tool; ORF, open reading frame; TM, transmembrane; XMIV, Xenopus MHC-linked Ig superfamily.
0022-1767/06/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
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
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.
The Journal of Immunology
lesser extent, bird MHCs are highly derived. In addition, analysis
of the Xenopus MHC has revealed that some major immune genes
seem to have emerged at the level of amphibians and has uncovered some new Ig superfamily (Igsf) genes that are activating or
inhibitory receptor candidates, similar to those first discovered on
NK cells (21–23).
Materials and Methods
cDNA sequence database searches for MHC genes
We obtained accession numbers for genes listed in the human MHC, excluding pseudogenes, from the Wellcome Trust Sanger Institute web site
(具www.sanger.ac.uk典). Basic local alignment search tool (BLAST)p and
tBLASTn were performed on the National Center for Bioinformatics Institute (NCBI) web site (具www.ncbi.nlm.nih.gov典) with either full-length
amino acid sequences or domain-by-domain in the X. laevis, X. tropicalis,
and/or EST_Others databases using the BLOSUM 45 matrix. Genes with
E-values of ⬍0.05 were further confirmed by BLASTp or BLASTx
searches in the vertebrate databases using the BLOSUM 45 matrix. When
no positive result was obtained, we further searched Xenopus EST databases in the Wellcome Trust Sanger Institute using the BLOSUM 50
matrix.
We began this study with BLASTn searches of X. tropicalis version 3.0
(estimated genomic coverage of 7.4⫻) at the Department of Energy Joint
Genome Institute (JGI; 具www.jgi.doe.gov/xenopus典) with MHC genes that
were isolated over the past 10 years (class I (24), class II (25, 26), TAP1
(10), TAP2 (27), PSMB8 (28), PSMB9 (29), Ring3 (30), C4 (31), Factor B
(32), HSP70 (33), and RXRB (34, 35)). In most cases, X. laevis genes were
used for the searches because most genes were cloned from this species,
and we were fortunate that usually there is enough sequence similarity in
coding regions between X. laevis and X. tropicalis to permit isolation of the
orthologues across species. Most scaffolds were large enough to contain
multiple genes, and thus we used various MHC candidate genes found in
the EST databases to screen other scaffolds containing the X. tropicalis
orthologues of the human MHC genes. Individual scaffolds were then retrieved from the JGI browser window, and all “fgenesh” entries and EST
hits were examined manually. To confirm the gene annotation, we searched
all predicted genes by BLASTx in the NCBI vertebrate database, using the
BLOSUM 45 matrix. In cases when we did not find Xenopus genes in the
EST databases, we searched EST databases using reconstructed nucleotide
sequences from the scaffolds. We tried to follow the nomenclature used in
the map to the HUGO Gene Nomenclature Committee (36) and its
database (37).
X. laevis cDNA library screening
We isolated two genes that have important roles in the mammalian immune
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
cDNA library made from mixture of spleen and intestine mRNA. The C2
probe was made by PCR using primers taken from EST entry (BX853282)
corresponding to nucleotide positions 42– 462, from a X. laevis cDNA
library made from mixture of liver, spleen, and thymus mRNA (10). The
PCR amplicons were cloned into the TA cloning vector (Invitrogen Life
Technologies) and sequenced. Both library screenings and washings were
conducted under high stringency conditions (38). Positive clones were isolated and sequenced in their entirety. The sequences are deposited to GenBank, and accession numbers are given as DQ268506 for X. laevis DM␤
and DQ268507 for X. laevis C2.
Phylogenetic trees
The deduced DM␣ (EST clone, AAH61681) and DM␤ amino acid sequences were aligned using Clustal X, and Neighbor-Joining bootstrapping
trees (1000 trial runs) were made and viewed in the TreeView 1.6.6 program (39). The deduced X. laevis and X. tropicalis (reconstructed from
scaffold) C2 amino acid sequences were also aligned with factor B and C2
of tetrapod species, bony and cartilaginous fish Bf/C2, whose assignment
to Bf or C2 is not clear, and lamprey and invertebrate Bf/C2 are considered
to represent the preduplication Bf/C2 state (40, 41). For both trees gaps
were included, and multiple substitutions were not taken into account.
with HindIII or SacI, and fragmented DNA was separated on an agarose gel
and blotted onto membranes. The DNA amount was increased proportionally 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
from X. laevis spleen and intestine, and the sequence was confirmed. Primers used for amplification were as follows: 5⬘-AAA GTG GAA CAG CCT
GAG CG-3⬘ and 5⬘-CAT CAC ATG CAC AAT GGT TCC-3⬘. Hybridization was performed under low stringency conditions (30% formamide;
6 ⫻ SSC) at 42°C for overnight, and washed in 2 ⫻ SSC, 1% SDS at room
temperature, followed by 2 ⫻ SSC, 0.1% SDS at 55°C (38). The same blot
was later washed under high stringency conditions (0.2 ⫻ SSC, 0.1% SDS
at 65°C) to eliminate low-homology signals.
Results
Database mining
The chicken DM␣1 and ␤1-encoding exons (obtained from
AL023516) were used to search databases for the Xenopus DM
genes; the deduced amino acid sequences of these regions of the
bird sequences were found to be more specific for DM compared
with their ␣2 or ␤2 Igsf domains, which more readily selected
classical class II sequences in BLAST searches. We and others (S.
Beck, personal communication) have done exhaustive searches in
the EST and genomic databases for teleost DM genes and could
not identify them, suggesting either that teleosts have lost the DM
genes or they arose in the tetrapod lineage after its divergence from
bony fish. The Xenopus sequences were used in a phylogenetic
analysis, and the trees solidify the hypothesis that the DM class II
genes are as old as classical class II␣ and class II␤ (Ref. 42 and
Fig. 1). Thus, we think it is more likely that these genes have been
lost in teleosts and will be found in the cartilaginous fish.
From the initial EST searches with human MHC genes (fulllength amino acid sequences), we found most of the Xenopus
housekeeping genes with significant E-values. Each gene was further confirmed by BLASTx for their orthology. Using these genes
found in the EST databases, we then BLAST-searched the X. tropicalis scaffolds version 3.0 (典www.jgi.doe.gov/xenopus具). All scaffolds so-identified were then inspected for open reading frames
(ORF), which were manually verified and then used to rescreen the
GenBank database (see percentage of identities in Table I). During
this process, we identified other genes on the scaffolds that were
then used to screen the EST databases.
A total of 122 ORFs were found on the eight genomic scaffolds
(Tables I and II) encompassing 3.65 Mbp of which 110 genes were
annotated that showed significant similarity to genes in the databases. Twelve genes had no database match (denoted as ORF). At
least one gene on each X. tropicalis scaffold shown in Fig. 2 has
been rigorously analyzed for MHC linkage previously (24 –33)
(Y. Ohta and M. F. Flajnik, unpublished data for FABGL, DAXX,
and FLOT1), and thus we are certain that all of these scaffolds are
in the Xenopus MHC.
We could not decide by phylogenetic analyses whether DDAH
and NOTCH were orthologues of the human MHC-encoded
DDAH2 and NOTCH4 or their paralogues found on human chromosomes 1, 9, and 19 (5). Their location within the MHC makes
it likely that they are the orthologues of the MHC-encoded human
genes. Conversely, several genes were found in the Xenopus MHC
that are present on different chromosomes in the human, sometimes in paralogous regions (Table I and red numbered loci in Fig.
2). This is likely due to differential silencing of genes after divergence from the common ancestor. These subjects are further described and discussed in more detail below.
Southern blotting
Extended class II region
Genomic DNA from different Xenopus species (2n–12n), or from siblings
in a family ( f/g ⫻ f/r) with known MHC haplotypes (27), was digested
All 15 functional genes in the human extended class II region and
3 of 4 genes flanking this region were found in ⬃415- and 200-kb
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
Data-mining the X. tropicalis genome project
3675
3676
SYNTENIC INTEGRITY OF MHC THROUGH EVOLUTION
regions of two X. tropicalis scaffolds, respectively; the gene density is ⬃24 kb/gene and ⬃40 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
Class III region
Forty-five human genes listed in the human class III region were
found on five scaffolds spanning ⬃2 Mbp (Fig. 2), suggesting that
like the extended class II region, the class III region is old and
extremely well conserved. Because the NOTCH gene is in scaffold
1316, where the majority of genes are in the class I region, the
class III region is contiguous with the class I region. Like the
extended class II region, there seems to have been at least three en
bloc inversions between C4 and PPT2, STK19 and C6orf29, and
HSP70 and CSNK2B. There are also potential translocations (e.g.,
ATP6V1G2, BAT1, and NEU1). PCR was used to identify the gap
between scaffolds 895 and 1207. A 0.8-kb fragment was sequenced
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 1. Phylogenetic analysis of vertebrate MHC molecules demonstrates an ancient origin of DM. The extracellular domains for the fulllength EST for DM␣ (GenBank accession no. AAH61681; A) and our
full-length clone for DM␤ (ABB85336; B) were used in the construction of
the trees. Genetic distance is shown as a bar on the bottom. Accession nos.
for each sequences are as follows: human DM␣ (AAH11447), mouse DM␣
(NP_034516), rat DM␣ (CAA89831), cow DM␣ (BAA11171), chicken
DM␣ (CAA18966), quail DM␣ (BAC82512), Xenopus DM␣
(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␣
(AAF99282), catfish II␣ (AAD39871), zebrafish II␣ (NP_001007049),
nurse shark DAA (AAA49310), human DM␤ (NP_002109), mouse DM␤2
(A55242), rat DM␤ (CAA89832), cow DM␤ (BAA11172), rabbit DM␤
(AAB53264), chicken DM␤1 (CAA18968), chicken DM␤2 (CAA18967),
quail DM␤1 (BAC82514), quail DM␤2 (BAC82513), human DO␤
(AAA59717), mouse A␤2 (AAA51637), chicken II␤ (AAS00716), quail
II␤ (BAC82510), Xenopus II␤ (BAA02845), medaka II␤ (BAA94279),
catfish II␤ (AAB67871), carp II␤ (CAA64709), trout II␤ (AAD53026),
shark II␤ (L20274), human A1 (NP_002107), mouse H2-K (AAA80451),
rat RT1␣ (XP_579224), chicken B-F (CAA18972), Xenopus I␣
(AAA16064), nurse shark UAA (AAC60347).
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.
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 ⬃217 kb on scaffold 1109, whereas the class I region (five genes, from class Ia to
TAP2) is ⬃274 kb on scaffold 1316. From Southern blotting analysis, 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
scaffold is ⬃244 kb, seemingly too large compared with intergenic
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
are within an intron length of each other.
A BTNL-II gene (butyrophilin-like MHC class II-associated),
located at the border of the mammalian class II and class III regions (43), was found neither in the EST databases nor the
genomic scaffolds. However, other BTN genes are found in the
human class I region. The BTN genes are Igsf members (44) that
display notable sequence similarity to other MHC genes, particularly to human MOG and bird B-G (13) (see below).
The Journal of Immunology
3677
Table I. List of genes, scaffold numbers, and database accession no. found in the databases
Gene
Scaffold
Region flanking the extended class II subregion
ZBTB9
726
C6orf82
726
PHF1
726
Extended class II subregion
KIFC
726
DAXX
726
ZNF297
726
TAPBP
726
RGL2
726
HKE2
726
C6org11
726
B3GALT4
726
RPS18
726
VPS52
726
RING1
726
HSD17B8
726
SLC39A7
726
Col11A2
Classical class II subregion
DO
BRD2
DM␣
DM␤
Class II␣
Class II␤
PSMB9
TAP1
PSMB8
TAP2
C6orf10
Class III subregion
NOTCH4
GPSM3
PBX2
726
917
917
No hit
1109
1109
1109
917
1109
917
1109
1316
1316
1316
1316
No hit
1316
1026
1026
RNF5
AGPAT1
1026
1026
EGFL8
PPT2
C6orf31
KFBPL
CREBL1
1026
1207
1026
1026
1026
TNXB
CYP21A2
C4
STK19
1026
1026
1026
895
DOM3Z
SKIV2L
RDBP
BF
895
1207
1207
1207
C2
895
1207
ZBTB12
BAT8
C6orf29
1207
1207
1207
NEU1
895
HSP70
547
X. laevisa
Percentage of
Identities/aab
No hit
No hit
No hit
BC087446 (F)
BC078516 (F)
AF130453 (F)
54/196 (Full)
73/109 (Full)
54/571 (Full)
CR848323 (F)
BX759743
No hit
No hit
AL594530
AL633707
BX737105
No hit
AL960564
BX758938
AL628421
CR760242 (F)
BX721639
AL968958
AL868786
U82809 (F)
BC079997
BJ042776
CF283998
No hit
BC084766 (F)
BC077337 (F)
BP688393
BC068873 (F)
BJ057872
BC081039 (F)
No Hit
BQ733936
AAB40402
48/229 (Gen)
51/538 (Gen)
31/193 (Part)
41/734 (Gen)
75/156 (Full)
60/551 (Full)
38/186 (Gen)
98/131 (Full)
72/621 (Gen)
Q66J69
67/251 (Full)
73/161 (Gen)
BC073179
AAH73179
No hit
CB942366
BF845697
67/1476 (Gen)
No hit
BX750317
No hit
No hit
CR760040
CF524557
BC087775
NM_001003660
No hit
U51449
BC061681 (F)
DQ268506 (F)
AF454378 (F)
NA
AAB18943
38/229 (Full)
31/241 (Full)
AAL58434
D13684 (F)
D87687 (F)
BAA02841
BAA19759
No hit
AB033151
No hit
No hit
AY204552 (F)
D44540 (F)
AY204554 (F)
No hit
AAP36718
BAA07945
AAP36720
NA
No hit
No hit
AL679273
BX733561
AL848378
AL959082
BX758119
CR761323 (F)
No Hit
BX730761
BX741700
AL878690
AL872993
No hit
No hit
No hit
AL866392
AL680203
CR761194
AL648835
BX705721
AL658338
BQ519832
BX711550
BX739487
CV811291
BX772914
AL882026
AL656289
AL867952
BX730249
BX736306
CX414897
No hit
BP703429
BC071048 (F)
38/1426 (Gen)
53/62 (Gen)
84/357 (Full)
CA793548 (F)
BC081085 (F)
80/120 (Full)
71/265 (Full)
No hit
BC059297 (F)
BC074389
No hit
No hit
42/281
66/283
64/225
40/259
53/402
BX846582
BC079793 (F)
D78003 (F)
BC087397 (F)
40/1103 (Gen)
45/460 (Full)
BAA11188
45/266 (Full)
BC088900 (F)
CV077949
No hit
D49373 (F)
53/392 (Full)
56/341 (Gen)
78/135 (Gen)
BAA08371
DQ268507
39/752 (Full)
BC072114 (F)
BX854837
BC073678 (F)
58/468 (Full)
69/558 (Gen)
67/703 (Full)
BC074166 (F)
P12890
X01102 (F)
(Full)
(Full)
(Gen)
(Gen)
(Gen)
P02827
(Table continues)
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
RXRB
X. tropicalisa
3678
SYNTENIC INTEGRITY OF MHC THROUGH EVOLUTION
Table I. Continued
Percentage of
Identities/aab
X. tropicalisa
X. laevisa
LSM2
VARS2
547
547
BP708188 (F)
BC084762 (F)
AAH90606
66/1211 (Full)
C6orf26
547
AW644535
36/149 (Part)
MSH5
CLIC1
DDAH2
LY6G6C
LY6
BAT5
CSNK2B
BAT4
APOM
BAT3
547
547
547
547
547
547
547
895
895
895
BP691385
AY277695 (F)
BC078574 (F)
No hit
No hit
BC077872 (F)
BC083993 (F)
BJ045925
BC078609
BC060479 (F)
57/512 (Gen)
AAH59765
55/278 (Full)
31/100 (Full)
30/123 (Full)
AAH75571
93/234 (Full)
39/361 (Gen)
45/188 (Gen)
51/1185 (Full)
BAT2
895
BX753796 (F)
AL967562
BX730148
CF224182
CF224183
No hit
BC059765 (F)
BC075381 (F)
BX687777 (F)
AL958403 (F)
BC075571 (F)
BC077003 (F)
BG487159
No hit
BX764202
BG348638
BG486156
AL955051
AL966216
BX698872
AL633000
AL792183
BX732414
BX716126
BX771103
No hit
No hit
AL661130
No Hit
BC061280 (F)
BC051009 (F)
37/2232 (Full)
BQ398178
BU905532
No hit
BJ614008
CD254193
BC084004 (F)
34/152 (Gen)
35/207 (Gen)
38/159 (Gen)
34/392 (Gen)
68/117 (Gen)
AAH61280
No hit
BX769629
AL646254
No hit
AL866886
CR760749
BC074549 (F)
AL969933
BX752964
BX779269
No hit
BX738291
AL632457
BX696867
BC087624 (F)
No hit
BX740904
AL633731
AL871337
AL629506
AL628261
BX44344
No hit
BX727166
BX731212
No hit
No hit
No hit
AW782810
BF613095
BC060755 (F)
AF545659 (F)
NA
40/332 (Gen)
32/712 (Gen)
60/930 (Full)
AAN86277
BC049004 (F)
BP681667
AAH75549
44/246 (Gen)
BC073486 (F)
BJ636557
BP709014
53/253 (Full)
48/145 (Gen)
89/529 (Gen)
No hit
BC089285 (F)
BP705701
BC074405 (F)
45/204 (Full)
55/429 (Full)
55/201 (Gen)
Q6GLQ4
BC081034 (F)
BJ614782
No hit
87/600 (Full)
61/618 (Gen)
78/629 (Gen)
No hit
No hit
49/291 (Gen)
48/309 (Gen)
LTB
895
TNF
895
LTA
895
NFKBIL1
895
ATP6V1G2
895
BAT1
895
Classical and extended class I subregion
POU5F1
No hit
TCF19
547
C6orf18
547
DDR1
547
FLOT1
547
TUBB
MDC1
547
547
NRM
KIAA1949
DHX16
547
547
547
C6orf136
C6orf134
MRPS18B
PPP1R10
547
547
547
547
ABCF1
GNL1
GABBR1
547
547
726
Olfactory receptor
Olfactory receptor
726
726
Gene
Scaffold
Human
chromosome
no.
X. tropicalisa
Genes not found in the human MHC, but in the Xenopus scaffoldsa
KIAA1720
726
1
AL774574
408 ribosomal protein S5
726
X
BX718023
CR848217
RASA1
726
5
No Hit
HGMA1L1
726
X
BX741519
Thymopoietin
917
12
No hit
MAP1
1109
14
CX40492
Carnitine O-acetyltransferase 1026
9
BC063356 (F)
X. laevisa
Percentage of
Identities/aab
BC084800 (F) 62/398 (Full)
BC054263 (F) 96/203 (Full)
CD253980
BX849417
No hit
BC073201
BC072849 (F)
70/708 (Gen)
43/91 (Gen)
31/107 (Gen)
26/361 (Gen)
50/587 (Full)
(Table continues)
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
Scaffold
Gene
The Journal of Immunology
3679
Table I. Continued
Gene
CENPA
PSMB10
PDE4DIP
Similar to CTGF
NPDC1-like
RGS3
Scaffold
Human
chromosome
no.
1026
1026
1207
2
16
1
547
547
895
6 (not MHC)
9
9
Scaffold
X. tropicalisa
AL863713
No hit
AL633679
BX700846
No hit
No hit
No hit
Percentage of
Identities/aab
X. laevisa
BC092389 (F)
BC056039 (F)
No Hit
67/90 (Full)
62/276 (Full)
22/350 (Gen)
BG234029
No hit
CA788699
40/132 (Gen)
30/120 (Gen)
36/176 (Gen)
X. tropicalis
X. laevis
Unknown genes (ORF) found in the Xenopus scaffolds
726
AL635951
726
No hit
726
BX751117
1109
No hit
BX688173
AL963961
BX781893
BX717682
No hit
AL868311
No hit
No hit
a
Percentage of Full-length DNA sequences are noted as (F).
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.
b
from both ends, confirming that the gap between scaffolds 895 and
1207 is short and contains a single intron of a factor B gene.
In the MHC of all teleosts so far studied, the three (or more)
immunoproteasome genes are tightly linked to class I genes (Refs.
18, 20, 45– 47; also see Fig. 5), and thus we were surprised to find
the third immunoproteasome gene PSMB10 in the class III region.
Because teleost MHC genes are found in many linkage groups and
spread onto different chromosomes, PSMB10 consequently may
have remained in the class I region in bony fish as a result of
translocation of ancestral class III region genes out of the MHC
(12, 15, 48) and coevolution via “functional clustering” of immunoproteasome and class I genes (49 –51). In contrast, because most
genes have maintained their ancient synteny in the Xenopus MHC,
it is likely that early in evolution PSMB10 indeed was located in
the class III region. Alternatively, PSMB10 translocated out of the
class I region in Xenopus, and its present location is a derived
characteristic. We await studies of the elasmobranch class I region
to elucidate the original location of PSMB10.
We found a gene similar to the complement C2 gene in the
scaffold 1207 near the factor B gene. Phylogenetic analysis
strongly supports that the gene is more similar to C2 than to factor
B (Fig. 3). In teleost fish, the Bf/C2 genes are often duplicated, and
one of these genes encodes a protein that, like C2, functions in the
classical complement pathway (48, 52–54). However, upon phylogenetic tree analysis, most of the teleost genes form a monophyletic cluster independent of tetrapod Bf and C2 clusters. Thus,
whereas it is still not clear whether the Bf/C2 gene duplication and
functional differentiation predated the emergence of teleosts, our
data clearly demonstrate that the Bf/C2 duplication predated the
appearance of amphibians. The three TNF members encoded in
the human class III region (LTA, LTB, and TNF) are found in the
Table II. Genes found in the Xenopus MHC
Category
Ag processing/presentation
Inflammation
Leukocyte maturation
Complement
Immune regulation
Stress response
Ig superfamily
Olfactory receptors
Nonimmune genes
Genes not found in the human MHC
Unknown genes
Total number of genes
Number
of Genes
11
5
3
3
3
1
6
2
64
13
12
122
Genes
Class I, class II␣, class II␤, DM␣, DM␤, PSMB8, PSMB9, PSMB10,
TAP1, TAP2, TAPBP
ABCF1, DAXX, LTA, LTB, TNF␣
DDAH1/2, LY6, LY6G6C
BF, C2, C4
NFKBIL1, RXRB, FKBPL
HSP70
XMIV
PSMB10 and others
ORF
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
1109
1316
547
547
547
895
895
895
No hit
No hit
No hit
CD099547
BP700625
No hit
No hit
No hit
No hit
No hit
No hit
CB942034
No hit
3680
SYNTENIC INTEGRITY OF MHC THROUGH EVOLUTION
Table III. Update on scaffold assembly
Scaffolds
Version 3.0
726 (656,544 bp)
917 (462,912 bp)
1109 (310,645 bp)
1316 (205,920 bp)
1026 (374,626 bp)
1207 (255,515 bp)
1207 (255,515 bp)
895 (482,803 bp)
547 (899,709 bp)
Version 4.0
396 (1,113,890 bp)
396 (1,113,890 bp)
895 (310,644 bp)
1038 (205,919 bp)
744 (484,856 bp)
744 (484,856 bp)
1175 (141,455 bp)
752 (471,867 bp)
488 (901,819 bp)
FIGURE 2. Comparison of the X. tropicalis MHC to the human MHC
(modified from Ref. 4) reveals extraordinary conservation. Partial human
MHC genes are listed as the template in the center and the Xenopus MHC
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
superscripts. Igsf genes unique to the Xenopus MHC on scaffolds 547 and
895 are shown in red boxes. Transcriptional orientations are indicated as
gradients and arrows on the right bottom.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
Xenopus MHC, suggesting that the synteny is old, and that nonMHC 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
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,
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
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 ⬍22–26%), as
The Journal of Immunology
3681
EST databases (BC073304, BC074259) when screening with the
human gene (E-value of ⬍e⫺39). 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
mentioned above. It is possible that the scaffold containing Xenopus NKp30 (data not shown) could be between scaffolds 547 and
895, although it seems unlikely because this would be too large of
a disruption (⬃2 Mb) in the midst of the other densely packed
class III genes. Furthermore, searches of the X. tropicalis genome
with Igsf domains only selected these MHC scaffolds, suggesting
that all XMIV members are in the MHC. All of the Xenopus species
(2n–12n) have multiple copies of XMIV genes, with no obvious
increase in gene numbers in the higher-order polyploids (Fig. 4C),
like for many other immune genes (Ref. 58 and unpublished data).
The following class III genes were not detected on the scaffolds
or in the EST databases: AGER, C6orf48, C6orf27, C6orf25,
LY6G6E, LY6G6D, LY6G5C, LY6G5B, C6orf47, and LST1. The
LY6 family members in human seem to be derived from recent
duplications and thus would not be expected to be found in Xenopus. The AIF1 paralogue, C9orf58, was found in the X. laevis
Categories of genes in the Xenopus MHC
Next, we classified genes found in the Xenopus MHC by their
functions (Table II). We found genes belonging to each category as
detailed in the human MHC such as those involved in the following: Ag processing for class I and class II molecules, inflammation,
leukocyte maturation, complement, immune regulation, Igsf, and
heat shock protein (HSP). Again, the overall MHC architecture is
well conserved. In the human MHC, ⬃28% of the expressed transcripts are potentially associated with immunity (4). In the Xenopus MHC, 32 genes (26.2%) fall into this category, also quite
similar to that of human. Thirteen genes were found in Xenopus
that are not in the human MHC, five of which are encoded on
MHC paralogous regions: KIAA1720 and ODE4DIP on human
chromosome 1, and RGS3, Carnitine acetyltransferase, and
NPDC1-like on human chromosome 9. As described above for
AIF1, the likely explanation for this finding is differential silencing
(in these cases) on the MHC paralogous regions in Xenopus. Furthermore, as mentioned above PSMB10 is found in the Xenopus
MHC, whereas it is located in humans on chromosome 16. Previous work in teleosts suggested that PSMB10 was originally located
in the MHC class I region, and subsequently translocated onto a
separate chromosome in the mammalian genome (51). Interestingly, we found carnitine acetyltransferase in the vicinity of the
constitutive proteasome subunit and direct homologue of PSMB10
and PSMB7, further supporting the idea that PSMB8, -9, and -10
arose from duplication of PSMB5, -6, and -7, with subsequent
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 3. Phylogenetic tree of Factor B and C2 sequences identifies
Xenopus C2. The tree was constructed by the Neighbor-Joining Method
based on an alignment of amino acid sequences using Clustal X version
1.81. Numbers indicate the bootstrap values, supporting the depicted partitioning 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
musculus Bf (NM_008198); Mumu C2 (NM_013484); Xela BfA, X. laevis
BfA (BAA06179); Xela BfB (BAA08371); Xela C2, X. laevis C2
(ABB85337); Omny Bf-1, Oncorhynchus mykiss Bf-1 (AAC83699); Omny
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
(BAA34707); Cyca Bf/C2-A3 (BAB32650); Trsc Bf, Triakis scyllium Bf
(BAB63203); Leja Bf, Lethenteron japonicum Bf (I50807); and Stpu Bf,
Strongylocentrotus purpuratus Bf (AAC79682).
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
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.
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
genes was indeed identified, near to the XNC genes (data not
shown). These data demonstrate that the class I-BTN association
is old.
3682
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
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
CN328971. Sequence numbers correlate
with the scaffold and location. ORFs for
XMIV1 and -4 contain two Igsf domains
and are designated as ⫺1 and ⫺2. Evolutionary conserved amino acids, ITIM,
and positively charged residues in the
TM and cytoplasmic (CYT) regions are
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 ⫻ 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
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.
SYNTENIC INTEGRITY OF MHC THROUGH EVOLUTION
The Journal of Immunology
translocation of PSMB5 and -6 (51). Synteny of PSMB10 and carnitine acetyltransferase in the Xenopus MHC suggests that differential silencing resulted from the presence of PSMB7-carnitine
acetyltransferase in the primordial MHC. Two olfactory genes
were found, but it is not clear whether these genes are orthologous
to those in the human MHC. There are 12 unknown genes of which
nine genes are found in the EST database, suggesting functional
genes.
Discussion
Genes involved in immune responses evolve rapidly, likely to
combat rapidly evolving or emerging pathogens (61, 62). This
rapid evolution of immune genes can be obstructive when pursuing
orthologous genes in divergent species; BLAST searches of the
genome using amino acid sequences from other species often resulted in “no hit.” Sometimes, even using the relatively closely
related X. laevis did not result in identification of the X. tropicalis
sequences. By contrast, searches using genes in large families such
as TAP1 and TAP2 resulted in too many hits because the conserved
ATP-binding domain selects other ABC transporter genes as well.
Similar problems were observed with the paralogous genes. Vertebrate genome analysis had revealed that there are three other
gene complexes similar to the MHC (63). We were usually successful in identifying which of the paralogues was orthologous to
the human MHC counterpart, but sometimes it was not clear. For
example, we could not identify Xenopus DDAH as either DDAH2
(human MHC) or DDAH1 (chromosome 9), and thus named the
Xenopus clone DDAH1/2 in Fig. 2. This was true of NOTCH as
well, where different exons seemed to be orthologous to the different NOTCH paralogues; therefore, we could not confidently
identify the MHC-encoded gene as NOTCH4. These phenomena
can be explained as differential subfunctionalization or neofunctionalization 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
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, suggesting that the sequence may not be entirely accurate in these
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 genome assembly were released (version 4.0 and 4.1, coverage 7.65
genome equivalents). The sizes and general organization are almost identical with the previous version except that two scaffolds
were connected (Table III).
From our previous work using recombinant X. laevis, we ordered the nonmammalian class I region as class II, TAP/LMP, class
I/C4 (6, 10). However, as shown in Fig. 2, the order of the genes
in the X. tropicalis scaffolds is class II, class I, TAP/LMP, C4.
Again, this could either be due to an assembly error in X. tropicalis
or alternatively to genomic re-organization that happened during
tetraploidization. The frog used for the scaffold assembly was heterozygous for the two class I region lineages that are found in all
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
wild-caught animals (10), suggesting that there is a block in recombination between genes in these lineages, perhaps because of
major sequence modifications in noncoding regions, recently
shown to be true in medaka (65). Unfortunately, the assembly was
complete for only one of the lineages (lineage A) in this particular
region, so we will have to wait to test this hypothesis.
There are two large clusters of histone and tRNA genes in the
human extended class I subregion. It is proposed that the MHC
may have hitchhiked with these clusters (or vice versa) to maximize transcriptional activity (4). Unfortunately, our analysis of Xenopus MHC did not include this extended class I region to examine
whether the cluster of histone and tRNA genes is an evolutionary
conserved feature of MHC. We await future versions of the genome project to examine this question.
In the vicinity of the human MHC, there are 34 olfactory-receptor loci, 14 of which are potentially functional. Sperm-expressed olfactory-receptor genes may be functionally involved in
the selection of spermatozoa by the female (sperm receptor selection hypothesis), as well as many other functions (66). Thus far, we
have found two olfactory receptors in the MHC and XNC scaffolds
(data not shown). Therefore, it will be interesting to determine
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 5. Genomic organization in the Xenopus class I region compared
with the medaka (47) and the chicken (13) MHCs. Transcriptional orientations
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.
3683
3684
Acknowledgments
We thank Louis Du Pasquier for discussions about the data.
Disclosures
The authors have no financial conflict of interest.
References
1. Kumanovics, A., T. Takada, and K. F. Lindahl. 2003. Genomic organization of
the mammalian MHC. Annu. Rev. Immunol. 21: 629 – 657.
2. Driscoll, J., M. G. Brown, D. Finley, and J. J. Monaco. 1993. MHC-linked LMP
gene products specifically alter peptidase activities of the proteasome. Nature
365: 262–264.
3. Antoniou, A. N., S. J. Powis, and T. Elliott. 2003. Assembly and export of MHC
class I peptide ligands. Curr. Opin. Immunol. 15: 75– 81.
4. Horton, R., L. Wilming, V. Rand, R. C. Lovering, E. A. Bruford, V. K. Khodiyar,
M. J. Lush, S. Povey, C. C. Talbot, Jr., M. W. Wright, et al. 2004. Gene map of
the extended human MHC. Nat. Rev. Genet. 5: 889 – 899.
5. Flajnik, M. F., and M. Kasahara. 2001. Comparative genomics of the MHC:
glimpses into the evolution of the adaptive immune system. Immunity 15:
351–362.
6. Nonaka, M., C. Namikawa, Y. Kato, M. Sasaki, L. Salter-Cid, and M. F. Flajnik.
1997. Major histocompatibility complex gene mapping in the amphibian Xenopus
implies a primordial organization. Proc. Natl. Acad. Sci. USA 94: 5789 –5791.
7. Kelley, J., L. Walter, and J. Trowsdale. 2005. Comparative genomics of major
histocompatibility complexes. Immunogenetics 56: 683– 695.
8. Vogel, G. 1999. Frog is a prince of a new model organism. Science 285: 25.
9. Kobel, H. R., and L. Du Pasquier. 1986. Genetics of polyploid Xenopus. Trends
Genet. 12: 310 –314.
10. Ohta, Y., S. J. Powis, R. L. Lohr, M. Nonaka, L. Du Pasquier, and M. F. Flajnik.
2003. Two highly divergent ancient allelic lineages of the transporter associated
with antigen processing (TAP) gene in Xenopus: further evidence for co-evolution
among MHC class I region genes. Eur. J. Immunol. 33: 3017–3027.
11. Koch, C. 1986. A genetic polymorphism of the complement component factor B
in chickens not linked to the major histocompatibility complex (MHC). Immunogenetics 23: 364 –367.
12. Graser, R., V. Vincek, K. Takami, and J. Klein. 1998. Analysis of zebrafish Mhc
using BAC clones. Immunogenetics 47: 318 –325.
13. Kaufman, J., S. Milne, T. W. Gobel, B. A. Walker, J. P. Jacob, C. Auffray,
R. Zoorob, and S. Beck. 1999. The chicken B locus is a minimal essential major
histocompatibility complex. Nature 401: 923–925.
14. Sato, A., F. Figueroa, B. W. Murray, E. Malaga-Trillo, Z. Zaleska-Rutczynska,
H. Sultmann, S. Toyosawa, C. Wedekind, N. Steck, and J. Klein. 2000. Nonlinkage of major histocompatibility complex class I and class II loci in bony
fishes. Immunogenetics 51: 108 –116.
15. Naruse, K., S. Fukamachi, H. Mitani, M. Kondo, T. Matsuoka, S. Kondo,
N. Hanamura, Y. Morita, K. Hasegawa, R. Nishigaki, et al. 2000. A detailed
linkage map of medaka. Oryzias latipes: comparative genomics and genome evolution. Genetics 154: 1773–1784.
16. Juul-Madsen, H. R., T. S. Dalgaard, B. Guldbrandtsen, and J. Salomonsen. 2000.
A polymorphic major histocompatibility complex class II-like locus maps outside
of both the chicken B-system and Rfp-Y-system. Eur. J. Immunogenetics 27:
63–71.
17. Kuroda, N., F. Figueroa, C. O’hUigin, and J. Klein. 2002. Evidence that the
separation of Mhc class II from class I loci in the zebrafish, Danio rerio, occurred
by translocation. Immunogenetics 54: 418 – 430.
18. Phillips, R. B., A. Zimmerman, M. A. Noakes, Y. Palti, M. R. Morasch, L. Eiben,
S. S. Ristow, G. H. Thorgaard, and J. D. Hansen. 2003. Physical and genetic
mapping of the rainbow trout major histocompatibility regions: evidence for duplication of the class I region. Immunogenetics 55: 561–569.
19. Shiina, T., S. Shimizu, K. Hosomichi, S. Kohara, S. Watanabe, K. Hanzawa,
S. Beck, J. K. Kulski, and H. Inoko. 2004. Comparative genomic analysis of two
avian (quail and chicken) MHC regions. J. Immunol. 172: 6751– 6763.
20. Shiina, T., J. M. Dijkstra, S. Shimizu, A. Watanabe, K. Yanagiya, I. Kiryu,
A. Fujiwara, C. Nishida-Umehara, Y. Kaba, I. Hirono, et al. 2005. Interchromosomal duplication of major histocompatibility complex class I regions in rainbow
trout (Oncorhynchus mykiss), a species with a presumably recent tetraploid ancestry. Immunogenetics 56: 878 – 893.
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,
N. Wagtmann, and C. C. Winter. 1997. Killer cell inhibitory receptors: diversity,
specificity, and function. Immunol. Rev. 155: 135–144.
23. Vivier, E., and M. Daeron. 1997. Immunoreceptor tyrosine-based inhibition motifs. Immunol. Today 18: 286 –291.
24. Shum, B. P., D. Avila, L. Du Pasquier, M. Kasahara, and M. F. Flajnik. 1993.
Isolation of a classical MHC class I cDNA from an amphibian: evidence for only
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.
Evolution of the MHC: isolation of class II ␤-chain cDNA clones from the amphibian Xenopus laevis. J. Immunol. 150: 2831–2843.
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. Immunol. 26: 735–750.
27. Ohta, Y., S. J. Powis, W. J. Coadwell, D. E. Haliniewski, Y. Liu, H. Li, and
M. F. Flajnik. 1999. Identification and genetic mapping of Xenopus TAP2 genes.
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
linkage to MHC. J. Immunol. 155: 1964 –1971.
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.
30. Salter-Cid, L., L. Du Pasquier, and M. Flajnik. 1996. RING3 is linked to the
Xenopus major histocompatibility complex. Immunogenetics 44: 397–399.
31. Mo, R., Y. Kato, M. Nonaka, K. Nakayama, and M. Takahashi. 1996. Fourth
component of Xenopus laevis complement: cDNA cloning and linkage analysis of
the frog MHC. Immunogenetics 43: 360 –369.
32. Kato, Y., L. Salter-Cid, M. F. Flajnik, M. Kasahara, C. Namikawa, M. Sasaki,
and M. Nonaka. 1994. Isolation of the Xenopus complement factor B complementary DNA and linkage of the gene to the frog MHC. J. Immunol. 153:
4546 – 4554.
33. Salter-Cid, L., M. Kasahara, and M. F. Flajnik. 1994. Hsp70 genes are linked to
the Xenopus major histocompatibility complex. Immunogenetics 39: 1–7.
34. Marklew, S., D. P. Smith, C. S. Mason, and R. W. Old. 1994. Isolation of a novel
RXR from Xenopus that most closely resembles mammalian RXR ␤ and is expressed throughout early development Biochim. Biophys. Acta 1218: 267–272.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
whether there are larger clusters of olfactory genes near the Xenopus MHC genes and determine their tissue and ontogenic
distribution.
The chicken MHC contains putative NK receptors in the C-type
lectin family that are most related to genes in the mammalian NK
cell complex (NKC) (13, 67). This finding not only demonstrates
a common evolutionary origin of the NK cell complex and MHC,
but also suggests coevolution of the linked NK cell and class I
receptors (13, 68). Similarly, the discovery of XMIV genes most
related to Ag receptor genes in the Xenopus MHC suggests that
they could coevolve with polymorphic class I and/or class II molecules. In addition, based on the fact that class I, class II, and Ag
receptor genes all have the specialized Igsf C1-type domain (69,
70), it seems likely that the ancestral Ag receptor genes were genetically linked to the MHC-presenting genes. It is possible that
the Igsf genes encoded in the Xenopus MHC are relics of such
original Ag receptors.
As described throughout the text, close linkage of class I-presenting genes and processing genes is found in all nonmammalian
vertebrates (Fig. 5). Interestingly, similar to what has been described recently in the chicken (71), the Xenopus TAP genes TAP1/
TAP2 are in opposite transcriptional orientations and may use a
bidirectional promoter. However, the MHCs in birds and teleost
fish have been extensively modified (5, 7, 12, 14 –16). A few of the
genes in the extended class II and class I regions have maintained
their synteny in bony fish (47, 72), but by and large their MHC
genes have been scattered throughout the genome. The teleosts
studied to date have small genomes, and their MHC genes may be
indicative of general modifications that have taken place for other
syntenic groups (15). This may be true of the chicken and quail as
well, in which the MHC is encoded on a microchromosome where
genes and intergenic distances have been greatly shortened compared with most vertebrates (13, 19). Birds have seemingly dispensed altogether with immunoproteasome genes and perhaps
other housekeeping genes. In contrast, this current study (and our
previous predictions) has shown that Xenopus is much similar to
human and allows for an understanding of the common MHC ancestor, at least at the level of amphibian emergence. By contrast,
the mammalian MHC has a peculiar organization in which the
class I genes are not closely linked to TAP and PSMB genes.
The loss of this linkage seems to be accompanied by the loss of the
genetic dimorphism of the linked class I/PSMB/TAP genes observed
in amphibian, teleosts, and cartilaginous fish (10, 64, 65, 73). Thus,
the amphibian seems to be the best model to study evolution of the
vertebrate MHC.
SYNTENIC INTEGRITY OF MHC THROUGH EVOLUTION
The Journal of Immunology
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
isotypes of B/C2-A expressed in different tissues. Dev. Comp. Immunol. 26:
533–541.
Savan, R., T. Kono, D. Igawa, and M. Sakai. 2005. A novel tumor necrosis factor
(TNF) gene present in tandem with the TNF-␣ gene on the same chromosome in
teleosts. Immunogenetics 57: 140 –150.
Kono, T., J. Zou, S. Bird, R. Savan, M. Sakai, and C. J. Secombes. 2005. Identification and expression analysis of lymphotoxin-␤ like homologues in rainbow
trout Oncorhynchus mykiss. Mol. Immunol. In press.
Greenberg, A. S., D. Avila, M. Hughes, A. Hughes, E. C. McKinney, and
M. F. Flajnik. 1995. A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks. Nature 374: 168 –173.
Courtet, M., M. Flajnik, and L. Du Pasquier. 2001. Major histocompatibility
complex and immunoglobulin loci visualized by in situ hybridization on Xenopus
chromosomes. Dev. Comp. Immunol. 25: 149 –157.
Holland, P. W., J. Garcia-Fernandez, N. A. Williams, and A. Sidow. 1994. Gene
duplications and the origins of vertebrate development. Dev. Suppl. 125–133.
Flajnik, M. F., M. Kasahara, B. P. Shum, L. Salter-Cid, E. Taylor, and
L. Du Pasquier. 1993. 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. 12: 4385– 4396.
Hughes, A. L., and M. Yeager. 1997. Molecular evolution of the vertebrate immune system. Bioessays 19: 777–786.
Trowsdale, J., and P. Parham. 2004. Mini-review: defense strategies and immunity-related genes. Eur. J. Immunol. 34: 7–17.
Kasahara, M. 1998. What do the paralogous regions in the genome tell us about
the origin of the adaptive immune system? Immunol. Rev. 166: 159 –175.
Flajnik, M. F., Y. Ohta, A. S. Greenberg, L. Salter-Cid, A. Carrizosa,
L. Du Pasquier, and M. Kasahara. 1999. Two ancient allelic lineages at the single
classical class I locus in the Xenopus MHC. J. Immunol. 163: 3826 –3833.
Tsukamoto, K., S. Hayashi, M. Y. Matsuo, M. I. Nonaka, M. Kondo, A. Shima,
S. Asakawa, N. Shimizu, and M. Nonaka. 2005. Unprecedented intraspecific
diversity of the MHC class I region of a teleost medaka, Oryzias latipes. Immunogenetics 57: 420 – 431.
Ziegler, A., G. Dohr, and B. Uchanska-Ziegler. 2002. Possible roles for products
of polymorphic MHC and linked olfactory receptor genes during selection processes in reproduction. Am. J. Reprod. Immunol. 48: 34 – 42.
Rogers, S. L., T. W. Gobel, B. C. Viertlboeck, S. Milne, S. Beck, and J. Kaufman.
2005. Characterization of the chicken C-type lectin-like receptors B-NK and
B-lec suggests that the NK complex and the MHC share a common ancestral
region. J. Immunol. 174: 3475–3483.
Parham, P. 1999. Immunogenetics. Soaring costs in defence. Nature 401:
870 – 871.
Williams, A. F., and A. N. Barclay. 1988. The immunoglobulin superfamily:
domains for cell surface recognition. Annu. Rev. Immunol. 6: 381– 405.
Du Pasquier, L., I. Zucchetti, and R. De Santis. 2004. Immunoglobulin superfamily receptors in protochordates: before RAG time. Immunol. Rev. 198:
233–248.
Walker, B. A., A. van Hateren, S. Milne, S. Beck, and J. Kaufman. 2005. Chicken
TAP genes differ from their human orthologues in locus organisation, size, sequence features and polymorphism. Immunogenetics 57: 232–247.
Sambrook, J. G., R. Russell, Y. Umrania, Y. J. Edwards, R. D. Campbell,
G. Elgar, and M. S. Clark. 2002. Fugu orthologues of human major histocompatibility complex genes: a genome survey. Immunogenetics 54: 367–380.
Ohta, Y., E. C. McKinney, M. F. Criscitiello, and M. F. Flajnik. 2002. Proteasome, transporter associated with antigen processing, and class I genes in the
nurse shark Ginglymostoma cirratum: evidence for a stable class I region and
MHC haplotype lineages. J. Immunol. 168: 771–781.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
35. Sharpe, C. R., and K. Goldstone. 1997. Retinoid receptors promote primary neurogenesis in Xenopus. Development 124: 515–523.
36. Povey, S., R. Lovering, E. Bruford, M. Wright, M. Lush, and H. Wain. 2001. The
HUGO Gene Nomenclature Committee (HGNC). Hum. Genet. 109: 678 – 680.
37. Wain, H. M., M. J. Lush, F. Ducluzeau, V. K. Khodiyar, and S. Povey. 2004.
Genew: the Human Gene Nomenclature Database, 2004 updates. Nucleic Acids
Res. 32: D255–D257.
38. Bartl, S., M. A. Baish, M. F. Flajnik, and Y. Ohta. 1997. Identification of class
I genes in cartilaginous fish, the most ancient group of vertebrates displaying an
adaptive immune response. J. Immunol. 159: 6097– 6104.
39. Page, R. D. 1998. GeneTree: comparing gene and species phylogenies using
reconciled trees. Bioinformatics 14: 819 – 820.
40. Nonaka, M., M. Takahashi, and M. Sasaki. 1994. Molecular cloning of a lamprey
homologue of the mammalian MHC class III gene, complement factor B. J. Immunol. 152: 2263–2269.
41. Smith, L. C., L. A. Clow, and D. P. Terwilliger. 2001. The ancestral complement
system in sea urchins. Immunol. Rev. 180: 16 –34.
42. Flajnik, M. F., K. Miller, and L. Du Pasquier. 2003. Evolution of the immune
system. In Fundamental Immunology, 5th ed. W. E. Paul, ed. Lippincott Williams
& Wilkins, Philadelphia, pp. 519 –570.
43. Stammers, M., L. Rowen, D. Rhodes, J. Trowsdale, and S. Beck. 2000. BTL-II:
a polymorphic locus with homology to the butyrophilin gene family, located at
the border of the major histocompatibility complex class II and class III regions
in human and mouse. Immunogenetics 51: 373–382.
44. Tazi-Ahnini, R., J. Henry, C. Offer, C. Bouissou-Bouchouata, I. H. Mather, and
P. Pontarotti. 1997. Cloning, localization, and structure of new members of the
butyrophilin gene family in the juxta-telomeric region of the major histocompatibility complex. Immunogenetics 47: 55– 63.
45. Michalová, V., B. W. Murray, H. Sültmann, and J. Klein. 2000. A contig map of
the Mhc class I genomic region in the zebrafish reveals ancient synteny. J. Immunol. 164: 5296 –5305.
46. Clark, M. S., L. Shaw, A. Kelly, P. Snell, and G. Elgar. 2001. Characterization
of the MHC class I region of the Japanese pufferfish (Fugu rubripes). Immunogenetics 52: 174 –185.
47. Matsuo, M. Y., S. Asakawa, N. Shimizu, H. Kimura, and M. Nonaka. 2002.
Nucleotide sequence of the MHC class I genomic region of a teleost, the medaka
(Oryzias latipes). Immunogenetics 53: 930 –940.
48. Sultmann, H., A. Sato, B. W. Murray, N. Takezaki, R. Geisler, G. J. Rauch, and
J. Klein. 2000. Conservation of Mhc class III region synteny between zebrafish
and human as determined by radiation hybrid mapping. J. Immunol. 165:
6984 – 6993.
49. Hughes, A. L. 1997. Evolution of the proteasome components. Immunogenetics
46: 82–92.
50. Murray, B. W., H. Sultmann, and J. Klein. 1999. Analysis of a 26-kb region
linked to the Mhc in zebrafish: genomic organization of the proteasome component ␤/transporter associated with antigen processing-2 gene cluster and identification of five new proteasome ␤ subunit genes. J. Immunol. 163: 2657–2666.
51. Clark, M. S., P. Pontarotti, A. Gilles, A. Kelly, and G. Elgar. 2000. Identification
and characterization of a ␤ proteasome subunit cluster in the Japanese pufferfish
(Fugu rubripes). J. Immunol. 165: 4446 – 4452.
52. Sunyer, J. O., I. Zarkadis, M. R. Sarrias, J. D. Hansen, and J. D. Lambris. 1998.
Cloning, structure, and function of two rainbow trout Bf molecules. J. Immunol.
161: 4106 – 4114.
53. Nakao, M., Y. Fushitani, K. Fujiki, M. Nonaka, and T. Yano. 1998. Two diverged
complement factor B/C2-like cDNA sequences from a teleost, the common carp
(Cyprinus carpio). J. Immunol. 161: 4811– 4818.
54. Nakao, M., M. Matsumoto, M. Nakazawa, K. Fujiki, and T. Yano. 2002. Diversity of complement factor B/C2 in the common carp (Cyprinus carpio): three
3685