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Adam et al. BMC Genomics 2010, 11:424
http://www.biomedcentral.com/1471-2164/11/424

Open Access

RESEARCH ARTICLE

The Giardia lamblia vsp gene repertoire:
characteristics, genomic organization, and
evolution
Research article

Rodney D Adam*1, Anuranjini Nigam2, Vishwas Seshadri2, Craig A Martens3, Gregory A Farneth3, Hilary G Morrison4,
Theodore E Nash5, Stephen F Porcella3 and Rima Patel6

Abstract
Background: Giardia lamblia trophozoites colonize the intestines of susceptible mammals and cause diarrhea, which
can be prolonged despite an intestinal immune response. The variable expression of the variant-specific surface
protein (VSP) genes may contribute to this prolonged infection. Only one is expressed at a time, and switching
expression from one gene to another occurs by an epigenetic mechanism.
Results: The WB Giardia isolate has been sequenced at 10× coverage and assembled into 306 contigs as large as 870
kb in size. We have used this assembly to evaluate the genomic organization and evolution of the vsp repertoire. We
have identified 228 complete and 75 partial vsp gene sequences for an estimated repertoire of 270 to 303, making up
about 4% of the genome. The vsp gene diversity includes 30 genes containing tandem repeats, and 14 vsp pairs of
identical genes present in either head to head or tail to tail configurations (designated as inverted pairs), where the two
genes are separated by 2 to 4 kb of non-coding DNA. Interestingly, over half the total vsp repertoire is present in the
form of linear gene arrays that can contain up to 10 vsp gene members. Lastly, evidence for recombination within and
across minor clades of vsp genes is provided.
Conclusions: The data we present here is the first comprehensive analysis of the vsp gene family from the Genotype
A1 WB isolate with an emphasis on vsp characterization, function, evolution and contributions to pathogenesis of this
important pathogen.
Background
Giardia lamblia (syn. G. duodenalis, G. intestinalis) is an
anaerobic protist that is medically important as a common cause of intestinal infection and diarrhea [1].
Humans and other susceptible mammals become
infected when cysts are ingested from contaminated
water or food and excyst into trophozoites in the proximal small intestine. These trophozoites replicate and
cause the symptoms of diarrhea. Infections with Giardia
are frequently prolonged and malabsorption with weight
loss may last for months in the absence of treatment,
despite an immune response that would be expected to
eradicate the infection. One of the possible reasons for
* Correspondence: adamr@u.arizona.edu
1

Departments of Medicine and Immunobiology, University of Arizona College
of Medicine, Tucson, AZ, USA

the persistence of infection is antigenic variation of the
variant-specific surface proteins (VSPs).
A single trophozoite expresses only a single member of
this protein family at any one time [2], but may switch
expression from one VSP to another in vitro at a rate
which has been estimated at once every 6 to 13 generations [3]. Antigenic variation has also been identified in
gerbils [4], mice [5,6], and humans [7], and occurs during
the process of encystation/excystation [8,9]. Antigenic
variation occurs in the absence of alteration of DNA
sequence or chromosomal location of the vsp genes [1012], and likely occurs by epigenetic mechanisms involving
histone acetylation status [12] and/or RNAi [13].
The VSP proteins are all cysteine-rich and have frequent CXXC motifs [1]. They have a 14 to 17 amino acid
N-terminal signal peptide which is predicted to direct the
protein to the trophozoite surface, upon which it is

Full list of author information is available at the end of the article
© 2010 Adam 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.

Adam et al. BMC Genomics 2010, 11:424
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cleaved from the remainder of the peptide [14,15]. Most
of the mature VSP protein diffusely coats the outside of
the trophozoite [16].
The C-terminus concludes with a nearly invariant
CRGKA motif that most likely remains in the cytoplasm
[17]. Immediately adjacent to the CRGKA motif is a
hydrophobic domain that likely forms the membrane
anchor for the VSP [17,18]. The entire C-terminal conserved region is about 38 amino acids in length, but the
upstream portions of the VSPs can be highly different,
allowing the organism to expose very different antigens to
the host.
Despite the similarities in the N-terminal signal peptide, the CXXC motifs, and the conserved C-terminus,
other features of the vsp genes vary substantially, such as
the presence or absence of tandem repeats. VspA6 is
about 5.6 kb in length, with the initial 99 bp followed by
23+ copies of a 195 bp tandem repeat, then about 1.3 kb
of additional sequence that ends with the highly conserved 3' end [10]. Tandem repeats have also been
reported in other vsps [11,19-21]. The vspC5 gene is over
2 kb in length and begins with 66 bp at the 5' end followed
by over 20 copies of a 105 bp tandem repeat that
extended to the conserved 3' end [11]. Thus, almost the
entire portion of this molecule that is assumed to be
exposed to the extracellular environment consists of tandem repeats.
One vsp gene has been described that is encoded as a
duplicated pair. The two copies of the vsp1267 ORF exist
as an inverted pair in a tail to tail orientation with approximately 3 kb of intervening DNA [17].
Previous studies have shown that vsp genes fall into
related groups or families. One example is vspA6 and the
related genes, vspA6-S1 and A6-S2; all sequenced from
the WB isolate [22]. VspA6-S2 differs from vspA6 by its
possession of a 201 bp tandem repeat that is similar to the
195 bp repeat of vspA6. VspA6-S1 is nearly identical to
vspA6 with only scattered substitutions outside the
repeat region, but has only a bit more than one copy of
the 195 bp tandem repeat, rather than the approximately
23 copies found in vspA6. Interestingly, vspA6-S1 has also
been sequenced from a Genotype A1 Peruvian isolate
called G3 M [23], and both sequences were identical, suggesting that across Genotype A1 isolates, some vsp genes
may not be changing rapidly.
An additional example of vsp genes from Genotype A1
isolates falling within related families includes several
genes that are similar to TSA417 [24], including tsp11
[25-27], such that homology is apparent throughout the
entire sequence. In addition, there are vsps with near
identity in the upstream non-coding region followed by
substantial divergence within the coding region [28].
A genomic evaluation of the vsp genes has been made
possible by the sequencing of the genome of the WB iso-

Page 2 of 14

late [29] 10× coverage (greater than 98% coverage) [30].
The G. lamblia isolates obtained from humans were originally divided into three groups; 1, 2, and 3. These groups
are now designated as Genotypes/assemblages A1, A2,
and B, respectively. Genotype A1 is a highly homogeneous group and is about 98-99% identical to Genotype
A2. Relatively little sequence data is available for Genotype A2. Genotype B is only about 80-90% identical to
Genotype A1 and has been proposed as a separate species [31-33]. Although the genome of the Genotype B isolate, GS, has been reported, the coverage was not
sufficient to fully describe the vsp repertoire. Therefore,
the current manuscript will focus on the Genotype A1
vsp repertoire from the WB genome, which was completed at 10× coverage and assembled into 306 contigs,
the largest of which is nearly 1 Mb in size [30].

Results and Discussion
Identification of vsp genes

All the vsp genes are cysteine-rich with frequent CXXCs
motifs present throughout their length [34]. However, the
Giardia genome also encodes other cysteine-rich proteins including three cyst-wall protein (CWP) genes [35]
and 61 High Cysteine Membrane Proteins (HCMPs) [36].
The HCMPs also have CXXC motifs, but in addition, they
have frequent CXC motifs, which are distinctly uncommon in the VSPs. Most notably, the vsp genes differ from
these other genes encoding cysteine-rich proteins by
their possession of a highly conserved 3' end that concludes with the translated amino sequence, CRGKA. For
vsps that were complete at the 3' end, a great majority
contained the encoded CRGKA motif in its entirety,
while a few genes matched at three or four residues.
Using these criteria, we identified a total of 303 vsp
genes in the current WB assembly, of which 228 were
complete and 75 were in partial or incomplete ORF format. The 228 complete genes include 10 pairs of identical
coding inverted gene pairs, so many of the analyses of
complete genes included 218 sequences. (A complete list
of the vsp genes along with their features is shown in
Additional file 1). Of the 75 partial genes that were considered in some of the analyses, 32 were incomplete at the
3' end and 43 were incomplete at the 5' end. Since the
genome coverage is >98%, it is unlikely that any vsp genes
were missed completely, although it is possible that some
were excluded from the assembly because of the potential
for complete or near 100% sequence identity to other vsp
genes. The total number of 303 vsp genes is probably a
high estimate for the total genomic repertoire since it is
likely that some of the genes that are incomplete at the 5'
end should or could have been paired with genes that are
incomplete at the 3' end. Therefore, for the purposes of
discussion, we have estimated the total number of vsp
genes to be 228 + 43 or 271 total. These estimates are

Adam et al. BMC Genomics 2010, 11:424
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consistent with earlier range estimates of 150 to 300 that
were based on DNA hybridization studies [3].
The 303 genes have been numbered sequentially, based
upon their contig number and location. The contigs were
numbered sequentially by decreasing size. Therefore, vsp
genes whose names include large numbers indicate that
these vsps are found on smaller contigs. Those vsps that
have a genomic context as duplicated, inverted pairs and
that therefore have identical coding regions, are each
given the same numerical designation, but these names
are followed by ".1" or ".2" sub-designation to highlight
this difference.
The vsp coding regions comprise approximately 3% of
the total genome. The inclusion of the upstream intergenic areas, which may be tasked with control of vsp gene
expression, increased the value to greater than 4%. Thus,
the vsp genes comprise at least 3-4% of the total Giardia
genome, which is consistent with estimates from a previous Giardia genome sampling survey [37].
The presence of a candidate Inr

All of the vsp genes previously described in the literature
have had the following DNA consensus sequence, PyAatgTT, at the beginning of the coding region, where atg represents the initiation codon. We have shown in transient
transfection studies that this consensus sequence is
required for efficient expression of luciferase from a vsp
promoter region (unpublished). Therefore, this consensus region surrounding the start codon is a candidate Initiator Element (Inr). We analyzed the vsp gene sequences
for the presence of the PyAatgTT sequence and found
that fewer than half of the genes contained this Inr
sequence. In other words, 107, or 41% of the 260 vsp
genes that were complete at the 5' end contained the
PyAatgTT sequence. Specifically, 65 vsp genes contained
putative Inrs of "CAatgTT", while 42 vsp genes contained
the sequence "TAatgTT". As described above and below,
many of the vsp genes were found in linear arrays in the
genome. Of the 25 vsp genes that represented the first vsp
gene within the linear arrays, 12 contained the putative
Inr. In surprising contrast, none of the downstream members of the linear arrays contained this sequence. In total,
107 (72%) of the 148 complete vsp genes that were not
present as downstream members of linear arrays contained the conserved Inr sequence. For those vsps containing the putative Inr, the Inr was usually (74%) the first
observed or most 5' oriented ATG start codon of the
gene. None of the subsequent downstream, in-frame
encoded ATGs contained a complete PyAatgTT
sequence. Further investigation and analysis will be
required to determine the role the Inr plays in vsp expression across the multitude of vsp variety and whether the
presence or absence of the Inr plays a role in in vivo vsp
expression.

Page 3 of 14

vsp genes without an ATG start codon may represent
pseudogenes

Three of the 218 vsp genes with complete sequences
available had no in frame ATG that could be found. These
three genes were identified by their possession of the conserved 3' sequence concluding with an encoded CRGKA
and were all found in the middle of large contigs. They
were relatively short vsp-encoding sequences with base
pair encoding regions of 224, 329, and 902 bases following the upstream stop codon. There were no obvious differences between these three genes and those containing
an ATG. Because of their internal contig location and the
absence of any mis-assembly evidence, we believe these
three genes may represent inactive, truncated vsp
pseudogenes.
Vsp size range, CXXC motifs, tandem encoded repeats and
recombination analysis of vsps

Vsp size range and CXXC motifs were analyzed within
the 218 unique vsps. The data demonstrated a wide range
in vsp size, from 222 to 6777 base encoding pairs. CXXC
motif analysis found an average of 12.6 encoded CXXCs
motifs per kb (range 3.6 to 18.6 per kb). The vast range in
vsp size along with differing numbers of CXXC motifs
implies a diverse range of function or performance within
the vsp family. More work remains to be performed in
order to analyze the context of VSP protein structure,
function and/or surface expression in the context of vsp
gene size and the presence of high or low numbers of
CXXC motifs.
Tandem repeats (TR) are a feature of some VSPs and
have been shown to be immunodominant in the case of
the 195 bp repeat domain found in VspA6 [19]. Thus, the
number and type of repeats encoded in vsps are likely to
play an important role in modulating, directing or avoiding the host immune response.
We identified a total of 30 complete vsp genes containing TR (Fig 1). The sequences of the TR were different
and unique for each of the 30 genes identified (see the
phylogenetic alignment of the tandem repeats in Additional file 2). The nucleotide sequences of the individual
repeats ranged in size from 105 to 516 bp in length. The
number of copies of the TR ranged from two, to more
than 20 per vsp. It is important to note that the repeat
copy numbers should be viewed as approximate because
vsp genes with larger numbers of repeats may have been
artificially condensed by the genome assembly process. In
addition, there is evidence of allelic variability in repeat
copy number for at least two of the previously identified
repeat-containing vsp genes vspA6 and vspC5 [10,11,28].
vsp Conserved 3' domain

A mechanism by which expression status of multiple vsps
could be monitored simultaneously would be valuable in

Adam et al. BMC Genomics 2010, 11:424
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Page 4 of 14

A
Vsp67 (VspA6)
2

1

3

4

B
Vsp122
1

Vsp90 (VspC5)
1

2

4

2

4

.5kb

Figure 1 Examples of vsp genes with tandem repeats. Three examples of vsp genes with tandem repeats are shown. The ORFs are divided into 3 or 4 sections, numbered 1 through 4 on the diagrams. For
these 3 genes, the first section consists of 66 to 99 bp beginning with
the start codon and encoded leader peptide, and extending to the tandem repeats. The second section consists of multiple tandem repeats,
which for two of the vsp genes (B and C), extends up to the conserved
3' region. The third section (A only) is nonrepetitive DNA extending
from the tandem repeats to the conserved 3' region. (1) Vsp67 (also
CRP170, vspA6) has approximately 22 copies of a 195 bp tandem repeat [10]. (2) vsp122 has three copies of a 516 bp repeat, by far the largest repeat found. (3) vsp90 (vspC5) has approximately 25 copies of a
105 bp tandem repeat [11].

the continued study of this interesting gene family. The
conserved 3' end has enough similarity among vsps to
allow the development of "universal amplification primers". Therefore, we analyzed the conserved 117 nucleotides of the 3' terminus in the 254 vsp genes for which
this sequence was available to determine whether there
was sufficient diversity within the 3' end to allow unique
identification of most vsps. Among these 254 genes, there
were 190 distinct 3' sequences. These 190 sequences
encode 99 different amino acid sequences. Thus, the
great majority of vsp genes have unique 3' conserved
regions. The nucleotide alignments are shown in Additional file 3 and the amino acid alignments are shown in
Additional file 4.
DNA alignments and evidence for DNA recombination
between vsp genes

It was previously proposed that gene duplication followed
by divergence was one of the mechanisms by which the
current number and diversity of the vsp repertoire was
generated [22]. The sequence diversity of the DNA
sequences is illustrated by the phylogenetic tree shown in
Additional file 5). Since prior studies suggested recombination among vsps [28], we performed a systematic
search for evidence of horizontal movement of DNA
sequences in the form of recombination between vsp
genes. Recombination analysis of all 218 full length vsps
was not possible due to the wide range of sequence divergence across the entire gene family (data not shown).
However, a more focused approach by (1) utilizing the C-

terminus conserved region as an alignment reference
point for all vsps, and (2) gradually adding additional, longer upstream sequences allowed us to identify minor
clades of vsps for running alignments, generating trees,
and testing for evidence of recombination. We used the
Sawyers test in the Geneconv package of algorithms for
these minor clades analyses. For each incremental
increase of adjacent upstream 3' sequence, alignments
and trees produced greater diversity and longer branching patterns, which correlated with the increased diversity seen in the 5' coding regions of the vsps. Clades and
subgroups within clades were identified for various
lengths of upstream sequence, all anchored with the 3'
conserved region (data not shown). The first minor clade
analyzed for recombination involved a group of vsp genes
with similarity along the 234 bp comprising the 3' terminus. In the phylogenetic tree, these 12 vsp sequences
actually separated into two related minor groups or
branches (all within Clade II of the three major clades of
vsp genes; see Alignments and phylogenetic tree analysis
section and Fig 2). The percent polymorphism for these
12 genes as a group was 74.36% (Table 1). The two subgroups consisted of eight and four vsps genes with 64.52%
and 25.21% polymorphisms, respectively (Table 1).
Recombination analysis demonstrated significant recombination at Bonferroni corrected values of 0.0311 and
0.0071 respectively, within each of the two subgroups. No
significant recombination could be detected between the
group of eight and the group of four genes.
The longest sequence that we could find extending
upstream from the 3' terminus, with a sufficient number
of vsp genes containing related sequence was 657 bp,
found in 13 genes. These 13 vsp genes demonstrated
23.28% polymorphism and showed significant recombination between them (p = 0.0365) in this region.
We then asked if by shortening the upstream sequence
down from 657 bp, whether we could find minor clades
containing at least two subgroups of vsps that could then
be tested in a manner similar to what was attempted for
the first 234 base pair region studied (in other words,
recombination across sub-groups). Shortening the
sequence down to 546 bases allowed us to find seven vsp
genes that separated into two subgroups of three and four
members each. Here, a Bonferroni-corrected (BC) Pvalue for recombination was found within the clade of
seven sequences (0.0114), demonstrating evidence for
recombination across the two minor groups. Also, evidence of recombination was found within the individuals
of the three member clade (0.0002 BC-corrected) and
within the four member subgroup (0.0195). The Dualbrothers recombination algorithm confirmed the recombination data between these seven members and allowed
discovery of several high mutation frequency regions at
~25 bp and 250 bp (Additional file 6A). These high fre-

Page 5 of 14

Table 1: Recombination analysis
DNA polymorphism analysis
BP

GAPS

Sawyers test for recombination

GROUP

Number of
VSPs

POLYMORPH
ISMS (%)

SINGLE VAR
SITES

Pi-Value

Inner frags

Best inner p-val

Best inner
BC-val

Outer frags

Best outer p-val

Best outer
BC-val

All (1&2)

12

234

0

174 (74.36)

42

0.3403

26

0.0000

0.0007

0

N/A

N/A

clade1

8

234

0

151 (64.52)

85

0.2599

16

0.0000

0.0002

1

0.0027

0.0311

clade2

4

234

0

59 (25.21)

50

0.1467

4

0.0008

0.0073

4

0.0011

0.0071

longClade

13

657

0

153 (23.28)

17

0.0865

41

0.0000

0.0000

1

0.0365

0.3755

All (3 & 4)

7

546

0

143 (26.19)

14

0.1347

10

0.0000

0.0002

1

0.0013

0.0114

clade3

3

546

0

95 (17.4)

95

0.1160

2

0.0000

0.0002

2

0.0000

0.0002

clade4

4

546

0

110 (20.14)

72

0.1178

6

0.0048

0.0268

2

0.0195

0.1069

Adam et al. BMC Genomics 2010, 11:424
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Clade 1 contains vsp genes; 41, 180, 49, 48.1, 204, 31, 43, and 38.
Clade 2 contains vsp genes: 95, 18, 21 and 23
longClade contains vsp genes: 279, 235, 272, 111, 248, 223, 191, 83, 234, 271, 222, 216 and 137
Clade 3 contains vsp genes: 217, 139 and 138
Clade 4 contains vsp genes: 270, 261, 290 and 62
Clade 5 contains vsp genes: 150, 140, 153 and 151
Clade 6 contains vsp genes: 245, 82 and 210.

Adam et al. BMC Genomics 2010, 11:424
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Page 6 of 14

Alignments and phylogenetic tree analysis of 218 complete
VSP proteins and vsp DNA sequences

III
I

II

0.5

Figure 2 Unrooted phylogenetic tree of the translated amino
acid sequences of all 218 vsp genes with complete sequences. Full
length vsp proteins were aligned, the alignment manually corrected,
and an unrooted tree produced. Predominate clades are listed as I, II,
and III and colored Green, Blue and Red. Many of the 218 vsp names are
not shown on this tree due to confined space at the ends of the branch
points. Representative members of each clade are shown. The bar at
the bottom of the figure signifies branch length related to number of
substitutions per residue analyzed.

quency mutation regions could be bracketing DNA fragments and assisting in their horizontal movement, while
keeping the intervening sequence intact. A 552 base pair
fragment was also discovered for seven other vsp genes,
and in this analysis, BC values of .0001 for the seven
members again demonstrated recombination across two
minor subgroups. In addition, a 0.0000 value within the
four member group demonstrated recombination within
this four member subgroup. Within the three member
subgroup, no recombination could be detected, probably
due to the low amount of sequence polymorphism
observed across these three members (7.24%). Dualbrothers analysis confirmed evidence of recombination
across the 552 bases of these seven genes (Additional file
6B). In summary, evidence exists that recombination has
occurred within conserved but divergent regions of the
vsp genes. Evidence also exists that discreet high-mutation-frequency regions bracket low-frequency regions,
implying protection or movement of functionally important regions during recombination.

The 218 full length Giardia WB VSP proteins were
aligned and an unrooted tree produced (Fig 2). The
majority of the 218 VSP protein sequences fall into three,
well-defined clades labeled I, II and III (Fig 2). Three VSP
proteins, whose branches are colored black (vsps3, 13,
and 122) lie between clades II and III and contain
sequences common to both of those clades. This tree
appears to suggest an expansion of three functional
groupings of VSP proteins within the WB genome. Of the
30 vsp genes containing tandem repeats, 27 fell within
clades II and III, while the remaining three consisted of
the three vsp genes that were between clades II and III.
We also performed a protein alignment using two copies
of the tandem repeats, taken from each of the 30 TR-containing genes and discovered that they clustered into several distinct clades (Additional file 2). Since a previous
report described a vsp gene (vspG3M/vspA6-S1; vsp175)
that was highly similar along the entire gene with vspA6
(vsp67), but contained only one copy of the 195 bp repeat
[19], we theorized that degenerated sequence forms of
the conserved tandem repeats may exist within nonrepeat-containing vsp genes. We performed a BLAST
analysis using single copies of each of the 30 TR to query
the non-repeat-containing vsp genes. Nine of the 30 demonstrated >80% nucleotide identity over a stretch of at
least 80 bp to 10 of the non-repeat-containing genes. This
result suggests that additional divergent homologies
exist, that other vsp genes may have contained similar
repetitive motifs that diverged over time, or that recombination between repetitive motifs and non-repeat-containing vsp genes might be occurring.
In order to compare protein to DNA phylogenetic relatedness, and to determine if the DNA sequences differed
substantially from the protein sequences, the DNA
sequences encoding these 218 vsp genes were aligned and
a Neighbor Joining tree with boot strap values at significant nodes was produced (Additional file 5). A color coding scheme illustrating the three dominant clades
produced from the protein alignment along with those
associated numerical clade designations is shown in
Additional file 5. Much of the conservation and clade designation seen in the protein-based tree is apparent in the
DNA sequence-based tree. Further characterization and
finishing of the partial vsp sequences will determine if
these predominant grouping trends hold true for all vsp
genes and their protein encoding sequences within the
WB genome.
Protein motifs may imply potential VSP function

The protein sequences of all complete vsp genes were
analyzed for the presence of motifs that might provide

Adam et al. BMC Genomics 2010, 11:424
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clues to their function. A zinc finger motif has been
described as being encoded by a subset of the vsp genes
[38], and zinc binding has been documented for VSPs,
although in substoichiometric amounts [39]. Therefore,
we were especially interested to see how commonly the
zinc finger motif was found across the entire repertoire of
218 complete WB VSPs. Using Pfam 22.0 to search the
218 complete VSPs for protein motifs, we found 36 VSPs
that had weak hits (E value between 1 and 1e-5) to the
C3HC4 type zinc finger (RING finger), which is a
cysteine-rich domain of 40-60 residues, has the consensus sequence: C-X2-C-X(9-39)-C-X(1-3)-H-X(2-3)-C-X2C-X(4-48)-C-X2-C where × is any amino acid and coordinates two zinc ions. Since we found only weak hits to a
Zinc RING finger motif, we hypothesized that the VSPs
contain a novel Giardia-specific Zinc finger. To elucidate
this further we searched for the previously described
motifs [39], namely, CxxCHxxCxxC and CxxCxxxCxxC.
Table 2 shows data for the number of these two motifs
found within the 218 complete WB VSPs and the alternative amino acids found in the 5th position of the CxxCxxxCxxC motif. One or both of these motifs was present in
151 vsps with 73 copies of the CxxCHxxCxxC motif in 67
VSPs and 255 copies of the CxxCxxxCxxC motif present
Table 2: Zinc finger motifs
Fifth AA

#

Charge

H

73

basic

N

60

polar

D

54

acidic

G

50

polar

A

33

nonpolar

P

18

nonpolar

S

17

polar

T

15

polar

V

5

nonpolar

Q

2

polar

E

1

acidic

Total

328

Page 7 of 14

in 149 VSPs. Only two vsps had the CxxCHxxCxxC, but
not the CxxCxxxCxxC motif. More experiments are
needed to determine if the CxxCHxxCxxC and/or CxxCxxxCxxC motif is required for zinc binding or if there is
a difference in their zinc binding properties. Interestingly,
the CxxCHxxCxxC motif does not seem to overlap with
the weak RING finger domain identified by Pfam, providing further evidence of a putative novel zinc or other
metal, or other substrate-binding domain within those
VSPs.
Of the 218 complete VSPs, 182 (83%) have a GGCY
motif. The average number of GGCYs in these 182 genes
is 1.4 per VSP, with a maximum of five occurring in one
vsp. Most GGCY motifs are in the carboxyl portion of
VSPs but some exist closer to the N terminus. The function of the GGCY motif is not known, some data suggest
that it may be immunogenic. For example, a 12 amino
acid epitope containing this motif induced late appearing
antibodies in neonatal mouse infections [40], and antibodies to a larger fragment containing the GGCY motif
caused trophozoite detachment and aggregation [41].
In addition to the weak RING finger domain, three
VSPs had very good (E value <1e-20) hits to the BmKX
domain, a short bioactive peptide present in the venom of
scorpions and thought to act as a potassium channel
blocker [42]. Five additional VSPs showed moderate (E
value between 1e-5 and 1e-20) hits to the Lamin_EGF
domain (2 VSPs) and the EGF_CA domain (3 VSPs).
Many other VSPs had weak hits to these three domains
and several additional domains (TIL and Furin-like) that
are also cysteine rich. Whether these similarities are of
biological significance remains to be determined, but it is
interesting to speculate that some of these data implies
diversity of vsp function.
The VSPs share a high degree of similarity of approximately 38 amino acids encoded by the 3' terminus of the
genes. Hidden Markov Modeling (HMM) to identify
membrane-spanning regions suggested that the C-terminal CRGKA motif is inside the membrane, while the 23
amino acid upstream and adjacent to the CRGKA span
the membrane, and the remainder of the VSP is extracellular (data not shown). This result is consistent with prior
hydrophilicity analyses [17]. As previously discussed
above, the CRGKA encoding C-terminal tail was used to
identify all known vsp members within the current WB
genome assembly and is found in all reported vsp genes,
suggesting an important function in VSP biology. Many
VSPs are palmitoylated [43,44], and in vivo, palmitic acid
binds specifically to the cysteine of the CRGKA tail controlling segregation of the VSPs to the detergent-resistant
domains within the plasma membrane [45]. Arginine
deiminase binds specifically in vivo to the arginine of the
CRGKA and catalyzes its citrullination [46]. These biochemical studies in addition to the highly conserved

Adam et al. BMC Genomics 2010, 11:424
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nature of the CRGKA motif suggest an important biological role. Thus, it is of interest that eight vsps have amino
termini that differ from the CRGKA at one or two positions; CHKKA, CRGKL, CRSKA, GRRKA (2 examples),
FCGKA, YRGKA, and WRGKA (Additional file 1), and
six of these eight lack either the cysteine or the arginine
or both. Experimental studies will be required to determine whether these alternate N-termini are compatible
with observed or hypothesized VSP function.
Genomic organization and context of the vsp genes in the
WB genome

The vsp genes were distributed among all five chromosomes (Table 3). Of the 196 vsp genes that could be
assigned to a specific chromosome, the density ranged
from 8 vsp genes per Mb on chromosome 1 to 29 vsp
genes per Mb on chromosome 4 (based on the total size
determined by optical mapping). The distribution of vsp
genes along chromosome 4 is shown in Fig 3. Many of the
vsp genes existed independently on the chromosome with
no other vsp genes in the near proximity. However, the
majority of vsp genes were not randomly distributed, but
were clustered into head to head or tail to tail arrangements, or as linear arrays (LAs) of vsp genes (see Fig 4 for
examples).
Genes present in inverted or tandem pairs (H-H, T-T, H-T)

Vsp gene pairs in Giardia can appear as an inverted pair
of genes with or without identical reading frames (Table
4). Fourteen of the 19 inverted pairs consisted of two
identical ORFs. Conversely, there were no examples of
identical genes that were not in this inverted pair
arrangement. The one example reported in the literature,
(vsp1267), consisted of two identical ORFs in tail to tail
orientation, separated by approximately 3 kb [17]. In the
current study, we discovered 13 additional pairs of identical genes present in head to head (4) or tail to tail (9) configurations with 2190 to 3847 bp of intervening DNA
between the paired ORFs (Table 5). Outside the coding
regions of these identical pairs, the 5' and 3' flanking
sequences of the two members of a pair were nearly identical up to a point of abrupt divergence which ranged
from 139 bp to 729 bp away from the coding regions
(Table 5).
The occurrence of identical pairs of genes was especially problematic during genome assembly in that this
redundancy prematurely terminated the contigs. In fact,
vsp1267 (vsp98.1) was not identified as a pair by the
genome assembly, but was found as a single gene at the
end of a contig. Only two of the 14 pairs with identical
members were greater than 10 kb away from the end of
any given contig. Whether other inverted pairs were
missed by the assembly is not known but quite possible.

Page 8 of 14

An additional five pairs of genes where the two paired
members contained different ORFs were discovered in
head to head (4) or tail to tail (1) orientation and were
separated by approximately 3 kb of intervening DNA in
all cases. For four of these five pairs, the two members of
the pair were from different clades, implying translocation from other perhaps singular genomic locations into
this tandem arrangement. We believe that the 14 identical pairs have arisen purely by gene duplication and
transposition events. It is possible that these gene duplication events are relatively recent in the evolutionary history of the vsp gene family and that over time, genetic
diversity through recombination between identical or
distant vsp genes may produce new vsp genes. The paired,
identical vsps and non identical paired vsps remain an
interesting, but cryptic observation of the vsp gene family.
vsp genes present as linear arrays (LA)

Over half the vsp genes found in the genome lie within
the genomic context of vsp linear arrays that consist of
two or more adjacent vsp genes (Table 4). The intervening
regions between the genes in these linear arrays averaged
60 bp (ranging from overlapping encoded regions to 241
bp of intergenic sequence). Apart from these linear
arrays, there were only two other head to tail pairs of vsp
genes that were less than 5 kb apart, but containing
greater than one kb of intergenic sequence between the
two genes. For these two non-linear array pairs, the two
members of a pair had no significant sequence similarity.
Therefore, this paired association may be random.
Most of the linear arrays were in very short contigs,
such that they frequently included only two vsp genes,
and often terminated one or both ends of the contig on
which they were located. In fact, 97 of the 102 vsp genes
in contigs <10 kb in length were in linear arrays. In terms
of the contigs themselves, vsp linear arrays accounted for
the majority of coding capacity of 56 of the contigs < 10
kb in length, even after some were joined by directed,
manual sequencing efforts. In the larger contigs, there are
several examples of linear arrays at the ends of adjacent
contigs (see Fig 3), suggesting that these adjacent linear
arrays may indeed represent even longer arrays than what
is observed here. The possibility of longer arrays also is
supported by the observation that the longest array discovered to date (10 vsp genes) is on the end of contig 12
that is adjacent to the end of contig 32 (optical mapping
data not shown), which has two vsp genes in a linear array
(Fig 3). Therefore, while the Giardia linear arrays
described here are impressive in their quantity, length
and gene numbers, much longer arrays may exist in the
genome.
As noted above, the first vsp of a linear array often
showed the typical features of vsp genes that have been

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Page 9 of 14

Table 3: Vsp chromosomal distribution
Chromosome
(size Mb)

vsps

#/Mb

5 (4.43)

65

15

4 (2.79)

81

29

3 (1.94)

18

9

2 (1.50)

20

13

1 (1.46)

12

8

described in the literature, including an extended noncoding region upstream and a putative initiator element
(Inr). Fig 3 shows the spacing within the linear array and
the variable placing of the first ATG after the nearest
upstream stop codon. In fact, vsp14 provides an example
of a sequence that contains a typically conserved 3'
region, but which lacks an initiation codon. Vsp13 is the
first member of a linear array on chromosome 4. The
coding region for Vsp 13 is followed by a stop codon, 38
bp, another stop codon, and 224 bp of coding region for
vsp 14, which also contains the conserved encoded
CRGKA, C-terminal domain. This short, stop-containing
intergenic region suggests that vsp 13 may not be
expressed. The frequency of these linear array formations
along with the lack of an Inr sequence in these down-

Chromosome 4 vsp distribution
vsp 13-14
TR

0

TR

1Mb
TR

c2

c15

stream members and the occurrence of short, intergenic
or overlapping encoding regions between these downstream vsp genes leads us to hypothesize that these
downstream members may not be actively expressed,
either individually or in an operon-like fashion. A potential hypothesis for their occurrence or genesis is that they
may serve as cassettes for recombination with the first
vsp gene in the linear array (that contains functional
upstream expression sequence) or distant, expressible vsp
genes in the genome. In both scenarios, the generation of
new antigenic or functional vsp genes would be the
potential outcome of such a scenario. Pursuant to this
hypothesis, we discovered that in certain linear arrays,
members within a linear array were sometimes more
closely related to each other phylogenetically than to vsp
genes elsewhere in the genome, or in other linear arrays
(data not shown). We also discovered that for some linear
arrays, members within the array were far more phylogenetically distant to each other than they were to genomically distant vsp genes (data not shown) suggesting either
active recombination to generate significant sequence
diversity or translocation of entire vsp coding regions into
linear array formations. Except for 13 of the 26 linear
arrays containing at least two complete genes, all members of a linear array are from the same phylogenetic
clade (Data not shown). The longest array (Fig 3) had nine
complete sequences, and eight of these vsp genes were
from Clade I while one vsp gene was from Clade II (Fig
4C). Among the eight Clade I vsps in this linear, there
were two pairs (vsp60/65 and vsp 61/64) in which the two
members of each pair were the closest relatives to each
other on the phylogenetic tree, implying previous gene
duplication, but little to no distant translocation events.
For example, vsps61 and 64 demonstrated 94% nucleotide
sequence identity, including long regions of 100% iden-

c8

TT

1Mb

TR

2Mb
c2 (cont)

c26
c21

c32

TR

c56

A

TR

36.1

c12

36.2

36 VSP Inverted pair
300

2.788 Mb
c35

TR

c36
c31

TR

c38
c44

900

1200

3600

3900

4200

168.1

c49
c52

600

c46

168.2

168 VSP Inverted pair
400

Figure 3 Distribution of vsp genes along chromosome 4. The contigs were initially assigned to individual chromosomes by mapping
end-sequenced BACs to PFG separations of chromosomes and subsequently refined by optical mapping. Chromosome 4 is the most densely populated with vsp genes (29/Mb). Each vsp gene is noted by a
vertical line, which is above the horizontal chromosome line for those
with a forward orientation in relationship to the chromosome direction and below for those oriented in a reverse direction. The contigs
from Assembly 14 [30] are shown below with arrows indicating the orientation of the contig. Vsps with tandem repeats are noted with "TR"
and those present in linear arrays are shown as filled in boxes with each
section representing one vsp gene. TT indicates tail to tail arrangement.

4500

B

2Mb

C
linear
array

A

56
0K

800

A
57
1K

1200 1600 2000

A
58

2K

3K

A

59
4K

5K

4800 5200 5600

2400

A

6K

60
7K

8K

61
9K

10K

6000 6400

A
62
11K

6800 7200

A

12K

A
63

13K

14K

A
64

15K

16K

65
17K

18K

Figure 4 Patterns of genomic organization of vsp genes. Genes
present as inverted pairs are shown in A and B. These are in tail to tail
orientation, and overall there were 10 paired identical tail to tail dual
gene sets and 4 paired identical head to head dual gene sets. A linear
array of vsps 57 to vsp65 is shown in C. Gene 59 is a clade II representative while the other vsp genes (57, 58, and 60-65 are all Clade I representatives.

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Table 4: Vsp gene patterns
Pattern
Inverted identical pairs

All vsps

Complete sequences only

14 pairs

Head to head

4

4

Tail to tail

10

10 (17 genes, but 3 of the paired members were incomplete)

Other head to head

10 (5 pairs)

10 (5 pairs)

Other tail to tail

4 (2 pairs)

3 (2 pairs)

Linear array

164

99

Overlapping linear array

5

5

Total vsp genes

303

228

tity. These closely related pairs suggest that gene duplication and divergence have played a role in expansion of the
linear arrays. In addition, sequence drift and recombination-driven sequence divergence may have also played
roles in the formation of these arrays. We speculate that
these linear arrays contain genes that are nonfunctional,
but either provide a reservoir for expanding the vsp gene
repertoire, or alternatively, represent the remains of discarded vsp genes.
Rarity of telomeric vsp genes

For other eukaryotic, non-Giardia microbial pathogens,
variant surface antigen genes are present and are frequently located in sub-telomeric locations. For example,
the VSG genes of African trypanosomes have been the
most intensively studied variable surface antigen genes
and they can be subtelomeric or chromosome-internal in
their location. Trypanosome chromosome-internal VSG
genes are expressed only when they are duplicated into
subtelomeric expression locations [47,48]. An additional
example of variable surface antigen genes can be found in
Plasmodium falciparum parasites which express one of a
family of var genes that encode proteins that are involved
in adherence to host cells. Variation in expression from
one var gene to another can change the binding characteristics of the organism. The P. falciparum genome has
been completely sequenced and assembled, and the 61
var genes have all been assigned to their chromosomal
locations. While the var genes can be chromosome-internal, nearly two thirds of them are subtelomeric [49]. Acti-

vation of subtelomeric or chromosome-internal var genes
occurs by epigenetic mechanisms in the absence of DNA
recombination [49,50].
In contrast, Giardia's vsp family appears to be very different from the trypanosome VSG or plasmodium var
gene families. First of all, only three vsp genes have been
found in subtelomeric locations, most likely, all at one
end of chromosome 5 [51]. In addition, there is no evidence for duplicative transposition of vsp genes to subtelomeric or other locations as a requirement for vsp gene
expression in Giardia. As an example, for two different
vsp genes (vspA6 and vspC5), the chromosomal locations
have been determined in organisms expressing these
genes and in antigenic variants that have lost expression
of these genes. In both cases, the chromosomal location
did not change with antigenic variation [10,11], and in
both cases, the vsps were chromosome-internal. Thus,
Giardia differs from these other protists in that a subtelomeric location for the vsps is relatively uncommon, and is
clearly not required for expression.

Conclusions
This description of the vsp genes and their encoded proteins from the WB isolate gives a picture of a repertoire
that has been expanded by duplication and recombination to the extent that expression of different VSPs may
facilitate differences in pathogenesis or ecological niches.
This description will provide important tools for better
understanding of the biology of these enigmatic proteins
and the trophozoite surfaces on which they are expressed.

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Table 5: Divergence points of identical pairs of genes
Gene #

5' Divergence
(nt above start codon)

3' Divergence
(nt beyond stop codon)

Distance between ORFs

26

169

-

2190

36

729

203

3102

162

561

-

3847

168

158

141

3302

1

-

559

2814

7

-

318 (572)

2687

20

-

541

2759

47

-

354

2721

48

-

515

3222

53

-

486

2670

87

-

514

2393

107

-

529

2638

126

-

530

2748

Head to head

Tail to tail

Methods
Vsp gene identification

Identification of vsp genes in the WB genome was performed by a combination of three approaches, (1) Called
ORFs that were identified by the automated BLAST
search as variant-specific surface proteins or trophozoite
surface antigens, (2) called ORFs with at least two CXXC
motifs in the encoded sequence, and (3) BLAST searches
of the genome assembly with the conserved 3' 110 bp.
Overall, the third approach was the most efficient at identifying all vsps except those ORF-coding regions where
the sequence was incomplete and lacking the 3' conserved regions. Another set of cysteine-rich genes has
been identified in Giardia (HCMP), which also have
CXXC motifs, occasional CXC motifs that are not found
in the vsp genes, but which collectively lack the 3' conserved region [36]. Therefore, we have compared the lists

of identified vsps with other cysteine-rich genes to eliminate the possibility of dual category assignment of genes.
Ultimately, the presence of the CRGKA motif at the Cterminus was the defining feature for vsp-designated
genes; for all vsp designated genes three of the five
encoded amino acids of the CRGKA motif must be present in the 3' coding end of the gene.
Genomic mapping and chromosomal localization using the
BACs, extensive chromosomal hybridizations, and optical
mapping

DNA sequences from individual clones were assembled
into contigs by ARACHNE after editing with CONSED at
described [30]. The assembled contigs and supercontigs
were compared directly to the BAC scaffolding. Many of
the BACs had been labeled and hybridized to PFGE blots
of Giardia chromosomes, allowing placement of the
supercontigs onto specific chromosomes. The chromo-

Adam et al. BMC Genomics 2010, 11:424
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some location of contigs has subsequently been verified
and expanded upon by data generated from the use of
optical mapping technologies. For optical mapping,
whole chromosomes from Giardia lamblia WBC6 were
isolated from trophozoite cell pellets using pulsed field
gel electrophoresis. Agarose plugs containing the chromosomes were sent to OpGen (Gaithersburg, MD) for
mapping. Optical mapping was done with MluI restriction enzyme and the observed fragments compared to the
MluI sites predicted in the assembled contigs using MapSolver v. 2.1.1. Sixty-two contigs representing 10.4 MB
uniquely mapped to one of the five chromosomes.
Directed sequencing

Since many of the contigs were prematurely terminated at
or near vsp sequences, we also subjected some of the
clones that aligned to these problem ends of contig
regions to directed sequencing. These extended clone
sequences were then used to join or extend contigs when
possible and to extend or complete vsp gene sequences.
In addition to the libraries used for the genome project,
an additional library was constructed from WB DNA that
was partially digested with SauIIIA1, cloned into pUC18,
and hybridized to the vsp 3' conserved region. Approximately 670 clones were subjected to vector-junction
sequencing. Following analysis, additional directed
sequencing was performed for 308 clones that had the
potential of adding to the existing vsp sequence data.
Search for motifs

The VSP amino acid sequences were analyzed using Pfam
23.0 [52] to search for Zinc finger motifs. An e-value cutoff of 10 was used to capture VSPs with weak hits to Zinc
fingers. Very weak Zinc finger domains were identified in
181 VSPs.
Alignments of vsp genes and recombination testing

Full length VSP proteins and the N-terminal 38 amino
acids for all VSPs were aligned using MUSCLE 1.1. Full
length vsp genes, the 3' 117 nucleotides for all vsps, and
two of the tandem repeat encoding sequences of the tandem repeat-containing genes were aligned using CLC
Genomic Workbench 3.2 (Katrinebjerg, Denmark).
Alignment trees were constructed using the Neighbor
Joining method with 1000 bootstraps. The alignments
were transferred into MacClade program (Maddison D.R.
and W.P. Maddison. 2003; MacClade 4 analysis of phylogeny and character evolution, version 4.06 Sinauer Associates, Sunderland Mass.) and manually corrected. DNA
polymorphism analysis and recombination analysis were
performed with the DNasp package of algorithms http://
www.ub.es/dnasp. Sawyer's test was performed with
GENECONV
http://www.math.wustl.edu/~sawyer/
mbprogs/ as previously described [53]. Additional
recombination analysis was performed using the Dualbrothers algorithm [54].

Page 12 of 14

Additional material
Additional file 1 List of all vsp genes with detailed information as follows: A. VSP number, B. Clade number (of the three major clades). A
blank indicates either an incomplete sequence or that this is one of the
three genes without a clade assignment, C. Name used in prior reports in
the literature, D. Prior Genbank number, when available, E. gene # from the
WB genome (the number that is given should be preceded by the prefix
GL50803_), F. Features including TT (tail to tail), HH (head to head), IP
(inverted pair of identical ORFs; the "a" or "b" refers to the first or second
member of the pair, TR (tandem repeats; the following number indicates
the number of nucleotides per repeat in the reported sequence, LA (linear
array; it is followed by O for overlapping genes, G. An "x" is used to note the
members of linear arrays that were first in the array, H. Number of nt/tandem repeat, I. Rep # refers to the number of tandem repeats, J. Chromosomal location, K. Compl refers to whether the sequence contains the
complete open reading frame; c indicates complete, 3 indicates missing 3'
sequence and 5 indicates missing 5' sequence, L. Inr indicates whether or
not a gene contains the putative Inr and whether it is a TAATGTT or CAATGTT, M. Alternative C-terminal 5 amino acid motif, in place of CRGKA, N.
number of amino acids in the translated sequence, O. The molecular mass
of the putative protein in kDA, P. The number of cysteines in the encoded
sequence, Q. % cysteines in the encoded protein, and R. Number of CXXC
motifs in the encoded protein.
Additional file 2 Rooted phylogenetic tree of the DNA sequences of
the 30 tandem repeat regions. Two of the tandem repeat DNA sequence
domains from all of the known and complete tandem repeat-containing
genes were aligned and a Neighbor Joining method tree was constructed
with 1000 bootstraps. Names of the respective tandem repeat-containing
genes are shown where TR stands for Tandem Repeat. This tree suggests
that some commonality and difference of the tandem repeats exist relative
to each other, implying sequence divergence or possible recombinatorial
actions driving the variety of TRs.
Additional file 3 Nucleotide alignment of the 3' regions. The 3'-terminal 117 nucleotides (including the stop codon) for all 218 complete VSPs
were aligned and the alignment manually corrected. Colored shading indicates conservation of nucleotides at particular positions across all vsps. At
the bottom of the figure is the consensus sequence from this alignment
where vertical box height correlates with frequency of occurrence of that
nucleotide.
Additional file 4 Amino acid alignment of the 3' regions. The C-terminal 38 amino acids for all 218 complete VSPs were aligned and the alignment manually corrected. Colored shading indicates conservation of
residues at particular positions across all vsps. Dashes indicate absence of a
residue. At the bottom of the figure is the consensus sequence from this
alignment where vertical box height correlates with frequency of occurrence of that residue.
Additional file 5 Rooted phylogenetic tree of the DNA sequences
encoding all 218 full length vsp sequences. Full length vsp genes were
aligned, manually corrected and a rooted tree created using the Neighbor
Joining method with 1000 bootstraps. Colored clade designation along
with numerical I, II, and III designations are based upon the unrooted tree
shown in Fig 2. The three vsps (vsps 3, 13, and 122) lying between clade II
and III are shown in black. The bar at the bottom of the figure signifies
branch length related to number of substitutions per base position analyzed.
Additional file 6 DualBrothers recombination alignment of sub
clades. A) Dualbrothers analysis of clade 3 and 4 as defined in the Recombination table. B) Dualbrothers analysis of clade 5 and 6 as defined in the
Recombination table. For each panel the x-axis represents the positions in
the sequence alignment, the y-axis shows the Bayesian posterior probability of the inferred tree topologies (Topologies) with each colored line indicating a distinct topology (4 shown for A and 3 shown for B), the green line
representing the sum of remaining tree topologies. Transition:transversion
and expected divergence rates are shown along with an identity bar graph
and base positional differences relative to a consensus (colored bars in horizontal grey bars).
Authors' contributions
RA coordinated the project and writing and did a portion of the directed
sequencing. AN compiled and analyzed vsp sequences. VS compiled and ana-

Adam et al. BMC Genomics 2010, 11:424
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lyzed vsp sequences. CM performed bioinformatics and sequence searching
and comparison analysis. GF performed vsp Recombination analysis. HM provided the optical mapping data. TN directed sequencing of additional vspcontaining clones and assisted with the analysis and writing. SP coordinated
directed sequencing efforts and analyses including recombination, motif and
phylogenetic analyses. RP worked on compiling and analyzing vsp sequences.
All authors read and approved the final manuscript.
Acknowledgements
This work was funded in part by NIH/NIAID grant AI43273 and in part by the
Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases, National Institutes of Health. The funding bodies had no role in
data analysis, writing or decision for submission. We appreciate the work of
Anita Mora on the graphics for the figures.
Author Details
1Departments of Medicine and Immunobiology, University of Arizona College
of Medicine, Tucson, AZ, USA, 2Dept of Immunobiology, University of Arizona
College of Medicine, Tucson, AZ, USA, 3Genomics Unit, Research Technologies
Section, RTB, NIAID, NIH, Rocky Mountain Laboratories, Hamilton, MT, USA,
4Josephine Bay Paul Center, MBL, Woods Hole, MA, USA, 5Laboratory of
Parasitic Diseases, NIAID, National Institutes of Health, Bethesda, MD, USA and
6Dept of Immunobiology, University of Arizona College of Medicine, Tucson,
AZ, USA
Received: 11 February 2010 Accepted: 9 July 2010
Published: 9 July 2010
© 2010 Adamavailable from: http://www.biomedcentral.com/1471-2164/11/424
This is an Open2010, 11:424 BioMed Central Ltd.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.
BMC article is et al; licensee distributed under
Genomics Access article

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doi: 10.1186/1471-2164-11-424
Cite this article as: Adam et al., The Giardia lamblia vsp gene repertoire: characteristics, genomic organization, and evolution BMC Genomics 2010, 11:424

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