Reconstructing an Ancestral Mammalian
Immune Supercomplex from a Marsupial Major
Histocompatibility Complex
Katherine Belov
1*
, Janine E. Deakin
2
, Anthony T. Papenfuss
3
, Michelle L. Baker
4
, Sandra D. Melman
4
,
Hannah V. Siddle
1
, Nicolas Gouin
5
, David L. Goode
3
, Tobias J. Sargeant
3
, Mark D. Robinson
3
, Matthew J. Wakefield
3
,
Shaun Mahony
6
, Joseph G. R. Cross
2
, Panayiotis V. Benos
7
, Paul B. Samollow
8
, Terence P. Speed
3
,
Jennifer A. Marshall Graves
2
, Robert D. Miller
4*
1 Centre for Advanced Technologies in Animal Genetics and Reproduction, Faculty of Veterinary Science, The University of Sydney, Camden, Australia, 2 ARC Centre for
Kangaroo Genomics, Research School of Biological Sciences, The Australian National University, Canberra, Australia, 3 The Walter and Eliza Hall Institute of Medical Research,
Parkville, Australia, 4 Department of Biology, University of New Mexico, Albuquerque, New Mexico, United States of America, 5 Department of Genetics, Southwest
Foundation for Biomedical Research, San Antonio, Texas, United States of America, 6 National Centre for Biomedical Engineering Science, National University of Ireland,
Galway, Ireland, 7 Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America,
8 Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, United States of
America
The first sequenced marsupial genome promises to reveal unparalleled insights into mammalian evolution. We have
used the Monodelphis domestica (gray short-tailed opossum) sequence to construct the first map of a marsupial major
histocompatibility complex (MHC). The MHC is the most gene-dense region of the mammalian genome and is critical to
immunity and reproductive success. The marsupial MHC bridges the phylogenetic gap between the complex MHC of
eutherian mammals and the minimal essential MHC of birds. Here we show that the opossum MHC is gene dense and
complex, as in humans, but shares more organizational features with non-mammals. The Class I genes have amplified
within the Class II region, resulting in a unique Class I/II region. We present a model of the organization of the MHC in
ancestral mammals and its elaboration during mammalian evolution. The opossum genome, together with other
extant genomes, reveals the existence of an ancestral ‘‘immune supercomplex’’ that contained genes of both types of
natural killer receptors together with antigen processing genes and MHC genes.
Citation: Belov K, Deakin JE, Papenfuss AT, Baker ML, Melman SD, et al. (2006) Reconstructing an ancestral mammalian immune supercomplex from a marsupial major
histocompatibility complex. PLoS Biol 4(3): e46.
Introduction
The major histocompatibility complex (MHC) is a multi-
gene complex critical to vertebrate immunity. The MHC is
the most gene-dense and polymorphic region of the
mammalian genome and is associated with resistance to
infectious diseases, autoimmunity, transplantation, and re-
productive success [1]. Loci contained within the MHC have
been historically grouped into three classes of genes called
Class I, II, and III. The three classes of loci are distinguished
based on both structure and function of their encoded
proteins. Class I molecules can be divided into classical and
non-classical molecules. Classical Class I (Class Ia) loci are
ubiquitously expressed and encode receptors that typically
bind and present endogenously synthesized peptides to
antigen specific CD8
þ
cytotoxic T cells. Non-classical Class I
(Class Ib) loci encode molecules that often perform functions
other than antigen presentation. Class Ib loci may be located
outside the MHC, and tend to be non-polymorphic and not
ubiquitously expressed. Classical Class II genes encode
receptors that present exogenously derived peptides to
CD4
þ
helper T cells, whereas non-classical Class II genes
participate in antigen presentation pathways. The MHC also
contains several genes encoding molecules that participate in
the processing of peptides for presentation to the immune
system. The Class III genes encode a variety of immune and
non-immune system–related molecules, most of which are
not involved in antigen presentation, and include cytokines
and components of the complement system. The three classes
of loci are been used to define regions within the MHC, i.e.
Class I, II, and III MHC regions.
Comparative analyses of the MHC organization across
distantly related species has revealed lineage specific rear-
rangements within the region and changes in gene complex-
ity. Detailed information on MHC organization is currently
available for seven species of eutherian (‘‘placental’’) mam-
mals, two birds, five teleost fish, and sharks [2–5]. There are
Academic Editor: Hidde L. Ploegh, Whitehead Institute for Biomedical Research,
United States of America
Received July 7, 2005; Accepted December 12, 2005; Published January 31, 2006
DOI: 10.1371/journal.pbio.0040046
Copyright:
2006 Belov et al. This is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author
and source are credited.
Abbreviations: BAC, bacterial artificial chromosome; bp, base pair; Class Ia,
classical class I; Class Ib, non-classical class I; LRC, leukocyte receptor complex; MHC,
major histocompatibility complex; NK, natural killer; NKC, natural killer complex;
ORF, open reading frame
* To whom correspondence should be addressed. E-mail: kbelov@camden.usyd.
edu.au (KB); rdmiller@unm.edu (RDM)
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Marsupial MHC

major differences in organization and complexity between
eutherian and non-mammalian MHC regions, and recon-
structing the evolutionary history of this region has been
difficult. The highly complex eutherian MHC is ordered along
the chromosome as the Class I–III–II regions. The eutherian
MHC is large and gene dense; for example, the human MHC
contains 264 genes and pseudogenes over the 3.6 Mb region.
In non-mammals, the MHC generally contains fewer genes
than is found in mammals and the Class I and II regions are
adjacent. Teleost fish are the exception. Their Class I and II
regions are unlinked. Of the MHC regions completely
sequenced, the chicken MHC is the least complex, containing
only 19 genes over 92 kb [6].
In eutherians, the Class I region contains a set of frame-
work genes whose presence and order are conserved among
species; and between this framework, the Class I genes have
expanded and diversified [7]. These Class I region framework
genes have not been reported in the MHC of non-mammals.
In eutherians, the Class II region contains the antigen
processing genes (TAP1, TAP2, PSMB8, and PSMB9), which
process endogenously synthesized peptides for presentation
on Class I molecules. In non-mammals, however, the antigen
processing genes are found in the Class I region, and their
proximity is thought to have influenced Class I gene evolution
[8].
Analysis of MHC structure in mammals distantly related to
eutherians (marsupials and monotremes) would bridge a 200
million y gap between eutherians and non-mammalian
vertebrates [9], and lead to a new understanding of the
evolutionary forces that shaped the complex eutherian MHC.
The availability of the opossum genome sequence provides
the first opportunity to bridge this phylogenetic gap and
provide insight into the evolutionary history of the mamma-
lian MHC. Here we report that the opossum MHC region is
similar to the eutherian MHC in both size and gene
complexity; however, it also contains organizational features
more like those found in non-mammals, revealing a likely
ancestral organization in mammals. This analysis is the
deepest comparison of the MHC region within mammals
undertaken to date.
Results/Discussion
The 3.6 Mb MHC region on human Chromosome 6
contains 140 genes between flanking markers MOG and
COL11A2 [10]. We found that the opossum MHC region
bounded by the same flanking markers spans 3.95 Mb and
contains 114 genes, recognized by homology to known genes
from other species and/or the presence of open reading
frames (ORF). Eighty-seven of these genes are shared with
human MHC (Figure 1). A list of putative opossum MHC gene
transcripts and our opossum MHC genome browser contain-
ing annotation are located at http://bioinf.wehi.edu.au/
opossum. The opossum MHC is located on Chromosome 2q
near the centromere, oriented with MOG proximal (Figure 2)
[11]. Physical mapping of 19 bacterial artificial chromosome
(BAC) clones, corresponding to loci spaced along the entire
scaffold, confirmed the accuracy of the assembly.
The opossum MHC is similar in size and gene content to
the MHC of eutherian mammals. However, the organization
of the opossum Class I, II, and III regions is different from
that of eutherians and shows more similarity to the
organization seen in birds and amphibians. Ostensibly, the
main difference between the opossum MHC and that of
eutherians is the position of the Class I genes (Figure 3). The
opossum MHC has (1) a Class I/II region that contains
interspersed Class I and II genes, (2) a ‘‘framework region’’
that is composed of only the framework genes in the
opossum, but which also includes Class I genes in eutherians,
(3) a Class III region with gene content and order highly
conserved with eutherians, and (4) two extended regions that
flank the MHC, corresponding to the eutherian extended
Class I and II regions, and containing a very similar gene
content and order.
In the opossum, Class I and II regions are adjacent and
interspersed rather than being separated by Class III (Figure
3). This unique arrangement of Class I and Class II loci has
not been described in any other mammalian species. The
proximity of Class I and II genes in opossum as well as non-
mammalian vertebrates implies that Class I and II genes were
originally located close together in the mammalian ancestor
(Figure 4). This conclusion is further supported by the
presence of Class I pseudogenes in the human Class II region,
and the presence of both functional and non-functional Class
I genes in the rodent Class II region (Figure 3) [1].
In humans and mice, the MHC contains a region referred
to as the Class I framework region due to the presence of a set
of non-Class I or II genes, amongst which the Class I loci are
interspersed [7]. The content and gene order of these
framework genes are conserved between mice and humans.
Remarkably, the opossum MHC contains a homologous
cluster of framework genes (including MOG, PPP1R11,
TRIM26, TRIM39, GNL1, POU5F1, and BAT1) next to the
Class III region opposite the Class I/II region. These genes are
in the same order as in the eutherian Class I framework
region, but they lack the interspersed Class I loci (Figures 1
and 4). This implies that a block of Class I framework genes
was established near the MHC locus prior to the translocation
of the Class I genes to this region in eutherians. Framework
genes have not been reported in the MHC of non-mammals,
but it is likely that the association of the framework region
genes is ancient and that the framework region moved into
the MHC en masse, given that five framework region genes
appear on the same scaffold as Class III genes in Xenopus
tropicalis (Ensembl scaffold_547) (unpublished data).
In Figure 4, we present a model to explain how MHC
Figure 1. Map of the Opossum MHC Coordinates Are Relative to scaffold_42:14,700,000 from MonDom2
The size and complexity of the opossum MHC is similar to that of eutherian mammals, but the organization resembles that seen in non-mammals. The
Class I and Class II regions are adjacent and somewhat interspersed. The antigen processing genes are closely linked to the Class I genes. The framework
region does not contain Class I genes, as it does in eutherians. Like eutherians, the opossum MHC does not contain the third inducible proteasome
subunit gene, PSMB10. Some genes present in the human MHC have not been found on the opossum scaffold_42 (MAS1L, the histone cluster, C6orf12,
HCG2P4, RANP1, C6orf205, C6orf1, DHFRP, HCP5, HCG9P1, PPIP9, LST1, NCR3, AIF1, LY6G5B, LY6G6D, LY6G5E, C6orf10, RPL32P1, PPP1R2P1, HTATSF1P,
MYL8P, and LYPLA2P1).
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organization evolved from a simple ancestral form to the
complex forms seen today in therian mammals. We propose
that in the MHC of a therian ancestor of marsupials and
eutherians, Class I and Class II loci were located together at
one end of the region, along with the antigen processing
genes. A similar hypothesis was suggested previously based on
studies of MHC organization in non-mammals [12]. Adjacent
to the Class I and II regions was a gene-rich Class III region
that already contained most of the genes present in human,
mouse, and opossum MHC. The framework region, devoid of
most or all Class I genes, assembled on the opposite side of
Class III. The extended regions are present in the opossum as
well as in eutherians, and therefore must have been present in
the ancestral form. Studies have identified extended region
genes in close proximity to MHC genes in teleost fish, despite
the overall non-linkage of Class I, II, and III genes in these
species [3,13,14].
The eutherian MHC Class I–III–II structure exemplified by
rodents and primates evolved relatively recently. Class I genes
must have relocated across the Class III region and
interspersed between the framework genes after the diver-
gence of marsupials and eutherians, but prior to the
divergence of primates and rodents (;60 million y ago)
[15]. This process gave rise to the eutherian Class I region. It is
unclear how or why the Class I genes relocated, but Class I
loci appear to ‘‘migrate’’ in different species along with their
frequent expansions and contractions. Specifically, Fukami-
Kobayashi et al. [16] have suggested that long interspersed
nuclear element (LINE) sequences can trigger genome frag-
ment duplications, producing pairs of duplicated genome
fragments. Perhaps, a series of duplicated genome fragments
inserted themselves between framework genes in ancient
eutherian mammals and have since been evolving via
expansions and contractions in their new location.
The opossum MHC is unique in that Class I and II genes are
interspersed and closely linked to antigen processing genes.
The Class I expansion has occurred within the Class II region.
The opossum MHC Class I/II region contains 11 putative Class
I and ten Class II genes (predicted coding sequences available
at http://bioinf.wehi.edu.au/opossum). Class II loci include the
non-classical DMA and DMB genes, whose homologs are
found in birds and eutherians. Three marsupial-specific
classical Class II gene families are present; DA, DB [17], and
a newly discovered family that we have designated DC
(Figures 1 and 5).
Of the 11 Class I loci in the opossum MHC, only one; UA1,
is known to have all the characteristics expected of a classical
Class Ia locus by being both ubiquitously expressed and
highly polymorphic. UA1 transcripts have been detected in all
tissues tested by RT-PCR and account for all previously
described Class Ia cDNAs [18] (Figure 6A). The level of UA1
polymorphism is also comparable to that of human HLA-A
Figure 2. Fluorescence In Situ Hybridization of Opossum MHC-
Containing BAC Clones on Opossum Metaphase Chromosomes
(A) Co-localization of BACs containing MOG (162N8) (green) and COL11A2
(27I16) (red) to show the orientation of the main MHC region at the
centromeric region of 2q.
(B) Co-localization of MHC-linked Class I UG (323O1) BAC to the
centromeric region of 2q (green) and putative non-classical Class I UB/
UC (253C16)–containing BAC to the telomeric region of 2p (red).
(C) Localization of CD1 (969K11) to Chromosome 2p.
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Marsupial MHC

(N. Gouin, P. B. Samollow, M. L. Baker, and R. D. Miller,
unpublished data). Expression of a single classical Class Ia
gene in the opossum is unusual for a mammal, but not
unprecedented in vertebrates. For example, both the chicken
and Xenopus laevis have a dominantly expressed single func-
tional Class Ia molecule [19,20]. Unlike X. laevis, the opossum
Class Ia gene, UA1, does not appear to have allelic lineages.
Two of the Class I loci (UA2 and UH) appear to be
pseudogenes, because they lack a predicted ORF and have not
been found expressed in any of the tissues examined (data not
shown). Two other loci (UF and UL) have predicted ORFs, but
their transcription has not been detected in any tissue so far
and their functionality remains unknown. Five of the
remaining Class I loci (UE, UK, UJ, UI, and UM) are all
transcribed in the thymus (Figure 6B); however, each have
tissue-specific expression, suggesting they are likely Class Ib
in nature (S. D. Melman, M. L. Baker, and R. D. Miller,
unpublished data). UG is transcribed in all tissues tested,
including thymus, but the peptide binding sites are not
polymorphic clearly suggesting it is a Class Ib gene (data not
shown; N. Gouin, M. L. Baker, P. B. Samollow, and R. D.
Miller,unpublished data). Overall, the majority of the 11
opossum Class I loci are transcribed. Since transcription is
detected in the thymus, these have the potential to
participate in T-cell selection, although other functions in
thymic differentiation and T-cell development and regula-
tion can not be ruled out.
The expressed Class I loci in the opossum Class I/II region
are highly diverse, sharing as little as 49% nucleotide identity,
and at most 83%, over exons 2, 3, and 4 among loci. A
phylogenetic analysis of the Class I loci, including Class Ia and
Ib loci from other species, is shown in Figure 7. Despite the
sequence divergence of opossum Class I loci, they are
phylogenetically related and probably evolved from common
ancestral loci. This observation raises some questions about
one of the current theories explaining the general absence of
non-classical Class I genes within the MHC of non-mammals.
It has been suggested that proximity of Class I genes to the
antigen processing genes has constrained their divergence
[12]. In eutherians, loss of this tight association by movement
Figure 3. Comparative Map of the MHC Organization in Mammals
The color code is the same as in Figure 1. Orthologous genes are indicated by connecting lines. Dashed lines and question marks represent unknown or
uncertain data for a given gene or portion of the genome. The total number of genes is reported for each region or sub-region (pseudogenes are not
included). Map is not drawn to scale. Unless specified, the small boxes represent single genes. This map was generated using data from [2,47]. The
asterisk in the figure indicates that the duplication of DMB concerns only the mouse.
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of the Class I genes away from the antigen processing genes
may have resulted in increased plasticity that led to
fluctuations in gene number and function and allowed Class
Ib genes to reside in the MHC [12]. However, in the opossum,
antigen processing genes have not constrained the diversifi-
cation of the adjacent classical and non-classical Class I genes.
It is unclear what selective advantage, if any, might have been
gained by the separation of Class I from Class II or antigen
processing genes in eutherians. Close linkage has been
implicated in co-evolution of Class I genes and antigen
processing genes [19,21–23]. Perhaps the Class I genes in M.
domestica have evolved to be less constrained by their
proximity to the antigen processing machinery, allowing
them to duplicate and diversify in close linkage to the TAP
and PSMB genes. Alternatively, co-evolution with the antigen
processing machinery may have severely restricted Class I
evolution in this marsupial, perhaps resulting in only a single
locus performing the classical role.
Class I loci, UB and UC, were previously assumed to be
linked to the MHC due to their high levels of sequence
similarity to UA1 [18], but surprisingly they are not found on
the scaffold containing the MHC region (Figure 1) and have
been localized to the telomere of Chromosome 2p, distant
from the MHC at 2q centromere (Figure 2). Localization of
Class I genes outside the MHC implies that these genes may
have a non-classical role. In eutherians, the Class I loci lying
outside the MHC are among the most divergent from Class Ia
genes [2]. However, in non-mammals, genes closely related to
Class Ia have been found outside the MHC, and in sharks,
Class Ib genes found outside the MHC share very high levels
of similarity to the Class Ia genes [8]. These non-mammalian
genes have been designated Class Ib without elucidation of
their functional roles, based on levels of expression and
polymorphism. Currently, we do not have information about
polymorphism levels of UB and UC, but their relatively low
expression levels [24] may indicate evolution towards non-
classical Class Ib functions. UB and UC are both flanked by
marsupial-specific retroelements of the CORE-SINE type [24],
which would be consistent with the role of such elements in
Class I gene mobility [16] and may explain the recent
relocation of UB and UC outside of the MHC [24]. The high
level of sequence similarity of UA, UB, and UC raises the
possibility that Class Ia genes can maintain their function
when unlinked to the MHC.
Comparisons between MHC sequences of distantly related
mammals highlight the conservation of the most important
regulatory sequences, namely the SXY DNA motifs. Tran-
scription of most MHC Class I and II genes is largely
regulated by the Class II transactivator (CIITA), which
interacts with several transcription factors, particularly those
that bind to this motif [25,26]. Conservation of promoter
elements in opossum Class I genes has been reported
previously [24]. Using computational methods, we were able
to identify SXY motifs upstream of most opossum MHC Class
I and II genes. Eight SXY motifs were identified within 273
base pairs (bp) of the coding start in the opossum Class II
genes (Table 1). Overall, these motifs were found to be
conserved between eutherians and the opossum (Figure 8A).
Eight SXY motifs were also identified upstream of opossum
Class I genes (Table 1). We were not able to identify the SXY
motif in genes UK, UF, and UM. Furthermore, the S motifs in
the promoters of genes UH, UI, and UL appear to be weak
with respect to the eutherian pattern. This suggests that the
opossum Class I SXY regions have diverged from their
corresponding eutherian motifs (particularly in the X motif;
Figure 8B) more than the Class II SXY regions have. This is
not unexpected given that Class II genes (classical and non-
classical) are typically co-expressed whereas the non-classical
Class I genes tend to evolve novel functions.
Perhaps most significantly, our data also suggest an ancient
relationship between the MHC and the natural killer complex
(NKC), which contains C-type lectin natural killer (NK) cell
receptor loci [27]. This relationship is drawn from the
presence of two genes within the opossum MHC, MIC and
OSCAR. Opossum MIC is the most distant homolog to the
polymorphic human Class I genes MICA and MICB found to
date (Figure 7). The MIC genes are Class I–related genes that
encode ligands for NKG2D, a C-type lectin NK receptor [28].
MIC genes are not found within the MHC of rodents. Instead,
rodents have closely related genes, known as MILL.Ina
phylogenetic analysis, the opossum MIC is basal to a clade
containing human MICA/B and the mouse MILL1/2 genes
(Figure 7). The function of rodent MILL genes is not yet
known [29], but our results support a common evolutionary
Figure 4. A Model of the Evolution of the Mammalian MHC
Organization of the MHC in the mammalian ancestor is similar to that of
non-mammals. Class I and II regions are adjacent and the antigen
processing genes are found within Class I. Class Ib genes are located
outside the MHC but on the same chromosome. The framework and
extended regions have assembled. The framework gene order is
conserved in both the opossum and eutherians. After the divergence
of the eutherian lineage, Class I genes relocated to the framework region.
MYA, million years ago.
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origin of MIC and MILL in eutherians. The presence of MIC
in the opossum MHC, and its apparent absence in non-
mammals, implies that MIC-like genes appeared before
marsupials and eutherians diverged, and uniquely evolved
into MILL in rodents.
The osteoclast-associated receptor (OSCAR) was first
discovered as a receptor on mouse osteoclasts [30], but it
has recently been shown to participate in antigen uptake and
processing for Class II molecules in dendritic cells [31].
OSCAR (also known as polymeric immunoglobulin receptor 3)
is located within the leukocyte receptor complex (LRC) of
humans, chimps, mice, and rats [32]. The presence of an
OSCAR homolog within the opossum MHC is surprising.
Using Genscan, we confirmed that the opossum OSCAR
homolog contains an intact ORF and a predicted promoter.
Human and opossum OSCAR share 47% identity at the amino
acid level and are reciprocal best hits in BLAST searches
(opossum OSCAR against human Refseq: best hit
Figure 5. Phylogenetic Tree of the MHC Class II Genes
(A) MHC Class IIB gene phylogeny based on full length amino acid sequences. Prior studies have named DAB and DBB [17]. Here we report a new Class
IIB gene family, DCB. The DMB genes were used as the outgroup. DAB was not found in scaffold_42 and a cDNA sequence was used for the analysis.
Physical mapping localizes DAB BACs to the centromeric region of 2q, and it appears this gene was not sequenced or was unable to be assembled.
(B) MHC Class II A gene phylogeny. Location of IIA genes near IIB genes in scaffold_42 allowed designation of IIA genes to class IIB gene families: DA, DB,
and DC. Bootstrap values are too low to be able to ascertain orthology with eutherian gene families.
DOI: 10.1371/journal.pbio.0040046.g005
Figure 6. RT-PCR Results Demonstrating Class I Expression
(A) RT-PCR results demonstrating Class I UA1 expression in brain (B), gut (G), kidney (K), liver (Lv), lung (Lg), skin (Sk), spleen (Sp), and thymus (T).
(B) RT-PCR results demonstrating expression of Class I UE, UI, UJ, UK, and UM in the thymus.
Similar results were found for UG (not shown). Controls (water) were negative for all primer combinations (not shown).
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Marsupial MHC

NP_573399.1 OSCAR isoform 4, e-value ¼ 7e
66). Further,
the presence of OSCAR in the opossum MHC suggests that
involvement in antigen processing may be its original
function.
MHC Class I molecules are ligands for NK cell receptors, so
these two gene families must co-evolve. Keeping up with the
rapid evolution of the MHC loci in response to pathogenic
pressures is thought to have resulted in the independent
evolution of two vertebrate NK receptor families, the C-type
lectin and Ig superfamily types. In humans, the C-type lectin
NK receptors are found on Chromosome 12 within the NKC
[27]. The second NK receptor family contains the killer cell
Ig-like receptors (KIR) and is encoded in the LRC on human
Chromosome 19 [27]. The recent discovery of C-type lectin
NK receptor genes in avian MHC [6] supports an ancestral
association of the MHC and the C-type lectin genes of the
eutherian NKC. Just as birds provide an ancestral link
between the MHC and NKC [33], the two aforementioned
opossum genes, OSCAR and MIC, provide links between the
MHC and LRC. OSCAR is in the MHC in opossum (Figure 1)
but in the LRC in humans and rodents. MIC genes are in the
MHC of opossums and humans, whereas the related MILL1/2
genes are in the LRC of rodents [34]. These observations
support the existence of an ancestral genomic region in
amniotes that probably contained MHC Class I loci and NK
cell receptor genes of both the KIR and C-type lectin forms.
This organization would have allowed both classes of NK
receptors to co-evolve with their MHC ligands.
Recently, CD1 genes were linked to the MHC of chickens,
and may have been part of the primordial MHC [35,36].
Although a clear evolutionary relationship is evident between
eutherian MHC Class I genes and CD1, CD1 is not located
within the eutherian MHC. The marsupial homolog of CD1
has been identified in the opossum genome (M. L. Baker, S. D.
Melman, and R. D. Miller, unpublished data). It is located on a
separate scaffold (scaffold_13) from that containing the
MHC and maps to Chromosome 2p (Figure 2). CD1, like the
NK receptors, probably moved out of the MHC after the
separation of mammals and birds but prior to the separation
of eutherians and marsupials.
Comparative analyses of the MHC region in opossum and
other species supports the idea that at one time in vertebrate
evolution there was a single ‘‘immune supercomplex’’ of
genes that contained MHC Class I and II, antigen processing
genes (TAP and PSMB), CD1, and C-type and Ig-type NK
receptor genes [37]. This complex is no longer found in any
living species analyzed so far, but clues of its existence remain
in extant genomes.
Materials and Methods
Sequence analysis and annotation. All results presented in this
paper are based on the MHC-containing scaffold from the
preliminary assembly of theMonodelphis domestica genome, MonDom2,
released by the Broad Institute. The contig N50 length is 111 kb and
the scaffold N50 length is 54 Mb (J. Chang, personal communication).
MonDom2 is an interim assembly of unordered scaffolds. A final M.
domestica assembly with ordered scaffolds that are anchored to
chromosomes is in preparation. Similarity features were identified
by aligning all of the human proteins from the extended MHC that
are represented in the RefSeq collection (Release 11, http://www.ncbi.
nlm.nih.gov/RefSeq) against the opossum genome using TBLASTN
[38] and extracting best hits. Known opossum MHC Class I and II
genes were located by aligning their transcripts with the opossum
genome assembly using BLASTN [38]. To exclude alignments with
shared domains in other genes, a heuristic approach that identified
the shortest chain of BLAST HSPs (highest-scoring segment pairs)
having the best protein coverage was implemented. A single scaffold,
scaffold_42, was found to contain most of the genes expected in the
MHC. Known MHC transcripts from other marsupial species,
including tammar wallaby (Macropus eugenii) and brushtail possum
(Trichosurus vulpecula) were aligned with opossum scaffold_42 using
TBLASTN and best hits were extracted. Gene predictions were made
by running GENSCAN [39] on scaffold_42. To visualize these four
feature annotation tracks, a MHC genome browser, based on
GBROWSE [40], was set up. All features were clustered spatially
(based on sequence position) using a custom PYTHON (http://www.
python.org) script and these cluster features were hand curated. If a
Figure 7. Phylogenetic Tree of the MHC Class I Genes
MHC class I phylogeny based on nucleotide alignments corresponding to
exons 2, 3, and 4 of MHC Class Ia and Ib loci from M. domestica and
representative vertebrate species.
DOI: 10.1371/journal.pbio.0040046.g007
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Marsupial MHC

known opossum gene was present in a cluster, that gene replaced the
cluster in the curated annotation. Class I cDNA sequences, obtained
using 59 and 39 RACE, were aligned with scaffold_42 using BLAT
[41]. Class III genes were annotated by extracting sequence from the
cluster neighborhood and GENOMESCAN [42] was used together
with the orthologous human protein. Class II and framework region
genes were annotated using a combination of GENSCAN, alignment
of orthologous proteins from multiple species with the predicted
protein and hand curation of the putative gene. The annotation of
the extended regions is based, in general, on similarity features only.
BAC isolation and physical mapping. Overgos were designed for
scaffolds with multiple BLAST hits to MHC genes in MonDom1.0
using the Overgo Maker program (http://genomeold.wustl.edu/tools/
?overgo¼1) and manually. High-density filters from the male opossum
BAC library VMRC-6 (http://bacpac.chori.org/opposum6.htm) were
probed with labeled overgos to identify MHC BAC clones. Overgo
labeling and filter hybridizations were carried out using the BACPAC
hybridization protocol (http://bacpac.chori.org/overgohyb.htm).
Overgos are as follows: 2447_ova, 59-CAAAGGGAAGTGAGCA
GAACCATG-39 and 59-CTGTATACATGGCTCTCATGGTTC-39;
2447_ovb, 59-ATGTGTTGTGCCTGAGGTTGTAGC-39 and 59-
CACTTCTAGGCCCAATGCTACAAC-39; 11936_ova, 59-AAAGGG
GAATTCTGGGGCATGAAG-39 and 59-CAGCGGTCCTCCTC
TACTTCATGC-39; 11936_ovb, 59-CCAGGAGGACAGCATAAGTA
GAAG-39 and 59-CTACCTAGGAGGTAGTCTTCTACT-39;
14804_ova, 59-CTTATCAGAGGCTAGCAGAGCTAA-39 and 59-
CCTCCTCTGATTCTTTTTAGCTCT-39; 14804_ovb, 59-
GTGCCCAAGGAACTTTCCAAATAC-39 and 59-CCCTGACACCTT
CATAGTATTTGG-39; 15208_ova, 59-CCAGATAGGCTGAT
GAGCCTTTAC-39 and 59-TTGAAGACCATTGCATGTAAAGGC-39;
15208_ovb, 59-GGTCACCTCAAAGAGTACTGGGTT-39,and
15208_3ovb, 59-CCAAGTTAACTCATCTAACCCAGT-39;
16657_5ova, 59-ATAAGGAATCCTGGGCCTGAGGAT-39 and 59-
TCATGGCTGCTCCTCTATCCTCAG-39. The overgos used to isolate
BAC 323O1 were: 59-GGCTGAGGGATGGAGAGGAACAGC-39 and
59-AATTCGGTGTCCTGGAGCTGTTCC-39. The overgos used to
isolate BAC 253C16 were Mdo3OVF, 59-CCTGCCGAGATCTCCCT
GACGTGG-39,andMdo3OVR,59-CCTCGCCATCCCGCAGC
CACGTCA-39.
A BAC containing the M. domestica CD1 locus was isolated from the
opossum BAC library VMRC-18 (http://bacpac.chori.org/opposum6.
htm). This clone (BAC 969K11) was identified because it contained
BAC-end sequences that flank CD1 in scaffold_13 in the current M.
domestica genome sequence assembly. Internal sequencing of BAC
969K11 was performed to confirm the presence of CD1.
PCR primers for each scaffold end were used to further screen
putative positive BACs. Resulting products were cloned into pCR 4-
TOPO cloning vector (Invitrogen, Carlsbad, California, United States)
and sequenced using vector primers M13 forward and reverse.
Primers: 2447_aF, 59-AAAGGAGGGACTGTTGGAGTAAGC-39, and
2447_aR, 59-TCTTGGCTCTTCAGACACACTATCC-39; 2447_bF,
59-GTTGATGAATGTGTTGTGCCTGAG-39, and 2447_bR, 59-CCA
GAACCCCTTTAGTGCCTATC-39; 11936_aF, 59-CCACATCCTATT
CATCTTTGACCC-39, and 11936_aR, 59-GGCAATGCTGGT
GACCTTCTAC-39; 11936_bF, 59-TGTGGGTTGGGTAGAGTG
GAATC-39, and 11936_bR, 59-GCTTCTGCTGTTTTTATGGGCAC
-39; 14804_aF, 59-TTGCCAGAGATTTCCCCAAAG-39,and
14804_aR, 59- CATTATGCCTAAACTGTGTGCCC-39; 14804_bF,
59-GGCTCAGAGAATGTAATGGGAGTG-39, and 14804_bR, 59-
GCACAGGAACAGTTGAACAGTAAGC-39; 15208_aF, 59-CACTGC
CAAACTTAGACTCTTCCC-39, and 15208_aR, 59-TGACCACC
CAAAAGCCTTGAG-39; 15208_bF, 59-TATTCGGTCACCACACA
GAGCC-39, and 15208_bR, 59-GCTTGCCATTCTCCAAAGGG-39;
16657_aF, 59-TTGGGTGCTTCAGTCAGAGAGTG-39,and
16657_bR, 59-TAGGAAAGAGGGATGCTGGGAG-39.
MHC-positive BAC clones (85D1, 162N8, 27I16, 158D1, 169A24,
175J22, 207O10, 121D2, 78J18, 34E18, 53E6, 58H11, 323O1, 256G22,
249P7, 255G18, 258J24, 278M18, 32301, 256G22, 249P7, 255G18, and
278M18) and the CD1-positive clone (969K11) were differentially
labeled and co-hybridized to metaphase chromosomes. BAC DNA (1
lg) was labeled by nick translation with either biotin-16-dUTP or
digoxygenin-11-dUTP (Roche Diagnostics, Basel, Switzerland). La-
beled probes were precipitated with 1 lg sheared opossum genomic
DNA (size range between 200 and 700 bp) to suppress repetitive
elements, and 50 lg salmon sperm DNA which acted as a carrier for
the precipitation. Metaphase chromosomes from a male M. domestica
fibroblast cell line were prepared [43] and hybridized as described
previously [44]. Fluorescence signals were captured on a Zeiss
Axioplan epifluorescence microscope (Carl Zeiss, Thornwood, New-
York, United States) equipped with a CCD (charge-coupled device)
camera (Spot RT; Diagnostic Instruments, Sterling Heights, Michigan,
United States) and merged with DAPI images using IPlab imaging
software (Scanalytics, Rockville, Maryland, United States).
Class I expression analysis. Analysis of Class I gene expression in
Table 1. Coordinates of the Opossum Class I and Class II Loci Located within the MHC
Name Class Strand Start SXY Start SXY End Relative Position
(SXY End to Gene Start)
DMA IIA þ 15520875 15520657 15520723
152
DMB IIB þ 15537756 15537419 15537483
273
DAA IIA
16449681 16449871 16449819
138
DCB IIB þ 16676304 16676125 16676182
122
DBB1 IIB þ 16143787 16143569 16143635
152
DBB2 IIB þ 16274807 Not found Not found n/a
DCA IIA
16673321 16673514 16673441
120
DBA1 IIA
16173517 16173713 16173664
147
DBA2 IIA
16391327 16391523 16391457
130
DXA IIA
15939128 Not found Not found n/a
UH I þ 15572303 15572099 15572188
115
UK I þ 15592656 Not found Not found n/a
UF I þ 15610609 Not found Not found n/a
UI I þ 15651694 15651656 15651724 þ30
UG I
15703369 15703519 15703457
88
UJ I þ 15713254 15713092 15713160
94
UA1 I þ 15841508 15841335 15841402
106
UL I þ 16004483 16004249 16004313
170
UE I þ 16045439 16045276 16045344
95
UM I þ 16067883 Not found Not found n/a
UA2 I þ 17048709 17048528 17048604
105
The locus name is indicated in the first column. The Class is indicated in the second column, with A and B indicating the alpha and beta chains of Class II, respectively. The transcription orientation (þor
strand) and coding start coordinates
shown are relative to scaffold_42 of the MonDom2.0 assembly. The start and end coordinates of the SXY motif locations are shown.
n/a, not applicable.
DOI: 10.1371/journal.pbio.0040046.t001
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Marsupial MHC

different tissues was done using reverse transcription PCR (RT-PCR).
Total RNA was extracted using Trizol (Invitrogen) following
manufacturers recommended protocols. Thymus, brain, and spleen
RNA were extracted from tissues taken from a 9-wk-old male M.
domestica. All other tissues were from an adult (1-y-old) female. RNA
was treated with TURBO DNA-free (Ambion, Austin, Texas, United
States) to remove contaminating DNA. RT-PCR using total RNA
samples from tissues shown in Figure 6 was performed using the
GeneAmp RNA PCR Core Kit with oligo-dT priming following
manufacturers recommended protocols (Applied Biosystems, Foster
City, California, United States). The primers used for each Class I
locus were designed to amplify exons 2 through 4, with the exception
of UA1, which amplifies exons 2 to 3, and UJ, which amplifies exons 3
to 4. The primer sequences were: UA1, 59-GCTCGGGGACTCG
CAGTTCATCTCG-39 and 59-CCATCTGCAGGTACTTCTTCAGC
CAC-39; UE, 59-CTGAACCGAGGTTCACAGCTGTA-39 and 59-
GCTCACTTCCAGAGAGCATCTCC-39; UI, 59-AGAGTACTTCGA
CAGCCACAGCGCT-39 and 59-CCTCTTCCTGACCTGAAGTCAAA
GA-39; UJ, 59-GCAACTTCAGGCGCGGGTTTAAAAG-39 and 59-
CGGTACTGGTGATGGGTCACTCCTG-39; UM, 59-ATGCGAGTC
AGAGCACCGAGATTGG-39 and 59-CTGAGTCAGAGGTGA
TATGGCGGGT 39 and UK, 59-GGGAGACCGCTCAGACTTTCGAA
39 and 59-CTTCATGGCTAATGTGATGAGTG-39.
The size of the PCR products for UA1, UE, UI, UJ, UK, and UM are
487, 710, 666, 478, 393, and 267 bp respectively. The specificity of
each RT-PCR amplification was confirmed by direct cloning (TOPO-
TA cloning kit; Invitrogen) and sequencing (BigDye Terminator Cycle
Sequencing Kit v3; Applied Biosystems) of the PCR products.
Sequencing reactions were run on an ABI 3100 and chromatograms
were analyzed using the Sequencher ver 4.5 program (Gene Codes,
Ann Arbor, Michigan, United States).
Phylogenetic analysis. Sequence alignments were made by first
aligning amino acid translations to establish gaps corresponding to
codon position. The MHC Class II trees were constructed using
neighbor joining using pairwise deletion with Jones-Taylor-Thornton
matrix (JTT) matrix model and 100 bootstrap replicates using MEGA
3. Species included are nurse shark (Gici), zebrafish (Brre), chicken
(Gaga), echidna (Taac), platypus (Oran), rednecked wallaby (Maru),
gray short-tailed opossum (Modo), brushtail possum (Trvu), rabbit
(Orcu), cat (Feca), mouse (Mumu), mole rat (Naeh), human (Hosa).
Class IIB Genbank references are as in [17,45]. Class IIA Genbank
accession numbers are listed below in Accession Numbers.
The MHC Class I tree was constructed using Maximum Parsimony
with 1,000 bootstrap replicates using the MEGA 3 program (http://
www.megasoftware.net). The overall tree topology was reproduced
using the Neighbor Joining and Minimal Evolution models. M.
domestica Class I loci were named in the following manner: UA1 was
identified as the locus encoding the previously identified Class Ia
transcripts (e.g., Modo3 included in this tree [18]), and UA2 is a locus
with high nucleotide identity (94% over exons 2, 3, and 4) to UA1;
UA1 and UA2 are the only two Class I loci within the MHC similar
enough to be considered two members of the same family. UB and UC
were previously described [24] and named, and are not in the MHC
(Figure 2). UE through UM are individual loci sharing 49% to 83%
nucleotide identity over exons 2, 3, and 4 in a pairwise comparison.
Species abbreviations are as in Figure 5 with the addition of rhesus
macaque (Mamu), cottontop tamarin (Saoe), pig (Susc), cow (Bota),
and rat (Rano).
Figure 8. Analysis of the SXY Promoter Regions of MHC Class I and II Genes
(A) SXY motifs in the MHC Class II genes. The LOGOs [48] of the corresponding position-specific scoring matrix models are presented. The height of each
stack of symbols (y-axis) represents the information content in each position of the DNA sequence in log
2
terms (bits of information) with a maximum
value of 2. (B) SXY motifs in the MHC Class I genes.
DOI: 10.1371/journal.pbio.0040046.g008
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Marsupial MHC

Analysis of SXY promoter regions. Known eutherian SXY motifs
were used to build models to scan the promoters of the opossum
genes. SXY motifs from 24 eutherian MHC Class II genes were
collected and position-specific scoring matrix models [46] were
constructed for each individual S, X, and Y motif. The distance
between the individual motifs was also taken into consideration. The
genes we used as training set were the human genes HLA-DOA, HLA-
DMA, HLA-DPA1, HLA-DQA1, HLA-DRA (distal), HLA-DRA, HLA-DOB,
HLA-DMB, HLA-DQB1, HLA-DRB1, and HLA-DRB3; the mouse genes
H2-DMa, H2-Ea, H2-Oa, H2-Ab1, H2-DMb1, and H2-Eb1; and the rat
genes RT1-Da, RT1-DOa, RT1-DMa, RT1-Db1, RT1-Bb, RT1-DOb, and
RT1-DMb. These models were subsequently used to scan the
promoters of the following ten opossum Class II genes: DBA1,
DBA2, DAA, DBB1, DBB2, DCA, DCB, DMA, DMB, and DXA1.
‘‘Promoters’’ refers to 5 kb upstream and 1 kb downstream of the
coding start.
Using the methodology described above, we also generated a model
for the Class I SXY motif using data from 27 eutherian genes: the
human genes HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G; the
mouse genes H2-M10.1, H2-M10.2, H2-M10.3, H2-M10.4, H2-M10.5,
H2-M10.6, H2-M2, H2-M3, H2-Q10, H2-Q7, H2-T10, and H2-T17; and
the rat genes RT-CE10, RT1-CE11, RT1-CE13, RT1-CE15, RT1-CE16,
RT1-CE3, RT1-CE4, RT1-CE5, and RT1-CE7. A second model was
constructed from two previously characterized Monodelphis MHC
Class I–related genes: UB and UC [24]. The promoters of the 11
Monodelphis genes (i.e., UH, UK, UF, UI, UG, UJ, UA1, UL, UE, UM, and
UA2) were scanned using these models.
Supporting Information
Accession Numbers
The GenBank (http://www.ncbi.nlm.nih.gov/Genbank) accession num-
bers for the genes and gene products discussed in this paper are
scaffold_42 (CH465496); brushtail possum FcRN (AF191647) and
TrvuUB (AF359509); chicken Gaga (AF013491) and (AY357253);
cottontop tamarin Saoe-G (M63952); cow Bota (X80936); gray short-
tailed opossum Modo-3 (AF125540), ModoUB (AF522352), and
ModoUC (AF522352); human DMA (NP006111), DNA (M26039), DPA
(M27487), DQA (M26041), DRA (M6033), FcRN (AF220542), HFE
(AF115265), HLA-A (U03862), HLA-B (X91749), HLA-Cw (U06487),
HLA-E (BC002578), HLA-F (BC009260), HLA-G (M32800), MICA
(AY204547), and MICB (U95729); mouse DNA (M95514), DQA
(M21931), DRA (U13648), FcRN (D37874), K
b
(U47328), MILL1
(NM_153749), and MILL2 (NM_153761); nurse shark Gici
(M89950); pig Susc (AF014002); platypus Oran (AY112715); rabbit Orcu
(K02441); rat DMA (NP942036), DNA (H004806), DPA (AH004805),
DQA (X14879), DRA (Y00480), and RT1 (X90376); rednecked wallaby
DAA (previously DRA) (U18109), DBA (previously DNA) (U18110), and
MaruUB01 (L04952); rhesus macaqueMamu-A (AJ542571) andMamu-B
(AF157402); and zebrafish Brre (L19445).
Acknowledgments
The authors would like to thank the Broad Institute, Cambridge,
Massachusetts, for making M. domestica genome sequence data
available.
Author contributions. KB and RDM conceived and designed the
experiments. KB, JED, ATP, MLB, SDM, HVS, NG, DLG, TJS, MDR,
MJW, SM, JGRC, PVB, PBS, TPS, JAMG, and RDM performed the
experiments and analyzed the data. ATP and DLG contributed
analysis tools. KB and RDM wrote the paper. JED, ATP, and NG
prepared figures.
Funding. This work was supported by grants from the Australian
Research Council (KB and JAMG), the National Health and Medical
Research Council (ATP and TPS), the National Institutes of Health
(NIH) (RR-014214, PBS), the National Science Foundation (PVB and
RDM), the NIH Institute Development Award Program of the
National Center for Research Resources (MLB and RDM), The
Southwest Foundation Forum (NG), and the University of Sydney
(KB).
Competing interests. The authors have declared that no competing
interests exist.
&
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PLoS Biology | www.plosbiology.org March 2006 | Volume 4 | Issue 3 | e460328
Marsupial MHC