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Genome Biology 2005, 6:R92
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2005Bekpenet al.Volume 6, Issue 11, Article R92
Research
The interferon-inducible p47 (IRG) GTPases in vertebrates: loss of
the cell autonomous resistance mechanism in the human lineage
Cemalettin Bekpen
*
, Julia P Hunn
*
, Christoph Rohde
*
, Iana Parvanova
*
,
Libby Guethlein
*§
, Diane M Dunn
†
, Eva Glowalla
*¶
, Maria Leptin
*‡
and
Jonathan C Howard
*
Addresses:
*
Institute for Genetics, University of Cologne, Zülpicher Strasse 47, 50674 Cologne, Germany.
†

Eccles Institute of Human Genetics,
University of Utah, Salt Lake City, UT 84112-5330, USA.
‡
Informatics & Systems Groups, Sanger Centre, The Wellcome Trust Genome Campus,
Hinxton, Cambridge, CB10 1SA UK.
§
Department of Structural Biology, Stanford University Medical School, Stanford, CA 94305, USA.
¶
Institute for Microbiology and Immunology, University of Cologne Medical School, 50935 Cologne, Germany.
Correspondence: Jonathan C Howard. E-mail:
© 2005 Bekpen et al.; licensee BioMed Central Ltd.
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 work is properly cited.
Vertebrate p47 GTPases<p>A survey of p47 GTPases in several vertebrate organisms shows that humans lack a p47 GTPase-based resistance system, suggesting that mice and humans deploy their immune resources against vacuolar pathogens in radically different ways.</p>
Abstract
Background: Members of the p47 (immunity-related GTPases (IRG) family) GTPases are
essential, interferon-inducible resistance factors in mice that are active against a broad spectrum of
important intracellular pathogens. Surprisingly, there are no reports of p47 function in humans.
Results: Here we show that the p47 GTPases are represented by 23 genes in the mouse, whereas
humans have only a single full-length p47 GTPase and an expressed, truncated presumed pseudo-
gene. The human full-length gene is orthologous to an isolated mouse p47 GTPase that carries no
interferon-inducible elements in the promoter of either species and is expressed constitutively in
the mature testis of both species. Thus, there is no evidence for a p47 GTPase-based resistance
system in humans. Dogs have several interferon-inducible p47s, and so the primate lineage that led
to humans appears to have lost an ancient function. Multiple p47 GTPases are also present in the
zebrafish, but there is only a tandem p47 gene pair in pufferfish.
Conclusion: Mice and humans must deploy their immune resources against vacuolar pathogens in
radically different ways. This carries significant implications for the use of the mouse as a model of
human infectious disease. The absence of the p47 resistance system in humans suggests that
possession of this resistance system carries significant costs that, in the primate lineage that led to

humans, are not outweighed by the benefits. The origin of the vertebrate p47 system is obscure.
Background
It is generally assumed that the immune system of the mouse
is a good experimental model for that in humans. However,
several studies suggest that immune mechanisms have been
evolving rather differently in the human and mouse lineages
(for review, see Mestas and Hughes [1]). The p47 (immunity-
related GTPases (IRG) family; see Nomenclature, below)
GTPases present a uniquely striking example of this
divergence.
Published: 31 October 2005
Genome Biology 2005, 6:R92 (doi:10.1186/gb-2005-6-11-r92)
Received: 4 June 2005
Revised: 7 September 2005
Accepted: 7 October 2005
The electronic version of this article is the complete one and can be
found online at />R92.2 Genome Biology 2005, Volume 6, Issue 11, Article R92 Bekpen et al. />Genome Biology 2005, 6:R92
In mice the interferon-γ-inducible p47 GTPases constitute
one of the most powerful resistance systems against several
important intracellular pathogens [2-4]. The proteins localize
on intracellular membrane systems in interferon-induced
cells, some (IGTP, IIGP1) favoring the endoplasmic reticulum
[5,6] and others (LRG-47, GTPI) the Golgi membranes [6,7]
(for names of individual IRG GTPases see Additional data file
1). Infection or phagocytosis, however, initiates redistribution
of the p47 GTPases to the phagocytic vacuole [6-8]. The p47
GTPases probably act specifically against vacuolar pathogens.
Thus, Gram-positive and Gram-negative bacteria, mycobac-
teria, and protozoal pathogens are all resisted by the p47
GTPases, whereas no viral target has yet been confirmed.

The p47 GTPase IIGP1 is a low-affinity nucleotide binding
protein with a slow GTP turnover [9]. At high protein concen-
trations and in the presence of GTP, IIGP1 oligomerizes and
increases GTP turnover by up to 20-fold. These properties are
distinct from those of the classical signaling GTPases and are
reminiscent of the dynamins and p65 (GBP-1) GTPases
[10,11]. The crystal structure of IIGP1 exhibits a H-Ras-1-like
nucleotide-binding domain flanked by amino-terminal and
carboxyl-terminal helical domains that are unknown in other
GTPases [12]. This basic structure is probably common to the
whole family. However, the divergent sequences of published
p47 GTPases [13] and the patterns of susceptibility in knock-
out strains (for reviews, see Taylor [2] and MacMicking [3,4])
show that the proteins are highly diversified. Thus, a sub-
group of three proteins (the GMS GTPases) have a radical
substitution (the substitution of Methinine (M) for Lysine
(K)) in the conserved P-loop G1 motif of the nucleotide bind-
ing site (Walker A motif) and correlated sequence variation
elsewhere in the G-domain [13], implying a distinct catalytic
mechanism for GTP hydrolysis. In the case of IIGP1 and LRG-
47, the cell biology of the two proteins is distinct; IIGP1 asso-
ciates with the endoplasmic reticulum membrane primarily
through an amino-terminal myristoylation sequence,
whereas LRG-47 associates with Golgi membrane via an
amphipathic helix in the subterminal domain [6]. We recently
showed that IIGP1 participates in a novel effector mechanism
in Toxoplasma gondii infected astrocytes involving vesicula-
tion and ultimately destruction of the parasitophorous vacu-
ole membrane [8]. In contrast, there is evidence that LRG-47
is involved in accelerated acidification of the phagocytic vac-

uole containing Mycobacterium tuberculosis [8].
The p47 GTPases are thus a functionally diverse resistance
system with many signs of adaptive divergent evolution. Sur-
prisingly, there are no reports of p47 GTPase function in
humans. To address this imbalance, we analyzed the p47
GTPase gene family in depth. We conclude that although the
mouse has 23 p47 GTPases, of which up to 20 may be func-
tional in resistance, the resistance system is entirely absent
from humans. This finding carries important implications for
our understanding of human and mouse immunity to vacu-
olar pathogens.
Results
Genomic organization of the p47 GTPase (Irg) genes of
the C57BL/6 mouse
There are 23 p47 GTPase (Irg) genes in the C57BL/6 mouse,
including the six previously known members of the family
[13], localized on chromosomes 7, 11 and 18 (Figure 1a,b; also
see Figure 7a). (For the nomenclature of the Irg genes, see
Nomenclature (below) and Additional data file 1). Two of the
mouse Irg sequences, namely Irga5 and Irgb7, are clearly
pseudo-genes (see legend to Figure 1b). The remaining 21 Irg
genes are intact across the GTP-binding domain, although
Irga1, Irga8, and Irgb10 are carboxyl-terminally truncated
relative to the majority, and no transcripts of Irga7 and Irgb8
have yet been found. Thus, the number of potentially func-
tional Irg genes is not six but rather 21 in the C57/BL6 mouse.
The nucleotide and protein sequences of these genes can be
found on our home page [14].
Genomic positioning and phylogenetic relationship of mouse Irg GTPasesFigure 1 (see following page)
Genomic positioning and phylogenetic relationship of mouse Irg GTPases. (a) Disposition of the 23 Irg genes on the mouse karyotype. Individual Irg genes

are listed in correct gene order in each cluster. (b) Positioning and orientation of Irg genes in the mouse chromosome 11 and 18 clusters. Positions of
genes refer to the location in Mouse ENSEMBL release (v28.33d.1, February 2005) [61] of the first G of the glycine codon of the G1 motif (GKS or GMS)
of the GTP-binding domain of each gene. The segments of the chromosome 11 cluster indicated with square brackets are regions of uncertain structure.
Gene orientation is given by black arrows. The shaded region of the chromosome 11 map is a duplication introduced in Mouse ENSEMBL v28.33d.1
(February 2005) in an attempt to resolve a region of high ambiguity indicated by the longer square bracket. In our view this duplication does not resolve
the ambiguities consistently, and we see no justification at present for the duplicated Irgb5 and Irgb6 genes. The sibling genes Irgb3 and Irgb4 differ by only
nine nucleotides; in this case, however, the independent existence of the two genes is proved by the proximity of the PA28βψ retropositioned pseudo-
gene to Irgb3 but not to Irgb4, in addition to consistent sequence differences. We have left the duplication of the Irgb5/Irgb6 region in the map for
consistency of the base numbering with this release of ENSEMBL. *Indicates minor sequence differences presumably due to sequencing errors. (c)
Unrooted tree (p-distance based on neighbour-joining method) of nucleotide sequences of the G-domains of the 23 mouse Irg GTPases, including the two
presumed pseudo-genes Irga5 and Irgb7. The sources of all Irg sequences are given in Additional data file 1, and the nucleotide and amino acid sequences
themselves are collected in the p47 (IRG) GTPase database from our laboratory website [14]. (d) Phylogenetic tree of the amino acid sequences of the G-
domains of 21 mouse Irg GTPases rooted on the G-domain of H-Ras-1 (accession number: P01112). The products of the two presumed pseudo-genes
Irga5 and Irgb7 are excluded from the analysis.
Genome Biology 2005, Volume 6, Issue 11, Article R92 Bekpen et al. R92.3
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Genome Biology 2005, 6:R92
Figure 1 (see legend on previous page)
(a)
(b)
(c)
(d)
Irgb3
Irgb4
Irgb8
Irgb1
Irgb6
Irgb2
Irgb5
Irgb9

Irgb10
Irga6
Irga1
Irga2
Irga4
Irga7
Irga3
Irga8
Irgd
Irgc
Irgm1
Irgm2
Irgm3
H-Ras-1
0.1

48.500 57.835 Mb
Irgb8 Irgb1 Irgb2
506.504 535.038529.186 555.881547.950
Irgm1
LRG47
699.281 735.402
Irgd
IRG47
Irgb7Irgb6
TGTP
710.592 800.523 818.817 832.237 kb
Irgb10 Irgm3
IGTP
Irgm2

GTPI
Irgb3
585.853 588.017
Irgb4
659.863 679.291
Irgb5
607.444
Irgb5
627.410
Irgb6*
TGTP*
//
905.741 957.685 kb
Irga8
60.730
60.970 Mb

Irga2 Irga3
736.672 761.222
Irga1
877.357
Irga5
Irga7
Irga6
IIGP
kb 0 10 20 30 40 50 60 70 80 110 140 150 160 170 180 190 200 210 220 23090 100 120 130
kb 0 10 20 30 40 50 60 70 80 110 140 150 160 170 180 190 200 210 220 23090 100 120 130 10
20
30
Irga4

Irgb9
/
/
0.05
786.364
815.677
840.662
Pa28
Mouse chromosome 18
Mouse chromosome 11
240
40
0
Irgb3
Irgb4
Irgb8
Irgb1
Irgb6
Irgb2
Irgb7Ψ
Irgb5
Irgb9
Irga6
Irga1
Irga2
Irga5Ψ
Irga3
Irga8
Irga4
Irga7

Irgb10
Irgd
Irgc
Irgm1
Irgm3
Irgm2
R92.4 Genome Biology 2005, Volume 6, Issue 11, Article R92 Bekpen et al. />Genome Biology 2005, 6:R92
The complex block of 13 genes on chromosome 11 contains
the most divergent sequences (Figure 1c,d; Additional data
file 2), including all three GMS (Irgm) GTPases [13], suggest-
ing that this cluster is relatively ancient. In contrast, the eight
Irga genes clustered on chromosome 18 are also clustered
phylogenetically, suggesting more recent divergence, proba-
bly from a translocated member of the Irgb (TGTP) cluster on
chromosome 11. The isolated Irg gene on chromosome 7,
Irgc, is an ancient root with no obvious systematic relation-
ship to the other subfamilies. Within the chromosomal clus-
ters, more recent duplication events are apparent. The sibling
pair Irgb3 and Irgb4 differ by only nine nucleotides in the
open reading frame. The genes Irgb1, Irgb3, Irgb4, and Irgb8
appear to have been duplicated in tandem with Irgb2, Irgb5,
and Irgb9, respectively. The pattern of divergence in the
mouse p47 tree suggests an old gene family that has under-
gone a succession of duplication-divergence cycles over time
- a pattern of evolution that is still actively continuing in sev-
eral of the subfamilies.
The structure of p47 GTPase genes and their splicing
patterns
The open reading frame of Irg genes is typically encoded on a
single long 3' exon (Figure 2a) behind one or more 5'-untrans-

lated exons. However, in one splice form of Irgm1 and one
splice form of Irgm2 the initial methionine is encoded at the
3' end of the penultimate exon (also see the legend to Figure
2). The closely related Irgb1 and Irgb4 genes are exceptional
in apparently occurring only as tandem transcripts in-frame
with their respective closely linked upstream genes Irgb2 and
Irgb5. If translated, such transcripts would generate 94 kDa
polypeptides containing two distinct full-length p47 GTPase
units. For the sequence phylogenies and alignments (Figure
1c,d; also see Figure 4, below), we provisionally treat these
separate p47 units as independent genes. It remains to be
seen whether the third tandem gene pair, Irgb9 and Irgb8, is
also expressed as a tandem transcript. That Irgb1, Irgb3, and
possibly Irgb8 are normally expressed in tandem with an
upstream gene is also consistent with the absence both of
autonomous transcripts of these exons and of interferon-
inducible promoter elements (see below).
Identification of interferon-stimulatable elements in
putative promoters of Irg genes
The basis for interferon-inducible expression of the mouse
p47 GTPases has previously been investigated only for Irgd
(IRG47) [15], in which an active interferon-stimulated
response element (ISRE) was found upstream of the putative
transcriptional start point. A GAS (γ-activated sequence) site
was predicted in the putative promoter region of Irgm1 (LRG-
47) [8]. Most of the transcribed p47 genes on chromosomes 11
and 18 exhibit multiple perfect interferon-inducible genomic
motifs, both ISRE and GAS elements (Figure 2b; Additional
data file 3). The sequences and relative positions of the GAS
and ISRE elements vary, both classes of site are not present in

all promoters, and the orientations of the two components are
also variable. Thus, the association of interferon-inducible
elements with Irg genes is presumably ancient and has been
retained against the disruptive forces of spontaneous genome
evolution. No further immunity-related inducible elements
such as NFκB sites were found to be associated with the
ISRE/GAS motifs. Irgd and Irga6 are both transcribed from
alternative 5'-untranslated exons, each furnished with an
independent promoter. In both genes the initial methionine is
encoded at the beginning of the long 3' exon, so that the two
transcripts of each gene generate identical proteins. Both
putative promoters of Irgd and Irga6 have interferon-induc-
ible elements. As noted above, genes Irgb1, Irgb4, and Irgb8
are probably expressed only as the 3' ends of tandem tran-
scripts with Irgb2, Irgb5, and Irgb9, respectively. No dedi-
cated 5'-untranslated exons could be identified for these
downstream domains. Using RT-PCR we were able to show
clear induction of eight further genes (Irga2, Irga3, Irga4,
Irga8, Irgb1, Irgb2, Irgb5, and Irgb10) in addition to the six
(Irga6 (IIGP), Irgb6 (TGTP), Irgd (IRG-47), Irgm1 (LRG-
47), Irgm2 (GTPI) and Irgm3 (IGTP)) assayed by Boehm and
coworkers [13] in L929 fibroblasts stimulated with
interferon-γ in vitro (Figure 3a).
The isolated p47 gene, Irgc, on chromosome 7 is a clear
exception. No clustered or isolated ISRE or GAS elements
could be identified up to 10 kilobases (kb) 5' of the putative
transcription start of this transcribed gene, and Irgc was not
induced in interferon-stimulated fibroblasts (Figure 3b,
panel i left). A weak Sox-related element was detected in the
proximal promoter region. In view of the close homology of

Irgc to the interferon-inducible Irg genes, we considered
whether Irgc is induced in tissues of mice 24 hours after
infection with Listeria monocytogenes [13,16]. No induction
of Irgc was detected in liver, lung, or spleen after 50 cycles of
amplification, whereas Irga2, used as a positive control, was
induced in all three tissues (Figure 3b; panel i right). How-
Genomic and promoter structure of mouse Irg GTPasesFigure 2 (see following page)
Genomic and promoter structure of mouse Irg GTPases. (a) Genomic structure of mouse Irg genes. Green blocks indicate coding exons and blue blocks
indicate 5'-untranslated exons. Orange arrows identify putative promoter regions. Stars identify exons shown to be excluded in alternative splice forms.
The scale bar is measured in base pairs up to the first base of the long coding exon. Note the presence of two promoters for Irga6 and Irgd. (b) Interferon
response elements in the promoter regions of mouse Irg genes. γ-Activated sequences (GAS; pale blue blocks) and interferon-stimulated response element
(ISRE; red blocks) sequences were identified in the promoters shown in panel a (also see Additional data file 7). Dark blue blocks downstream of each
promoter represent the most 5' exon. The yellow block identifies a putative Sox1 transcription factor binding site in the proximal promoter region of Irgc.
The scale bar is measured in base pairs from the first base of the 5' exon.
Genome Biology 2005, Volume 6, Issue 11, Article R92 Bekpen et al. R92.5
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Figure 2 (see legend on previous page)
(a)
(b)
1,000
2,000
6,000
9,000
11,000
13,000
3,000
4,000
5,000
7,000

8,000
10,000
12,000
14,000
Irgb2/b1
Irgb6
0
6,000
(TGTP)
Irgc
11.2-2nd Exon
Irgb5/b4
6,000
Irgb2
Irgb10
Irgb9
Irga1
Irga2
20,500
Irga3
Irga4
Irga8
(IIGP)
Irga6
Irgd
6,000
Irgm1
Irgm2
Irgm3
(LRG47)

(GTPI)
(IGTP)
(IRG47)
11,750
Irgb5
Irgb1
Irgb4
-1,000
-750
-500 -250 +1
-1,200
Exon 1
(TGTP)
(IRG47)
Irgd(p2)
(IIGP)
Irga6(p2)
Irgb2
Irgb6
Irgc
Irgb5
Irgb10
Irgb9
Irga1
Irga2
Irga3
Irga4
Irga8
Irgd(p1)
Irgm1

Irgm2
Irgm3
(LRG47)
(GTPI)
(IGTP)
Irga6(p1)
R92.6 Genome Biology 2005, Volume 6, Issue 11, Article R92 Bekpen et al. />Genome Biology 2005, 6:R92
ever, Irgc, unlike Irga2, was constitutively expressed in the
mature mouse testis (Figure 3b; unpublished data). We con-
clude that mouse Irgc is expressed in a tissue-specific manner
and is not induced by infection.
The coding sequences of the p47 GTPases
In Figure 4 we present the predicted translation products of
the 21 intact p47 GTPase genes, and reconstructed partial
sequences of the two pseudo-genes, Irga5ψ and Irgb7ψ,
aligned on the secondary structures of Irga6 [12] and H-Ras-
1 [17]. The full alignment confirms a number of major features
that are already apparent from the previously published
alignment of six family members [13] and consolidates the
definition of the p47 GTPases as a distinctive sequence fam-
ily. Especially noteworthy are novel features of the amino-
and carboxyl-termini, which were not apparent before.
Eleven of the proteins, including six of chromosome 18 Irga
gene products and Irgb2, Irgb5, Irgb9 and Irgb10, carry the
amino-terminal myristoylation signal MGxxxS [18]. This
sequence in Irga6 (IIGP1) is indeed myristoylated in vitro
[19] and in vivo, and, as expected, favors binding of the pro-
tein to membranes [6]. No other membrane attachment
sequences or lipid modification motifs are apparent in p47
GTPase sequences, despite the documented attachment of

several of these proteins to membranes [5,6,16]. Irgb2, Irgb5,
Irgb7, Irgb9 and Irgc have carboxyl-terminal extensions up to
65 residues in length compared with the canonical IIGP1
sequence.
The p47 GTPase genes of the human genome
Only two IRG sequences, both transcribed, are present in
humans (or chimpanzee), one (IRGC) on chromosome 19
(19q13.31) and the other (IRGM) on chromosome 5 (5q33.1).
Human IRGC is more than 85% identical at the nucleotide
level and 90% at the amino acid level to the isolated mouse
gene Irgc. IRGM encodes an amino- and carboxyl-terminally
truncated G-domain homologous to the Irgm (GMS) sub-
family of mouse p47 GTPases. Predicted protein products of
IRGC and the IRGM gene fragment are included in an
extended phylogeny (Figure 5) and alignment (Figure 6) of
the vertebrate IRG proteins.
The IRGC mouse and human genes sit in chromosomal
regions syntenic between chromosomes 7 and 19, respectively
(Figure 7a) and are clearly orthologous. The proximal
promoter region of human IRGC is largely conserved with
that of mouse Irgc. However, as in the mouse, no interferon
response elements are found either in the proximal conserved
region or in divergent regions up to 10 kb upstream of the
transcriptional start (data not shown). Human IRGC, like
mouse Irgc, is not inducible in vitro by interferons, is not
expressed detectably in brain or liver, but is strongly
expressed in adult testis (Figure 3b, panel ii). As in the mouse,
a weak Sox element is present in the proximal promoter of
human IRGC.
The human genomic segments syntenic to the mouse chro-

mosome 11 and chromosome 18 IRG gene clusters both
mapped to human 5q33.1, suggesting that the interferon-
inducible IRG proteins were once encoded in a single block
ancestral to the human chromosome 5 region (Figure 7b).
IRGM maps only 80 kb away from the closest syntenic
marker DCTN4. IRGM is transcribed in unstimulated human
tissue culture lines HeLa and GS293 (Figure 8a), with no
increase after interferon induction. Polyadenylated tran-
scripts of IRGM occur with five 3' splicing isoforms extending
more than 50 kb 3' of the long coding exon (Figure 8b). The
transcripts have a 5'-untranslated region of more than 1,000
nucleotides that corresponds largely to the U5 region of an
ERV9 repetitive element [20]. The promoter region corre-
sponds to the ERV9 U3 LTR (long terminal repeat) without
interferon response elements, and three of the five splice
forms have exon-intron boundaries downstream of the puta-
tive termination codon, normally a signal for rapid RNA deg-
radation [21].
Interferon responsiveness of mouse and human p47 (IRG) GTPaseFigure 3 (see following page)
Interferon responsiveness of mouse and human p47 (IRG) GTPase. (a) Interferon (IFN)-γ responsiveness of eight new mouse Irg genes. Inducibility of eight
further Irg genes (also see Boehm and coworkers [13]) in L929 fibroblasts induced for 24 hours with IFN-γ, demonstrated by RT-PCR. D refers to a
positive control genomic DNA template; O refers to a negative control of the same genomic template after DNAse1 treatment; and + and - refer to RT-
PCR on DNAse1-treated RNA templates from IFN-γ-induced and IFN-γ-noninduced cells, respectively. The sibling genes of the Irgb series could not be
individually amplified because of their close sequence similarity. The identities of the amplified genes responding to interferon induction, indicated by
vertical arrows, were subsequently established by sequencing of multiple clones from the PCR product. (b) Irgc is not induced by interferon or infection
but is constitutively expressed in testis. (i, left) Mouse L929 fibroblasts were induced for 24 hours with IFN-β or IFN-γ or left uninduced (-). Irgc could not
be detected by RT-PCR even after 50 amplification cycles in L929 cells. Irga2 after 50 cycles was used as a positive control for the interferon-induced L929
RNA. RNA from mouse testis provided a positive control for Irgc. (i, right) RT-PCR for Irgc and Irga2 (50 and 30 amplification cycles respectively) on RNA
from tissues of uninfected mice (-) or mice infected 24 hours previously with Listeria monocytogenes (+). Irga2 was induced in all tissues and Irgc in none.
RNA from mouse testis provided a positive control for Irgc, which is detected after 50 cycles. Testis expression of Irga2 was barely detected after 30

cycles (compare with i, left, showing Irga2 in testis after 50 cycles). (Panel ii, left) Human IRGC is not induced by 24 hours of stimulation with IFN-β or IFN-
γ in human cell lines (induction of GBP-1 [accession number P32455] was assayed as a positive control) and (Panel ii, right) is constitutively expressed only
in human testis. GAPDH was used as control.
Genome Biology 2005, Volume 6, Issue 11, Article R92 Bekpen et al. R92.7
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Figure 3 (see legend on previous page)
(a)
(b)
IFN - γ
Irga2
+-
D0
Controls
+-
D0
+-
D0
+-
D0
+-
D0
+-
D0
+-
D0
+-
D0
IFN - γ
Controls

Irga3 Irga4 Irga8
Irgb10
Irgb1,3,4,8
Irgb2,5,9
+-
D0
Irga7
Live
r
Brai
n
Testis
no DNA
GAPDH
IRGC
50 Cycles
622 bp
27 Cycles
495 bp
(i)
IRGC
GAPDH
GBP-1
-
- -
1 fibroblast
O
THP-1
Hela
Testis

IFN
50 Cycles
27 Cycles
622 bp
428 bp
27 Cycles
495 bp
no DNA
- + - - +
50 Cycles
30 Cycles
Liver Lung
Spleen Testis
+
Listeria
Irgc
Irga2
(ii)
622 bp
963 bp
no
DNA
-
IFN
Testis
50 Cycles
50 Cycles
Irgc
Irga2
622 bp

963 bp
no
D
NA
β
β

β
γ
γ

γ
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At the protein level the shortest isoform of IRGM is shorter
than a canonical G-domain, being truncated in the middle of
β-strand 6 just before the G5 sequence motif, which interacts
with the guanine base of the bound nucleotide (Figures 6 and
8b; also see Ghosh and coworkers [12]). The longer isoforms
are terminated by short sequence extensions that are
unrelated to known GTPase domains. A rabbit antiserum
raised against recombinant human IRGM produced in
Escherichia coli failed to detect signal by immunofluores-
cence or Western blot in human cell lines (data not shown).
IRG genes of the dog
Is the mouse (order Rodentia) or the human (order Primata)
the exception? We looked for IRG genes in a third order of
mammals, the Carnivora. We recovered a total of eight IRG
genes from the public genome database of the dog Canis
familiaris (Figures 5 and 6) as well as a partial sequence of a
9th gene (not shown). Of these, one (not shown) is a pseudo-

gene by a number of criteria, another is clearly dog IRGC,
whereas the partial sequence is novel but most closely related
to IRGC. The remainder assort into segments of the phylog-
eny already established for the interferon-inducible mouse
IRG genes (Figure 5). Both GMS and GKS genes are repre-
sented and are inducible by interferon in dog MDCK epithe-
lial cells (Additional data file 4). The three dog GMS genes
appear to have diversified independently from the mouse
GMS genes (Figure 5). As in humans and mouse, dog IRGC
was not induced by interferon-γ (Additional data file 4). Over-
all, the IRG gene status of the dog clearly resembles that of
mouse rather than that of humans.
IRG genes in fish genomes
IRG GTPases are at least as old as the vertebrates. We have
identified at least two distinct irg genes in the freshwater
pufferfish Tetraodon nigriviridis, a closely linked pair of irg
genes in the saltwater pufferfish Fugu rubripes, and at least
11 partially clustered irg genes in the zebrafish Danio rerio
(Figures 5 and 6, and Additional data file 5). The fish irg
genes fall into separate clades from the mammalian genes
(Figure 5). A specific IRGC homolog is not immediately
apparent. GMS subfamily IRGM genes are absent from fish.
The pufferfish and zebrafish irgf genes have one intron iden-
tically positioned at the end of helix 4 of the G-domain (indi-
cated on Figure 6; also see Additional data file 5). This intron
is 81 bp long in both pufferfish species but is substantially
longer in the zebrafish genes. The distinct irge subfamily of
the Danio irg genes are intronless in the open reading frame,
like mammalian IRG genes.
IRG homologs with divergent nucleotide-binding

regions: the quasi-GTPases
The mouse, human and zebrafish genomes encode proteins
that are homologous to the IRG GTPases but are radically
modified in the GTP-binding site. The mammalian protein
FKSG27 (IRGQ), a protein of unknown function that is 70%
conserved between man and mouse, is extended amino-ter-
minally relative to a p47 GTPase by about 100 residues
encoded on three short exons. The remaining 420 residues,
encoded on a single long exon, are clearly homologous to and
colinear with the IRG proteins (Figure 6 and Additional data
file 6), especially in the amino- and carboxyl-terminal parts of
the exon. The region of lowest similarity is in the G-domain,
and conserved GTP-binding motifs are lacking (Figure 6, and
Additional data files 6 and 7). Thus, FKSG27 (IRGQ) is not a
GTPase despite its phylogenetic relationship to the IRG pro-
teins. FKSG27 (IRGQ) is closely linked to IRGC in humans
and mouse (Figure 7a).
The zebrafish genome contains three IRG homologs with
more or less modified GTP-binding motifs (irgq1-irgq3; Fig-
ures 5 and 6, and Additional data file 7). Their homology to
IRG genes is stronger than that of FKSG27 (IRGQ), but as
with FKSG27 (IRGQ) their function as GTPases is doubtful.
The irgq1 gene is clustered on a single BAC clone with four
apparently normal irge genes and immediately downstream
of a truncated p47 gene, irgg, with which irgq1 is transcribed
as the carboxyl-terminal half of a tandem transcript. Thus,
the hypothetical protein product would be a carboxyl-termi-
nally truncated p47 GTPase, linked at its carboxyl-terminus
to a similarly truncated p47 homolog probably without
GTPase function.

We propose to term the modified IRG proteins without
GTPase function 'quasi IRG' proteins, hence IRGQ. IRGQ
sequences reveal their phylogenetic relationship to the IRG
proteins, but they are nevertheless more or less radically
Amino acid alignment of the mouse Irg GTPasesFigure 4 (see following page)
Amino acid alignment of the mouse Irg GTPases. Sequences of all 23 mouse Irg GTPases showing the close homology extending to the carboxyl-terminus,
aligned on the known secondary structure of Irga6 (indicated in blue above sequence alignment). The sequences of notional products of the two pseudo-
genes Irga5 and Irgb7 have been partially reconstructed; premature terminations are indicated by red highlighting. In the C57BL/6 mouse the sequence of
the Irga8 gene is damaged by an adenine insertion, indicated by the red highlighted K at position 204. (The sequence given after this point is that given after
correcting the frameshift, and is identical to that of the CZECHII [Mus musculus musculus] sequence BC023105 that lacks the extra adenine.) The
turquoise-highlighted M in M1 and M2 are initiation codons that are dependent on alternative splicing (also see Figure 2a); the unusual methionine residues
in the G1 motif of GMS proteins are highlighted in green. The blue background Q residue of Irgb5 and Irgb2 at positions 405 and 396 indicate the point at
which tandem splicing occurs to Irgb4 and Irgb1, respectively. Canonical GTPase motifs are indicated by red boxes.
Genome Biology 2005, Volume 6, Issue 11, Article R92 Bekpen et al. R92.9
comment reviews reports refereed researchdeposited research interactions information
Genome Biology 2005, 6:R92
Figure 4 (see legend on previous page)




1 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
| | | | | | | | | | | | | | | |

160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320
| | | | | | | | | | | | | | | | |

330 340 350 360 370 380 390 400 410
| | | | | | | | |

Irga6
Irga1
Irga2
Irga4
Irga7
Irga5
Irga3
Irga8
Irgd
Irgm1
Irgm2
Irgm3
Irgb3
Irgb4
Irgb8
Irgb1
Irgb6
Irgb10
Irgb2
Irgb7

Irgb5
Irgb9
Irgc
H-Ras-1

N
S5
H4
αE
S6
αG
αF
αd
H3 H5
αH αI αJ αK
αL
C
Irga6
Irga1
Irga2
Irga4
Irga7
Irga5

Irga3
Irga8
Irgd
Irgm1
Irgm2
Irgm3
Irgb3
Irgb4
Irgb8
Irgb1
Irgb6

Irgb10
Irgb2
Irgb7
Irgb5
Irgb9
Irgc
H-Ras-1
1 MGQLFS SPKSD-ENNDLPSSFTGYFKKFNTGRKIISQEILN
1 MGQLFS LLKN KCQFLVSSVAEYFKKFKKIVIIILQEVTT
1 MGQLFS SRRS EDQDLSSSFIEYLKECEKGINIIPHEIIT
1 MGQLLS DTSKTEDNEDLVSSFNEYFKNIKTE-KIISQETID
1 MDQLLS DTSKNEDNDDLVSSFNAYFKNIKTENKIISQETID
1 MGQLFS GTSK SEALCSSFTEYFQKFKVENKIISQEIST
1 MGQLFS HIPKDEDKGNLESSFTEYFRNYKQETKIISEETTR
1 MGQLFS NMPKDEDKGILESSFTEYFRNYKQETKIISEETTR
1 MDQFISAFLKGASENSFQQLAKEFLPQYSALISKAGGMLSPETLT
1 MKPSHSSCEAAPLLPNMAETHYAPLSSAFPFVTS YQTG-SSRLPEVSR
1 MPTSRVAPLLDNMEEAVESPEVKEFEYFSDAVFIPKDGNTLSVGVIK
1 MDLVTKLPQNIWKTFTLFINMANYLKRLISPWSKSMTAGESLYSSQNSSSPEVIE
1 MAQLLVFSFENFFKNFKKESKILSEETIT
1 QHPPLHTATCQPSSSRPSRLTAQLLVFSFENFFKNFKKESKILSEETIT
1 MAQLLVISFENFFKNFKKESKILSEETIT
1 QHPPLNTATCQTSTGRTSQITAQLLEFNFKNFFKNFKKESKILSEETIT
1 MAWASSFDAFFKNFKRESKIISEYDIT
1 MGQSSS KPDAKAHNMASSLTEFFKNFKMESKIISKETID
1 MGQTSS STSPPKEDPPLT FQVKTKVLSQELIA
1 XPFWFVPPLGTIDICQDWVKLPLLHPLQRRILLLTFQMKTKILSQELIT
1 MGQTSS STPPPKEDPDLTSSFGTNLQNFKMKTKILSQELIA
1 MGQTSS STLPPKDDPDFIASFGTNLQNFKMKTKILSQELIA
1 MATSRLPAVPEETTILMAKEELE

1

LIELRMRKGNIQLTNSAISDALKEIDSSVLNVAVTGETGSGKSSFINTLR-GIGNEEEGA
SIELDMKKENFQEANSAICDALKEIDSSLVNVAVTGETGSGKSSFINTLR-GIGHEEEGA
SIEINMKKGNIQEVNSTVRDMLREIDNTPLNVALTGETGSGKSSFINTLR-GIGHEEGGA
LIKLYLNKGNIHGANSLIRDMLREIDNTPINIAVTGESGAGKSSLINALI-GIGPEEEGA
LIELHLNKGNIHGANSLIREALKNIDNAPINIAVTGESGVGKSSFINALI-GTGPEEEGA
LIELYLTLGDVQQANNAITYALRXLARTPQNVALIGESGRGKYSFINVFR-GLDMKRKM-
SIELCLKRGDFQRANSVISDALKNIDNTPINIAVTGESGAGKSSLINALR-EVKAEEESA
SIELCLKKGDIQRANSIISDALKNIDNAPINIAVTGESGAGKSSLINALR-EIKAEEESA
GIHKALQEGNLSDVMIQIQKAISAAENAILEVAVIGQSGTGKSSFINALR-GLGHEADES
STERALREGKLLELVYGIKETVATLSQIPVSIFVTGDSGNGMSSFINALR-VIGHDEDAS
RIETAVKEGEVVKVVSIVKEIIQNVSRNKIKIAVTGDSGNGMSSFINALR-LIGHEEKDS
DIGKAVTEGNLQKVIGIVKDEIQSKSRYRVKIAVTGDSGNGMSSFINALR-FIGHEEEDS
LIESHLEDKNLQGALSEISHALSNIDKAPLNIAVTGETGTGKSSFINALR-GVRDEEEGA
LIESHLEDKNLQGALTEISHALSNIDKAPLNIAVTGETGTGKSSFINALR-GVRDEEEGA
LIESHLEDKNLQGALSEISHALSNIDKAPLNIAVTGETGTGKSSFINALR-GVRGEEEGA
LIESHLENKNLKEALTVISHALSNIDKAPLNIAVTGETGTGKSSFINALR-GISSEEKDA
LIMTYIEENKLQKAVSVIEKVLRDIESAPLHIAVTGETGAGKSTFINTLR-GVGHEEKGA
SIQSCIQEGDIQKVISIINAALTDIEKAPLNIAVTGETGAGKSTFINALR-GIGHEESES
SIESSLEDGNLQETVSAISSALGDIEKVPLNIAVMGETGAGKSSLINALQ-GVGDDEEGA
FIELYLEDGNLXETVSAISSALGDIEKVPLNIAVMGETGAGKSSLINALQ-GTGADEDGV
FIESSLEDGNLQETVSAISSALGGIEKAPLNIAVMGETGAGKSSLINALQ-GVGDDEEGA
FIESSLEDGNLRETVSAISSALGGIEKAPLNIAVMGETGAGKSSLINALQ-GVGDDEEGA
ALRTAFESGDIPQAASRLRELLANSETTRLEVGVTGESGAGKSSLINALR-GLGAEDPGA
MTEYKLVVVGAGGVGKSALTIQLIQNHFVDE

A-KTGVVEVTMERHPYKH-PNIP NVVFWDLPGIGSTNFPPNTYLEKMKFY-EYDFFI
A-KTGVVEATMERHPYKH-PNMP NVVFWDLPGIGSTKFPPKTYLEKMKFY-EYDFFI
A-HTGVTDKTKERHPYEH-PKMP NVVFWDLPGTGSEDFQPKTYLEKMKFY-EYDFFI

A-EVGVIETTMKRTSYKH-PKIE TLTLWDLPGIGTQKFPPKTYLEEVKFK-EYDFFI
A-EVGVIETTMKRNFYKH-PKIE TLTLWDLPGIGTQKFPPKTYLEEVKFK-EYDFFI
A-TVGVVETTMNRTPYRN-PNIP NVIIWDLPGIGTTNFPPKHYLKKMQFYVMYDFFI
A-EVGVTETTMKVSSYKH-PKVK NLTLWDLPGIGTMKFQPKDYLEKVEFK-KYDFFI
A-EVGVTETTMKVYSYKH-PKVK NLTLWDLPGIGTKKFPPKTYLETVEFK-KYDFFI
A-DVGTVETTMCKTPYQH-PKYP KVIFWDLPGTGTPNFHADAYLDQVGFA-NYDFFI
A-PTGVVRTTKTRTEYSS-SHFP NVVLWDLPGLGATAQTVEDYVEEMKFS-TCDLFI
A-PTGVVRTTQKPTCYFS-SHFP YVELWDLPGLGATAQSVESYLEEMQIS-IYDLII
A-PTGVVRTTKKPACYSSDSHFP YVELWDLPGLGATAQSVESYLEEMQIS-TFDLII
A-PTGVVETTMKRTPYPH-PKLP NVTIWDLPGIGSTTFPPQNYLTEMKFG-EYDFFI
A-PTGVVETTMKRTPYPH-PKLP NVTIWDLPGIGSTTFPPQNYLTEMKFG-EYDFFI
A-PTGVVETTMKRTPYPH-PKLP NVTIWDLPGIGSTNFQPQNYLTEMKFG-EYDFFI
A-PTGVIETTMKRTPYPH-PKLP NVTIWDLPGIGSTNFPPQNYLTEMKFG-EYDFFI
A-PTGAIETTMKRTPYPH-PKLP NVTIWDLPGIGTTNFTPQNYLTEMKFG-EYDFFI
A-ESGAVETTKDRKKYTH-PKFP NVTIWDLPGVGTTNFKPEEYLKKMKFQ-EYDFFL
AASTGVVHTTTERTPYTY-TKFP SVTLWDLPSIGSTAFQPHDYLKKIEFE-EYDFFI
TAPVGVVYTTIEKKSYPY-AKFP SAILWELPAIGFHHFQPHDYLKKIKFE-EYDFII
AASTGVVHTTTERTPYTY-TKFP SVTLWDLPGIGSTAFQPHDYLKKIEFE-EYDFFI
AASTGVVHTTTERTPYTY-TKFP SVTLWDLPGIGSTAFQPHDYLKKIEFE-EYDFFI
A-LTGVVETTMQPSPYPH-PQFP DVTLWDLPGAGSPGCSADKYLKQVDFG-RYDFFL
YDPTI EDSYRKQVVIDGETCLLDILDTAGQEEY SAMRDQYMRT GEGFL

153 IISATRFK KNDIDIAKAISMMK-KEFYFVRTKVDSDITNEADGKPQTFDKEKVL
152 IISATCFK KNDIDLAKAISMMK-KEFYFVRTKVDTDLRNEEDFKPQTFDKEKVL
152 IISATRFK KNDIDLAKAIGIMK-KEFYFVRTQVDSDLRNEEDFKPQTFDREKVL
153 IVSATRFT KLELDLAKAITNMK-KNYYFVRTKVDIDVENERKSKPRTFEREKAL
154 IVSSTRFT KHELDLAKAIGIMK-KNYYFVRTKVDIDLENERKSKPRTFDREKTL
151 IVSATCFR KNDIDLSKAVVMIK-KKDFLLRTKEDIDIENENX
154 IVSSSRFT KLELDLAKATRIMK-KNYYFVRSKVDCDLDNEKKSKPRNFNRENTL
154 IVSATRFT NHEIELAKAIRIMK-KNYYFVRSKVDFDLYNEEKSKPRNFNRKNTL

158 IISSSRFS LNDALLAQKIKDAG-KKFYFVRTKVDSDLYNEQKAKPIAFKKEKVL
160 IIASEQFS SNHVKLSKIIQSMG-KRFYIVWTKLDRDLSTS VLSEVRLL
159 IVASEQFS LNHVKLAITMQRMR-KRFYVVWTKLDRDLSTS TFPEPQLL
168 IVASEQFS SNHVKLAITMQRMR-KRFYVVWTKLDRDLSTS TFPEPQLL
142 IISATRFK EIDAHLAKTIEKMN-TKFYFVRTKIDQDVSNEQRSKPRSFNRDSVL
162 IISATCFK EIDAHLAKTIEKMN-TKFYFVRTKIDQDVSNEQRSKPRSFNRDSVL
142 IISATRFK EIDAHLAKAIAKMN-TKFYFVRTKIDQDVSNEQRSKPKSFNRDSVL
162 IISATRFK EIDAHLAKAIAKMN-IKFYFVRTKIDQDISNEQRSKPKSFNRDSVL
140 IISATRFK ENDAQLAKAIAQMG-MNFYFVRTKIDSDLDNEQKFKPKSFNKEEVL
152 IISSARFR DNEAQLAEAIKKMK-KKFYFVRTKIDSDLWNEKKAKPSSYNREKIL
146 IVSAIRIK QSDIELAKAIVQMN-RGLYFVRTKTDSDLENEKLCNPMRFNRENIL
162 VS-AGRIK HSDVELAKAIVQMN-RGLYFNRTKTDIDLKNEKLYNPMRFNRENTL
155 IVSSGRFK HNDAELAKAIVQMN-RSFYFVRTHTDLDLMVVKRSNPRRFNRENTL
155 IVSSGRFK HNDAELAKAIVQMN-RSFYFVRTHTDLDLMVVKLSDPRKFNKENIL
136 LVSPRRCG AVESRLASEILRQG-KKFYFVRTKVDEDLAATRSQRPSGFSEAAVL
79 CVFAINNTKSFEDIHQYREQIKRVKDSDDVPMVLVGNKCDLAARTVE

QDIRLNCVNTFRENGIAEPPIFLLSNKNVCHYDFPVLMDKLISDLPIYKRHNFMVSLPN
QDIRLNCVNTFKENGIAEPPIFLISNENVCHYDFPVLMDKLISDLPDYKRHNFMLSLPN
QDIRLNCVNTFRENGIAEPPIFLISNKNVCHYDFPVLMDKLISDLPVFKRQNFMFSLPN
KQIQSYSVKIFNDNNMAVPPIFLISNYDLSDYDFPFLVDTLIKELHVQKRHNFMLSLPN
KQIQSYAMNTFSDNNMAIPPIFMVSNYDLSKYDFPVMMDTLIKDLHAEKRHNFMLSLPG

NQVRNSYLDTFRESKIDEPQVFLISNHDLSDYDFPVLMDTLLKDLPAEKRQNFLLSLPN
NQIRNSYLDTFRESKIDEPQVFLISNHDLSDYDFPVLMDTLLKDLPAEKRHNFLLSLPN
QQIRDYCVTNLIKTGVTEPCIFLISNLDLGAFDFPKLEETLLKELPGHKRHMFALLLPN
QNIQENIRENLQKEKVKYPPVFLVSSLDPLLYDFPKLRDTLHKDLSNIRCCEPLKTLYG
QSIQRNIRDSLQKEKVKEHPMFLVSVFKPESHDFPKLRETLQKDLPVIKYHGLVETLYQ
QSIQRNIRENLQQAQVRDPPLFLISCFSPSFHDFPELRNTLQKDIFSIRYRDPLEIISQ
KKIRDDCSGHLQKALSSQPPVFLVSNFDVSDFDFPKLETTLLRELPSHKRHLFMMSLHS

KKIRDDCSGHLQKALSSQPPVFLVSNFDVSDFDFPKLETTLLRELPAHKRHLFMMSLHS
KKIRDDCSGHLQKVLSSQPPVFLVSNFDVSDFDFPKLENTLLRELPAHKRHLFMMSLHS
KKIKDECLGLLQKVLSSQPPIFLVSNFDVSDFDFPKLETTLLKELPAHKRHLFMMSLHS
KNIKDYCSNHLQESLDSEPPVFLVSNVDISKYDFPKLETKLLQDLPAHKRHVFSLSLQS
EVIRSDCVKNLQNANAASTRGFL-SLKLX
KSIRICLSSNLKERFQQEPPVFLVSNFDVSDFDFPKLESTLLSQLPAYKHQIFMSTLQV
KSLQICISSNLKECFHQEPPVFLVSNFDVSDFDFPKLESTLLSQLPAYKHQIFMRTLQV
KQIRHTISSMLKEVTHQEPPVFLVSNFDVSDFDFPKLESTLLSQLPAYKHHMFMLTLPI
EQIRNSISNILKEVTHQEPPVFLVSNFDVSDFDFPNLESTLLSQLPAYKHHMFMLTLPI
QEIRDHCTERLRVAGVNDPRIFLVSNLSPTRYDFPMLVTTWEHDLPAHRRHAGLLSLPD
SRQAQDLARSYGI PYIETSAKTRQGVEDAFYTLVREIRQHKLRKLNPPDESGP


ITDSVIEKKRQFLKQRIWLEGFAADLVNIIPSLTFLLDSDLETLKKSMKFYRTVFGVDET
ITDSVIETKRQSLKQRHWLQGFAGVLLSYLH
ITDSVIEKKRNFLRWKTWLEGFADGLL SFFLESDLETLEKSMKFYRTVFGVDDA
FTDQAIDRKYKATQQFIWLEAFKIGVVAIFPVLGNLRNKDMKKIKNTLNYYQKIFGVDDE
ITEAAIDRKHKATQQIVWLEAFNVGLLANFPVTGILGDNDVKKLEKSLNYYRKIFGVDDE

ITEAAIQKKYNSTKQIIWLQATKDGLLATVPVVGILKDLDKERLKKRLDYYRDLFGVDDE
ITEAAIQKKYNSTKQFIWLQAMKDGLLATVPVVGILKDLDKERLKRSLDYYRDLFGIDDE
ISDASIELKKHFLREKIWLEALKSAAVSFIPFMTFFKGFDLPEQEQCLKDYRSYFGLDDQ
TYEKIVGDKVAVWKQRIANESLK NSLGVRDDDNMGECLKVYRLIFGVDDE
VCEKTVNERVESIKKSIDEDNLH TEFGISDPGNAIEIRKAFQKTFGLDDI
VCDKCISNKAFSLKEDQMLMKDLEA AVSSEDDTANLERGLQTYQKLFGVDDG
VTETAIARKRDFLRQKIWLEALKAGLWATIPL-GGLVRNKMQKLEETLTLYRSYFGLDEA
VTETAIARKRDFLRQKIWLEALKAGLWATIPL-GGLVRNKMQKLEETLTLYRSYFGLDEA
VTETAIDRKRDFLRQRIWLEALKAGVWTTIPL-GGLVRDKMQKLEETLTLYRSYFGLDEA
VTETTIARKRDFLRQKIWLEALKAGLWATIPL-GGLVRDKMQKLEETLTLYRSYFGLDEA
LTEATINYKRDSLKQKVFLEAMKAGALATIPL-GGMISDILENLDETFNLYRSYFGLDDA

VINAIVDRKRDMLKQKIWKESIMPRAWATIPS-RGLTQKDMEMLQQTLNDYRSSFGLNEA
VINAIVDWKRDMLKQKVWKESTTPRAWATIPS-LGLTQKDMEMLQQTLNDYRSSFGLDEA
VTDSTIDRKRDMLKQKVWKESTMPRAWATIPS-LGLTQKDMEMLQQTLNDYRSSFGLDEA
VTDSTIDRKRDMLKQKIWKESIMPRAWATIPS-RGLTQKDMEMLQQTLNDYRSSFGLDEA
ISLEALQKKKDMLQEQVLKTALVSGVIQALPVPGLAAAYDDALLIRSLRGYHRSFGLDDD
GCMSCKCVLS

Irga6
Irga1
Irga2
Irga4
Irga7
Irga5
Irga3
Irga8
Irgd
Irgm1
Irgm2
Irgm3
Irgb3
Irgb4
Irgb8
Irgb1
Irgb6
Irgb10
Irgb2
Irgb7
Irgb5
Irgb9

Irgc
H-Ras-1
325 SLQRLARDWEI-EVDQVEAMIKSPAVFKPTDEETIQERLSRYIQEFCLANGYLLP

318 SLQRLARAWEIDQVDQVRAMIKSPAVFTPTDEETIQERLSRYNQEFCLANGYLLP
325 SLELVAKDFQV-PVEQVKKTMKTPHLLKKYREETFRNDFKKLVSTFG RLL
326 SLELVAKDFQV-PVEQVKEIMKSPHLLKTNGKETLGEKLLKYLEKFETATGGLL

326 SLMFMAKDAQV-PVELLIKNLKSPNLLKCK-EETLEELLLNCVEKFASANGGLL
326 SLMFIAKDAQV-PVELLKIKLKSPYLLELE-EETLGGLILNCVEKFASANGGLL
330 SIKEIAEKLGA-PLADIKGELKCLDFWSLVKDNSIIAQATSAAEAFCAVKGGPES
316 SVQQVAQSMGTVVMEYKDNMKSQNFYTLRREDWKLRLMTCAIVNAFFR-LLRFL
316 SLHLVALEMKNKHFN TSMESQETQRYQQDDWVLARLYRTGTRVGSIGFDYMK
327 SLQQVARSTGRLEMGSRALQFQDLIKMDRRLELMMCFAVNKFLRLLESSWWYGLWN
333 SLENIAKDFNV-SVNEIKAHLRFLQLFTKNNDMSFKEKLLKYIEYISCVTGGPL
314 SLENIAKDFNV-SVNEIKAHLRSLQLLTKNNDMSFKEKLLKYIEYISCVTGGPL
313 SLENIAKDFNV-SVNEIKAHLRSLQLLTKNNDMSFKEKLLKYIEYISCVTGGPL
333 SLENIAKDFNV-SVNEIKAHLRSLQLLTKNNDMSFKEKLLKYIEYISCVTGGPL
311 SLENIAQDLNM-SVDDFKVHLRFPHLFAEHNDESLEDKLFKYIKHISSVTGGPV

317 SLENIAEDLNV-TLEELKANIKSPHLFSDEPDTSLTEKLLKYIGNP
332 SLKNIAEDLNV-TLEELKANIKSPHLLSDEPDTSLTEKLLKYIGNP
326 SLENIAEDLNV-TLEELKANIKSPHLLSDEPDTSLTEKLLKYIGNP
326 SLENIAEDLNV-TLEELKANIKSPHLLSDEPDTSLTEKLLKYIGNP
308 SLAKLAEQVGK-QAGDLRSVIRSPLANEVSPETVLRLYSQSSDGAMRVARAFERGIPVFG


KNSFLKEIFYLKYYFLDMVTEDAKTLLKEICLRN

KN-HCREILYLKLYFLDMVTEDAKTLLKEICLRN

AVGLYFPAIYYLQLHILDTVTEDAKVLLRWKYSKPRSNSTYP
AVGLYFRKTYYLQLHFLDTVTEDAKVLLRWKYSKPRSNSTYP

AAGLYFRKTYYLQFHFLDTVAEDAKVLLKAAQTHFAHSF
AAGLYFRKTYYLQFHFLETVAEDAKVLLKEAY
SAFQALKVYYRRTQFLNIVVDDAKHLLRKIETVNVA
PCVCCCLRRLRHKRMLFLVAQDTKNILEKILRDSIFPPQI
CCFTSHHSRCKQQKDILDETAAKAKEVLLKILRLSIPHP
VVTRYF RHQRHKLVIEIVAENTKTSLRKALKDSVLPPEIH
ASGLYFRKTYYWQSLFIDTVASDAKSLLNKEEFLSEKPGSCLSDLPEYWETGMEL
ASGLYFRKTYYWQSLFIDTVASDAKSLLNKEEFLSEKPGSCLSDLPEYWETGMEL
ASGLYFRKTYYWQSLFIDTVASDAKSLLNKEEFLSEKPGSCLSDLPEYWETGMEL
ASGLYFSKTYYWQSLFIDTVASDAKSLLNKEEFLSEKPGSCLSDLPEYWETGMEL
AAVTYYRMAYYLQNLFLDTAANDAIALLNSKALFEKKVGPYISEPPEYWEA

YFSKVFHLQNYFIDTVASDAKIILSKEELFTEQVSSFNSKASPYREESVGKVFPVSPGSTFL
YFSKVFHLQNYFIDTVASDVKIILSKEELFTEQVSSFNSKASPYREESVGEVFPVGPGSTFL
YFSKVFHLQNYFIDTVASDVKIILSKEELFTEQVSSFNSKASLYREESVGKVFPVGPGSTFL
YFSKVFHLQNYFIDTVASDVKIILSKEELFTEQVSSFNSKASPYWEESVGKVFPVGPGSTFL
TLVAGGISFGTVYTMLQGCLNEMAEDAQRVRIKALEEDEPQGGEVSLEAAGDNLVEKRSTGEGTSEEA

FHFFEMFQSDSDKLCHVHVLLLLTSWGLSGETVT
FHFFEMFQSDSDKLCHVHVLLLLTSWGLSGETVT
FHFIEMFQSDSDELCHVHVLLLLTSGGLSSETVT
FHFFEMFQSDSDKLCHVHVLLLLTSWGLSGETVT
PLSTRRKLGLLLKYILDSWKRRDLSEDK


GXXXXGK/M
S
G1
SWI
G2

DXXG/SWII
G
3

N/TXXD
G4

SAK
G5

S2
αA

αC
H2B
H1
S3
αB
H2
H2A
S1
S4
S4
413
295
406
416
421
191
417
410
420
409
407
423
421
441

421
441
415
232
458
473
467
467
463
189
310
R92.10 Genome Biology 2005, Volume 6, Issue 11, Article R92 Bekpen et al. />Genome Biology 2005, 6:R92
modified, primarily in the nucleotide binding site. In view of
the substantial divergence between the IRGQ genes and func-
tional p47 GTPases, it was unexpected not to find close
homologs of the Danio irgq sequences in either the Fugu or
Tetraodon genomes. The evolution and diversity of the Danio
irgq genes is apparently linked to the evolution and diversity
of the GTPase-competent IRG sequences.
IRG homologs outside the vertebrates
No unambiguous IRG homologs have been found outside the
vertebrates. However, two possibly related sequence were
recovered from the Caenorhabditis elegans genome, and sev-
eral groups of putative GTPases of unknown function exist in
the bacteria that have sequence features reminiscent of IRG
GTPases. Perhaps the most striking of these are found in the
Cyanobacteria (see Additional data file 1 for accession num-
bers for these sequences). Among other features, all of these
sequences have in common with the IRG GTPases the pres-
ence of a large hydrophobic residue in place of the familiar

catalytic Q61 of H-Ras-1, but this feature is far from diagnos-
tic for the IRG GTPases [22]. Despite several suggestive char-
acteristics of these invertebrate and bacterial GTPase
sequences, it is not possible on the basis of sequence criteria
alone to establish their phylogenetic relationship with verte-
brate IRG proteins.
Discussion
The p47 GTPases (IRG proteins) are an essential resistance
system in the mouse for immunity against pathogens that
enter the cell via a vacuole. In this study we reached several
unexpected conclusions about the evolution of the system.
First, the IRG resistance system, despite its importance for
the mouse, is absent from humans because it has been lost
during the divergent evolution of the primates. Second, the
IRG resistance system is at least as old as the bony fish but
missing in the invertebrates. Finally, the IRG proteins appear
to be accompanied phylogenetically by homologous proteins,
here named IRGQ proteins, that probably lack nucleotide
binding or hydrolysis function, and that may form regulatory
heterodimers with functional IRG proteins. We consider
these points in order.
The argument for the absence of the IRG resistance system in
humans relies on several findings. The system is reduced
from 23 genes in mouse to one full-length gene and a
transcribed G-domain in humans, and the residual genes lack
the character of functional resistance genes. Thus, IRGC is
highly conserved in humans, dog and mouse, is not interferon
or infection inducible, and is expressed constitively in mature
testis. IRGM, although clearly derived from a typical GMS
subfamily resistance gene, is transcribed constitutively from

an endogenous retroviral LTR, is unresponsive to interferon,
and appears to be structurally damaged in several ways.
We argue that the IRG resistance system has been lost from
primates (the situation in chimpanzee is identical to that in
Extended phylogeny of the G domains of IRG and related proteinsFigure 5
Extended phylogeny of the G domains of IRG and related proteins. The
phylogeny relates all of the IRG sequences described in this report and
reveals the distinct clades on which the nomenclatural fine structure is
based. All except the mouse sequences are labeled with the species of
origin. Dog IRG sequences are found in the B, C, D and M clades, and
human sequences only in clades C and M. The mouse and human quasi-
IRG proteins, IRGQ (FKSG27), could not be included in the phylogeny
because they are so deviant in the G-domain (see Figure 6 and Additional
data file 6).
(dog)
(dog)
(dog)
()Fugu
(zebrafish)
(zebrafish)
(zebrafish)
(zebrafish)
(zebrafish)
IRGD
(dog)
IRGC
(dog)
irgf7
()Tetraodon
irgf8

()Tetraodon
IRGM
(human)
Irgb3
Irgb4
Irgb8
Irgb1
Irgb6
Irgb2
Irgb7
Irgb5
Irgb9
Irgb10
IRGB12 (dog)
IRGB11 (dog)
Irga1
Irga2
Irga6
Irga4
Irga7
Irga3
Irga8
Irgd
Irgc
(mouse)
IRGC (human)
irgf1
irgf2
irgf3
irgf4

irgf6
irgf5
irg g
irge4
irge2
irge6
irge3
irge1
irge5
Irgm2
Irgm3
Irgm1
IRGM6
IRGM5
IRGM4
irgq2
irgq1
irgq3
H-Ras-1
0.2
(zebrafish)
(zebrafish)
(zebrafish)
(zebrafish)
(zebrafish)
(zebrafish )
(zebrafish)
(zebrafish)
(zebrafish)
()Fugu

Genome Biology 2005, Volume 6, Issue 11, Article R92 Bekpen et al. R92.11
comment reviews reports refereed researchdeposited research interactions information
Genome Biology 2005, 6:R92
humans; unpublished data) rather than gained by the murine
rodents (including rat; unpublished data) on the following
grounds. First, like the mouse, the dog genome has several
complete, interferon-inducible IRG genes in addition to
IRGC. Second, humans and chimpanzees possess a degraded
member of the GMS subfamily of IRG proteins, confirming
that this distinctive subfamily, present and functional as
resistance genes in dog and mouse, was widely distributed at
the origin of the mammalian radiation. Finally, the IRG sys-
tem is present in bony fish, representing ancient vertebrates.
Rapid expansion and contraction of multigene families asso-
ciated with pathogen resistance has frequently been docu-
mented in both animals and plants [23-28]. In all of these
cases, however, the resistance mechanism itself has been
retained as its protein mediators have evolved or even, in the
natural killer receptor case, been replaced by a different
molecular species [29]. The IRG case may be different
Extended alignment of the vertebrate IRG proteinsFigure 6
Extended alignment of the vertebrate IRG proteins. Individual sequences are given in full and are labeled as in Figure 5. Unusual residues in the G1 motif
are highlighted (M of the GMS proteins in green and two deviant residues in the zebrafish irgq sequences in pink). The essential structural relationship
between IRG genes and quasi-IRG genes is apparent in the alignment despite the modified G-domains. For mouse and human IRGQ the long carboxyl-
terminal coding exons that contain the p47 homology were used for the alignment. In human IRGQ the sequence
ENPKGESLKNAGGGGLENALSKGREKCSAGSQKAGSGEGP was removed from the alignment between positions 210 and 211 (highlighted in turquoise)
to prevent extensive gap formation. The position of the intron present in pufferfish and zebrafish irgf genes is indicated by two adjacent residues
highlighted in blue.

FPVLMDKLISDLPIYKRHNFMVSLPNITDSVIEKKRQFLKQRIWLEGFAADLVNI-IPSLT

FPKLETKLLQDLPAHKRHVFSLSLQSLTEATINYKRDSLKQKVFLEAMKAGALAT-IPLGG
FPKLEETLLKELPGHKRHMFALLLPNISDASIELKKHFLREKIWLEALKSAAVSF-IPFMT
FQSLETTLLKELPAHKRHIFMQYLPNITESAIDRKRDSLRQKVWLEAVKAGASAT-IPFMG
FQSLETTLLRELPSHKRHIFMQYLPIVTEATIDRKRDCLRQKVWLEAIKAGASAS-IPLVG
FPRLEETLLKELPVHKRHIFALLLPNLSYTSIEMKRAFFKEKIWLDALKSSALSF-IPFMA
FPKLRDTLHKDLSNIRCCEPLKTLYGTYEKIVGDKVAVWKQRIANESLK N

FPELRNTLNRDISDIRYCGPLKNLSHTYEKVISDKVTMFRGKIASKSF D
FPELRDALNRDISDIRYCGPLENLSDTCEKIINDKVTSFQEQIGSKTFQ D
FPELRKSLHRDISNIGYRGHLENLTHTCEKVINGKVTTLQGQIGSKSFQ D
FPMLVTTWEHDLPAHRRHAGLLSLPDISLEALQKKKDMLQEQVLKTALVSGVIQA-LPVPG
FPTLVSTWEHDLPSHRRHAGLLSLPDISLEALQKKKAMLQEQVLKTALVLGVIQA-LPVPG
FPLLMSTWEHDLPAHRRHAGLLSLPDISLEALQKKKDMLQEQVLKTALVSGVIQA-LPVPG
FQKFISTFKDEVFKIRAEEFSGFLDKMLHGGWLKAR
FQKLVNTLEEELPKNKRFALIQSLPVYSLETLTKKITYFKKLIWLNAVGAGVGAF-PPIPG
FQKLVNTLEEELPKNKRFALIQSLPVYSLEALTKKITYFKKLIWLNAVGAGVGAI-APIPG
FEELQNTLAEELPVHKRNALLQAWPVCSAASLEMKIKMFEGVIWAASLASAGIAV-VPLPG
FN-LVGTLESELSDQKGFALVQSVPVYSLAMLEKKKALLEKFIWLAALASSACTL-VPNQF
FQTFVDTLEKQLPDHKRDALILSLPIYSSKILEEKIEIFMKQTWSAAVASGSVAV-VPVPG
FQTFVDTLEKQLPDHKRDALILSLPIYSSKILEEKIEIFMKQTWSAAVASGSVAV-VPVPG
LNLLQDRMEKELPQHKRRVLMLALPNITLEINEKKKKALEENIRKVAFLSACVAL-FPLPG
LNLLEEKMEEELPQHKRRVLLLALPNITQEINEKKKEALGQNIGKVAILSACVAA-VPIPG
LNLLQEMMEKEILKCKRILLKSALLNVKQEVIEQRKDTLKRNIERVTEQSVAITD-VHLPG
FQLLQERMETELPLHKRRVLMLALPNVSLDVIKKKKEVLEKDIAKVAFISATVSA-VPIPG
FLTFLEVMRGDLPEIRAHALLLALPTFSSSLVTQKKDAFKALVWAAASLSGGVSA-IPVPL
FVSLESALSSDLNTIRTSAFAYYIARTVKENL
LFLVSANYPETLDLAKLKGMLKAAIPSHKKVALARYVSKQLDEDVFWKRSDSCKFM
FHETHETLERELPEHKRNVLLVAMPNISLEIIEKKKEAFKSKIPLWAFVSAAGAV-VPVPG
FHLLYETLEREFPEHQRDVLLVAMSNISLEINGKKKEAFKSKIPYWALVSSVGAL-VPVPG
FHRLHATLERELPEHKRDALLFAMPNMSLEIIEKKKEAFKSKIPHYAFVSAACAA-VPVPG

FHRLHATLERELPEHKRDALLVSLANMSLEIIKKKKEAFKSKIPHYAFVSAACAA-VPLPG
LPGLGTWLQHALPTAQAGALLLALPPASPRAARRKAAALRAGAWRPALLASLAAAAAPVPG
LPGLCEWLRRALPPAQAGALLLALPPASPSAARTKAAALRAGAWRPALLASLAAAAAPLPG
DAFYTLVREIRQHKLRKLNPPDESGPGCMSCKCVLS

DIRLNCVNTFRENGIAEPPIFLLSNKNVCHYD
NIKDYCSNHLQESLDSEPPVFLVSNVDISKYD
QIRDYCVTNLIKTGVTEPCIFLISNLDLGAFD
KIRNDCITQLQNVKVCDPQVFLVSNLDLSSYD
KIRNDCVKHLMEANMSDAQVFLVSSFELSDYD
QIRDNCLANLSNIGVPEPCIFLVSNFDLDDFD
NIQENIRENLQKEKVKYPPVFLVSSLDPLLYD
-IRENVLENLQKERVCEY
NIQENIQENLQKERVFEPIIFLVSSFEPLLHD
NIWENIQETLQKVGVCEPIIFLVSSFEPLLHD
NIRENIRETLHKEGVCEPIIFLVSSFNPFLHD
EIRDHCTERLRVAGVNDPRIFLVSNLSPTRYD
EIRDHCAERLREAGVADPRIFLVSNLSPARYD
EIRDHCAERLRVAGMTDPRIFLVSNLSPARYD
QMRQDCEKYLKEKKLD-PHIFLVSTHDTHNYE
KIREDCKVNLLK LNISKIFLISSFHLERYD
KIREDCKVNLLK VRISKIFLISSFHLERYD
TIREDCLKNLKQ LGDPKVFLISSFDLEKYD
AIREDCYRNLKE VGNPKVFLISSFDLRKYD
HIRENCHRNLKD IDDPHAFLICSFELHKYD
HIREDCHRNLKD MDDPHAFLICSFELHKYD
SIRKDCINGLRKIGIEDPIVFLISGWELSKYD
VIREDCVNGLRKIGIEDPVVFLISNFELGKYD
SIREDCENGLRKIGIEYPVVFLISGWDLGKYD
NIRDKCKSELSKI-VKDPAVFLISCNELNKYD

AKKAASLDVLKAEGVPLPKVFLVQPSALEKLD
GLRAQYTQELQREKLSEQQMFLINSQDRSAFD
VISMVDLIEDKAVEEVRQWTEKVLSKLDIQQS
RIRDNCKKGLLNAGVQA-QVFVLSNFELQRYD
LIRENCKRGLLNAGLQA-QVFLLSSFELQRYD
LIRENCKEGLLKEGVQAPQVFLLSNFELRRHD
LIRENCKEGLLKEGVQAPQVFLLSNFELRRHD
DSGCTAARSPEDELWEVLEEAPPPVFPMRPGG
DSERAAALSPEDETWEVLEEAPPPVFPLRPGG
SRQAQDLARSYGI PYIETSAKTRQGVE
154 ISATRFK KNDIDIAKAISMMK-KEFYFVRTKVDSDITNE ADGKPQTFDKEKVLQ
141 ISATRFK ENDAQLAKAIAQMG-MNFYFVRTKIDSDLDNE QKFKPKSFNKEEVLK
165 ISSSRFS LNDALLAQKIKDAG-KKFYFVRTKVDSDLYNE QKAKPIAFKKEKVLQ
154 ISSTRFT INDAQLATAIRKMK-KNFYFVRSKVDSDLYNL KRTKPSDFNKDEILL
155 ICATRFK INDVQLATAIKKMK-KNFYFVRSKVDSDLYNL KRIKPREFNKDEILQ
156 ISSSRFS LNDALLAQNIKEIG-KKFYFVRTKVDNDLYNE EKSKPMSFKRERVLQ
161 IASEQFS SNHVKLSKIIQSMG-KRFYIVWTKLDRDLSTS VLSEVRLLQ
117 VASAQFS MNHVMLAKTAEDMG-KKFYIVWTKLDMDLSTG ALPEVQLLQ
130 IASEQFS MNLVKLAKAIQVLG-KRFYIVWTKLDRDLSTS ALLKERLLQ
149 IASEQFS MNLVKLVKAIQRQG-KRFYIVWTKLDRDLSTR VLPEEQVLQ
149 IASEQFS MNLVKLVKSIQGQG-KRFYIVWTKLDRDLSTC VLSEEQLLR
137 VSPRRCG AVESRLASEILRQG-KKFYFVRTKVDEDLA ATRSQRPSGFSEAAVLQ
139 VSPRRCG AVETRLAAEILCQG-KKFYFVRTKVDEDLA ATRTQRPSGFREAAVLQ
139 VSPRRCG AVETRLASEILRQG-KKFYFVRTKVDEDLA ATRTQRPSGFSEAAVLQ
132 VISERVR ENNMLLVDEIDKRK-KPFYFIRTKIDNDVKSQRR KSKFSETQALE
148 VTSERFR ENDIELAKAINKSN-KLFYFIRTKIDNDVR AESNKRNFDERVLLD
146 VTSERFR ENDIELAKAIKKSN-KLFYFIRTKIDNDVR AESYKRNFDEPMLLD
123 LNSERFM QNDVMLAKEIRKQK-KNFYFVRSKIDNDIS AEQRKKTFDEQRVLC
126 ISSERFK ENDVYLAKEIQKKQ-KRFYFVRNKIDNDIC SVANGK-INEQQLLC
158 ISSERFK ENDIMLANAIKERK-KLFYFLRSKIDNDIH AESHRKDFDEQKVLS

157 ISSERFK ENDIMLANAIKERK-KLFYFLRSKIDNDIH AESHRKDFDEQKVLS
130 IASDRFR ECHTQLAKEIMRMG-KKFYFVRSKIDASIT AEKKKKNFDQKKTLD
130 IASDRFK ECHTHLAKEIMRMG-KKFYFVRSKIDASIT AEKRKKNFDLKKTLD
130 IASDRFR ECHTQLAKGIMRMG-KKFYFVRSKIDASIT AEKKKKNFDQKKTLD
128 ISSDRFK EHHSLLAEEIVRLR-KTFYFVRSKIDQSID SEKYKKTFDQEKMLD
122 LTSTDRP SANSVAVWKEVRSL-QKETVYFVLLAS VKDTEKSLE
133 IVSDWEK VRHVKLAKEVEKLR-KHYLLVQTKVDSCLQTQG DLCCEETEILD
135 SL SANAF SSSEGQQVASVLAL-CDVYILVSPLRVRLRTIQL-LQQASSMGKECYL-
149 ISDTCFR KNDVKLAKEIQKMG-KKFYFVRSKVDDDLLN AQRSQRDFDPEQTLS
122 ISETRFR ENDVKLAKEIQKMG-KKFYFVRSKVDNDLQS EQRYQRDFDPEKTLS
160 ISATRFR ENDVKLAKEIQKMG-KKFYFVRSKVDNDLQN AQRSQRNFDAEQTLA
160 ISATRFR ENDVKLAKEIQKMG-KKFYFVRSKVDNDLQN AQRSQRNFDAEQTLA
169 APTEENWAQVRSLVSPDAPLVG VRTDGQGEDPPEVLEEEKAQNASDGNSGDARSEGKKAGIG
170 APTEKDWAQVQALLLPDAPLVC VRTDGEGEDPECLGEGKMGPGKAGSEGLQQVVGMKKSGGG
80 VFAINNTKSFEDIHQYREQIKRVKDSDDVPMVLVGNKCDLAART VE
AKTGVVEVTMERHPYKH-PNIP NVVFWDLPGIGSTNFPPNTYLEKMKFYEYDFFII
APTGAIETTMKRTPYPH-PKLP NVTIWDLPGIGTTNFTPQNYLTEMKFGEYDFFII
ADVGTVETTMCKTPYQH-PKYP KVIFWDLPGTGTPNFHADAYLDQVGFANYDFFII
APIGAVETTFDRTEYKH-RKFP NVTLWDLPGVGTTTFHPQEYLEKMKFREYDFFII
APTGPVETTFLRKAYKH-PKFP NVTFWDLPGIGTTSFQPQDYLEKMVFREYDFFII
ASVGVVETTMKKTPYQH-PKYP KVTFWDLPGTGTPNFHPHEYLEMVEFATYDFFII
APTGVVRTTKTRTEYSS-SHFP NVVLWDLPGLGATAQTVEDYVEEMKFSTCDLFII
PPTELVKATQRCASYFS-SHFS NVVLWDLPGTGSATTTLENYLMEMQFNRYD-FIM
APTGVVKTTQIPTCYSY-PHFP NVELWDLPGTGAGTQSLENYLEEMKFSWYDLFII
APTGVVRTTQVPTCYSS-SHFP YMELWDLPGTGTGTQSLENYLEKIHFSQYDLFII
APTGVVRTTQIPTCYSF-SDIP NVELWDLPGTGAATQNLETYLEEMQFSKYDLFII
ALTGVVETTMQPSPYPH-PQFP DVTLWDLPGAGSPGCSADKYLKQVDFGRYDFFLL
ALTGVMETTMQPSPYPH-PQFP DVTLWDLPGAGSPGCPADKYLKQVDFSRYDFFLL
ALTGVVETTMQPSPYPH-PQFP DVTLWDLPGAGSPGCPADKYLKQVDFGRYDFFLL
AQTGAIETTKQATMYQQ-SNLP HIRLWDLPGMGTPSFASKSYVKMMNFDLYDMFMV

ASTGTTETTMKPNMYEH-PFMP NVKIWDLPGIGSPKFRAKKYLKDVNFHMYDFFLI
AFTGTTETTMKPNMYEH-PFMP NVKIWDLPGIGSPKFRAKKYLKDVNFHMYDFFFI
APTGVTETTMEPNMYEH-PAMP NVKIWDLPGIGSPNFKADKYLKDVKLKNYDFFII
APTGLTETTKKATMYTH-PTKP NVRLWDLPGIGTPNFKANQYLKDVKFETYDFFII
APTGVTETTLVPTMYRH-PTMP NIELWDLPGTGSPKFKAKKYLKDVKLETFDFFII
APTGVTETTLVPMMYKH-PTMP NVELWDLPGTGSPKFKAKKYLKEVKLETFDFFII
AETGPVETTMEPEVYIH-PKYH NVKVWDLPGIGTPNFKADEYLELVEFERYDFFII
AETGVVETTMEPKAYNH-PKIQ HVKVWDLPGIGTPNFKADEYLQQVEFERFDFFII
AKTSSVVTTAEPEVYFH-PKYE NVKLWDLPGIGTPNFKADKYLELVEFERYDFFII
AETGFEETTMEPKDYIH-PNFK NVRLWDLPGIGTPNFKAKDYLKLVKFERYDFFII
AQNPPSAAPEELAVFTN-PKHP DFRLWDLPPISSDANFKPEDYIERFKATRYNAII
AGASISNPALS PVYP DVRFWDISGIEAV-MDYSVFEMKQAMKCYDFYII
ALTGVTETTKEAVEYAL-PDSH NIRFWDLPGLGKIG DLS
APVGVVETTVDVKEYPH-PDYP NVSLWDLPGIGTTKFPADEYLKLVGFEKFDFFII
APVGVVETTAEVKEYPH-PNYP NVSLWDLPGIGTTKFPADEYLKLVGFEKFDFFII
APTGVVETTTEVRAYPH-PSYP NVTLWDLPGIGTTRFPADQYLKHVGFERFDFFII
APTGVVETTTEVRAYPH-PSYP NVTLWDLPGIGTTRFPADQYLKHVGFERFDFFII
APTGPTP YPA-PERP NVVLWTVPLGPTATSP AVTPHPTHYDALILVTPG
VPTAPTP FPA-PERP NVVLWTVPLGHTGTATTAAAASHPTHYDALILVTPG
-YDPTI EDSYRKQVVIDGETCLLDILDTAGQEEY SAMRDQYMRT GEGFLC

LIELRMRKGNIQLTNSAISDALKEIDSSVLNVAVTGETGSGKSSFINTLR-GIGNEEEG-A
LIMTYIEENKLQKAVSVIEKVLRDIESAPLHIAVTGETGAGKSTFINTLR-GVGHEEKG-A
GIHKALQEGNLSDVMIQIQKAISAAENAILEVAVIGQSGTGKSSFINALR-GLGHEADE-S
SIEKSLKEGNLQKAVSDINKALKDIDNAPLSIAVTGESGTGKSSFINALR-GVGHDEEG-A
TIQSHLEKGDLQSAFSAINDALRDIDNAPLNIAVTGESGTGKSSFINALR-GMGHDEEG-A
RIQAALKEAKLKDVADIIEESLVAAENAPLDVAVIGESGTGKSSFINALR-GLSYEEEG-S
-TERALREGKLLELVYGIKETVATLSQIPVSIFVTGDSGNGMSSFINALR-VIGHDEDA-S
NVEKASADGNLPEVISNIKETLKIVSRTPVNITMAGDSGNGMSTFISALR-NTGHEGKA-S
NIEKALGGRKLLEVVPMVRETLERASSVPLRIAVTGDSGNGMSSFINALR-GIGHDEED-S

NIEKALGDGKLLEVVSMIRETLETVSSAPVSIAVTGDSGNGMSSFINALR-EIGHDEKD-S
NIEKALGEGKLLDMVSVVRETLETASSVPVSIAVTGDSGNGMSTFINALR-KIGHNEED-S
ALRTAFESGDIPQAASRLRELLANSETTRLEVGVTGESGAGKSSLINALR-GLGAEDPG-A
ALRTAFESGDLPQAASHLQELLASTESIRLEVGVTGESGAGKSSLINALR-GLEAEDPG-A
ALRSAFESGDIPQAASRLRELLASSQSIRLEVGVTGESGAGKSSLINALR-GVGAEDPG-A
TIRDVFAGESPETIPHRLISLLEVFDRFKIDIAVTGDSGAGKSSLINAIL-GLKPDDKG-A
KTRKLK DKLTELENVTLNMAITGMTGAGKSSFVNALR-GLRDDDEG-A
KTTKLK DKLTELENVTLNMAITGMTGVGKSSFVNALR-GLRDDDKD-A
ATAKAK ESFDQFMNVSLNIAVTGKTGSGKSSFINALR-GLKDDDEG-A
AAVKAK EELDRLDSVTLNIAVTGEAGAGKSSFINALR-DLSDEDEN-S
PDVHLNSSAEYINEMECVIEQNKQLGNVTLHVAVTGSTGAGKSSFINAIR-GLTSDDEN-A
MECVIEQNKQLGNVTLHVAVTGSTGAGKSSFINAVR-GLTSDDEN-A
AVNTIK EYLKQQDLVELNIGVTGESGSGKSTFVNAFR-GLGDEDEG-S
AVSRIR EYLRKQDLVELNVGVTGESGSGKSTFVNAFR-GLGDEDEG-S
AFGTIS NYFKETSLV-LNIGVTGESGSGKSTFVNAFR-GLGDEDEG-S
AINTIK ECLRKQDLVELNIGVTGESGSGKSTFVNAFR-GLGNEEKG-S
NLLETLKESIEKNNISDIRDALEDMLISRINIAIAGERNAEKATFINSLR-GLSQEDEG-A
ITKLQNMYKSTGFGAAKVSAVLEALSHFQLDVAVLGETGSGVSTLVNALV-GLENEESS-G
QISKLSQTRDFTDNPSKLQAILGALDHFRLDVGVLGETGCGSSSLINALL-GLKNSNET-A
AAAKIK ELLDNPSNATLNIGITGESGSGKSSFVNAFR-GVDHKDEKEA
AAAKIK ELLDNTSNTTLNIGITGEAGSGKSSFVNAFR-GVDDRDEK-A
AVDKIK KLLERAANTPLNIGITGESGSGKSSFVNAFR-GVDHQDNQ-A
AVDKIK KLLEKRANTPLNIGITGESGSGKSSFVNAFR-GVDHRDNQ-A
AVREAFETGGLEAALSWVRAGLERLGSARLDLAVAGTTNVGLVLDMLLGLDPGDPGAAPAS
AVREAFETGGLEAALSWVRSGLERLGSARLDLAVAGKADVGLVVDMLLGLDPGDPGAAPAS
MTEYKLVVVGAGGVGKSALTIQLIQNHFVDE

N/TXXD
G4

SAK
G5

H2B
H1
S2
S3
αA
310

αB
αC
H2 H2A
S1
S4
S5
H4
αE
S6
αF
αd
H3
H5
αJ αK
GXXXXGK/M
S
G1
SWI
G2

DXXG/
S
WII
G3

N
1 MGQLFSSPKSD-ENNDLPSSFTGYFKKFNTGRKIISQEILN
1 MAWASSFDAFFKNFKRESKIISEYDIT
1 MDQFISAFLKGASENSFQQLAKEFLPQYSALISKAGGMLSPETLT
1 MGQSS-STPSHKTGGDLASSFGKFFKDFKLESKILSQEAIT
1 MGQSPPSTPSNRNGGDLASSFDKFFKEFKLDSKIISQETIS
1 MDKFMCDFLVGKN FQQLAINFIPHYTTLVNKAGGIIASENLD
1 MKPSHSSCEAAPLLPNMAETHY APLSSAFPFVTSYQTGSSRLPEVSRS-
1 MEAM
1 LHCFFPLLQVTPLLSDVTQPTHSLHTPLLTSSNYDMPYNMGWSSLSKETAI
1 MTQPNHSLHIPLSTSFTSIVPYNMGWTVLPKATAT
1 MAQPTQSLHTPSPTSFTSTVPYHKGGSILSESGAM
1 MATSRLPAVP EETTILMAKEELE
1 MATSKLPVVPGEEENTILMAKERLE
1 MATSKLRAVPGEEETTILMAKEELE
1 MFFSRLCMPAKVQEDHLG
1 MPEKEEDKNENLYIISSEFLDIMSNATDDPDSISEDMKEVIDAKPKE
1 KEEEDENENLYIVSSEFINIMSNATDDPDSISVDMKEVIDAKPNE
1 METQDP-AIAEAVQASGESTLEK
1 MTDDSSADM-NFSGALQRLGESDPNA
1 MKIQKQKQELSNSSKPDTHSHSTAKENV-SLKSANTVQVEHIYEM
1
1 MATFEDYCVITQEDLDDIKDSISTQDLPS
1 MDILEDYDIITQNDLEEIKESISTEDLPT
1 VDALEHLYEIKVEDKLKEIKEILYTQDLPT

1 MSNISQKVVLLFAEQEELVDLRKAISTQDLPT
1 MADVIKGL
1 MLHGGWLKARYATQHVQQTEKLETED
1 MAIQCTHRICSYLTNSLFFRFVVSTALRSMKINQDDLD
1 MADSSDIVEIKEALRNNNQAL
1 MADSSDFAEIKEALQNNNQAL
1 -MVNVCVCYITVGLSVGMISRLSDFYIVTVGFALCVQVIMADSLDTTEIKEALQNNNQAL
1 -MVNVCVCYITVGLSVGMISRLSDFYIVTVGFALCVQVIMADSLDTTEIKEALQNNNQAL
1 RLLPPAQDGFEVLGAAELE
1 RLLPPAQDGFEVLGAAELE
1
Irga6 319
Irgb6 305
Irgd 324
IRGB12 (dog)
319
IRGB11 (dog) 320
IRGD (dog) 321
Irgm1 310
IRGM (a) (human)
IRGM6 (dog) 278
IRGM5 (dog) 298
IRGM4 (dog) 298
Irgc 302
IRGC (human) 304
IRGC (dog) 304
irgg1 (zebrafish)
irge1 (zebrafish) 309
irge5 (zebrafish) 307
irge3 (zebrafish) 284

irge4 (zebrafish) 285
irge2 (zebrafish) 319
irge6 (zebrafish) 318
irgf1 (zebrafish) 293
irgf3 (zebrafish) 293
irgf2 (zebrafish) 293
irgf4 (zebrafish) 290
irgq2 (zebrafish) 276
irgq1 (zebrafish)
irgq3 (zebrafish)
irgf7 (Tetraodon) 308
irgf8 (Tetraodon) 285
irgf6 (Fugu) 351
irgf5 (Fugu) 351
Irgq1 303
IRGQ1(human) 343
H-Ras-1(human)
TIQERLSRYIQEFCLANGYLLP KNSFLKEIFYLKYYFL
SLEDKLFKYIKHISSVTGGPV AAVTYYRMAYYLQNLFL
SIIAQATSAAEAFCAVKGGPE SSAFQALKVYYRRTQFL
SLGEKLLRYVEKFCSVSGGLI ATGVYFRKIFYLQNYFL
PLGEKLLKYVEKFCSVSGGPI AAGIYFRKIYYLKNYFL
SIAEKAMKCVECYCSVNGGLP STIFQFFKIYFLHLKFI
KLRLMTCAIVNAFFRLLRFLP CVCCCLRRLRHKRML

AVSCMNCNTSSCLYTILRYIPL LGDFIINFLRKWKHRRLL
ALSCMNCKTASYLYSILSYIPF LGDTVINYLRVWKHRHFL
ALSWMNCNAASYLYSVLSYIPI LGTTGIHYLKWWSQGHLL
VLRLYSQSSDGAMRVARAFERG IPVFGTLVAGGISFGTVYTMLQGCL
VLRLYSQSSDGAMRVARAFERG IPVFGTLVAGGISFGAVYTMLQGCL

VLRLYSQSSDGAMRVARAFEKG IPVFGTLVAGGISFGTVYTMLQGCL

LIDMMSNPVIAITKTLGTIMAL LPG GALPAGGAAVASVHYLLNVGL
LMDMISNPVIAIAVTLGTIMAL LPG GALPAGGTAVATVHYLLNVGL
ALTRLQVSGTLVVLFSAEYVAS LVPGVGSVAAAGLSFGTTYYLLRSGL
RISPMAKPVKSLEDLLDSKNLAVN VQNTADAFRNSHTNLTRAL
KLTNKELSALTSKEAAVKFAWS MVPVVGSIKTAQMSYSTTLNLLRTGV
KLTNKELSALTSKEAAVKFAWS MVPVVGSKKTAQMSYSTTLKLLRTGV
ILTLLGAASVLISEDAVELLVS FIPIIGSVVAGGLSYLTVSGMLKKAL
LLSLVGAVSVVGAESTVEYILS LVPILGTVVAGGLSYLTVSTMLRRAL
IINLLEAEVPKIEN EYFLS FMPFIGTEIKKIKSSVAVSSMLKTAL
ILALLSSATLVLGGMSVLAAESALEYFLSTIPLIGSVAAAAMSYKTITLMLKKTL
VKRRLAEAEKDTST ATTRLV ELAIPRQARSVSRSFTVMLQALNNAI


ITQTLNQTASVAGLMAAEEGLR FFPIFGTMIAGSLSCAVIYKALSDFL
IVKALSELASVAGLMAAEEGLR FIPIFGTMIAGTLSYAATYNALSDFL
ILKLLLQSAAVAGLMLAEEGLK FIPLFGTLVASTLSYKVTEKALLDFL
VLNLMSQSSAISSLTETRESYS FIPLFGIPVARKLSYEITERALHNFL
RLGSWAGEGTAGGAALSALSFL WPTGGAAATGGLGYRAAHGVLLQAL
RLGAWAGEGTAGGAALGALSFL WPAGGAAATGGLGYRAAHGVLLQAL

DMVTEDAKTLLKEICLRN
DTAANDAIALLNSKALFEKKVG PYISEPP EYWEA
NIVVDDAKHLLRKI-ETVNVA
EAVVSDAKVLLNKEEIFKETVGSGQAYLLQDVGIENRKSDATSS
DTVVSDAKVLLKKEEIFKDPVDSEQTYLHTNVGNENGKSDTSSS
NTVADDAKILLHKTLEILSHRR
FLVAQDTKNILEKILRDSIFPPQI

EIVAEDTRTILKKILKDSII
EIVAKDTRSIVKKILTDSII
EIVAEDTKTILKKILEDAII
NEMAEDAQRVRIKALEEDEPQGGEVSLEAAGDNLVEKRSTGEGTSEEAPLSTRRKLGLLLKYILDSWKRRDLSEDK
NEMAEDAQRVRIKALEDDEPQP-EVSLEVASDNGVEKGGSGEGGGEEAPLSTCRKLGLLLKYILDSWKKHD-SEEK
NEMAEDAQRVRIKALEEDEPQS-EVSLEAAGDNGVEKRGSGEGGCEEAPLSARRKLGLLLKYILDSWKKRDLSEEK


KEMADDTRKVLVVSQLA
REMADDTRKVLAISQLA
KELANVAREIRKEVLDSVR
NEMIKDMRQVLQVAGLDE
QDLAETAS
QDLAETAREVLKAAGVTGVY
NEIAEDARNVLMASLETEV
NDIAEDARNVLNASLETEV
NVIAEDIRNVI
NDLAKDAETVFKALLETEV
DDMGADAEKVVAMVTGERQ


EMLTDDAQYVFEKALRCMNSSV
KMLTEDAQNVFEKALRCMNSSV
HMLAEDAQNVFKRALCCMNSSV
DMLTEDAQDVYNRVINHINS
DEMLADAEAVLGPPEPNQ
DEMRADAEAVLAPPEPAQ

Irga6
Irgb6

Irgd
IRGB12 (dog)
IRGB11 (dog)
IRGD (dog)
Irgm1
IRGM (a) (human)
IRGM6 (dog)
IRGM5 (dog)
IRGM4 (dog)
Irgc
IRGC (human)
IRGC (dog)
irgg1 (zebrafish)
irge1 (zebrafish)
irge5 (zebrafish)
irge3 (zebrafish)
irge4 (zebrafish)
irge2 (zebrafish)
irge6 (zebrafish)
irgf1 (zebrafish)
irgf3 (zebrafish)
irgf2 (zebrafish)
irgf4 (zebrafish)
irgq2 (zebrafish)
irgq1 (zebrafish)
irgq3 (zebrafish)
irgf7 (
Tetraodon)
irgf8 (Tetraodon
)

irgf6 (Fugu)
irgf5 (Fugu)
Irgq1
IRGQ1 (human)
H-Ras-1 (human)
Irga6
Irgb6
Irgd
IRGB12 (dog)
IRGB11 (dog)
IRGD (dog)
Irgm1
IRGM (a) (human)
IRGM6 (dog)
IRGM5 (dog)
IRGM4 (dog)
Irgc
IRGC (human)
IRGC (dog)
irgg1 (zebrafish)
irge1 (zebrafish)
irge5 (zebrafish)
irge3 (zebrafish)
irge4 (zebrafish)
irge2 (zebrafish)
irge6 (zebrafish)
irgf1 (zebrafish)
irgf3 (zebrafish)
irgf2 (zebrafish)
irgf4 (zebrafish)

irgq2 (zebrafish)
irgq1 (zebrafish)
irgq3 (zebrafish)
irgf7 (Tetraodon)
irgf8 ( Tetraodon)
irgf6 (Fugu)
irgf5 (Fugu)
Irgq1
IRGQ1 (human)
H-Ras-1 (human)
FGVDETSLQRLARDWEIEVDQVEAMIKSPAVF-KPTDEE
FGLDDASLENIAQDLNMSVDDFKVHLRFPHLF-AEHNDE
FGLDDQSIKEIAEKLGAPLADIKGELKCLDFWSLVKDN-
FGLDDASLETIAKDLNVSVEKLKANLTSPHLLSVEKEDE
FGLDDISLKTIAKDLNVSVEKLKANLMFPHLLSVEKYDE
FGLDEKSVKGIAEKLDMSVEEIKSFTKSLDFWLLVKDD-
FGVDDESVQQVAQSMGTVVMEYKDNMKSQNFYTLRREDW

FGVDDESLQQIAQSMGKPMEEYRAIMKSRDLHTIIRGDW
FGVDDKSLQQMAQSMGKPMEEYRAIMKSQDVHTVLTGDW
FGVDDDSLQEVAQSMGKPKEEYKAIMKSQDLHTALAWDW
FGLDDDSLAKLAEQVGKQAGDLRSVIRSPLAN-EVSPET
FGLDDDSLAKLAEQVGKQAGDLRSVIRSPLAN-EVSPET
FGLDDDSLAKLAEQVGKQAGDLRSVIRSPLAN-EVSPET

FGLSNQALQVLSERVNKPVEVLNAAKTSRFKD-GVTDRI
FGLSNQALEVLSGRVNKPVKVLKAAKTSRFKD-GITEHI
FGLDDGSLARLSEKINK PLVGHLAKSKIAS-AIQEK-
LGLNEKSLKQLSERTNKPVSLLKLAIKSPVSL-AVLDRM
FGLDEKSIDKLSVRVNN LSLKAIRRSPLVV-AIGQK-

FGLDEKSIDKLSVRVNN PSLKAIRRSPLVV-AIGQK-
FGLDDPSLQKLCERSGKTVEELKSLMKSPLHH-GINPSS
FGLDDPSLQMLCERSGKTIEEFKSLMKSPLRG-GINPAS
FGLDDQSLQKLCERSGKTIEELKSLMKSPLCY-GINTSL
FNLDDESLQRLCDVSGKSLEEIKSLMKSPLKA-GIGSYS
LCLDDESLQRLARQRGL-DPAKLKALRTCALSVEVSKSE


FGLDTPSLQRLADTTGVQLTDLTSVIRSPLSLDNINAQL
FGLGRPSLQRLADTTGVQLTDLTSVIRSPLGLNIIDAEL
FGLDGPSLQRLADSTGVPLEDLTSVVRSPLSLNTIDKAF
FGLGRPSLQRLVAITGVPLVDLT-IISSPLTLDNINTDL
LGLEPAAVARRERALGLAPGVLATRTRFPGPVTRAEVEA
LGLEPTALARRERALGLASGELAARAHFPGPVTRAEVEA

αG

C
αL
FLLDSDLETLKKSMKFYRTV
MISD-ILENLDETFNLYRSY
FFKGFDLPEQEQCLKDYRSY
LINDNEVEKLEETLHLYRSY
YISDNDVETLKDTLTLYRSY
CFNGFDFPQQEKCLNLYQSH
SLGVRDDDNMGECLKVYRLI

TLGIWNADDLGECLIAYHLF
ILGIQDEDDLGQCLIAYHLF
ILGIQNANDLGEFLNAYHRL

LAAAYDDALLIRSLRGYHRS
LAAAYDDALLIHSLRGYHRS
LAAAYDDALLIRSLRGYHRS

VSLAVDYGIMKKFFKQVFMA
VSLAVEYVIMKKFFKQVFMA
LSAACDTGMVALFLTRCYFA
ISLITDKAILIVYLIGCHYA
LSMACDAAILLGFFTKCYYA
LSMACDAAILLGFFIKCYYA
LSISADIAIIAEELRKYYSA
LSVAVDLVIVKREIEIYYST
LSISVNVDIIAEELTKYYSE

LSVAVDVMIIKEETEKYFRG
VSSMVDATVGVRILVKAQIS


LSVAVDLSLIVGLVQQYKTS
LSVAVDLSLIAGLVQQYKTG
LSVAVDGALIAGVVQQYKTG
LSAAVDADLIAGVVQQYKTG
LGWACDVALLRGQLAEWRRA
LGWACDVALLRGQLAEWRRG


413
415
420
440

441
419
409
181
377
397
397
463
463
433
250
410
408
386
384
410
422
398
398
387
402
379
247
161
417
394
460
456
407
447

189
αΙ
αΗ
R92.12 Genome Biology 2005, Volume 6, Issue 11, Article R92 Bekpen et al. />Genome Biology 2005, 6:R92
because here the resistance mechanism itself has apparently
been lost during primate evolution.
It will be of interest and of considerable importance to ana-
lyze the different strategies by which humans, dog and mouse
deploy resistance mechanisms effective against vacuolar
pathogens. None of the known mechanisms active in humans
against vacuolar pathogens, namely nitric oxide and oxygen
radicals [30-32], tryptophan depletion [33,34], accelerated
acidification by Rab5a [35], cation depletion [36-38] or
autophagy [39,40], is missing from the mouse. Nevertheless,
it remains possible that the distinctive resistance actions of
the p47 GTPases [7,8] are performed by an unrelated and
thus far unidentified molecular machine in primates.
The loss of a highly evolved and complex resistance system
that is active against vacuolar pathogens needs an adaptive
explanation. The evolution of a successful avoidance strategy
by the pathogens is unlikely, because many different patho-
gens and pathogen classes are controlled by IRG proteins in
the mouse [2,4]. Nevertheless very recent evidence suggests
that Chlamydia spp. divergence between humans and mouse
may indeed be partially driven by differences in the deploy-
ment of p47 GTPases, in this case Irga6 (IIGP1) [41]. Human
Synteny relationships between the human and mouse IRG genes (a) Synteny between mouse chromosome 7 and human chromosome 19 in the region of the IRGC and IRGQ genesFigure 7
Synteny relationships between the human and mouse IRG genes (a) Synteny between mouse chromosome 7 and human chromosome 19 in the region of
the IRGC and IRGQ genes. The figures indicate distances from the centromere in megabases. The locations of three further syntenic markers are given.
Gene orientation is given by black arrows. (b) Complex synteny relationship between human chromosome 5 and mouse chromosomes 11 and 18 in the

regions containing the mouse Irg genes. Figures indicate distances from the centromere in megabases. The locations of IRG genes are shown in the yellow
panels. Positions of diagnostic syntenic markers are also indicated. Syntenic blocks are given in full color, and the rest is shaded.

Mouse chromosome 7

49.0 48.9 48.8 48.7
Kcnn4 Plaur Xrcc1
13.1 13.2 13.3 13.4

Irgc

CINEMA

Irgq

FKSG27

IRGC

CINEMA

IRGQ

FKSG27
170.559
Mb
Mb
Kb
Kb
271.086

789.088914.746
(a)
(b)
130 131 132 133 147 148 149 150 151 152 153 154 155 177 178 179 180 181
245 244




Mouse chromosome 18
63 62 61 6064
58 57 56 55 54 53 52 51 50 49 48 4759
246
135134
Irgm1 LRG47
Irgb8
Irgb9
Irgb1
Irgb2
Irgb3
Irgb4
Irgb5
Irgb6 TGTP
Irgb7
Irgd IRG47
Irga1
Irga2
Irga3
Irga4
Irga5

Irga7
Irga6 IIGP
Irga8
Mb
Irgb10
Irgm3

IGTP
Irgm2

GTPI
////
IRGM
Human chromosome 19
Mouse chromosome 11
Human chromosome 1
Human chromosome 5
Genome Biology 2005, Volume 6, Issue 11, Article R92 Bekpen et al. R92.13
comment reviews reports refereed researchdeposited research interactions information
Genome Biology 2005, 6:R92
Chlamydia trachomatis is controlled by IIGP1 in interferon-
treated mouse oviduct epithelial cells, whereas control of the
extremely closely related mouse C. muridarum is independ-
ent of interferon. However, a more plausible general model is
the evolution in the primate lineage of improvements to the
battery of parallel mechanisms, rendering the IRG system
redundant. Interestingly, in this context it was noted in the
Chlamydia study quoted above that interferon-stimulated
mouse oviduct epithelial cells did not express the important
resistance factor indoleamine deoxygenase, which is respon-

sible for tryptophan depletion, whereas this is well expressed
in interferon-stimulated human HeLa cells [41]. In general,
pathogen resistance mechanisms also carry costs, for exam-
ple autoimmunity and allergy, arising from the adaptive
immune system and many others arising from innate immu-
nity [42-46]. Indeed, the interferon-inducible dynamin-like
GTPases, the Mx proteins, which confer on mice strong resist-
ance to certain RNA viruses and have been considered func-
tionally related to the p47 GTPases [4,47-49], exist in a
balanced polymorphism in the wild over null alleles [50,51],
and have been lost spontaneously from all except two labora-
tory mouse strains [52]. It is not yet obvious what specific
costs might be associated with possession of the Mx or IRG
resistance systems.
The IRG proteins are well represented in the bony fish but,
although they are abundant and diverse in the zebrafish, in
Fugu there are only two very closely linked and similar genes
that are, indeed, annotated as a single tandem gene in
ENSEMBL (although we judge this not to be the case). Thus,
the available annotated fish genomes seem to mirror the IRG
situation in the mammals, with Fugu and Tetraodon reflect-
ing the reduced human case and Danio the complex dog and
mouse. However, it has not yet been reported whether any
Structure and expression of the human IRGM geneFigure 8
Structure and expression of the human IRGM gene. (a) (left panels) RT-PCR analysis of expression of IRGM in HeLa and GS293 cells. The b and c splice
variants were amplified simultaneously by the same primer pair (IRGMs1-rGMS). A different downstream primer (IRGMs1-r1) internal to all the 3' splice
forms was used to show differences in the overall expression level of IRGM in the two cell lines. No RT' indicates that no reverse transcriptase is included
in cDNA preparation. The band immediately below the IRGMc band in GS293 cell material, indicated with an asterisk, is a nonspecific band amplified only
in this cell line. The band was sequenced and is unrelated to IRGM. (right panel) Analysis of IRGM expression in human brain, liver and testis by RT-PCR.
GAPDH was used as a control. (b) Five splice forms of the IRGM gene have been identified, as indicated: IRGM(a)-IRGM(e). The promoter and 5'-

untranslated regions of the gene are associated with an ERV9 retroviral LTR. Scale-bar is given in base pairs.
IRGM(a)
385
385
385
30,529
30,529 20,255
17,161
Start Codon Stop Codon
ORF
Intron
385
1,402
20,255
29,250
100
3,436
385
1,402
17,161
29,250
3,436
IRGM(c)
IRGM(d)
IRGM(e)
U5 region
(ERV9)
RP
U3 region
(ERV9)

Promoter Region
IRGM(b)
Exon
HeLa
GS293
+-
+-
1
kb
Marker
+-
+-
500
IFN
- γ
*
Mc
Mb
IRGMc
IRGMb
GAPDH
27 Cycles
495 bp
IRGMc
IRGMb
38 Cycles
504 bp
641 bp
Live r
Brai n

T
estis
no
DNA
no RT
GS293
no DNA
HeLa
(a)
(b)
R92.14 Genome Biology 2005, Volume 6, Issue 11, Article R92 Bekpen et al. />Genome Biology 2005, 6:R92
fish IRG genes respond to infection [53]. Both Danio and the
pufferfish are Actinopterygian fish, in which where there is
increasing agreement that the genome has been amplified by
three rounds of duplication [54,55]. It is plausible that the
complex IRG representation in Danio may be attributed to
preservation of these genes on more than one of the potential
eight paralogons, whereas only a single copy carries IRG
genes in the pufferfish. Further clarification of this issue
awaits the completion of the genomes.
The phylogenetic origin of the IRG proteins is obscure.
Because the family is conserved at least down to the bony
fishes with little structural modification, the IRG genes are
not strictly fast-evolving and their basic conservatism makes
them easy to identify. Thus, their apparent absence from most
known invertebrate genomes is probably real. Although many
components of the adaptive immune system appear to have
evolved close to the chordate-vertebrate boundary [56], this
is not generally the case for innate immune mechanisms
[57,58] There seems to be no reason in principle why the IRG

system should not work in invertebrates because it is cell-
autonomous. However, the putative GTPase sequences that
we have recovered from C. elegans and from Cyanobacteria
are too distantly related outside the G-domain for a clear phy-
logenetic relationship to IRG proteins to be established from
sequence similarity alone, whereas the similarities within the
G-domains, although occasionally striking, are to some extent
forced by the maintenance of a highly conserved function,
namely regulated GTP hydrolysis. A stronger case for a
meaningful phylogenetic relationship between these proteins
and the vertebrate IRG proteins would follow from structural
evidence that they display the distinctive IRG fold exempli-
fied by mouse Irga6 (IIGP1) and from a detailed analysis of
their catalytic mechanism.
The basic unit of IRG protein function may be a dimer
because several genes we have identified occur in pairs in a
head-to-tail arrangement, are expressed as tandem tran-
scripts, and are presumably expressed as dimeric proteins.
This conclusion is consistent with the dimer of IIGP1 (Irga6)
observed in the crystal, shown by site-directed mutagenesis of
the dimer interface to be essential for GTP-dependent oli-
gomerization and cooperative hydrolysis [12]. However, a
second dimer interface is also required for oligomerization
(unpublished data), and which of the two dimer structures
the constitutive IRG dimers represent is of considerable
interest. Unlike the observed homodimer of IIGP1, the prod-
ucts of the two putative tandem genes in the mouse Irgb2/b1
and Irgb5/b4 are heterodimers, implying that the two IRG
subunits serve distinct functions in the protein. The same
may be true for the tandem pair of irg genes of Fugu, which

are annotated as a single tandem gene in ENSEMBL. These
latter genes have diverged very recently, because with three
exceptions they are identical at the nucleotide level over the
first 290 amino acids. However, they have diverged substan-
tially in the carboxyl-terminal region (Figure 6), suggesting a
recent selective force. If the two tandem IRG domains are
indeed expressed as a tandem protein (as favored by the
ENSEMBL annotation), then it will be as a heterodimer with
significant sequence variation at the carboxyl-terminus. The
extreme case of heterodimer differentiation in IRG tandem
genes may occur in Danio, in which gene irgg, a canonical
(although truncated) IRG gene, is apparently expressed in
tandem with the adjacent downstream gene irgq1, which is a
modified (and also truncated) quasi-GTPase gene that is
unlikely to function as a GTPase (Additional data file 7). In
this case the role of the irgq1 domain may be regulatory for
the amino-terminal irgg domain. Other IRGQ proteins may
also be regulators of IRG proteins, interacting with the func-
tional IRG proteins with a symmetry resembling one or other
of the two dimer structures. Thus IRGQ proteins would coe-
volve with IRG proteins. This would explain why the three
irgq genes of the zebrafish have no homologs in the puffer-
fishes, with their single tandem pair of irg proteins, and
recalls the recent observation that the GAP protein of the
small GTPase, Rap1, is itself probably derived from a GTPase
ancestor, retaining the G-domain structure but not the
sequence to reveal its origin [59].
Better understanding of the mechanism of action and regula-
tion of the p47 GTPases is needed before their complex evolu-
tionary history can be put in context. From the evidence we

present here, however, it is already clear that effective resist-
ance to vacuolar pathogens in humans and mouse must be
organized on radically different principles.
Nomenclature
We introduce here a general nomenclature on phylogenetic
principles for the p47 GTPases, based on the stem name IRG
(immunity-related GTPases). This stem was favored over
other possibilities because the name IRG-47 has priority in
the literature as the first description of a p47 GTPase [60].
The phylogenetic basis of the nomenclature is apparent from
Figure 5; each deep monophyletic clade is identified by a sin-
gle-letter suffix as IRGM or IRGC. The nomenclature pro-
posed here in Figure 5 and in Additional data file 1 has been
accepted by the gene nomenclature committees of human and
mouse, and by the zebrafish sequencing project. We have
tried throughout to use the different forms of gene name
accepted by the nomenclature committees for mouse (Irg)
human (IRG), dog (IRG) and zebrafish (irg). The nomencla-
ture of the IRGQ genes departs from the phylogenetic princi-
ple. The IRGQ nomenclature simultaneously recognizes the
affinity of these sequences to IRG genes and stresses their
anomalous GTP-binding domain features. It is, however,
highly unlikely that the IRGQ sequences of humans, mouse,
and fish represent a monophyletic group. It is more likely that
the IRGQ genes of each taxonomic group derive from IRG
genes of other clades of that group. This pattern is already
apparent by inspection of the irgq protein sequences of the
zebrafish in Figure 6, but it cannot be discerned from the G-
Genome Biology 2005, Volume 6, Issue 11, Article R92 Bekpen et al. R92.15
comment reviews reports refereed researchdeposited research interactions information

Genome Biology 2005, 6:R92
domain-based phylogeny shown in Figure 5 because of the
specific divergence of the G-domains of the irgq proteins.
Materials and methods
Use of database resources
All available public databases were extensively screened by
BLAST and related searches for sequences belonging to the
IRG family. In the case of the mouse, transcript sequences
derived from the C57BL/6 strain were given preference over
sequences of other and undefined strain origin, and com-
pared in all cases with genomic sequence available via the
ENSEMBL (v28.33d.1, February 2005) array of websites [61].
A systematic study of polymorphism has not yet been com-
pleted, but it is already clear that nearly all IRG sequences
derived from the CZECHII cDNA libraries (Mus musculus
musculus) differ from C57BL/6 sequences. These differences
make allocation of many CZECHII sequences to individual
clade members of the C57BL/6 mouse problematic.
Identification of certain Irg sequences with recognized gene
symbols was achieved through the Mouse Genome Initiative
web resources [62]. Where ambiguities persist in the mouse
genomic map, especially on chromosome 18 in the region of
IrgA6-IrgA8 (Mb60.878-60.958), and on chromosome 11 in
the region from PA28
βψ
to IrgB7
ψ
(Mb57.570-57.700), we
used primary BAC and cosmid sequences to reach a consen-
sus view.

Human and dog IRG sequences were identified from the
available public databases (ENSEMBL, National Center for
Biotechnology Information) and confirmed wherever
possible by multiple sequence comparisons at transcriptional
and genomic levels. Fugu material was obtained and analyzed
through [63-65]. Tetraodon sequence was initially assembled
from the GSS sequence database at National Center for Bio-
technology Information and subsequently from the
University of California at Santa Cruz compiled genome data-
base [66] via the BLAST server. Zebrafish sequence was
obtained from zebrafish genome resources at the Sanger Cen-
tre [67] and analyzed in an Acedb database using the Spandit
annotation tool.
Chromosomal locations and synteny analysis of mouse and
human chromosomes was initiated through ENSEMBL [68].
Further details were obtained through the Sanger Centre
[69]. Nucleotide sequences and translated open reading
frames of IRG family members used in this paper are given in
Additional data files 9 and 10, and can also be accessed at the
IRG family database at our laboratory [14].
Phylogeny and alignment protocols
Routine sequence analysis and local sequence database man-
agement was handled using DNA-Strider 1.3f12, Vector-Nti,
and MacVector 7.2. The identity and similarity matrix on pro-
tein and nucleotide sequences (Additional data file 2) are
based on GeneDoc (version number 2.6.002). Phylogenetic
analysis was conducted using the neighbor-joining method
[70], as implemented in the MEGA2 program [71]. We used
p-distances for constructing the phylogenetic trees. Reliabil-
ity of the neighbor-joining trees was examined using the boot-

strap test [72].
Alignments were performed via the BCM multiple alignment
programme suite [73] and EBI-ClustalW [74] using the
default options and manipulated according to the crystal
structure of IIGP1 [12]. Shading of alignments was performed
using Boxshade [75] and additional sequences were shaded
manually according to the default options of Boxshade.
Identification of transcription factor binding sites
Promoter regions (2 kb upstream of putative transcription
start point) were screened for putative transcription factor
binding sites with the Transcription Element Search System
[76,77], and the results were further analysed and confirmed
manually. Additional promoter analysis of Irgc (mouse Cin-
ema) and IRGC (human CINEMA) was performed with Con-
Site [78] based on phylogenetic footprinting [79].
RT-PCR on cells and tissues
C57BL/6J mice were obtained from the animal house at the
Institute for Genetics, University of Cologne. Listeria mono-
cytogenes infection was performed as described previously
[13]. Twenty-four hours after infection, the mice were killed,
and liver, lung and spleen were removed and snap frozen in
liquid nitrogen. Mouse L929 fibroblasts were stimulated for
24 hours with 200 U/ml interferon-γ or 200 U/ml interferon-
β (R&D System GmBH, Weisbaden-Nordenstadt, Germany
and Calbiochem-Novabiochem Corparation La Jolla, CA,
respectively). Human cell lines (Hela, GS293 (GeneSwitch™
-293, Invitrogen GmbH Karlsruhe, Germany), HepG2, T2,
THP1, MCF-7, SW-480, Primary foreskin fibroblast-HS27)
were stimulated for 24 hours with 2,000 U/ml interferon-β or
200 U/ml interferon-γ (PBL Biomedical Laboratories, NY,

USA and Peprotech/Cell concepts GmbH UmKirsh, Ger-
many, respectively). Total RNA was extracted from tissues
and cells using the RNAeasy mini kit (QIAGEN, Hilden, Ger-
many), except for testis, for which the RNAeasy Lipid Tissue
Kit (QIAGEN) was used. Poly(A) RNA was isolated from total
RNA using the Oligotex mRNA kit (QIAGEN). Total RNA
from human tissues was purchased from Biochain (Hayward,
CA, USA). cDNA was generated from mRNA and total RNA
using the Super Script First-Strand Synthesis System for RT-
PCR (Invitrogen, Carlsbad, CA, USA). The generated cDNAs
were screened for the presence of p47 (IRG) GTPase tran-
scripts by PCR. A list of the primers used is given in Addi-
tional data file 8. The amplified fragments were confirmed by
sequencing.
Additional data files
The following additional data are included with the online
version of this paper: A list of all IRG gene family members
R92.16 Genome Biology 2005, Volume 6, Issue 11, Article R92 Bekpen et al. />Genome Biology 2005, 6:R92
described in this paper (gives names, synonyms, accession
numbers and further information for each IRG gene; Addi-
tional data file 1); nucleotide and amino acid identities based
on G-domain of mouse Irg family (gives percentage of identity
on both protein and nucleotide level within the mouse Irg
family; Additional data file 2); ISRE and GAS elements of
mouse IRG family genes (contains the positions and exact
sequences of all ISRE and GAS elements found in putative
promoters of mouse IRG genes; Additional data file 3); induc-
ibility of Dog p47 (IRG) GTPases (shows interferon inducibil-
ity of members of the p47 (IRG) GTPases present in the dog;
Additional data file 4); genomic organization of Danio rerio

p47 (irg) GTPases (illustrates the genomic organization of all
p47 (irg) GTPases found in zebrafish to date; Additional data
file 5); protein similarity matrix of Irgc and Irgq (contains
comparison between the mouse p47 GTPase Irgc and the long
coding exon of the closely linked quasi-GTPase Irgq
(FKSG27); Additional data file 6); divergent nucleotide-bind-
ing motifs in quasi-GTPases (compares the nucleotide
binding motifs of quasi-GTPases to those of the classical
mouse p47 GTPases; Additional data file 7); a list of the prim-
ers used (contains the sequences of all primers used in this
study; Additional data file 8); nucleotide sequences of all IRG
family members (Additional data file 9); protein sequences of
all IRG family members (Additional data file 10).
Additional data file 1A list of all IRG gene family members described in this paperA list of all IRG gene family members described in this paper (gives names, synonyms, accession numbers and further information for each IRG gene)Click here for fileAdditional data file 2Nucleotide and amino acid identities based on G-domain of mouse Irg familyNucleotide and amino acid identities based on G-domain of mouse Irg family (gives percentage of identity on both protein and nucle-otide level within the mouse IRG family)Click here for fileAdditional data file 3ISRE and GAS elements of mouse IRG family genesISRE and GAS elements of mouse IRG family genes (contains the positions and exact sequences of all ISRE and GAS elements found in putative promoters of mouse IRG genes)Click here for fileAdditional data file 4Inducibility of Dog p47 (IRG) GTPasesInducibility of Dog p47 (IRG) GTPases (shows interferon inducibil-ity of members of the p47 (IRG) GTPases present in the dog)Click here for fileAdditional data file 5Genomic organization of Danio rerio p47 (IRG) GTPasesGenomic organization of Danio rerio p47 (IRG) GTPases (illus-trates the genomic organization of all p47 (IRG) GTPases found in zebrafish to date)Click here for fileAdditional data file 6Protein similarity matrix of Irgc and IrgqProtein similarity matrix of Irgc and Irgq (contains comparison between the mouse p47 GTPase Irgc and the long coding exon of the closely linked quasi-GTPase Irgq (FKSG27)Click here for fileAdditional data file 7Divergent nucleotide-binding motifs in quasi-GTPasesDivergent nucleotide-binding motifs in quasi-GTPases (compares the nucleotide binding motifs of quasi-GTPases to those of the clas-sical mouse p47 GTPases)Click here for fileAdditional data file 8A list of the primers usedA list of the primers used (contains the sequences of all primers used in this study)Click here for fileAdditional data file 9Nucleotide sequences of all IRG family membersNucleotide sequences of all IRG family membersClick here for fileAdditional data file 10Protein sequences of all IRG family membersProtein sequences of all IRG family membersClick here for file
Acknowledgements
We are greatly indebted to Lois Maltais of the Mouse Genome Database at
The Jackson Laboratory; Ruth Lovering, Gene Nomenclature Advisor,
HUGO Gene Nomenclature Committee (HGNC); and Yvonne Edwards of
the Fugu Genomics Project at the UK Human Genome Mapping Project
(HGMP) Resource Centre for their time and effort in developing a useful
nomenclature for the p47 GTPases. We are grateful to Kerstin Jekosch,
Informatics & Systems Groups, Sanger Centre, for help with analyzing and
annotating the zebrafish genes; to Cornelia Stein, Institute for Genetics,
Cologne for communicating unpublished zebrafish material; and to Natasa
Papic, Institute for Genetics, Cologne for assistance in editing the long
sequence alignments. This study was supported by the Centre for
Molecular Medicine, Cologne, and DFG grants SPP1110, SFB243 and
SFB635. Iana Parvanova was supported by the DFG Graduate College
'Genetics of Cellular Systems'; and Cemalettin Bekpen and Julia Hunn were
supported by the Cologne Graduate School in Genetics and Functional

Genomics. We are particularly grateful to the anonymous referee who
drew our attention to the candidate p47 GTPase sequence C46E1.3 in C.
elegans.
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