In silico identification of opossum cytokine genes suggests the complexity of the marsupial immune system rivals that of eutherian mammals.

Abstract

Cytokines are small proteins that regulate immunity in vertebrate species. Marsupial and eutherian mammals last shared a common ancestor more than 180 million years ago, so it is not surprising that attempts to isolate many key marsupial cytokines using traditional laboratory techniques have been unsuccessful. This paucity of molecular data has led some authors to suggest that the marsupial immune system is 'primitive' and not on par with the sophisticated immune system of eutherian (placental) mammals.
The sequencing of the first marsupial genome has allowed us to identify highly divergent immune genes. We used gene prediction methods that incorporate the identification of gene location using BLAST, SYNTENY + BLAST and HMMER to identify 23 key marsupial immune genes, including IFN-gamma, IL-2, IL-4, IL-6, IL-12 and IL-13, in the genome of the grey short-tailed opossum (Monodelphis domestica). Many of these genes were not predicted in the publicly available automated annotations.
The power of this approach was demonstrated by the identification of orthologous cytokines between marsupials and eutherians that share only 30% identity at the amino acid level. Furthermore, the presence of key immunological genes suggests that marsupials do indeed possess a sophisticated immune system, whose function may parallel that of eutherian mammals.

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BioMed Central
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Immunome Research
Open Access
Research
In silico identification of opossum cytokine genes suggests the
complexity of the marsupial immune system rivals that of eutherian
mammals
Emily SW Wong†1, Lauren J Young†2, Anthony T Papenfuss3 and
Katherine Belov*1
Address: 1Faculty of Veterinary Science, University of Sydney, Sydney, New South Wales, Australia, 2School of Chemical and Biomedical Sciences,
Central Queensland University, Rockhampton, Queensland, Australia and 3Division of Bioinformatics, The Walter and Eliza Hall Institute of
Medical Research, Melbourne, Victoria, Australia
Email: Emily SW Wong - emilyw@vetsci.usyd.edu.au; Lauren J Young - l.young@cqu.edu.au; Anthony T Papenfuss - papenfuss@wehi.edu.au;
Katherine Belov* - kbelov@vetsci.usyd.edu.au
* Corresponding author †Equal contributors
Abstract
Background: Cytokines are small proteins that regulate immunity in vertebrate species. Marsupial
and eutherian mammals last shared a common ancestor more than 180 million years ago, so it is
not surprising that attempts to isolate many key marsupial cytokines using traditional laboratory
techniques have been unsuccessful. This paucity of molecular data has led some authors to suggest
that the marsupial immune system is 'primitive' and not on par with the sophisticated immune
system of eutherian (placental) mammals.
Results: The sequencing of the first marsupial genome has allowed us to identify highly divergent
immune genes. We used gene prediction methods that incorporate the identification of gene
location using BLAST, SYNTENY + BLAST and HMMER to identify 23 key marsupial immune genes,
including IFN-γ, IL-2, IL-4, IL-6, IL-12 and IL-13, in the genome of the grey short-tailed opossum
(Monodelphis domestica). Many of these genes were not predicted in the publicly available automated
annotations.
Conclusion: The power of this approach was demonstrated by the identification of orthologous
cytokines between marsupials and eutherians that share only 30% identity at the amino acid level.
Furthermore, the presence of key immunological genes suggests that marsupials do indeed possess
a sophisticated immune system, whose function may parallel that of eutherian mammals.
Background
The marsupial and eutherian (placental) lineages
diverged approximately 180 million years ago. Marsupials
are chiefly distinguished from other mammals by their
unique reproductive strategies, with young born in an
immature state with only the most rudimentary neurolog-
ical and immunological systems [1]. At birth, the animal
manoeuvres its way to a waiting teat, where it attaches
until it reaches a state of maturity that allows it to function
independently. Marsupials possess lymphoid tissue and
cellular components that are structurally similar to those
of other mammals. Key antigen receptor and recognition
Published: 10 November 2006
Immunome Research 2006, 2:4doi:10.1186/1745-7580-2-4
Received: 26 June 2006
Accepted: 10 November 2006
This article is available from: http://www.immunome-research.com/content/2/1/4
© 2006 Wong et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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molecules including Major Histocompatibility (MHC)
Class I, II and III [2], T Cell Receptors alpha, beta, gamma
and delta [3,4], Toll-like receptors [5] and immunoglobu-
lins [6] have been characterized.
However, conventional experimental strategies using
degenerate primers for reverse-transcriptase polymerase
chain reaction (RT-PCR) and heterologous probes for
screening genetic libraries have only identified the most
phylogenetically conserved immune molecules, with
cytokines proving particularly difficult to isolate [7]. To
date, only eleven cytokines including one receptor have
been cloned from marsupials. They include tumour
necrosis factor alpha (TNF-α) [8,9], lymphotoxin (LT) -α
and -β [10,11], Interleukin IL-1β [12], IL-1R2 [7], IL-5
[13], IL-10 [14], leukemia inhibitory factor LIF; a member
of the IL-6 family [15] and three type I Interferon (IFN)
genes [16]. These cytokines show relatively high levels of
identity compared to their eutherian homologues. Previ-
ous attempts to isolate the more divergent T-cell derived
cytokines that orchestrate adaptive immunity such as IL-2,
IL-4 and interferon-γ have failed [7,17].
Identification of divergent marsupial immune genes is
important for two reasons. Firstly, unsuccessful attempts
to isolate T cell derived cytokines in the laboratory has led
some authors to suggest that the marsupial immune sys-
tem is 'primitive' and does not possess the level of com-
plexity demonstrated by eutherians such as humans and
mice. The fact that some T cell driven responses are also
aberrant adds to this argument. Marsupials appear to have
delayed skin graft rejection [18] and antibody class
switching [19], together with an apparent lack of an in
vitro Mixed Lymphocyte Response [20]. Elucidation of
genes involved in specific immunity will help us to deter-
mine whether the apparently 'simple' immune responses
generated by marsupials are genetically hardwired.
The second reason for identifying divergent immune
genes in the marsupial genome is to develop marsupial
specific immunological reagents. To date, most assay sys-
tems employed to characterise cells and their function rely
on eutherian reagents or culture techniques developed in
eutherian species. Where low levels of cross reactivity exist
between marsupials and these model species, the useful-
ness of the data generated from such assays is limited.
Identification of key cell markers, such as CD4 and CD8
will allow us to generate marsupial-specific reagents,
which would then be used to gain a better understanding
of the marsupial immune response.
Difficulties associated with identifying rapidly evolving
cytokines are not limited to marsupials. The chicken IL-2
gene took seven years of focused effort to identify [21],
and was eventually found using expression strategies and
not heterologous cloning techniques. The recent sequenc-
ing of the complete genomes of a large number of non-
eutherian vertebrates will expedite the isolation and char-
acterization of these immune genes in distantly related
species. However, currently automated annotation tech-
niques are not sensitive enough to identify many of these
molecules outside the eutherian lineage.
The first marsupial genome was recently sequenced by the
Broad Institute. The subject of this project, Monodelphis
domestica, is a South American opossum. It is a well-recog-
nised biomedical model in the study of comparative phys-
iology, immunogenetics, cancer development and disease
susceptibility. Two publicly available annotations of this
genome have been generated. Ensembl have produced a
gene build with their automatic pipeline [22], which relies
principally on GeneWise [23], while the UCSC genome
browser provides several annotation tracks with similarity
features and gene models, for example chained TBLASTN
alignments of human proteins, BLAT alignments of Ref-
Seq mRNAs, and Genscan [24] and N-SCAN [25] predic-
tions. With the exception of the Genscan predictions,
which are ab initio gene predictions based on genomic
sequence only, the gene builds rely on cross species
homology, as no large-scale opossum EST projects have
been completed yet and there are only 425 known opos-
sum protein sequences in GenBank. In most cases,
Ensembl and the UCSC genome browser were unable to
identify highly divergent cytokine genes such as IL-2, 4
and 13.
To overcome this shortcoming in the automated annota-
tion of the opossum genome and to start to address uncer-
tainties about immune function in marsupials, we have
adopted a manual, expert-curated approach to annotating
highly divergent genes. The critical first stage of this is the
careful identification of the genomic region containing
the gene. This is performed using a sensitive TBLASTN
search. HMMER [26] can also be useful at this stage. Fre-
quently, it is necessary to first narrow the search to the
syntenic region by identifying conserved flanking genes.
Having identified similarity features, gene prediction is
performed on genomic sequence extracted from the
region. The accuracy of gene prediction is dependent on
the prediction method. As with the automated annota-
tions, we favour gene predictors that incorporate informa-
tion from orthologous sequences into the prediction
process. In addition to GeneWise and N-Scan, there are
now several such methods available including Procrustes
[27], HMMgene,[28] GenomeScan [29], and Augustus+
[30]. Procrustes and the default GeneWise algorithm per-
form spliced alignment. Augustus+ uses an interesting
approach, which constrains predicted genes to incorpo-
rate user-supplied hints. However, it is not particularly

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convenient for manual use or use by biologists lacking
scripting skills. While not the only possible choice, we
have found GenomeScan to be both convenient and rea-
sonably accurate (based on comparison with known euth-
erian sequences). It is worth noting that there is another
class of gene prediction methods that obtain homology
information from syntenic regions of other genomes.
These include TwinScan [31], which is asymmetric and
predicts genes in one genome only and SLAM [32], which
simultaneously aligns two genomes and predicts genes in
both. These methods were unlikely to be useful in our
study as we were looking for genes that are highly diver-
gent and difficult to align at the genomic level. Finally, a
comparison of predicted results with known eutherian
sequences and curation of the result was undertaken if
required. Our success with this strategy suggests that this
method will be applicable to the identification of rapidly
evolving gene families in other distant vertebrate species.
Results
Overview
In silico searching revealed a total of 23 cytokine
sequences, all of which are described in the opossum for
the first time and 5 of which are novel for any marsupial
species (see Table 1). A number of critical cytokine recep-
tors are also identified, as are the sequences for the hall-
mark T cell cluster of differentiation markers, CD4 and
CD8.
The majority of genes reported in this study were identi-
fied using sensitive peptide BLAST searches (Table 2). The
most divergent genes, interleukins 2, 4 and 13, were iden-
tified using synteny searches. Properties of the putative
proteins identified in this study, predicted structures and
comparison with human sequences are summarised in
Tables 1 and 2. Sequence data of the predicted proteins
are available online [33].
Isolation of interleukins using BLAST and synteny searches
Interleukins 2, 4 and 21 and their common gamma chain
receptor were identified using both BLAST and syntenic
strategies. IL-21 was identified by a sensitive TBLASTN
search (e-value = 2e-18) on Chromosome 5:7034081–
7057815. The predicted protein is of similar size and con-
tains the same number of exons as human IL-21 [see
Additional File 1]. The signal peptide was predicted to be
encoded within the first 21 amino acids (score = 7.6, p =
0.06), with N-linked glycosylation sites predicted at posi-
tions 46 and 106 and O-linked glycosylation of threonine
predicted at position 55. Instability motifs (ATTTA) were
Table 1: Comparison of putative opossum and known human cytokine sequences. Opossum IFN-α genes were compared with 13
human IFN-α genes.
IdentityNumber of exons in open reading frame Number of amino acids % amino acid identity*
Cytokines
opossumhuman opossum human
Interleukin 2
Interleukin 4
Interleukin 5
Interleukin 6
Interleukin 10
Interleukin 12A
Interleukin 13
Interleukin 19
Interleukin 20
Interleukin 21
Interleukin 22
Interleukin 24
Interleukin 26
Interferon γ
Type 1 Interferons
IFN-α (seven genes)
IFN-β
IFN-κ
Cytokine receptors
Common cytokine receptor gamma chain (IL-2Rγ)
Interferon-γ receptor 2
T cell surface receptors
CD4
CD8
4
4
4
5
5
7
4
6
6
5
4
6
5
4
4
4
4
5
5
7
4
6
5
5
5
6
5
4
144
138
137
221
173
231
122
188
154
163
150
220
201
167
153
153
134
212
178
253
146
215
176
162
179
206
171
166
41.8
43.3
53.0
36.3
59.5
52.2
36.7
58.0
59.7
46.5
40.0
43.7
55.9
47.0
1
1
1
1
1
1
183
184
156
188–9 (range)
187
207
33–42 (range)
43.2
51.3
8
6
8
7
349
269
367
344
54.9
49.6
9
6
9
6
485
349
458
235
45.6
37.7
* compared with human sequence

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not found in the 3' UTR of the sequence before the
poly(A)+ signal.
Opossum IL-2 was found by searching genomic sequence
flanking IL-21, which is adjacent to, and has significant
structural homology with IL-2 in humans [see Additional
File 2]. This strategy was adopted since the alignment of
human IL-2 against the opossum genome using TBLASTN
resulted in no hits. A 395 kb region adjacent to IL-21 was
extracted and 15 genes within this region were predicted
with GENSCAN. The predicted gene most similar to IL-2
was identified using BLASTP. The sequence was extracted
and GenomeScan was used with an IL-2 orthologue to
obtain a more accurate prediction. Opossum IL-2 was
located on Chromosome 5:7191593–7196834 (Fig 1)
and contains several conserved residues essential for bio-
logical activities, including two cysteine residues that pro-
vide structural stability [34] and the amino acids leucine
and aspartic acid within helix A, which are crucial for
binding of the ligand to IL-2Rβ in humans [35]. Also well
conserved is a glutamine residue in the D helix, which is
directly involved in the binding of the IL-2Rγ chain [36].
Similar to the human sequence, the putative peptide is
142 amino acids in length and contains 4 exons. A signal
peptide that contains a potential O-linked glycosylation
site (position 13 – Thr) is predicted from positions 1–22
(score = 9.9, p = 0.03). A potential N-linked glycosylation
site, not found in humans or mice, but present in several
eutherians including the cat and dog, is found at position
101. Four mRNA instability motifs (ATTTA) are present
upstream of the poly(A)+ signal.
Opossum IL-2Rγ was identified using TBLASTN (e-value =
8e-119) (Table 1). It shares 61% amino acid similarity
with the human sequence [see Additional File 3].
IL-5 was identified on chromosome 1:307529660–
307531352. It shares 53.0% identity to human IL-5, and
86.7% identity to the tammar wallaby IL-5 [13] [see Addi-
tional File 4].
Synteny searches located the sequence for IL-4 [see Addi-
tional File 5]. RAD50 (GenBank accession no: AAB07119)
and kinesin-like protein KIF3A (GenBank accession no:
NP_008985) are situated adjacent to IL-4 and IL-13 in
humans. The area between these proteins in opossum was
extracted and GENSCAN predictions were searched with
BLASTP and FASTP for suitable matches. IL-4 was identi-
fied using FASTP and was located on Chromosome 1
(307752915–307754456). The predicted peptide is 138
amino acids in length (Fig 2). It has low levels of identity
to human IL-4 (30.8%). Two putative N-linked glycosyla-
tion sites were identified. SPScan was unable to predict a
putative signal sequence although two instability motifs
(ATTTA) were recognised in the 3' UTR region. Despite the
variation in sequence between the predicted opossum and
Table 2: Summary of putative opossum cytokine genes including search strategy, best hit, predicted glycosylation sites and signal
peptide information.
Identity Search StrategyBest HitNumber of predicted
glycosylation sites
Signal peptide
identified
Cytokines
referencee-valueN-glyO-gly opossum
Interleukin 2
Interleukin 4
Interleukin 5
Interleukin 6
Interleukin 10
Interleukin 12A
Interleukin 13
Interleukin 19
Interleukin 20
Interleukin 21
Interleukin 22
Interleukin 24
Interleukin 26
Interferon-γ
Type 1 Interferons
IFN-α (7 genes)
IFN-β
IFN-κ
Cytokine receptors
Common cytokine receptor gamma chain (IL-2Rγ)
Interferon-γ receptor 2
T cell surface receptors
CD4
CD8
synteny
synteny
BLAST
BLAST
BLAST
BLAST
synteny/HMMER
BLAST
BLAST
BLAST
BLAST
BLAST
BLAST
synteny
-
-
AAD37462.1
NP_112445.1
AAD01799.1
NP_032377.1
-
NP_001009940.1
NP_061194.2
Q9HBE4
NP_065386.1
NP_006841.1
NP_060872.1
-
-
-
4e-028
0.081
3e-011
9e-006
-
3e-008
1e-004
0.088
3e-005
2e-006
3e-009
-
1
2
1
2
2
1
0
3
0
2
1
2
0
2
1
0
0
3
0
1
0
0
0
1
0
0
0
0
Yes
Yes
Yes
Yes
Yes
No
No
Yes
Yes
Yes
Yes
Yes
No
Yes
BLAST
BLAST
BLAST
AAO37656.1
AAO37656.1
AAO37656.1
1e-019 to 9e-974 (range) 0–4 (range)
2
1
0
0
0
Yes
Yes
No
BLAST
BLAST
NP_000197.1
NP_005525.2
9e-019
3e-018
6
3
3
2
No
Yes
BLAST
BLAST
NP_000607.1
Q60965
5e-009
9e-012
4
1
0
8
Yes
Yes

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Alignment of IL-2 amino acid sequences
Figure 1
Alignment of IL-2 amino acid sequences. Diamonds denote functionally important residues [64]. Inverted triangles indi-
cate cysteine residues involved in disulfide bonds in human protein [64]. Squares above the alignment show predicted glyco-
sylation sites from the opossum sequence. Dots represent identity to Monodelphis domestica sequence. Sequences used for
alignment: Homo sapiens (NP_000577), Bos taurus (NP_851340), Sus scrofa (NP_999026), Mus musculus (NP_032392), Gallus gal-
lus (NP_989484), Canis familiaris (NP_001003305), Macaca fascicularis (Q29615), Felis cattus (AAC15974), Equus caballus
(CAA49190), Cervus elaphus (P51747), Capra hircus (AAQ10671), Ovis aries (NP_001009806), Oryctolagus cuniculus (O77620),
Peromyscus maniculatus (AAP04419), Rattus norvegicus (NP_446288), Anser cygnoides (AAR28994). Not all sequences are shown
in the figure.

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Alignment of IL-4 amino acid sequences
Figure 2
Alignment of IL-4 amino acid sequences. Inverted triangles indicate cysteine residues that form disulfide bonds in the
human protein [65]. Squares above the alignment show predicted N-linked glycosylation sites in the opossum sequence. Dots
represent identity to Monodelphis domestica sequence. Sequences used for alignment: Homo sapiens (NP_000580), Mus musculus
(NP_067258), Gallus gallus (NP_001007080), Equus caballus (P42202), Rattus norvegicus (NP_958427), Mesocricetus auratus
(Q60440).

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human IL-4 protein sequences, disulfide bonds that join
helix B to the CD loop and that are important for biolog-
ical activity are conserved [37].
IL-4 and IL-13 were identified simultaneously using a syn-
tenic approach since they sit adjacent in the human
genome [see Additional File 5]. Opossum IL-13 (Chromo-
some 1:307682382–307686155) is found 74.30 kb
upstream from opossum IL-4 and does not contain any
glycosylation sites. Alignment with mammal and chicken
protein sequences (Fig 3) revealed a truncation of 32
amino acids from the 5' end of the peptide in opossum IL-
13. This is probably due to incorrect gene prediction, a fact
supported by the absence of signal peptide and any insta-
bility motifs.
Opossum IL-6 was identified using a sensitive TBLASTN
search (e-value = 0.08). Opossum IL-6 is located on Chro-
mosome 8:296810942–296824133 and the PROSITE IL-
6 family motif (C-x(9)-C-x(6)-G-L-x(2)- [F,Y]-x(3)-L) is
conserved [see Additional File 6]. The signal peptide is
predicted from positions 1–28 (score = 8.1, p = 0.20) and
no instability motifs (ATTTA) are found in the 3' UTR.
Opossum IL-6 has maintained significant structural simi-
larities to human and other mammalian IL-6 genes
despite its comparatively low sequence identity. The
number and position of cysteine residues in opossum IL-
6 are identical to those found in eutherian and chicken
sequences. An arginine molecule in helix D that is
involved in IL-6β binding [38] is also conserved.
Opossum
260,626,803) was identified using a TBLASTN search and
is predicted to be 58% similar to its human orthologue
[see Additional File 7]. Cysteine residues are conserved
between the marsupial, eutherians and chicken
sequences.
IL-12 alpha chain (chr7:260,616,009–
IL-10 family members were identified in two clusters.
Chromosome 2 contained
113144942; [see Additional File 8]), IL-19 (113283404–
113294773; [see Additional File 9]), IL-20 (113319666–
113324608; [see Additional File 10]), IL-24 (113362216–
113377467; [see Additional File 11]) with identical head-
to-tail transcriptional orientation and organisation to
their human orthologues. Chromosome 8 contained IL-
26 (23485674–23494985; [see Additional File 12]) and
IL-22 (23457582–23460076; [see Additional File 13]).
The complete IL-22 open reading frame was not identified
since the 3' end (approximately 33 amino acids and 2
exons) fell in an unsequenced gap. However, conservation
of a predicted N-linked glycosylation site at N54 between
putative opossum IL-22 and human IL-22 (a site crucial
for IL-22 modulation during the inflammatory response)
suggests that this partial sequence is opossum IL-22. Both
IL-10
(113139397–
chicken and the amphibia contain IL-10 family members,
although only one IL-19-like ancestral gene replaces IL-
19, IL-20 and IL-24 in the chicken [39]. Orthology of the
IL-10 family cytokines with their eutherian counterparts
was confirmed by phylogenetic analysis [see Additional
File 14]. All putative IL-10 family members clustered
closely with their eutherian orthologs.
Isolation of cluster of differentiation markers using
TBLASTN
CD4 [see Additional File 15] and CD8 [see Additional File
16] were identified by TBLASTN search and found on
chromosome 8 (104157682–104183462) and chromo-
some 1 (716671734–716675645) respectively. Their
number of amino acids and potential glycosylation sites
are noted in Table 2. Neither we, nor Ensembl, were able
to successfully predict the terminal exons of these two
genes.
Isolation of interferons using BLAST, synteny and hidden
Markov models
Type I IFNs
Nine type I IFN coding sequences and 2 pseudogenes were
identified in the opossum genome using BLAST strategies.
Seven IFN-α genes, along with single copies of IFN-β and
IFN-κ were identified. Predicted opossum IFN-α
sequences share 68–78% identity and 78–99% similarity
at the amino acid level. IFN-α and -β genes were located
in a cluster on Chromosome 6, with IFN-κ situated
approximately 12 kb away (Fig 4). Phylogenetic analysis
revealed that opossum IFN-α sequences were interspersed
with known tammar wallaby IFN-αs (Fig 5).
Interferon gamma (IFN-γ) and interferon gamma receptor (IFN-γR2)
The signal transducing chain of the Interferon gamma
receptor was identified in the opossum genome on Chro-
mosome 4 (14328267–14355149). It shares 29–46%
amino acid identity with eutherian and chicken sequences
[see Additional File 17]. The ligand of IFN-γR2, IFN-γ, was
not identified in the genome, despite exhaustive searches
including searches using the Hidden Markov model
(HMM) containing predicted ancestral sequences. Accord-
ing to the gene organization in other vertebrates (includ-
ing birds and fish), IFN-γ should be adjacent to IL-22 and
IL-26 on chromosome 8. A large gap (9.6 kb) was located
in this region, suggesting that IFN-γ was simply not
sequenced, rather than being absent from the genome.
However, the availability of BAC end sequences generated
by the genome sequencing project did allow us to identify
a BAC (VMRC-18:653P7) that spanned this region.
Researchers at the Broad Institute, led by Kerstin Lind-
blad-Toh and April Cook kindly sequenced this BAC
(GenBank Accession: AC190119). IFN-γ was thus identi-
fied (Fig 6). It shares 47% amino acid identity with
human IFN-γ but predicted glycosylation sites are unique.

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Alignment of IL-13 amino acid sequences
Figure 3
Alignment of IL-13 amino acid sequences. Inverted triangles indicate cysteine residues that form disulfide bonds in the
human protein [66]. Dots represent identity to Monodelphis domestica sequence. Sequences used for alignment: Homo sapiens
(NP_002179), Mus musculus (NP_032381), Gallus gallus (NP_001007086), Rattus norvegicus (NP_446280), Canis familiaris
(NP_001003384), Sus scrofa (Q95J68).

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Level of confidence in our gene predictions
Where possible, gene predictions were verified by align-
ment with known marsupial cDNA sequences, and com-
pared to Ensembl gene predictions and UCSC similarity
features. For instance, a known cDNA sequence is availa-
ble for Trichosurus vulpecula (possum) IL-10 cDNA (Gen-
bank ref: AF026277). Our predicted opossum IL-10
protein shared 76% amino acid identity with possum IL-
10, and exon-intron boundaries match. However, despite
our use of robust methodologies, we are still not confi-
dent with prediction of the most divergent immune genes
sequences. Some doubt exists with our predications for IL-
4 and IL-13 and the terminal exons of IL-22, CD4 and
CD8. Characterisation of their cDNA, together with labo-
ratory-based assays will ultimately confirm the reliability
of the predictions reported here.
Discussion
Without EST and protein databases, annotation of dis-
tantly related mammalian species such as the marsupials
and monotremes is challenging. Neither Ensembl nor
UCSC were able to identify IL-2, 4, 13, 22 and IFN-γ. In
general, automated gene prediction missed key immune
genes because of their low levels of sequence similarity
with their eutherian orthologs. We suggest that future
studies focusing on in silico mining of divergent genes
should take into account gene location and features.
Application of this strategy allowed us to successfully
identify key immune genes in the opossum genome,
which traditional laboratory methods failed to isolate.
Discovery of key cytokines in the opossum genome sug-
gests that a re-examination of immune responses (espe-
cially T cell responses) is warranted in marsupials. The
peculiarities in class switching and in vitro T cell prolifera-
tion, which have previously been observed in marsupials
are largely controlled by T cells and their products. The
ability to discriminate between classic 'helper' T cells and
'cytotoxic' T cell families will now be possible due to the
identification of CD4 and CD8 sequences in the opossum
genome. Further, identification of cytokines normally
produced by these subsets in eutherian mammals will
allow us to investigate Th1 and Th2 profiles that orches-
trate immunity to intracellular and extracellular patho-
gens respectively.
There are a myriad of interactions between cytokines at
the cellular level, but the presence of a number of key
cytokines orchestrate the global immune response. For
example, when the macrophage-derived IL-12 is domi-
nant, Th1 responses predominate resulting in cell-medi-
ated immunity. When the B-cell growth factor IL-4 is
dominant, Th2 responses dominate and a humoral
immune response is activated [40]. Sequences for both of
these genes are present in the opossum genome, along
with other classical Th1- (IL-2, IFN-γ) and Th2- (IL-4, IL-
5, IL-6, IL-10 and IL-13) associated molecules.
The presence of key cytokines in a marsupial genome
strongly suggests that marsupials are capable of complex
immune responses comparable to those seen in eutherian
mammals. Knowledge of these gene sequences provides a
springboard for future studies. For instance, marsupials
appear to be susceptible to infection with intracellular
pathogens such as herpesvirus and mycobacterial spp
[41], indicative of impaired Th1 cytokine responses. The
availability of Th1 and Th2 cytokine sequences will allow
us to study IL-10 profiles, which are known to play a crit-
ical role in the survival of intracellular pathogens by
inhibiting the expression of inflammatory cytokines such
as IFN-γ and TNF. Meanwhile, studies of Th2 cytokines
may focus on protection against parasites. Both American
and Australian marsupials co-exist with a range of success-
ful parasites; opossums are reported to have natural
trypanosome infection rates of up to 100% [42] and carry
nematode burdens in the wild [43], whilst a variety of
Genomic organisation and transcriptional directions of type I IFNs on chromosome 6
Figure 4
Genomic organisation and transcriptional directions of type I IFNs on chromosome 6.
12 Kb
IFNK
IFNA7
IFNA6IFNA3IFNA1
IFNA2
IFNBIFNA4IFNA5

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Phylogenetic tree showing evolutionary relationship between type I interferon protein sequences
Figure 5
Phylogenetic tree showing evolutionary relationship between type I interferon protein sequences. Opossum
sequences are marked by diamonds. Sequences used: Homo sapiens IFN-α14 (NP_002163.1), IFN-α2 (NP_000596.2), IFN-β
(NP_002167), IFN-ω1 (P07352), IFN-κ(NP_064509.1); Mus musculus IFN-α2 (P01573), IFN-β (NP_034640), IFN-κ
(NP_954608.1); Sus scrofa IFN-δ (NP_001002832.1); Equus caballus IFN-α3 (CAA01292), IFN-ω2 (CAA01293); Bos taurus IFN-
ω1 (P07352), IFN-t (XP_874910); Ovis aries IFN-τ(CAA39783); Felis catus IFN-β (Q9N2J0); Macropus eugenii IFN-α1
(AAO37656), IFN-α2 (AAO37657.1), IFN-β (AAO37658.1); Gallus gallus IFN-α1 (CAA63214), IFN-β (NP_001020007).
SsuIFND
Hsa IFNA14
Hsa IFNA2
Eca IFNA3
Mmu IFNA2
Hsa IFNW1
Eca IFNW2
Bta IFNW
Oar IFNT
Bta IFNT
Mdo IFNA1
Meu IFNA1
Meu IFNA2
Placental IFNA and subtypes
Mdo IFNA2
Mdo IFNA3
Mdo IFNA4
Mdo IFNA5
Mdo IFNA6
Mdo IFNA7
Meu IFNB1
Mdo IFNB
Fca IFNB
Hsa IFNB1
Marsupial IFNA
Mmu IFNB
MmuIFNK
IFNB
Hsa IFNK
Mdo IFNK
IFNK
Gga IFNA, Gga IFNB
100
70
100
51
100
92
100
100
99
91
85
53
98
70
86
95
80
78
62
32
83
43
100
32
57
60
0.2

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Alignment of IFN-γ amino acid sequences
Figure 6
Alignment of IFN-γ amino acid sequences. Squares above the alignment show predicted N-linked glycosylation sites from
the opossum sequence. Dots represent identity to Monodelphis domestica sequence. Sequences used for alignment: Homo sapi-
ens (NP_000610.2), Felis cattus (P46402), Equus caballus (P42160), Equus asinus (O77763), Sus scofa (NP_999113.1), Ovis aries
(P17773), Mus musculus (NP_032363.1), Rattus norvegicus (NP_620235.1), Peromyscus maniculatus (AAP44086.1), Gallus gallus
(P49708), Danio rerio (NP_998029.1).

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helminth infections are common across a range of Aus-
tralian marsupials [44].
Allograft responses can now be studied due to the availa-
bility of sequence information for interleukins 2, 4, 21
and IL-2Rγ [45]. The opossum is an important model for
tumour immunology since it can be induced to accept
melanoma cells at both juvenile and adult life stages [46].
Both IL-2 and IL-24 are associated with melanoma
tumour suppression [47] in humans and it is now possi-
ble to study the role of these genes in the opossum model,
as well as in the maintenance of transmissible allograft
tumours in Tasmanian devil facial tumour disease [48].
Conclusion
Here we describe and apply a method to identify divergent
immune genes from the genome of a model marsupial,
Monodelphis domestica. We are now extending this analysis
to characterize the entire opossum immunome. We report
here that the opossum genome contains representatives
from the major vertebrate immune gene families. These
genes appear to be structurally similar, and therefore will
most likely prove to be functionally equivalent, to their
eutherian homologues. The way is now clear to further
probe the genes that orchestrate the marsupial immune
response and to investigate the role that these molecules
have on maintaining health and influencing disease sus-
ceptibility in this unique group of animals.
Methods
Data source
Draft sequencing of the genome of a female opossum
(Monodelphis domestica) has recently been completed by
the Broad Institute [49]. Analysis was performed on
assembly MonDom4 (January 2006).
Sequence identification
To optimise the chances of identifying previously undis-
covered sequences, our search strategy relied on a prelim-
inary database screen for sequence conservation, together
with positional analyses of the gene sequence relative to
other genes within the genome (synteny). Finally, the
putative sequence was analysed for the presence of biolog-
ically significant sites associated with both structure and
function in their eutherian homologues.
Similarity searching using BLAST
Sequence similarity searching (TBLASTN) was performed
with known eutherian sequences. Positive hits from the
BLAST search with good potential were extracted for fur-
ther structural analysis. When ambiguities existed
between alignments from BLAST results, each of the mul-
tiple hits were extracted and inspected. Assessment meth-
ods for ambiguous hits included tests for reciprocal-best-
hit where the aligned sequence was blasted against SWISS-
PROT and TrEMBL protein databases to confirm prelimi-
nary findings. Proteins discovered in BLAST searches were
used to mine additional homologues. To do this, param-
eters were optimised for sensitive searching. In order to
increase our ability to detect highly divergent sequences,
the BLOSUM 45 similarity matrix [50] was used. Addi-
tionally, application of soft-masking and the lowering of
the neighbourhood word threshold score to 9 increased
the chance of detecting homologous sequences that other-
wise might have been overlooked using default parame-
ters.
Synteny analysis
If the protein of interest was not detected by the initial
BLAST search, other methods were employed. Similarity
searches were performed with genes found in close syn-
tenic regions in the human genome. Syntenic regions were
extracted from the opossum database, and passed into
GENSCAN [24]. The predicted peptide sequences were
analysed by performing similarity searches against the
SWISS-PROT and TrEMBL databases using BLASTP and
FASTP [51]. In order to improve the accuracy of identified
cytokine sequences, the sequence was re-extracted from
the opossum database and the putative protein was re-
evaluated using GenomeScan [29]. Combined results
from GenomeScan and GENSCAN were compared with
documented structural features of the cytokine.
Additional methods for gene identification
For sequences that were not detected using the above
methods, a hidden Markov model (HMM) was built and
calibrated using the HMMER 2.3.2 package [52]. The
model was built as a multiple local alignment profile with
the Krogh/Mitchison substitution weight matrix [53] and
used to search the six-frame translation of the opossum
genome.
Ancestral sequences were included in the HMM. These
were calculated by programs in the Phylip package [54].
PRODIST was used to compute a distance matrix under
default settings. After this, the program NEIGHBOUR was
used to create a neighbour joining (NJ) tree from the
matrix. The tree was rooted with a teleost species. Follow-
ing this, ProML was set to produce ancestral sequences at
each of the NJ tree nodes.
Structural features
Once the gene of interest was located, exon/intron bound-
aries were identified using the gene prediction programs
GENSCAN [24] and GenomeScan [29]. Our experience
suggests that some caution is advisable in the interpreta-
tion of data from existing gene prediction software; exces-
sively long predicted genes ('thready' gene predictions)
due to mis-identification of first exons and merging of
adjacent genes, and unlikely predictions of splice sites

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(based on comparison with orthologous sequences) were
the most common problems we observed. Mindful of
these limitations, our gene predictions were compared
with known gene structures. The presence of signal pep-
tides was predicted by SPScan (Accelrys GCG) and estima-
tion of glycosylation sites were made with NetOGlyc 3.1
[55] and NetNGlyc 1.0 [56]. Finally, sequences were sub-
mitted to the PROSITE database [57] for detection of pro-
tein family motifs that would confirm gene identify.
Sequence alignments
Sequences from the opossum and other species were
aligned using ClustalW [58]. Accession numbers of
sequences used in analyses are shown in figure legends.
Sequence labels in the alignments are abbreviated by the
first letter from the genus with the first two letters from the
species name followed by the gene name. In figures, resi-
dues with functional importance are highlighted.
Phylogenetic analysis
Neighbour-joining (NJ) trees were constructed using the
Jones-Taylor-Thornton substitution model [59] and 500
bootstrap replicates in MEGA 3.1 [60]. The tree, con-
structed from amino acid sequences, was rooted using
chicken sequences.
Sequence identity
Sequence identity and similarity calculations were carried
out using GAP (Accelrys GCG), with the Needleman-
Wunsch alignment [61], except for IFN-α genes which
were calculated in GenDoc [62] using the BLOSUM 35
similarity matrix [50] for comparisons of human and
opossum IFN-α genes and BLOSUM 80 matrix [50] for
comparisons among opossum sequences. GCG, GENS-
CAN, BLASTP and FastA programs were accessed through
the Australian National Genomic Information Service
(ANGIS) [63].
Competing interests
The author(s) declare that they have no competing inter-
ests.
Authors' contributions
EW performed the bioinformatics studies and helped to
draft the manuscript
LJY wrote the final manuscript and participated in the
design of the study
ATP critically revised the bioinformatics data and co-ordi-
nated the design of the bioinformatics approach
KB conceived of the study and co-ordinated and helped
with the preparation of the final manuscript
All authors read and approved the final manuscript
Additional material
Additional File 1
Alignment of IL-21 amino acid sequences. Squares above the alignment
show predicted glycosylation sites from the opossum sequence. Residues
Asp33 and Gln145 are important for receptor binding in humans and are
denoted by a diamond [71]. Inverted triangles indicate cysteine residues
that are conserved across species. Dots represent identity to Monodelphis
domestica sequence. Sequences used for alignment: Homo sapiens
(Q9HBE4), Mus musculus (NP_068554.1), Gallus gallus
(NP_001020006.1), Canis familiaris (NP_001003347.1), Sus scofa
(Q76LU6), Bos taurus (Q76LU5).
Click here for file
[http://www.biomedcentral.com/content/supplementary/1745-
7580-2-4-S1.doc]
Additional File 2
Syntenic region between human chromosome 4q27 and opossum chro-
mosome 5, illustrating the gene cluster of interleukin 2 and 21. Tran-
scriptional directions are indicated by arrows.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1745-
7580-2-4-S2.doc]
Additional file 3
IL-2Rγ amino acid sequences. Conserved cysteine residues are marked
with an inverted triangle. Dots represent identity to Monodelphis
domestica sequence. Completely conserved residues are shaded.
Sequences used for alignment: Homo sapiens (NP_000197.1), Mus
musculus (NP_038591.1), Gallus gallus (NP_989858.1), Rattus
norvegicus (NP_543165.1), Canis familiaris (NP_001003201.1),
Sus scrofa (NP_999248.1), Bos taurus (NP_776784.1).
Click here for file
[http://www.biomedcentral.com/content/supplementary/1745-
7580-2-4-S3.doc]
Additional file 4
Alignment of IL-5 amino acid sequences. Dots represent identity to
Monodelphis domestica sequence. Sequences used for alignment:
Homo sapiens (NP_000870.1), Macaca mulatta
(NP_001040598.1), Bos taurus (NP_776347.1), Canis familiaris
(NP_001006951.1), Mus musculus (NP_034688.1), Macropus
eugenii (AAD37462.1), Gallus gallus (NP_001007085.1).
Click here for file
[http://www.biomedcentral.com/content/supplementary/1745-
7580-2-4-S4.doc]
Additional file 5
Syntenic region between human chromosome 5q23.3 and opossum
chromosome 1, illustrating the gene cluster of interleukin 5, 4 and 13.
Transcriptional directions are indicated by arrows.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1745-
7580-2-4-S5.doc]

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Additional file 6
Alignment of IL-6 amino acid sequences. Residues involved in receptor
binding in human IL-6 are denoted with diamonds. Cysteine residues con-
served among all species are marked with an inverted triangle. PROSITE
family motif is boxed. Dots represent identity to Monodelphis domes-
tica sequence. Sequences used for alignment: Homo sapiens
(NP_000591.1), Mus musculus (NP_112445.1), Oryctolagus cunic-
ulus (Q9MZR1).
Click here for file
[http://www.biomedcentral.com/content/supplementary/1745-
7580-2-4-S6.doc]
Additional file 7
Alignment of IL-12α amino acid sequences. Cysteine residues conserved
among all species are marked with an inverted triangle. Dots represent
identity to Monodelphis domestica sequence. Sequences used for align-
ment: Homo sapiens (NP_000873.2), Mus musculus
(NP_032377.1), Gallus gallus (NP_998753.1), Rattus norvegicus
(NP_445842.1), Ovis aries (NP_001009736.1) Canis familiaris
(NP_001003293.1).
Click here for file
[http://www.biomedcentral.com/content/supplementary/1745-
7580-2-4-S7.doc]
Additional file 8
Alignment of IL-10 amino acid sequences. Dots indicate identity to
Monodelphis domestica sequence. Sequences used for alignment:
Homo sapiens (NP_000563.1), Mus musculus (NP_034678.1),
Gallus gallus (NP_001004414.1), Trichosurus vulpecular
(AAD01799), Canis familiaris (NP_001003077.1), Sus scofa
(Q29055), Cervus elaphus(P51746).
Click here for file
[http://www.biomedcentral.com/content/supplementary/1745-
7580-2-4-S8.doc]
Additional file 9
Alignment of IL-19 amino acid sequences. Dots indicate identity to
Monodelphis domestica sequence. Sequences used for alignment:
Homo sapiens (NP_037503.2), Mus musculus (NP_001009940.1).
Click here for file
[http://www.biomedcentral.com/content/supplementary/1745-
7580-2-4-S9.doc]
Additional file 10
Alignment of IL-20 amino acid sequences. Dots indicate identity to
Monodelphis domestica sequence. Sequences used for alignment:
Homo sapiens (NP_061194.2), Mus musculus (NP_067355.1),
Tetraodon nigroviridis (AAP57416.1).
Click here for file
[http://www.biomedcentral.com/content/supplementary/1745-
7580-2-4-S10.doc]
Additional file 11
Alignment of IL-24 amino acid sequences. Dots indicate identity to
Monodelphis domestica sequence. Sequences used for alignment:
Homo sapiens (NP_006841.1), Mus musculus (NP_444325.1),
Rattus norvegicus (NP_579845.1), Tetraodon nigroviridis
(AAP57418.1).
Click here for file
[http://www.biomedcentral.com/content/supplementary/1745-
7580-2-4-S11.doc]
Additional file 12
Alignment of IL-26 amino acid sequences. Dots indicate identity to
Monodelphis domestica sequence. Sequences used for alignment:
Homo sapiens (NP_060872.1), Danio rerio (NP_001018635.1).
Click here for file
[http://www.biomedcentral.com/content/supplementary/1745-
7580-2-4-S12.doc]
Additional file 13
Alignment of IL-22 amino acid sequences. Dots indicate identity to
Monodelphis domestica sequence. Sequences used for alignment:
Homo sapiens (NP_065386.1), Mus musculus (NP_058667.1), Sus
scofa (AAX33671.1), Rattus norvegicus (ABF82262.1), Danio rerio
(NP_001018628.1).
Click here for file
[http://www.biomedcentral.com/content/supplementary/1745-
7580-2-4-S13.doc]
Additional file 14
Neighbour-Joining tree of IL-10 family ligand protein sequences
rooted by midpoint. JTT amino acid substitution matrix was used and
500 bootstrap replicates performed. Branches supported by bootstrap val-
ues over 70 are in bold. Opossum sequences are marked by triangles.
Sequences used for this analysis were Homo sapiens IL-10
(NP_000563.1), IL-19 (NP_715639.1), IL-20 (NP_061194.2), IL-22
(NP_065386.1), IL-24 (NP_006841.1), IL-26 (NP_060872.1); Mus
musculus IL-10 (NP_034678.1), IL-19 (NP_001009940.1), IL-20
(NP_067355.1), IL-22 (NP_058667.1), IL-24 (NP_444325.1); Rat-
tus norvegicus IL-24 (NP_579845.1); Sus scofa IL-10 (Q29055); Bos
taurus IL-10 (P43480); Trichosurus vulpecular IL-10 (AAD01799);
Gallus gallus IL-10 (NP_001004414.1); Cyprinus carpio IL-10
(BAC76885.1); Tetraodon nigroviridis IL-10 (CAD67786.1); Tak-
ifugu rubripes IL-10 (CAD62446.1) Danio rerio IL-26
(NP_001018635.1) and Monodelphis domestica. Sequences labels in
the tree are abbreviated by the first letter from the genus with the first two
letters from the specific name followed by the gene name.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1745-
7580-2-4-S14.doc]
Additional file 15
Alignment of CD4 amino acid sequences. Dots indicate identity to
Monodelphis domestica sequence. Sequences used for alignment:
Homo sapiens (NP_000607.1), Mus musculus (NP_038516.1),
Macaca mulatta (BAA09671.1) Felis cattus (NP_001009250.1), Rat-
tus norvegicus (NP_036837.1) Oncorhynchus mykiss
(AAY42068.1).
Click here for file
[http://www.biomedcentral.com/content/supplementary/1745-
7580-2-4-S15.doc]
Additional file 16
Alignment of CD8 amino acid sequences. Dots indicate identity to
Monodelphis domestica sequence. Sequences used for alignment:
Homo sapiens (NP_001759.3), Mus musculus (Q60965), Gallus
gallus (NP_990566.1), Canis familiaris (NP_001002935.1), Sus
scofa (NP_001001907.1), Rattus norvegicus (AAH88126.1).
Click here for file
[http://www.biomedcentral.com/content/supplementary/1745-
7580-2-4-S16.doc]

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Acknowledgements
This work was funded by the Australian Research Council (KB), Central
Queensland University (LJY) and the University of Sydney (KB). EW's PhD
scholarship is funded by the ARC Centre for Kangaroo Genomics and the
Jean Walker Trust. We thank the Broad Institute (especially Kerstin Lind-
blad-Toh and April Cook) for providing us with early access to the opos-
sum genome data and for sequencing an additional BAC which was
important for this study. We also gratefully acknowledge the encourage-
ment and support of Terry Speed, who contributed to the publication costs
of this manuscript.
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Additional file 17
Alignment of IFNGR-2 amino acid sequences. Cysteine residues con-
served among all species are marked with an inverted triangle. Dots indi-
cate identity to Monodelphis domestica sequence. Sequences used for
alignment: Homo sapiens (NP_005525.2), Mus musculus
(NP_032364.1), Gallus gallus (NP_001008676.1).
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7580-2-4-S17.doc]