Genome Res. 13:2005-2017, 2003
©2003 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/03 $5.00
Sixty Alleles of the ALS7 Open Reading Frame in Candida albicans: ALS7 Is a Hypermutable Contingency Locus
Ningxin Zhang1,
Annette L. Harrex3,
Barbara R. Holland2,4,
Lauren E. Fenton3,
Richard D. Cannon3 and
Jan Schmid1,5
1 Institute of Molecular BioSciences, Massey University, Palmerston North,
New Zealand
2 Institute of Fundamental Sciences, Massey University, Palmerston North,
New Zealand
3 Department of Oral Sciences and Orthodontics, University of Otago,
Dunedin, New Zealand
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ABSTRACT
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The ALS (agglutinin-like sequence) gene family encodes proteins
that play a role in adherence of the yeast Candida albicans to
endothelial and epithelial cells. The proteins are proposed as virulence
factors for this important fungal pathogen of humans. We analyzed 66 C.
albicans strains, representing a worldwide collection of 266
infection-causing isolates, and discovered 60 alleles of the ALS7
open reading frame (ORF). Differences between alleles were largely caused by
rearrangements of repeat elements in the so-called tandem repeat domain (21
different types occurred) and the VASES region (19 different types). C.
albicans is diploid, and combinations of ALS7 alleles generated
49 different genotypes. ALS7 expression was detected in samples
isolated directly from five oral candidosis patients. ORFs in the opposite
direction contained within the ALS7 ORF were also transcribed in all
strains tested. Isolates representing a more pathogenic general-purpose
genotype (GPG) cluster of strains tended to have more tandem repeats than
other strains. Two types of VASES regions were largely exclusive to GPG
strains; the remaining types were largely exclusive to noncluster strains. Our
results provide evidence that ALS7 is a hypermutable contingency
locus and important for the success of C. albicans as an
opportunistic pathogen of humans.
Candida albicans is a diploid yeast with a predominantly clonal
mode of reproduction (Tibayrenc
1997 ) that is an important opportunistic pathogen of humans
(Odds 1988). We recently
presented evidence of a ubiquitous general-purpose genotype (GPG) cluster of
C. albicans strains that are exceptionally successful as human
pathogens (Schmid et al.
1999). Two other studies have since provided evidence that this
genotype (as deduced from the position of GPG reference strains included in
the studies) was the most frequent among the infection-causing isolates they
analyzed (Botterel et al. 2001;
Bougnoux et al. 2002). We are
currently undertaking comparisons between GPG cluster strains and other
strains to identify physiological and genetic traits which are associated with
their increased pathogenicity, as a means to identify pathogenicity factors
relevant to interactions of C. albicans with the human host
(Schmid et al. 1995;
Giblin et al. 2001).
During a screen for genomic differences between GPG cluster strains and
other strains using amplified fragment length polymorphism (AFLP) analysis, we
discovered that the open reading frame (ORF) of the ALS7 gene
(Hoyer and Hecht 2000) showed
GPG cluster-specific sequence modifications. The gene is part of a family of
large agglutinin-like cell surface glycoproteins that mediate adherence of
C. albicans to host tissue (Gaur
and Klotz 1997; Chandra et al.
2001; Hoyer 2001;
Fu et al. 2002). ALS
genes are also present in Candida dubliniensis and Candida
tropicalis (Hoyer et al.
2001). C. albicans ALS genes have been shown to be
differentially expressed in a strain-dependent manner
(Hoyer et al. 1998a) in
response to parameters such as growth stage
(Hoyer et al. 1998b),
morphological transition between yeast and hyphal growth
(Hoyer et al. 1998a;
Murad et al. 2001), and
biofilm formation (Chandra et al.
2001). It has also been documented that alterations in repeat
regions of ALS genes lead to strain-specific variability in the Als
proteins (Hoyer et al. 1995).
Taken together these observations indicate that Als proteins provide C.
albicans with a large and flexible repertoire of similar but nonidentical
surface proteins. In other eukaryotic microbial pathogens such as
Plasmodium, Trypanosoma, and Giardia, such a repertoire of
proteins is considered important not only for adhesion but also for evasion of
host defenses (Nash 1997;
Ramasamy 1998;
Barry and McCulloch 2001). High
mutation rates in the genes encoding these proteins are a crucial part of
generating diversity. The term "hypermutable contingency genes"
was coined for such genes (Duncan et al.
1991; Moxon and Thaler
1997; Barry and McCulloch
2001; Bridges
2001). Aside from encoding cell surface proteins, hypermutable
contingency genes are defined by (1) being potentially highly mutable, and (2)
mutations which are not caused by error-prone DNA replication but are rather
specific, being brought about by recombination (or methylation) events,
involving oligonucleotide repeats or homo-ploymeric tracts
(Moxon and Thaler 1997;
Bridges 2001). ALS7
would be particularly well suited as a hypermutable contingency gene in C.
albicans; its ORF contains not only the tandem repeat domain,
characteristic of all ALS genes, but in addition a second repeat
region, the so-called VASES region.
We present here evidence, based on the allelic variation at the
ALS7 locus in a large set of strains representing the major
phylogenetic lineages in a worldwide collection of 266 infection-causing
isolates, that ALS7 is a hypermutable contingency locus. We also
describe restraints on the rearrangements of repeat units in the ALS7
ORF and expression data which allow conclusions to be drawn regarding the role
of ALS7 in the pathogenesis of candidosis in humans.
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RESULTS
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An AFLP Marks Alterations in the ALS7 Gene Specific to GPG
Cluster Strains
Following the discovery that a ubiquitous GPG cluster of C.
albicans causes candidosis 10–100 times more often than other
clusters of strains (Schmid et al.
1999), we used AFLP analysis
(Vos et al. 1995) for genomic
comparisons between GPG strains and other strains. We searched for AFLPs
highly specific to GPG strains as markers of loci contributing to
pathogenicity. In these comparisons we used two partially overlapping sets of
isolates, each set containing 25 GPG cluster strains and 16 strains not
belonging to the cluster (noncluster strains), representing a worldwide
collection of 266 infection-causing isolates
(Schmid et al. 1999). A list
of strains in the sets and their ALS7 alleles can be found in the
Supplemental Material (available online at
www.genome.org).
Set 1 contained isolates chosen on the basis that they best represent features
that distinguish GPG cluster and noncluster strains (see Methods for details).
Set 2 contained isolates that best represent the entire spectrum of GPG
cluster strains and the entire spectrum of noncluster strains, respectively
(this includes borderline strains with an uncertain affiliation; see Methods
for details). Laboratory strain SC5314, used to construct the Stanford genome
database
(http://www-sequence.stanford.edu:8080/bin/blastncontigs6),
was also included in both sets, as a GPG cluster strain
(Giblin et al. 2001), bringing
the number of GPG model strains to 26.
In a comparison between GPG cluster and noncluster isolates in Set 1, we
discovered a 947-bp AFLP fragment, MC5-c, present in 21 of 26 GPG cluster
strains (81%) and in one of 16 noncluster strains (6%). The fragment was also
present in 22 of 26 GPG cluster strains in Set 2 (85%) and three of 16 (19%)
noncluster strains in Set 2. AFLP products from two GPG cluster strains were
cloned and sequenced. Database searches showed homology to part of the
ALS7 gene in strain 1161 (accession no. AF201684
[GenBank]
;
Hoyer and Hecht 2000) and
SC5314 (bp 7513–1511 in contig 6–2514;
http://www-sequence.stanford.edu:8080/contigs/Contig6-2514).
Sixty Alleles of the ALS7 Gene and 49 ALS7
Genotypes in 66 C. albicans Strains
According to the definition of Hoyer
(2001), the ALS7 gene
contains three domains (Fig.
1), and two of these, named the tandem repeat domain and the
3'domain, contain repeats; the repeats in the 3' domain are
contained in the so-called VASES region. The GPG cluster-specific AFLP
fragment overlapped with the VASES region, and amplification of the region of
ALS7 corresponding to the AFLP fragment from noncluster strains gave
a variety of PCR products. This suggested that the repeat-containing parts of
the ALS7 ORF might undergo frequent rearrangements typical for
hypermutable contingency loci, as outlined in the Introduction. We therefore
decided to assess variability in both repeat regions for all of our model
strains.
The tandem repeat domain is composed of 108-bp units
(Hoyer and Hecht 2000;
Hoyer 2001). We amplified the
entire tandem repeat domain in different strains and used the size of the
amplicons to determine the number of repeat units present
(Fig. 2A). We used restriction
digestion of the amplicons to verify that the PCR products were indeed
composed of 108-bp units (Fig.
2B). The analysis of the VASES region was more complicated.
Although the region is named after a 15-bp repeat encoding the amino acid
sequence VASES (in addition there is a similar version encoding VTSES), it
contains three other repeat elements. We found that the region is usually
assembled from one or more units, each comprising an 87-bp repeat, followed by
a 138-bp repeat, followed by a stretch of VA/TSES repeats, followed by a
141-bp repeat. At the end of the region is one additional 87-bp repeat
(Fig. 1). We characterized the
VASES regions by first amplifying the entire region
(Fig. 2C) and then, using this
PCR product as a template, carrying out additional amplifications to deduce
arrangement of individual units (data not shown; see Methods for details).
From the product sizes we calculated the number of repeats in each unit.
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Figure 2 Characterization of ALS7 alleles by PCR and restriction analysis
in the 42 strains of model Set 1. GPG cluster strains are marked by a shaded
bar under the strain name, other strains by a hollow bar. (A) PCR
amplification of the tandem repeat domain, using primers MC5inpf and MC5repr.
(B) BsmAI digest of the products; a single BsmAI
site in each 108-bp repeat is predicted from the nucleotide sequence, and so
digestion of the PCR product should produce a 108-bp band plus three other
bands of very similar molecular weight: an 80-bp band from the 5'
flanking sequence and bands of 95 bp and 116 bp from the 3' flanking
sequence. In addition, there should be a 270-bp band from the 3'
flanking sequence. (C) Amplification of the VASES region, using
primers MC5spF andMC5pf. Numbers at the right of the figure give fragment
sizes in kb.
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Where PCR analysis indicated the presence of two different-sized tandem
repeat domains or VASES regions in the same strain, we also generated PCR
products spanning both the tandem domain and VASES region to derive the size
of the two alleles present (data not shown). We tested the accuracy of our
PCR-based derivations of arrangements of repeat units by comparing them with
the published sequences available for SC5314 and 1161 and with our sequences
obtained for three tandem repeat domains and 12 VASES regions (see below). In
all cases, the PCR-based estimates proved correct.
Our analysis revealed the presence of 60 alleles, distinguishable by PCR
analysis, in our 66 strains (Fig.
3) and 49 ALS7 genotypes (i.e., allele combinations; for
a list of genotypes, see Suppl. Material). None of these alleles was identical
to that described by Hoyer and coworkers
(Hoyer and Hecht 2000;
Hoyer 2001), bringing the
total number of alleles to 61 in 67 strains. We plotted the running sum of
alleles found in our analysis versus 1/(running sum) of the number of strains
analyzed by us (Fig. 4) to
obtain an estimate of the maximum number of alleles present in cluster and
noncluster strains. The estimate is between 30 and 40 for cluster strains and
 90 for noncluster strains. Given that there are only very few alleles
which are common to both groups of strains (see below), the total number of
alleles within the species is likely to exceed 100. It is important to note,
however, that the PCR analysis underestimates the diversity at the locus,
because the nucleotide sequences of the repeat units appearing identical in
size by PCR were not always completely identical (see below).
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Figure 4 Running sum of the number of different alleles found in cluster strains
(triangles, dashed line) and in noncluster strains (squares, solid line)
plotted against 1/(running sum) of strains analyzed. The intercept of the
trend lines with the y-axis gives an indication of the number of
alleles that should be observed if an infinite number of isolates were
analyzed.
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GPG Cluster-Specific Characteristics of ALS7 Alleles
Few ALS7 alleles were present in both GPG cluster and noncluster
strains, and two alleles, 16a201 and 17a103, accounted for approximately half
of the loci in GPG cluster strains, but were rare in noncluster strains.
However, a large number of alleles occurred in only one or two strains
(Fig. 5A). Likewise, no GPG
cluster and noncluster strain had the same ALS7 genotype (see list of
genotypes in Suppl. Material), and only two genotypes occurred at frequencies
exceeding 10% in either group (17a103/16a201 in 11% of GPG cluster strains and
17a103/17a103 in 17% of GPG cluster strains).
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Figure 5 (A) Frequency of alleles, (B) frequency of different
sized tandem repeat domains, and (C) frequency of different types of
VASES regions for GPG cluster strains (solid bars) and noncluster strains
(hollow bars) analyzed. Allele frequencies were calculated assuming that all
strains tested were diploid; that is, where PCR analysis suggested the
presence of only one allele in a strain, the allele was assumed to be present
in two copies. The two borderline strains (HUN92 and HUN122; see a list of
strains in Suppl. Material) were included in the calculations.
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Differences between GPG cluster strains and other strains were more
apparent when the tandem repeat domain and the VASES region were analyzed
separately (Fig. 5B,C). The
majority (76%) of GPG cluster strains had between 14 and 17 tandem repeats.
Only 25% of noncluster strains had between 14 and 17 repeats. Conversely, 50%
of noncluster strains had  10 repeats, but only 17% of GPG cluster strains
had 10 repeats. In the VASES region, GPG cluster strains predominantly
(83% of strains) had short versions, limited to a maximum of two stretches of
VA/TSES repeats. In contrast, the majority of noncluster strains (71%) had
alleles with 3 stretches of VA/TSES units. All of these differences
between GPG strains and noncluster strains were significant (z test,
P < 0.01).
Diversity Is Generated Using Restrained Arrangements of Conserved
Units
We wanted to know whether the individual repeat units in the repetitive
parts of the gene contained high numbers of random mutations, or whether
allelic diversity was mainly brought about by rearranging units whose
sequences were largely conserved. The degree of conservation of the units'
sequences would be an indication of the functional importance of the sequence
information contained in the units. We therefore analyzed nucleotide
substitution rates for the individual units that make up the variable regions,
namely the 108-bp repeats of the tandem repeat domain and the 15-bp, 87-bp,
138-bp, and 141-bp repeats making up the VASES region. The rates were
calculated by comparing all available sequences of a given unit. This included
comparisons between units within the same allele and comparisons between units
in different alleles. We drew on both previously published (GenBank) and
genome database sequences (C. albicans 1161 and SC5314,
respectively), plus sequence data that we generated
(Table 1; the table also
contains data for nonrepetitive parts of the gene). We also generated
neighbor-joining trees based on the sequences obtained for each type of repeat
unit. These could highlight instances where sequences of units were fully, or
highly, conserved between strains or instances where sequences of units at
particular positions were more conserved than at other positions
(Fig. 6).
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Figure 6 Neighbor-joining trees based on genetic distances (uncorrected p) between
sequences of individual 108-bp repeats (A), 15-bp VA/TSES repeats
(B), 87-bp repeats (C), 138-bp repeats (D), and
141-bp repeats (E). Repeats are labeled as follows: The first digits
indicate the strain (up to four digits; names are abbreviated if necessary),
followed by a blank space, followed by the name of the allele (using the same
nomenclature as in Fig. 3; in
some cases the name is incomplete because only one VASES region was cloned and
sequenced, and the allele to which it belonged was not determined), followed
by a Cif the strain belongs to the GPG cluster, and an N for noncluster. In
all panels except B, the next digits give the position of the repeat
in the domain, that is, the first unit of its kind in the domain has the
number 1. The last unit of its kind in a domain is labeled with an L as the
final letter. In B, the position of VA/TSES repeats is designated by
two numbers: The first describes the number of the stretch of VA/TSES units in
the region, and the second describes the position of the repeat in the
stretch; this is followed by an L if it is the last unit in the stretch (e.g.,
2.3L is the third and last unit in the second stretch of VA/TSES repeats in
the region).
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Generally speaking, all repetitive units showed higher nucleotide
substitution rates than nonrepetitive parts of the gene
(Table 1). The higher
substitution rates were, however, caused mainly by pronounced differences
between a few types of a particular repeat unit. These types could in
themselves be highly conserved, that is, a given type of unit could be found
in different strains and alleles, and certain types were often associated with
particular positions in the repeat regions.
In the tandem repeat domain (Table
1; Fig. 6A), both
the first and last 108-bp units were distinct and highly conserved (the first
unit had a nucleotide substitution rate comparable to that of nonrepetitive
regions in the gene). The intermediate repeats varied more, with repeat units
belonging to the same allele and from similar strains (e.g., SC3514 and
RIHO13, both belonging to the GPG cluster) generally grouping together.
However, there were instances where distant strains had very similar or even
identical units (e.g., the SC5314 units 4, 6, and 11were identical to unit 6
in strain1161).
In the VASES region, there were only two types of the 15-bp repeats,
encoding VASES and VTSES, after which the region is named
(Table 1;
Fig. 6B). Both were as
conserved at the nucleic acid level as the nonrepetitive parts of the gene. In
addition, each of the stretches of VA/TSES repeats comprising the VASES region
started with VTSESVASES; this arrangement was thus also as conserved as the
nonrepetitive parts of the gene. The probability of finding this arrangement
at the beginning of all stretches if VASES and VTSES units were mixed randomly
is less than 0.001 (X2 test; the calculation took into account the
frequencies with which each of the two units were encountered).
Both the first and last 87-bp units of the VASES region
(Table 1; Fig. 6C) were distinct from
other 87-bp units (the only exceptions to this rule were first and last units
in laboratory strain 1161). The units had an apparent nucleotide substitution
rate comparable to that of the nonrepetitive regions of the gene. Even for the
intermediate 87-bp units, identical versions were present in different strains
(e.g., the units from W132, OD8824, CLB44, RIHO13, and SC5314 at the bottom
left of the tree in Fig.
6C).
Some versions of the 138-bp unit from distantly related strains were
identical (Table 1;
Fig. 6D). There are three
groups containing eight, seven, and five completely identical repeats in the
tree shown in Figure 6D. The
nucleotide substitution rates in these groups (1/138 x number of units
in group) are 0.001, 0.001, and 0.002, respectively, which is less than, or
equal to, the rates determined for nonrepetitive parts of the gene.
The neighbor-joining tree for the 141-bp repeats
(Fig. 6E) shows two clusters of
genetically similar units predominating at the first and last positions of the
VASES region; the last repeats are almost fully conserved if strain 1161 is
omitted from the analysis.
The Ratio of Nonsynonymous to Synonymous Mutations in Repeat Units
Indicates That They Are Under Selection
As a test of the significance of repeat regions in terms of functionality
of the encoded protein, we compared repeat and nonrepetitive regions in terms
of the ratio of nonsynonymous (amino acid-altering) to synonymous, (silent)
mutations (dN/dS ratio). This ratio is
held to be an indicator of natural selection
(Yang and Nielsen 2002). A
ratio of <1.0 indicates purifying selection; that is, amino acid
substitutions are generally selected against because the vast majority will
incur selective disadvantages. A ratio >1.0 indicates positive selection;
that is, amino acid substitutions have a high potential of offering fitness
advantages. A ratio equal to 1.0 indicates neutral evolution; that is, the
absence of selection.
If repetitive parts of the ORF contribute to the functionality of the
protein, their dN/dS ratio should be
significantly different from 1.0. For the 108-bp, 87-bp, 138-bp, and 141-bp
repeats, the ratios were 0.38, 0.51, 0.50, and 0.63, respectively. For the
15-bp repeat, only one nonsynonymous mutation occurred, and
dN/dS was therefore equal to infinity.
We determined that these values were significantly different from 1.0
(P < 0.001; ztest; see Methods for details of analysis).
In contrast, the dN/dS ratios of the
nonrepetitive regions of the ORF were 0.07 (5' domain), 0.45 (3'
domain upstream of VASES region), and 0.91 (3' domain downstream of
VASES region). Comparing the two sets of ratios, it is apparent that the
repeat units roughly matched adjacent nonrepetitive parts of the ORF in terms
of dN/dS ratio.
Alleles Present in the Same Strain Are More Similar to Each Other
Than Expected by Chance
When we analyzed allele combinations present in individual strains, we
discovered that the two alleles in a given strain were significantly more
similar (in regard to both tandem repeat domain and VASES region) than would
be expected by chance; that is, if all alleles could combine randomly. We
detected this by comparing all observed differences between alleles within the
same strains with a matrix describing the differences that would be expected
if all alleles could be paired randomly, taking allele frequency into
consideration (Table 2). When
GPG cluster and noncluster strains were considered separately, only the tandem
domains in the same strain were significantly more similar to each other than
expected by chance (Table
2).
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Table 2. Observed Differences Between Two Copies of the ALS7 Locus in
the Same Strain and Differences Expected from Random Mixing of Alleles
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These results differ from the random assortment expected, regardless of the
outcome of the current debate on C. albicans's mode of reproduction:
clonal and/or (para)sexual. If C. albicans still undergoes
(para)sexual recombination, alleles should be mixed in the process. If sexual
recombination were absent, each of the two present-day alleles in a given
strain should be the result of independent divergence of separate clonal
lineages, each originating from a single allele in the ancestor of the species
(Tibayrenc 1997;
Hull et al. 2000;
Welch and Meselson 2000).
Two scenarios would explain why alleles in a given strain are more similar
to each other than expected by chance. In the first, homozygous ALS7
alleles, generated by chromosome loss followed by duplication of the remaining
chromosome (Janbon et al.
1999) or mitotic recombination leading to duplication of a part of
the chromosome (Whelan and Soll
1982), subsequently diverge due to rearrangements and point
mutations in the repeat regions. In the second scenario, two different alleles
are combined in the same strain as a result of (para)sexual events (either
recent or in the past); they are then altered through rearrangements of
repetitive regions so that the alleles become more similar (alternatively,
possession of highly similar alleles favors [para] sexual events between
strains). It should be possible to distinguish between the two scenarios,
based on point mutations in the nonrepetitive parts of the gene. Because the
frequency of the events leading to homozygosity exceeds the rate of nucleic
acid substitution in nonrepetitive DNA
(Whelan and Soll 1982;
Janbon et al. 1999), under
scenario 1 the nonrepetitive parts of two copies of the gene in the same
strain should be identical or near identical. At the very least they should be
significantly more similar to each other than to nonrepetitive parts of the
gene in other closely related strains. Under scenario 2, one would expect to
find point mutations distinguishing the nonrepetitive regions of the two
alleles in the same strain. We therefore sequenced nonrepetitive parts of the
two alleles, 16a201and 17a103, in strain RIHO13. We found eight point
mutations, which distinguished nonrepetitive parts of the two alleles. We also
found that both alleles were more similar to the nonrepetitive part of the
gene in the closely related strain SC5314 than to each other (seven and five
substitutions, respectively, separated the two alleles from the SC5314
sequence; both RIHO13 and SC5314 are GPG strains). It thus seems unlikely that
alleles 16a201 and 17a103 are derived from a recent common ancestral
allele.
Detection of ALS7 mRNA in C. albicans
The presence of ALS7 mRNA in RNA samples from C. albicans
strains grown in YEPD broth was investigated by two-step reverse
transcriptase-polymerase chain reaction (RT–PCR) amplification of the
VASES region (Fig. 7A). The RNA
samples from all twelve strains tested contained ALS7 mRNA of the
sizes expected based on characterization of the alleles, indicating that none
of the various ALS7 alleles were pseudogenes. Amplification of a
C. albicans EF1B fragment (spanning an intron) from these RNA
samples, and PCR amplification without the reverse transcriptase step,
indicated that amplicons were not due to DNA contamination of the samples.
ALS7 mRNA could also be detected in cells grown under a number of
other growth conditions. ALS7s amplicons were present after one-step
RT–PCR amplification of RNA samples from cluster strains AU90 and CLB42
and noncluster strains CLB44 and OD8824 grown on defined (YNB) medium, using
primers MC5spF and MC5spR. Cells grown under these conditions were in the
yeast morphology. There was no obvious difference between the expression
levels in cluster and noncluster strains. ALS7 mRNA could also be
detected by RT–PCR of RNA from the same strains when they were induced
to form filaments by first growing them in YNB medium without glucose at pH
6.5 for 3 h and then adding glucose to a final concentration of 8mM
(Holmes and Shepherd 1988).
There was no obvious difference in ALS7 mRNA expression between cells
grown as yeast or as mycelia, even though hypha-specific expression has been
demonstrated for ALS3 and ALS8
(Hoyer et al. 1998a;
Murad et al. 2001).
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Figure 7 Expression of ALS7. (A) RT–PCR amplification of
VASES region of ALS7 mRNA from GPG cluster and noncluster C.
albicans strains. Cells were grown in YEPD broth, and RT–PCR used
primers MC5spF and MC5allF. Numbers on the left indicate DNA fragment
size in kb. (B) Northern hybridization verifying full-size
transcripts (7.2 kb and 7.8 kb) in Hun68 cells grown in YPD plus bovine serum.
Varying amounts of mRNA were loaded, and blots were hybridized with a 2.1-kb
probe generated from the HUN68 VASES region (the same bands were seen when the
tandem repeat domain was used as a probe; data not shown), stripped, and
reprobed with a 1.1-kb actin probe. Exposure time was 60 min.
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ALS7 amplicons were also obtained after RT–PCR amplification
of RNA obtained from swabs of oral candidosis lesions taken from five
different patients. This indicated that ALS7 mRNA was expressed in a
situation where C. albicans is causing human disease. In each case,
transcripts corresponding to multiple noncluster alleles were present.
In all of the above experiments, the variable VASES region of the
transcript was amplified (using primers MC5spF and MC5spR) in order to confirm
that each allele was expressed. Although we were unable to generate a product
comprising the entire predicted transcript (even in CLB44 with a short 3a301
allele), it was possible to amplify various portions of ALS7 mRNA by
RT–PCR, including the N-terminal portion of the gene (nt 401–1319
of the 6006-nt ORF, using primers MC5N2F and MC5NR). This indicated that mRNA
of the expected size was present in C. albicans strains. We directly
confirmed the presence of full-length ALS7 mRNA in two strains,
SC5314 and Hun68, using Northern blots and probes that hybridized with either
the VASES region or the tandem repeat domain. Both probes hybridized to RNA of
a size expected for the full-length transcript. However, transcript
concentrations were low in both strains under three different growth
conditions tested (YPD medium, RPMI medium, and YPD medium with bovine serum
added; the latter two conditions induce ALS3 expression
[Hoyer et al. 1998a]). Based
on comparisons of the ALS7 hybridization signal intensity with that
of actin mRNA (see Fig. 7B for
example), taking into account effectiveness of transfer of mRNA from gel to
filter, and the size of hybridizing region of the mRNA molecules, we estimate
that the concentration of ALS7 mRNA was approximately 1%–2%
that of actin mRNA. This is comparable to the basal (uninduced) mRNA
concentration of an S. cerevisiae gene with a comparable function,
the AGA1 gene encoding the anchorage subunit of agglutinin
(Roy et al. 1991;
http://genome-www4.stanford.edu/cgi-bin/SGD/SAGE/querySAGE).
We note that detection of transcript by Northern hybridization required the
use of long probes and the loading of large amounts of mRNA (see Methods).
Initial attempts to detect ALS7 mRNA in a variety of strains by
Northern hybridization using shorter probes and loading less RNA failed,
although ALS3 message was detected (data not shown). This indicates
that low levels of transcription of ALS7 in cultures were not
restricted to SC5314 and Hun68.
Expressed Reverse ORFs Within ALS7
Analysis of available nucleotide sequences of ALS7 genes (SC5314,
1161, two alleles of RIHO13, and partial sequence of var1.7) revealed that
each contained ORFs, initiated with an ATG, in the opposite orientation to
ALS7. These were located almost entirely within the tandem repeat
domain. SC5314 had one reverse ORF of 517 amino acids (ALS7 nt
2807–1254; see Fig. 1).
The two alleles for GPG cluster strain RIHO13 each contained two adjacent
reverse ORFs that were in-frame and separated by a stop codon and 6 bp. The
lengths of the ORFs were 429 and 145 aa in allele 17a103, and 333 and 217 aa
in allele 16a1201. Noncluster strain var1.7 had one reverse ORF of 157 aa, and
noncluster strain 1161 had two adjacent, in-frame, reverse ORFs of 153 and 73
aa. An amino acid sequence alignment of all putative proteins encoded by the
reverse ORFs showed that they had a high degree of homology. Excluding
deletions, the average number of amino acid substitutions between two
sequences was 19±14, and the maximum was 44.
To determine whether the reverse ORFs could encode functional proteins, we
measured dN/dS ratios and investigated
their amino acid sequence similarity to other proteins. The
dN/dS ratio among the five reverse ORF
sequences was 0.67, significantly different from 1.0 (P < 0.001,
binominal test; see Methods for analysis details). In a BLAST search against
nonredundant GenBank protein sequences, the best match between the reverse
ORFs was in most cases C. albicans Als7p. The exceptions were the
reverse ORFs of var1.7 (Als7p was the second best match), the second reverse
ORFs in both RIHO13 alleles (no match with Als7p but with other proteins), and
the second ORF in 1161 (no significant match with any protein in the
database).
Further analysis suggested that the reverse ORFs could be transcribed. In
SC5314 there was a TATA box positioned 106 nt upstream of the reverse ORF, and
two CAAT boxes –118 and –146 relative to the start codon.
Significantly, both the TATA box and one of the CAAT boxes lie in the last
108-bp repeat of ALS7, which is distinct from other 108-bp repeats
(see above), and only the last 108-bp repeat has these features. There was
also a motif, TAG...TATGT...TTT, 44–46, 53–57, and 102–104
nt, respectively, downstream of the ORF stop codon, that matched the consensus
for S. cerevisiae transcription termination sequences
(Zaret and Sherman 1982). The
sequence TATAAA, 193 nt downstream of the tripartite transcription termination
signal, could possibly function as a polyadenylation motif. Similar promoter,
termination, and polyadenylation motifs were also present in both RIHO13
alleles and var1.7 (in the latter strain we did not sequence the region where
the termination and polyadenylation signals are predicted to lie, but they are
likely to be present because they are located in a nonrepetitive, highly
conserved part of the gene). Noncluster strain 1161, whose last 108-bp repeat
differs from that found in the other strains (see above) had only the
transcription termination signals downstream of the combined ORFs, but no TATA
or CAAT box upstream of the ORFs.
We investigated whether mRNAs corresponding to the reverse ORFs were indeed
present in cells, using four GPG cluster strains (SC5314, RIH013, Jam-2c,
OD8807) and four noncluster strains (Var1.7, W53, gaymc-c, YsU123).
RT–PCR was carried out as a two-step process. cDNA was synthesized from
RNA in two reactions, one using primer TRF which bound to, and copied,
ALS7 mRNA, the other using TRR which bound to, and copied, the
reverse ORF. As the predicted 5' end of the reverse ORF is within the
tandem repeat domain, primer TRF bound several times within this domain. TRF,
however, was specific for ALS7 mRNA and would only bind to the
reverse ORF mRNA with 7/23 mismatches. Likewise, TRR was specific for the
reverse ORF mRNA and would only bind to ALS7 mRNA with 11/24
mismatches. The cDNAs were then PCR-amplified using TRF and TRR as primers.
There was a ladder of amplicons in both the ALS7-specific and the
reverse ORF-specific cDNAs in all of the strains (results for four strains are
shown in Fig. 8), indicating
that both ALS7 and the reverse ORF mRNA were expressed in these
cells. The distance between the ladder bands was 108 bp, which is equivalent
to the repeat length in the tandem repeat domain. The lack of the two smallest
reverse ORF amplicons from SC5314 was due to point mutations in two of the
tandem repeats in this strain that prevented the TRF oligonucleotide binding
and priming amplification.
View larger version (81K):
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|
Figure 8 RT–PCR amplification of ALS7 and reverse ORF mRNA from
C. albicans strains. ALS7 cDNA was synthesized from RNA
using primer TRF and reverse ORF cDNA was synthesized from RNA using primer
TRR. cDNA was amplified by PCR using primers TRF and TRR. Numbers on the
left indicate DNA fragment size in base pairs.
|
|
We were not able to detect the reverse ORF mRNA on Northern blots. These
blots were probed with DNA probes corresponding to the entire tandem repeat
region (1.6 kb and 2.6 kb respectively in strains Hun68 and SC5314), which
should hybridize to both forward and reverse message. Taking into account the
strength of the signal for the ALS7 message at various amounts of
mRNA loaded, the concentration of reverse ORF transcript must be less than 15%
of that of the forward transcript.
|
DISCUSSION
|
|---|
Evidence That ALS7 Is a Hypermutable Contingency Locus
Highly variable surface proteins are found in many microbes, in particular
in pathogens such as Plasmodium, Giardia, and Trypanosoma
(Duncan et al. 1991;
Ramasamy 1998;
Ayala and Rich 2000;
Adam 2001). The genes encoding
these proteins have been termed `hypermutable contingency genes', and the
major mechanism underlying the protein variability is rearrangement of
repeat-containing parts of their ORFs
(Duncan et al. 1991;
Moxon and Thaler 1997;
Barry and McCulloch 2001;
Bridges 2001). ALS7
meets the criteria for hypermutable contingency loci in that (1) it is part of
a family of surface proteins (Hoyer
2001), (2) it contains repeat units which make it potentially
highly mutable, and (3) rearrangement of conserved units in repeat regions
generates allelic diversity far in excess of the diversity in both
nonrepetitive parts of the ALS7 ORF and far in excess of the
diversity reported for other C. albicans ORFs
(Bougnoux et al. 2002).
Biological Functions of ALS7 and ALS7
Variability
Hypermutable contingency loci generate phenotypic variants with a frequency
of approximately 10–3 switch/organism/population
doubling—the frequency is fairly constant among different microbes of
both eukaryotic and prokaryotic origin
(Barry and McCulloch 2001). The
resulting repertoire of phenotypes is believed to assist in the survival of
the organism by allowing it to rapidly respond to, and exploit, alterations in
unpredictable and/or hostile environments
(Duncan et al. 1991;
Moxon and Thaler 1997;
Barry and McCulloch 2001;
Bridges 2001). In the pathogens
Giardia, Plasmodium, and Trypanosoma, the biological role of
hypermutable surface proteins has been the subject of considerable study. It
is generally agreed that evasion of the immune system is one function of these
proteins. This is achieved by escaping an immune response through alteration
of immunogenic epitopes and also possibly by interfering with maturation of
antibody responses, because of cross-reactivity between different forms of
repeat domains (Duncan et al.
1991; Nash 1997;
Ramasamy 1998;
Adam 2001). Ramasamy
(1998) also discusses a
possible function as superantigens. However, it is also widely believed that
immune evasion is not the only function of these proteins
(Nash 1997;
Ramasamy 1998;
Adam 2001). Nash
(1997) suggests that the
nature of the genes encoding these proteins allows the rapid generation and
selection of sets of clonal lines which are best suited to adhere to different
host surfaces. Nash (1997)
also points out that highly variable surface proteins with repeat domains are
not restricted to pathogens but can be found in free-living amoeba, supporting
a role in adhesion, or other functions not related to immune selection. There
is also evidence of an important role of repeat-containing surface proteins in
adhesion of bacteria when colonizing human hosts (McNab et al.
1996,
1999;
Roos and Jonsson 2002).
Our analysis of ALS7 indicates a main role in adhesion rather than
in immune evasion for several reasons. First, expression levels seem to be low
under many growth conditions, and we would expect an `immunological decoy' to
be expressed at higher rates. Second, the constraints restricting the
arrangements of most of the repeat units would suggest a role beyond that of a
decoy. Third, if the diversity-generating repeats had mainly a decoy function,
one might expect them to have higher
dN/dS ratios than the remainder of the
ORF, because amino acid substitutions in them would increase diversity and
could be expected to have a high probability of enhancing fitness. This is not
what we observed. Rather, dN/dS ratios
of repeat units roughly matched those of adjacent nonrepetitive parts of the
ORF, suggesting that the parts of Als7p encoded by the repeats are under
similar functional restraints as the parts encoded by adjacent nonrepetitive
units. A role of Als7p in adhesion would also be in keeping with both
functional and expression data on other Als proteins
(Gaur and Klotz 1997;
Chandra et al. 2001;
Hoyer 2001;
Fu et al. 2002).
Similar arguments regarding biological role may apply to the putative
additional proteins, encoded by the reverse ORFs within the gene. However, we
have no direct evidence that the reverse ORFs are translated. Only the reverse
ORFs' low dN/dS ratio, and the
significant similarity between their predicted products and Als7p, suggest
that the reverse ORFs could encode proteins, and that their role may be
related to that of the Als proteins.
A case can be made that maintenance of particular arrangements of repeat
units in the ALS7 gene is driven by continuous selection in the human
host, and that therefore the role of the gene and its product(s) is specific
to the interaction between C. albicans and the human host. This
argument is based on differences at the ALS7 locus between the two
laboratory strains 1161 and SC5314 on the one hand and the remaining isolates,
none of which were propagated for prolonged periods outside the host (our
patient isolates are stored at –70°C and were not subcultured
extensively before analysis), on the other. The ALS7 allele of strain
1161 often violates restraints on the arrangements of repeat units common to
other strains (as illustrated in Fig.
6) and is missing transcription initiation signals for the reverse
ORFs. Likewise SC5314, although a GPG cluster strain, has a VASES region that
does not produce the GPG-specific AFLP product present in 90% of GPG patient
isolates. It is also unusual in that it only has a single reverse ORF. A
simple explanation for these findings is that the two laboratory strains,
while being subcultured in various laboratories, underwent modifications in
hypermutable sequence regions that would have been selected against in the
host. If so, it should be possible to generate, in the laboratory, clonal
lineages of a given strain with different ALS7 alleles and use
competition experiments between these lineages in an animal model to learn
more about the biological function of the repeat units and their
arrangements.
Transcriptional Control of ALS7 Expression?
Expression of ALS7 mRNA could be detected in C. albicans
cells cultured under a variety of conditions and in cells obtained directly
from oral candidosis lesions. It would be interesting to see whether
ALS7 mRNA can be detected in oral samples taken from people colonized
with C. albicans without clinical signs of candidosis (as has been
shown for some of the secreted aspartic proteinase genes;
Naglik et al. 1999). The level
of ALS7 expression in vitro was low and comparable to that of a gene
encoding a related yeast cell surface protein under uninduced conditions. It
seems likely, therefore, that under our in vitro experimental conditions,
ALS7 transcription was repressed. Although we were able to detect the
reverse ORF mRNA by RT–PCR, we were unable to detect the mRNA directly
on a Northern blot. This indicates that the reverse ORF mRNA is present in
cells, but at lower concentrations than ALS7 mRNA. The possibility
that it may play a role in the control of ALS7 expression
(Vanhee-Brossollet and Vaquero
1998) requires further investigation.
If the function of Als7p is in adhesion, why is its expression regulated to
such a low level under a variety of growth conditions? A likely explanation is
that Als7p has a highly specialized function. C. albicans, like many
other microorganisms, possesses several putative adhesins that enable it to
adhere to, and colonize, a variety of surfaces
(Cannon and Chaffin 1999). Many
of these adhesins are large-molecular-mass glycoproteins, the production of
which puts a metabolic burden on the cells. Constitutive Als7p expression may
constitute a higher cost to the cell than mechanisms regulating its synthesis.
An additional benefit of such regulation is that it will minimize the
development of neutralizing antibodies by the host. The idea of a specialized
function for ALS7 would make sense, given that it is part of a family
of related adhesins and given that several other ALS genes are known
to be transcriptionally activated by different sets of stimuli (Hoyer et al.
1998a,b;
Chandra et al. 2001;
Murad et al. 2001).
Evidence That Als7p Contributes to the Success of GPG Cluster
Strains
We began this study with the aim of identifying genes that contribute to
C. albicans pathogenicity, by studying polymorphisms specific to GPG
cluster strains—which cause infections significantly more often than
other C. albicans genotypes. The low genetic diversity among GPG
cluster strains suggests that these strains could be of fairly recent origin
(Schmid et al. 1999).
Therefore it is possible that some GPG cluster-specific polymorphisms are not
related to pathogenicity but are present merely because all of these strains
share a recent ancestor. GPG cluster-specific features of the ALS7
gene, however, are unlikely to be a result of derivation from a recent common
ancestor. Given that cluster strains have the same set of repeat elements as
noncluster strains, and given the high rates of intragenic recombination in
repeat regions (10–2 to
10–3 per generation;
Levinson and Gutman 1987;
Schug et al. 1998;
Ayala and Rich 2000), it is
unlikely that ancestral features of the gene would have been maintained in
cluster strains, unless they were continually selected for (this is also
supported by the analysis of the laboratory strains, especially 1161); that
is, unless they and the ALS7 gene that contain them are contributing
to the success of these strains as pathogens.
Related to this we would also note that, although we were able to
demonstrate genetic differences between GPG cluster strains from different
geographic regions using the Ca3 probe
(Schmid et al. 1999), we found
no indication that isolates from different geographic regions differ in regard
to their ALS7 genes (data not shown). Unless cluster-specific
features of the ALS7 genes are under constant selection, it is
difficult to explain why geographically separated and genetically distinct
lineages of GPG cluster strains are similar in regard to a hypermutable
sequence.
If particular features of the ALS7 gene product(s) contribute to
the success of cluster strains, they probably do not do so in isolation. There
is no apparent reason why noncluster strains could not generate alleles at
least very similar to those present in cluster strains. If so, the low
prevalence of such alleles in noncluster strains must mean that their
possession does not confer a selective advantage on noncluster strains, but
only on cluster strains. Therefore there must be cluster-specific
polymorphisms in other genes, which are required for the ALS7
polymorphisms to convey a selective advantage. Polymorphisms in other
ALS genes would be obvious candidates. Sequence and functional
analyses of allele repertoires of the different ALS genes in
individual patient isolates and functional analysis of strains with
experimentally altered sets of allelic repertoires could be a valuable
approach to elucidate the function of this gene family.
|
METHODS
|
|---|
Strains
Strains used are listed in Supplemental Material (available online at
http://www.genome.org).
Two overlapping sets of patient isolates (25 GPG cluster strains and 16
noncluster strains) were selected to represent a collection of 266
infection-causing isolates from 12 geographic regions in six countries,
genotyped using the probe Ca3 (Schmid et
al. 1999). Strains for set one were selected using the quartet
method (Schmid et al. 1999) as
follows: The Ca3-based genetic distances were calculated for all possible
quartets containing two GPG cluster strains and two noncluster strains from
the collection. For each GPG cluster strain, the number of times it was
closest to another cluster strain in these quartets was recorded. Likewise,
for each noncluster strain the number of times it was closest to the other
noncluster strain in these quartets was also recorded. The higher the quartet
value, the more distinct the GPG cluster strain was from noncluster strains
and the more distinct the noncluster strain was from GPG cluster strains.
After strains were thus ranked by quartet values, high-ranking GPG cluster and
noncluster strains from each geographic region were chosen as model strains.
Set 2 was chosen on the principle that the genetic distances (based on Ca3
finger-prints) between all GPG cluster strains and the 25 model GPG cluster
strains were as small as possible, and the genetic distances between all
noncluster strains and the 16 noncluster model strains were also as small as
possible, using algorithms developed by Holland
(2001). Because different
geographical regions were represented in the collection by different numbers
of isolates, a correction was made to ensure that each region had the same
impact in the process. This was achieved by weighting the contribution of each
strain to the distance calculations so that it was inversely proportional to
the number of isolates from the region. Laboratory strain SC5314 was added to
the sets as the 26th GPG cluster strain (based on Ca3 fingerprinting data;
Giblin et al. 2001).
General Molecular Biology Methods
Methods were generally based on those described by Ausubel et al.
(1994). All PCR reactions were
performed in a final volume of 20 µL containing 1 U Taq DNA
polymerase (QIAGEN), 4 µL of Q-buffer, and 1x PCR buffer supplied by
the manufacturer (QIAGEN), 10 pmol of each primer, 200 µM of each dNTP
(Roche Diagnostics), and 10–100 ng DNA. PCR conditions were varied
according to the primers used (a list of primers can be found in the Suppl.
Material), following the guidelines listed in Ausubel et al.
(1994). All PCR protocols
included a final 5-min extension step at 72°C. In PCR amplifications using
primers MC5pf and MC5outpr, a `touchdown' protocol was used
(Don et al. 1991); that is,
the annealing temperature was 10°C above the optimum annealing temperature
for cycles 1–6 and 5°C above the optimum for cycles 7–12 (the
total number of cycles was 36).
DNA Extraction, AFLP Analysis, and Isolation of the MC5-c AFLP
Product
C. albicans DNA was extracted from strains using the method of
Scherer and Stevens (1987) and
was used as a template for AFLP as described by Vos et al.
(1995). The AFLP used
MseI adapters only, and primers M-C (preselective amplification) and
M-CTC (see list of primers in Suppl. Material). AFLP products were separated
by electrophoresis in 5% (w/v) denaturing polyacrylamide gels
(Maxam and Gilbert 1980) and
visualized by silver staining (Promega Silver Staining System, Promega). The
GPG cluster-specific band MC5-c was excised from the gel, re-amplified using
Primer M-CTC, gel purified, cloned into pGEM-T® (Promega), and sequenced,
using universal primers M13F and M13R, on an ABI Prism 377 sequencer (Perkin
Elmer).
Characterization of VASES Regions
Initially the entire region was amplified using primers MC5pf and MC5outpr,
and its size assessed on 0.8% agarose gels, using 1Kb Plus DNA Ladder
(Invitrogen) and previously characterized VASES regions of known size as
molecular weight standards. The number of units of 366 bp (87-bp repeat +
138-bp repeat + 141-bp repeat) that would equate to this size, taking into
consideration that at the beginning of the region there will be an 87 + 138-bp
unit and at the end a 141 + 87-bp unit was calculated (see
Fig. 1 for general organization
of VASES regions). The PCR product was then used as a template for PCR
amplifications with primers MC5VApf and MC5VApr, and primers MC5spF and
MC5Vapf, to confirm the proposed structure and to determine the size of the
various stretches of 15-bp VA/TSES units in that region.
Calculation of dN/dS Ratios
The software PAML (Yang
1997) was used to determine the ratio of synonymous/nonsynonymous
changes (dN/dS) between pairs of
sequences (Yang and Nielsen
2000). PAML can only calculate ratios for sequences without
deletions, and in case of the reverse ORF only a 135-bp deletion-free region
present in all ORFs was therefore used for analysis. Because two identical
sequences or two sequences differing only by nonsynonymous mutations will
generate an undefined dN/dS value
(division by zero), such values cannot be combined with others to generate a
meaningful average; all averages reported in the text therefore exclude such
pairs. In order to include as many values as possible in statistical tests
aimed at determining whether the average
dN/dS for a sequence was  1.0, the
following approach was used: For each pair of sequences compared, the
resulting dN/dS was categorized as
<1, =1, or >1 (cases in which there were only nonsynonymous changes can
therefore be included as >1 in the analysis, and only cases where no
mutation distinguishes the two sequences are excluded). We then determined
whether the frequency of values >1 was significantly different from the
frequency of values <1, using the z test or binominal test. A
significant difference indicates that the values were not symmetrically
distributed around a value of 1.0, and that therefore the average ratio
observed was significantly different from 1.0.
RT–PCR and Northern Hybridization
RNA was extracted from C. albicans cells grown in media as
described in the Results section or from C. albicans cells isolated
directly from oral candidosis lesions. The lesions of patients presenting with
oral candidosis at University of Otago School of Dentistry clinics were wiped
with a sterile swab. All patients were HIV-negative. Ethical approval for the
procedure was obtained from the Ministry of Health Otago Ethics Committee
(protocol no. 99/11/095). The swab was immediately vortex mixed in 2 mL YEPD
broth, and a portion of the liquid was diluted and plated on YEPD agar
containing chloramphenicol (20 µg/mL) to confirm and quantify C.
albicans presence. The yeasts in the remaining YEPD broth were harvested
by centrifugation (3,000 g, 5 min) prior to RNA extraction. Total RNA
was isolated from C. albicans cells (1.8 x 109
cultured cells or all remaining cells isolated from oral swab) by the hot
phenol method of Schmitt et al.
(1990). For RT–PCR, RNA
samples were treated with DNAase I (DNA-free kit, Ambion) to remove traces of
DNA from the RNA samples. Purity of RNA samples was confirmed by RT–PCR
amplification using primers EF1BF and EF1BR that spanned the intron in C.
albicans EF1B. DNA present in the sample (containing the intron) gave an
amplicon of 891 bp; RNA gave an amplicon of 526. RT–PCR was carried out
as either a one-step reaction (OneStep RT–PCR kit, QIAGEN) or a two-step
process. The first step in the two-step process was reverse transcription of
RNA samples using Super-Script reverse transcriptase (Invitrogen) and a
specific primer or a poly-T primer. The RNA was removed from the cDNA with
RNase H, and the cDNA was amplified using Taq polymerase and specific primers.
For Northern hybridization, serial dilutions of mRNA (0.5–20µg)
obtained from total RNA using the Sigma GenElute mRNA isolation kit were
loaded on 1%agarose gels, containing 6% formaldehyde. RNA was transferred to
nylon membranes (Roche). Each blot was hybridized first with a probe
comprising the VASES region, then with a probe comprising the actin gene, and
finally with a probe comprising the tandem repeat domain. Double-stranded DNA
probes for each Northern blot were prepared from DNA from the corresponding
strain by DIG labeling with random priming (the double-stranded tandem repeat
domain probe would therefore hybridize with both forward and reverse
transcripts). Detection was via luminescence (DIG Nonradioactive Nucleic Acid
Labeling and Detection System; Roche). A lane of DIG-labeled RNA size markers
(1.4 kb–6.9 kb) was included on every blot.
|
Acknowledgements
|
|---|
This work was funded by a Marsden grant from the Royal Society of New
Zealand. Sequence data for C. albicans SC5314 was obtained from the
Stanford Genome Technology Center website at
http://www-sequence.stanford.edu/group/candida.
This sequencing was accomplished with the support of the NIDCR and the
Burroughs Wellcome Fund. We thank Mr. N. Firth (Department of Stomatology,
University of Otago) for obtaining the oral candidosis samples described in
this paper, and Lois Hoyer and John Tweedie for helpful comments on the
manuscript.
The publication costs of this article were defrayed in part by payment of
page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 USC section 1734 solely to
indicate this fact.
|
Footnotes
|
|---|
Article and publication are at
http://www.genome.org/cgi/doi/10.1101/gr.1024903.
[Supplemental material is available online at www.genome.org. The sequence
data from this study have been submitted to GenBank under accession numbers
AY170875
[GenBank]
–AY170888.]
4 Present address: Allan Wilson Centre for Molecular Ecology and
Evolution, Massey University, Palmerston North, New Zealand. 
5 Corresponding author.
E-MAIL
J.Schmid{at}massey.ac.nz;
FAX 64 (6) 350-5688.
|
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ABSTRACT
RESULTS
