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Previous Article | Next Article 
Journal of Bacteriology, October 2000, p. 5730-5736, Vol. 182, No. 20
Department of Microbiology and Institute of Cancer
Research, Columbia University, New York, New York 10032
Received 22 May 2000/Accepted 24 July 2000
The fungal pathogen Candida albicans is naturally
diploid, and current gene disruption strategies require two successive
transformations. We describe here a genetic construct
(UAU1) for which two copies may be selected. Insertion of
UAU1 into one genomic site, after a single
transformation, allows selection for segregants with two copies of the
insertion. Major classes of segregants are those carrying homozygous
insertion mutations and allelic triplications, which have two insertion
alleles and a wild-type allele. Thus nonessential and essential genes
may be distinguished rapidly through PCR tests for homozygosis and
triplication. We find that homozygous mutations may be isolated at
three nonessential loci (ADE2, RIM20, and
YGR189), while only allelic triplications were found at two
essential loci (SNF1 and CDC28). We have
unexpectedly isolated homozygous mutants with mutations at
CDC25; they are viable but defective in filamentation on
serum-containing medium. The UAU1 cassette is thus useful
to assess rapidly the essentiality of C. albicans genes.
Candida albicans is an
opportunistic fungal pathogen. It has been of experimental interest for
two main reasons. First, it is a significant pathogen that causes oral
mucosal infections, vaginitis, nosocomial bloodstream infections, and a
variety of deep tissue infections (26). Therefore, many
experimental studies have focused on pathogenesis, drug resistance, and
analysis of prospective drug targets. Second, it is a distant cousin of
the most well-characterized unicellular eukaryote, Saccharomyces
cerevisiae, so that the function of a C. albicans
gene may be suggested by its role in S. cerevisiae.
Yet, surprisingly, C. albicans may use gene products and
regulatory pathways in novel ways (4, 16, 17, 28, 34, 37).
The contrast between C. albicans and S. cerevisiae can provide unique insight into regulatory mechanisms, interpathway relationships, and general aspects of eukaryotic biology.
Molecular genetics has played an increasingly prominent role in studies
of C. albicans, particularly with the partial
genomic sequence as a tool for gene discovery. However, genetic
methods are cumbersome for two reasons (30). First, C. albicans strains are diploid (or of higher ploidy) and there is no
meiotic division, so that gene disruption mutants must be constructed
through two successive transformations. Second, with current
methods, disruption of one allele may be straightforward, but
disruption of the second allele is infrequent if the second disruption
construct is homologous to the first. These problems can make it
difficult to construct a homozygous mutant and to determine whether a
gene is essential for growth.
We describe here a genetic strategy that circumvents these
difficulties. It yields homozygous insertion mutations after a single
transformation. The strategy provides a rapid test for essential genes
and should thus accelerate drug target validation. In addition, the
strategy can provide a preliminary assessment of mutant phenotypes, and
it lends itself to large-scale analysis of gene function.
Plasmids.
The UAU1 (ura3-ARG4-ura3)
(Fig. 1A) cassette is carried in
plasmid pBME101, which was constructed as follows. Plasmid
pRS424ARG4-URA3-BH1 (4) was digested with
BsmI and SpeI, overhangs were blunted with Vent
polymerase, and blunt ends were ligated to form plasmid pBME98. This
plasmid was then digested with XhoI and SmaI,
filled in, and religated to remove a polylinker ClaI site to
yield plasmid pBME99. Five hundred base pairs of the 3' end of
URA3 was amplified from pRS424ARG4-URA3-BH1 with primers
Kpn5' and Xho3'-500 and ligated into the XhoI and
KpnI sites of pBME99, generating plasmid pBME101.
(Oligonucleotide sequences are listed in Table
1.)
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
A Single-Transformation Gene Function Test in Diploid
Candida albicans
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
View larger version (20K):
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FIG. 1.
Genetic properties of UAU1. (A) Conversion of
UAU1 to URA3. The UAU1 marker (top)
comprises an intact ARG4 gene flanked by URA3
deletion derivatives ura3 
3' and ura35'. The
URA3 segments are nonfunctional, so the UAU1
cassette confers an Arg+ Ura
phenotype. The
URA3 segments share 530 bp of homology and can thus
recombine to yield an intact URA3 gene. Recombination
excises the ARG4 gene and results in an Arg
Ura+ phenotype. (B) Anticipated use of UAU1 to
select for homozygous mutants (double-disruption selection). One allele
of a gene (here ADE2) is disrupted with a UAU1
insertion through transformation with selection for an Arg+
phenotype. Growth of the transformant yields rare recombinant
segregants in which the UAU1 insertion allele is homozygous.
Such segregants may be selected after they undergo recombinational
excision within one UAU1 cassette to yield a unique
Arg+ Ura+ phenotype. (C) Outcome of
double-disruption selection with an ade2::UAU1/ADE2
strain. Genotypes were determined with PCR primers depicted at the top.
The ade2::UAU1/ADE2 strain yielded two classes of
Arg+ Ura+ segregants. One class (homozygote)
was Ade, grew into red colonies, and yielded diagnostic
ade2::UAU1 and ade2::URA3 PCR products
(Fig. 2A, lanes 1, 2, 5, 7, and 9). The other class (allelic
triplication) was Ade+, grew into white colonies, and
yielded ade2::UAU1, ade2::URA3, and ADE2
PCR products (Fig. 2A, lanes 3, 4, 6, 8, and 10). The latter class is
depicted as a trisome as an example, but it may arise through
translocation, tandem duplication, or other genetic rearrangements.
TABLE 1.
Oligonucleotide sequences
XhoI-CDC28 and a
cdc28::UAU1 PCR product produced from template plasmid
pBME101 and primers cdc28 5'DR and cdc28 3'DR.
Strains.
The C. albicans strains (Table
2) are derivatives of strain BWP17
(ura3:: 
imm434/ura3::imm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG), which was
derived from strain CAI4 (7) as described previously
(44). The following UAU1 disruption heterozygotes
were constructed through transformation of strain BWP17 with the
restriction fragments indicated and selection for Arg+
transformants: strain BMY7, the NotI/EcoRV insert
fragment from plasmid pBME 102; strain BMY18, the
PvuII insert fragment from plasmid pBME105; and strain
BMY22, the PvuII insert fragment from plasmid pBME108. The
following UAU1 disruption heterozygotes were constructed
through transformation of strain BWP17 with the PCR product indicated
and selection for Arg+ transformants: strain BMY16, PCR
with primers cdc25 5'dr and cdc25 3'dr on template pBME101; and strain
DAY151, PCR with primers YGR189 5'DR and YGR 189 3' DR on
template pBME101. The UAU1 heterozygote BMY17 was
constructed by homologous integration of the UAU1 cassette into URA3 sequences of a rim20::URA3
allele. Specifically, we transformed strain DAY18 (originally called
Enx-het1 [44]), of genotype
rim20::URA3/RIM20
ura3::imm434/ura3::imm434
his1::hisG/his1::hisG arg4::hisG/arg4::hisG, with
the PCR product of template pBME101 amplified with primers 5-Detect and
3-Detect, selected for Arg+ transformants, and screened for
those that became Ura. (This integration reaction is the
reverse of the excision reaction diagrammed in Fig. 1A.) Integration of
UAU1 cassettes in heterozygotes was confirmed through
genomic DNA PCRs with the primer pairs listed in Table 2.
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Media and growth conditions. C. albicans and S. cerevisiae were routinely cultured in YPD plus uridine (2% Bacto Peptone, 1% yeast extract, 20% dextrose, and 80 µg uridine per ml). Selection was done on SD synthetic medium (6.7% yeast nitrogen base plus ammonium sulfate, without amino acids, and with 2% dextrose); auxotrophic supplements were added at standard concentrations (14) except that uridine was added at 80 µg per ml.
PCR detection. Genotypes at each locus were determined through PCR assays with the primers indicated in Table 2. Flanking primers (illustrated by Ade2amp5 and Ade2amp3 in Fig. 1C) were used to detect wild-type alleles and URA3 insertion alleles. However, we found that detection of UAU1 insertion alleles was unreliable with flanking primers, presumably because of the more efficient synthesis of the smaller products from wild-type and URA3 insertion alleles. Therefore, we used a flanking primer and an internal ARG4 primer (illustrated by Ade2amp3 and Arg4det in Fig. 1C) in a second PCR to detect the presence of UAU1 insertion alleles. PCR was performed with total yeast genomic DNA as described previously (44). Reaction mixtures were typically heated to 94°C for 5 min, followed by 33 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 3 min. After a final incubation at 72°C for 10 min, reaction mixtures were stored at 4°C.
Determination of recombination rates.
Recombination rates
were determined by the method of Lea and Coulson, as described by
Rattray and Symington (33). A UAU1 insertion
heterozygote was streaked onto YPD and grown at 30°C for 2 to 3 days.
Nine entire single colonies were used to inoculate separate 3-ml YPD
broth cultures that were grown to saturation at 30°C. One milliliter
of saturated culture was washed, suspended in 1 ml of sterile water,
and diluted for plating. One hundred microliters of the undiluted
suspension was plated on SC-Arg-Ura, 100 µl of a 1/100 dilution was
plated on SC-Ura, and 20 µl (in a total volume of 200 µl) of a
1/10,000 dilution was plated on YPD. Colony counts of these plates were
used to determine the median mitotic recombinant frequencies
(Ura+/total or Ura+ Arg+/total) for
each strain. Recombination rates (events per cell per generation) were
calculated according to the formula rate = (0.4343 × median
frequency)/(log N 
log N0),
where N is the total number of cells in the culture and
N0 is the initial number of cells (one cell)
that gave rise to the culture (33).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Selection for homozygous mutants.
We constructed a gene
disruption cassette, UAU1 (ura3-ARG4-ura3), that
can exist in two states (Fig. 1A). The cassette includes three
segments: ura33', a nonfunctional 3' deletion copy of
URA3; ARG4; and ura35', a nonfunctional 5'
deletion copy of URA3 that shares 530 bp of sequence
identity with the ura33' segment. The cassette expresses
ARG4, but not URA3, in this state
(UAU1). The 530 bp of homology between the ura3
segments permits recombination to yield an intact URA3 gene
and excision of ARG4. The resulting cassette expresses
URA3 but not ARG4. We reasoned that the cassette would permit identification of homozygous mutants (Fig. 1B). A heterozygous UAU1 insertion, introduced by transformation,
might occasionally become homozygous through gene conversion or a
mitotic recombination event. The homozygous mutant would be uniquely
capable of yielding segregants that express both ARG4
and URA3. Thus, homozygous mutants should be found among
Arg+ Ura+ segregants in a population
carrying a UAU1 insertion.
colonies. We grew 30 independent cultures of the
heterozygote and plated aliquots to isolate Ura+ and
Arg+ Ura+ segregants (Table 2).
Ura+ segregants arose a rate of 3.5 × 106 per division as uniformly white
Ade+ colonies. Arg+ Ura+ segregants
arose at a rate of 2.0 × 108 per division.
Among the Arg+ Ura+ colonies, 23% were red and
Ade. We used PCR analysis to determine the genotype of
one randomly chosen Arg+ Ura+ segregant from
each culture (Fig. 2B, lanes 1 to 10, and data not shown). We found 12 segregants of genotype ade2::UAU1/ade2::URA3, and these were phenotypically red and Ade. The 18 remaining segregants were of genotype
ade2::UAU1/ade2::URA3/ADE2 and were
phenotypically white and Ade+. These latter
Arg+ Ura+ Ade+ segregants
thus have three copies of the ADE2 locus; the third copy may
derive from trisomy or translocation (Fig. 1C). Our results indicate
that homozygous mutants can be isolated after a single transformation.
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Homozygote-trisome (HT) test for essential genes. Identification of essential genes in C. albicans is vital to assess prospective drug targets (30) and to consider more-refined functional tests (1). C. albicans genes have been considered essential if one allele can be disrupted but the second cannot. We reasoned that the UAU1 cassette might simplify and accelerate this assessment: a UAU1 insertion in nonessential genes yields both homozygous and triplication-bearing segregants, but a UAU1 insertion in an essential gene should yield only triplication-bearing segregants. Thus, we examined the consequences of UAU1 insertions in three likely essential genes: SNF1, CDC28, and CDC25.
Petter et al. have presented strong experimental evidence that SNF1 is essential for viability (29). We created a snf1::UAU1/SNF1 heterozygote and found that it produced Ura+ and Arg+ Ura+ segregants at rates similar to those for ADE2 and YGR189 insertions (Table 2). PCR genotyping of 30 independent Arg+ Ura+ segregants revealed that all were snf1::UAU1/snf1::URA3/SNF1 triplication derivatives (Table 2; Fig. 2D). Our failure to obtain snf1/snf1 homozygotes is consistent with the conclusion that SNF1 is an essential gene. CDC28 specifies a cyclin-dependent protein kinase, and activity of such kinases is vital for cell cycle progression in all eukaryotes (21). The C. albicans CDC28 gene complements corresponding defects in other yeasts (3, 35), so we inferred that C. albicans CDC28 may be essential. A cdc28::UAU1/CDC28 heterozygote produced Ura+ and Arg+ Ura+ segregants at rates comparable to those for other insertion heterozygotes; PCR genotyping revealed that 30 independent Arg+ Ura+ segregants were cdc28::UAU1/cdc28::URA3/CDC28 triplication derivatives (Table 2; Fig. 2E). These results support the idea that C. albicans CDC28 is an essential gene. The S. cerevisiae CDC25 gene is essential for viability; Cdc25p is an activator of Ras proteins (39). C. albicans CDC25 complements an S. cerevisiae cdc25 mutant (8), so we inferred that CDC25 may be essential in C. albicans as well. A cdc25::UAU1/CDC25 heterozygote produced Ura+ segregants at rates comparable to those for other insertion heterozygotes and produced Arg+ Ura+ segregants at a slightly lower rate (Table 2). PCR genotyping revealed that 2 of 30 independent Arg+ Ura+ segregants were of genotype cdc25::UAU1/cdc25::URA3 (Fig. 2F, lanes 3 and 8); the remaining 28 segregants were cdc25::UAU1/cdc25::URA3/CDC25 triplication derivatives (Table 2; Fig. 2F). In principle, the DNA segment deleted from the CDC25 locus might have been translocated to a different region of the genome in the two segregants. Such a translocation would be undetected in flanking-primer PCRs but should be detected with PCRs of internal segments. However, three internal primer pairs yielded no PCR product from genomic templates prepared from the cdc25::UAU1/cdc25::URA3 segregants (Fig. 3). Therefore, CDC25 is not essential for viability in C. albicans. A recent study indicates that C. albicans Ras2p is a positive regulator of serum-induced filamentation (6). The two cdc25/cdc25 homozygotes grew at a slightly reduced rate and were partially defective in filamentation on agar containing serum. These findings support the idea that Cdc25p promotes Ras2p activity and filamentation but indicate that CDC25 is not an essential gene.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
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Analysis of gene function in C. albicans is a critical step toward understanding its biology, pathogenesis, and vital processes. In the past, this analysis has required two successive transformations to create homozygous null mutations or conditionally expressed alleles (1, 7, 30, 43). These methods allow construction of precisely defined mutant strains that can be characterized via a battery of refined phenotypic tests. Here, we have described a double disruption selection strategy with a different purpose: to construct homozygous mutants rapidly for a preliminary assessment of gene function. Our strategy is not a replacement or a substitute for the careful crafting of homozygous mutants. Instead, it is a strategy that lends itself to large-scale analysis of gene function.
We have presented here an application of double-disruption selection that facilitates one important goal of C. albicans research: the identification of essential genes. We refer to this test as the HT test. The methodology fills an important need for basic research, because it is otherwise laborious to determine whether a gene is essential (29, 30), and current technology can yield conflicting results (5, 16, 22, 38). The methodology also fills an important need for genome-based pharmaceutical research, in which one must evaluate a large number of gene products as prospective drug targets (23). Finally, we expect that the technical simplicity of our method may encourage those who study a conserved gene in some other organism to test the homologous gene's function in C. albicans.
The results of HT tests were largely consistent with expectations based on prior studies: SNF1 and CDC28 are essential; ADE2 and RIM20 are not. However, the test also argued that CDC25 is not essential, because homozygous cdc25/cdc25 mutants were isolated. (The deletion that we constructed removes much of the domain that is necessary and sufficient for GDP-GTP exchange factor activity [15], so we can formally conclude only that Cdc25p exchange factor activity is not essential.) We note, though, that the cdc25/cdc25 homozygotes arose a lower rate than homozygous insertion mutations in other nonessential genes. This observation might be explained if viability of the cdc25/cdc25 mutant depended upon a genetic suppressor mutation, as is the case in S. cerevisiae (39). A second possibility is that CDC25 is completely dispensable, as is likely the case in Schizosaccharomyces pombe (19), but that the structure or position of the locus limits recombination. We note that the same ambiguities apply to traditional (two-transformation) gene disruption strategies: infrequent isolation of a targeted mutant may reflect either the inadvertent selection of the genetic suppressor or recombinational, rather than functional, properties of the locus. The ambiguities may be resolved by protein depletion experiments or use of dominant-negative mutants (1, 6). Thus, the advantage of the HT test is that it provides functional information rapidly that can justify more definitive but laborious experiments.
There is a circumstance
the case of preexisting triplicated
allelesin which the HT test might yield misleading results.
There are three loci for which triplicated alleles have
been reported, CHS2, FTR1, and
FKS1 (GSC1) (9, 22, 32), although
FKS1 (GSC1) is apparently not triplicated in all
isolates of strain CAI4 (5). One can envision that insertion
of the UAU1 cassette into CHS2, for example,
would yield a genetic structure consistent with a genotype of
chs2::UAU1/CHS2; the actual genotype would be
chs2::UAU1/CHS2/CHS2. Subsequent Arg+
Ura+ selection would yield exclusively segregants of
genotype chs2::UAU1/chs2::URA3/CHS2, not because
CHS2 is essential but because the strain initially had
triplicated alleles. Thus far, preexisting triplications have seldom
been observed in C. albicans, so we expect that this
limitation for the HT test will not outweigh its usefulness.
Our results with double-disruption selection raise an important question that we have not yet resolved: how large a DNA segment becomes homozygous in the Arg+ Ura+ homozygous disruption mutants? Given that natural C. albicans isolates are heterozygous for preexisting mutations (31, 40-42), it is possible that selection for homozygosity of a UAU1 insertion mutation may yield homozygosity of a linked mutation as well. In our small survey of known nonessential genes, we found only homozygotes that had phenotypes consistent with traditionally constructed mutants. However, the possibility that a linked mutation may become homozygous remains an important caveat for any conclusion derived solely from double-disruption selection.
Our estimates of mitotic recombination rates point to a
surprising conclusion: that heterozygous mutations become homozygous at
a rate of 2 × 103 per division. We calculate this
rate from the rate of production of detectable homozygotes (~1 × 108 per division) and the rate of recombination to
generate URA3 from UAU1 (~3 × 106 per division). This homozygosis rate is
102- to 103-fold higher than expected from
studies of S. cerevisiae (24) and violates
anecdotal common knowledge derived from use of Ura-blaster cassettes to
create gene disruptions (7, 43). One simple explanation is
that our estimate of the rate of recombination to generate
URA3 from UAU1 may be artificially low because of a selective advantage of Arg+ cells during growth in broth
culture. However, in coculture experiments, we have not detected such
an advantage (B. Enloe and A. P. Mitchell, unpublished results). A
second explanation is that our frequency estimates are in error because
of differences in growth dynamics on the selective plates. Thus, for
example, Ura+ colonies might arise only from
preexisting recombinants, while Arg+ Ura+
colonies might arise during growth after plating. A third
explanation is that the event that generates URA3 in a
UAU1 insertion heterozygote is not equivalent to the
event that generates URA3 in a homozygote or triplication
derivative. For example, a single recombination event might generate
two copies of the insertion mutation and simultaneously promote
conversion of UAU1 to URA3 in one of the copies.
Therefore, our rate estimates serve as an empirical guide, but we
remain skeptical that interchromosomal recombination is so frequent in
C. albicans.
Allelic triplications were detectable for all of the genes we examined, and we are uncertain of their genetic structure. One simple possibility is that they result from trisomy for the respective chromosome, perhaps in conjunction with an overall increase in ploidy. In support of this idea, we note that several different selections yield monosomic and trisomic C. albicans derivatives (12, 13, 27). A second possibility is that the triplications result from tandem duplication or translocation of a smaller genomic segment, a mechanism that also has experimental support (11, 20, 25). The UAU1 cassette may be a useful tool to define the genetic and environmental parameters that influence changes in gene dosage through either of these mechanisms.
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ACKNOWLEDGMENTS |
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We are grateful to Dana Davis, Teresa Lamb, Vincent Bruno, and Wenjie Xu for many helpful discussions and for comments on the manuscript and to an anonymous referee for suggesting that CDC25 may have undergone a transposition.
We thank the Burroughs Wellcome Fund (Mycology Scholar Award) and the NIH (grant PO1 AI37194) for financial support.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, Columbia University, 701 West 168th St., New York, NY 10032. Phone: (212) 305-8251. Fax: (212) 305-1741. E-mail: apm4{at}columbia.edu.
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Abstract
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Materials and Methods
Results
Discussion
References
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