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The yeast Candida albicans, a commensal colonizer and occasional pathogen of humans, has a rudimentary mating ability. However, mating is a cumbersome process that has never been observed outside the laboratory, and the population structure of the species is predominantly clonal. Here we discuss recent findings that indicate that mating ability is under selection in C. albicans, i.e. that it is a biologically relevant process. C. albicans strains can only mate after they have sustained genetic damage. We propose that the rescue of such damaged strains by mating may be the primary reason why mating ability is under selection.

The yeast Candida albicans can mate. However, in the natural environment mating may generate progeny (fusants) fitter than clonal lineages too rarely to render mating biologically significant: C. albicans has never been observed to mate in its natural environment, the human host, and the population structure of the species is largely clonal. It seems incapable of meiosis, and most isolates are diploid and carry both mating-type-like (MTL) locus alleles, preventing mating. Only chromosome loss or localized loss of heterozygosity can generate mating-competent cells, and recombination of parental alleles is limited. To determine if mating is a biologically significant process, we investigated if mating is under selection. The ratio of nonsynonymous to synonymous mutations in mating genes and the frequency of mutations abolishing mating indicated that mating is under selection. The MTL locus is located on chromosome 5, and when we induced chromosome 5 loss in 10 clinical isolates, most of the resulting MTL-homozygotes could mate with each other, producing fusants. In laboratory culture, a novel environment favoring novel genotypes, some fusants grew faster than their parents, in which loss of heterozygosity had reduced growth rates, and also faster than their MTL-heterozygous ancestors-albeit often only after serial propagation. In a small number of experiments in which co-inoculation of an oral colonization model with MTL-homozygotes yielded small numbers of fusants, their numbers declined over time relative to those of the parents. Overall, our results indicate that mating generates genotypes superior to existing MTL-heterozygotes often enough to be under selection., (Copyright © 2015 by the Genetics Society of America.)

The insertion of a suction catheter or a bronchoscope down an endotracheal tube increases the resistance to gas flow down the tube. The extent to which this occurs depends on the relative diameters of the endotracheal tube and the coaxially introduced catheter. This study utilises a laboratory model to quantify this effect, using a steady flow down an annulus between two tubes whose long axes lie co-axially. Two diameters of an endotracheal tube were modelled to represent flow down adult and neonatal endotracheal tubes; these were of internal diameter (d(o)) 6.3 mm and 3.2 mm, and of length (L) 555 mm. A steady flow of air was generated to pass through the model 'endotracheal' tube. Flowrates were calculated to give Re of approximately 5000 for the larger endotracheal tube, and of approximately 1300 for the smaller. These values correspond to clinically appropriate flowrates in adult and neonatal patients, respectively. The pressure drop deltaPo down the endotracheal tube was measured initially without any obstruction, using a calibrated pressure transducer. Catheters of diameter (d(i)) 0.8 mm, 1.6 mm, and 3.2 mm were introduced into the larger diameter endotracheal tube, while catheters of 0.8 mm and 1.6 mm were introduced into the smaller one, and flow was restored to its original value. The pressure drops deltaP down the endotracheal tubes were measured with the catheters introduced a length 'x' into the tube, to x = L/2 and to x = L. Results are compared with a theoretical calculation on the basis of laminar flow for concentric tubes. If a sampling tube or suction catheter is used down the length of an infant's endotracheal tube, the results show that for most values of do/di, there is a significant rise in deltaP/deltaPo. Where a flexible bronchoscope is used down an endotracheal tube or a telescope down a rigid bronchoscope, the value of deltaP/deltaP(o) may also increase unacceptably where d(o)/d(i) is low. The results show that for equal d(o)/d(i), and equal values of x, deltaP/deltaPo are lower for higher values of Re than for lower; and that for lower values of Re there is a more rapid increase in deltaP/deltaPo as x increases, than for higher Re, especially at low values of d(o)/d(i). This result quantifiably confirms clinical experience; that care must be taken in introducing a catheter down a neonatal endotracheal tube. Deviation of these results from the theoretical calculation is less for the smaller Reynolds numbers and smaller values of d(o)/d(i), because under these conditions the flow is more likely to be laminar, with a greater degree of concentricity.

The mechanisms and rates by which genotypic and phenotypic variation is generated in opportunistic, eukaryotic pathogens during growth in hosts are not well understood. We evaluated genomewide genetic and phenotypic evolution in Candida albicans, an opportunistic fungal pathogen of humans, during passage through a mouse host (in vivo) and during propagation in liquid culture (in vitro). We found slower population growth and higher rates of chromosome-level genetic variation in populations passaged in vivo relative to those grown in vitro. Interestingly, the distribution of long-range loss of heterozygosity (LOH) and chromosome rearrangement events across the genome differed for the two growth environments, while rates of short-range LOH were comparable for in vivo and in vitro populations. Further, for the in vivo populations, there was a positive correlation of cells demonstrating genetic alterations and variation in colony growth and morphology. For in vitro populations, no variation in growth phenotypes was detected. Together, our results demonstrate that passage through a living host leads to slower growth and higher rates of genomic and phenotypic variation compared to in vitro populations. Results suggest that the dynamics of population growth and genomewide rearrangement contribute to the maintenance of a commensal and opportunistic life history of C. albicans.

The major repeat sequence, discovered in the yeast Candida albicans, is a stretch of repeated DNA that occurs nine times in the haploid genome of this opportunistic fungal pathogen and probably a similar number of times in the genome of Candida dubliniensis. In C. albicans it constitutes 1-2% of the genome. Its occurrence is limited to those two species. Despite its major role as a genomic feature, its function, mode of expansion in size due to duplication of internal subunits, and its origin and mechanism of distribution throughout the genome are not understood, although it is associated with chromosome translocations, chromosome length polymorphisms and regulation of the yeast-hypha dimorphic transition. The polymorphism of the major repeat sequence has been exploited in epidemiology and taxonomic studies. This review describes its sequence, occurrence, use in epidemiology and examines the evidence for its role in chromosome dynamics.

Candida dubliniensis and Candida albicans, the most common human fungal pathogen, have most of the same genes and high sequence similarity, but C. dubliniensis is less virulent. C. albicans causes both mucosal and hematogenously disseminated disease, C. dubliniensis mostly mucosal infections. Pulse-field electrophoresis, genomic restriction enzyme digests, Southern blotting, and the emerging sequence from the Wellcome Trust Sanger Institute were used to determine the karyotype of C. dubliniensis type strain CD36. Three chromosomes have two intact homologues. A translocation in the rDNA repeat on chromosome R exchanges telomere-proximal regions of R and chromosome 5. Translocations involving the remaining chromosomes occur at the Major Repeat Sequence. CD36 lacks an MRS on chromosome R but has one on 3. Of six other C. dubliniensis strains, no two had the same electrophoretic karyotype. Despite extensive chromosome rearrangements, karyotypic differences between C. dubliniensis and C. albicans are unlikely to affect gene expression. Karyotypic instability may account for the diminished pathogenicity of C. dubliniensis.

The major repeat sequence (MRS) is known to play a role in karyotypic variation in Candida albicans. The MRS affects karyotypic variation by expanding and contracting internal repeats, by altering the frequency of chromosome loss, and by serving as a hotspot for chromosome translocation. We proposed that the effects of the MRS on translocation could be better understood by examination of the effect of the MRS on a similar event, mitotic recombination between two chromosome homologs. We examined the frequency of mitotic recombination across an MRS of average size (approximately 50 kb) as well as the rate of recombination in a 325-kb stretch of DNA adjacent to the MRS. Our results indicate that mitotic recombination frequencies across the MRS were not enhanced compared to the frequencies measured across the 325-kb region adjacent to the MRS. Mitotic recombination events were found to occur throughout the 325-kb region analyzed as well as within the MRS itself. This analysis of mitotic recombination frequencies across a large portion of chromosome 5 is the first large-scale analysis of mitotic recombination done in C. albicans and indicates that mitotic recombination frequencies are similar to the rates found in Saccharomyces cerevisiae.

The release of the diploid genomic sequence of Candida albicans and its recent community-based annotation have permitted a number of studies which have significantly advanced our understanding of the biology of this important human pathogen. These advances range from analysis of genomic changes to differential gene expression under a variety of conditions. A few general conclusions can be drawn from the data presently in hand; one can expect more and more new insights as the number and kind of experiments grows.

Candida albicans is the most common fungal pathogen of humans. The recent discovery of sexuality in this organism has led to the demonstration of a mating type locus which is usually heterozygous, although some isolates are homozygous. Tetraploids can be formed between homozygotes of the opposite mating type. However, the role of the mating process and tetraploid formation in virulence has not been investigated. We describe here experiments using a murine model of disseminated candidiasis which demonstrate that in three strains, including CAI-4, the most commonly used strain background, tetraploids are less virulent than diploids and can undergo changes in ploidy during infection. In contrast to reports with other strains, we find that MTL homozygotes are almost as virulent as the heterozygotes. These results show that the level of ploidy in Candida albicans can affect virulence, but the mating type configuration does not necessarily do so.

The size of the genome in the opportunistic fungus Candida albicans is 15.6 Mb. Whole-genome shotgun sequencing was carried out at Stanford University where the sequences were assembled into 412 contigs. C. albicans is a diploid basically, and analysis of the sequence is complicated due to repeated sequences and to sequence polymorphism between homologous chromosomes. Chromosome 7 is 1 Mb in size and the best characterized of the 8 chromosomes in C. albicans. We assigned 16 of the contigs, ranging in length from 7309 to 267,590 bp, to chromosome 7 and determined sequences of 16 regions. These regions included four gaps, a misassembled sequence, and two major repeat sequences (MRS) of >16 kb. The length of the continuous sequence attained was 949,626 bp and provided complete coverage of chromosome 7 except for telomeric regions. Sequence analysis was carried out and predicted 404 genes, 11 of which included at least one intron. A 7-kb indel, which might be caused by a retrotransposon, was identified as the largest difference between the homologous chromosomes. Synteny analysis revealed that the degree of synteny between C. albicans and Saccharomyces cerevisiae is too weak to use for completion of the genomic sequence in C. albicans.

The major repeat sequence (MRS) is found at least once on all but one chromosome in Candida albicans, but as yet it has no known relation to the phenotype. The MRS affects karyotypic variation by serving as a hot spot for chromosome translocation and by expanding and contracting internal repeats, thereby changing chromosome length. Thus, MRSs on different chromosomes and those on chromosome homologues can differ in size. We proposed that the MRS's unique repeat structure and, more specifically, the size of the MRS could also affect karyotypic variation by altering the frequency of mitotic nondisjunction. Subsequent analysis shows that both natural and artificially induced differences in the size of the chromosome 5 MRS can affect chromosome segregation. Strains with chromosome 5 homologues that differ in the size of the naturally occurring MRSs show a preferential loss of the homologue with the larger MRS on sorbose, indicating that a larger MRS leads to a higher risk of mitotic nondisjunction for that homologue. While deletion of an MRS has no deleterious effect on the deletion chromosome under normal growth conditions and leads to no obvious phenotype, strains that have the MRS deleted from one chromosome 5 homologue preferentially lose the homologue with the MRS remaining. This effect on chromosome segregation is the first demonstration of a phenotype associated with the MRS.

Candida albicans is a diploid yeast with a predominantly clonal mode of reproduction, and no complete sexual cycle is known. As a commensal organism, it inhabits a variety of niches in humans. It becomes an opportunistic pathogen in immunocompromised patients and can cause both superficial and disseminated infections. It has been demonstrated that genome rearrangement and genetic variation in isolates of C. albicans are quite common. One possible mechanism for generating genome-level variation among individuals of this primarily clonal fungus is mutation and mitotic recombination leading to loss of heterozygosity (LOH). Taking advantage of a recently published genome-wide single-nucleotide polymorphism (SNP) map (A. Forche, P. T. Magee, B. B. Magee, and G. May, Eukaryot. Cell 3:705-714, 2004), an SNP microarray was developed for 23 SNP loci residing on chromosomes 5, 6, and 7. It was used to examine 21 strains previously shown to have undergone mitotic recombination at the GAL1 locus on chromosome 1 during infection in mice. In addition, karyotypes and morphological properties of these strains were evaluated. Our results show that during in vivo passaging, LOH events occur at observable frequencies, that such mitotic recombination events occur independently in different loci across the genome, and that changes in karyotypes and alterations of phenotypic characteristics can be observed alone, in combination, or together with LOH.

One hundred and twenty Candida albicans clinical isolates from the late 1980s and early 1990s were examined for homozygosity at the MTL locus. Of these, 108 were heterozygous (MTLa/MTLalpha), whereas seven were MTLa and five were MTLalpha. Five of the homozygous isolates were able to switch to the opaque cell morphology, while opaque cells were not detectable among the remaining seven. Nevertheless, all but one of the isolates homozygous at the MTL locus were shown to mate and to yield cells containing markers from both parents; the non-mater was found to have a frameshift in the MTLalpha1 gene. In contrast to Saccharomyces cerevisiae, C. albicans homozygotes with no active MTL allele failed to mate rather than mating as a cells. There was no correlation between homozygosity and fluconazole resistance, mating and fluconazole resistance or switching and fluconazole resistance, in part because most of the strains were isolated before the widespread use of this antifungal agent, and only three were in fact drug resistant. Ten of the 12 homozygotes had rearranged karyotypes involving one or more homologue of chromosomes 4, 5, 6 and 7. We suggest that karyotypic rearrangement, drug resistance and homozygosity come about as the result of induction of hyper-recombination during the infection process; hence, they tend to occur together, but each is the independent result of the same event. Furthermore, as clinical strains can mate and form tetraploids, mating and marker exchange are likely to be a significant part of the life cycle of C. albicans in vivo.

Electrophoretic karyotype studies have shown that clinical isolates of Candida albicans have extensive chromosome length polymorphisms. Chromosome translocation is one of the causes of karyotypic variation. Chromosome translocation events have been shown to occur very frequently at or near the major repeat sequence (MRS) on chromosomes. The MRS consists of the repeated sequences RB2, RPS and HOK, and the repeated sequences are considered to be the template for recombination. To investigate which element of the MRS is important for chromosome translocation, we constructed three cassettes, each containing a URA blaster and sequences homologous to one of the repeats, for insertion into the MRS region on the chromosomes. The ura3 strain STN22u2, which shows a stable, standard karyotype, was transformed with each construct. Insertion events with each cassette occurred at almost all chromosomes. Insertion into the RB2 repeat, but not into the RPS repeat, was accompanied by chromosome translocation in some transformants: chromosome translocations between chromosomes R and 7 and chromosomes 1 and 7 were found, as well as deletions of 7A and 7C from chromosome 7. We conclude that the insertion at the RB2 region may initiate chromosome translocation in C. albicans., (Copyright 2004 John Wiley & Sons, Ltd.)

Single-nucleotide polymorphisms (SNPs) are essential tools for studying a variety of organismal properties and processes, such as recombination, chromosomal dynamics, and genome rearrangement. This paper describes the development of a genome-wide SNP map for Candida albicans to study mitotic recombination and chromosome loss. C. albicans is a diploid yeast which propagates primarily by clonal mitotic division. It is the leading fungal pathogen that causes infections in humans, ranging from mild superficial lesions in healthy individuals to severe, life-threatening diseases in patients with suppressed immune systems. The SNP map contains 150 marker sequences comprising 561 SNPs and 9 insertions-deletions. Of the 561 SNPs, 437 were transition events while 126 were transversion events, yielding a transition-to-transversion ratio of 3:1, as expected for a neutral accumulation of mutations. The average SNP frequency for our data set was 1 SNP per 83 bp. The map has one marker placed every 111 kb, on average, across the 16-Mb genome. For marker sequences located partially or completely within coding regions, most contained one or more nonsynonymous substitutions. Using the SNP markers, we identified a loss of heterozygosity over large chromosomal fragments in strains of C. albicans that are frequently used for gene manipulation experiments. The SNP map will be useful for understanding the role of heterozygosity and genome rearrangement in the response of C. albicans to host environments., (Copyright 2004 American Society for Microbiology)

We present the diploid genome sequence of the fungal pathogen Candida albicans. Because C. albicans has no known haploid or homozygous form, sequencing was performed as a whole-genome shotgun of the heterozygous diploid genome in strain SC5314, a clinical isolate that is the parent of strains widely used for molecular analysis. We developed computational methods to assemble a diploid genome sequence in good agreement with available physical mapping data. We provide a whole-genome description of heterozygosity in the organism. Comparative genomic analyses provide important clues about the evolution of the species and its mechanisms of pathogenesis.

Although increases in chromosome copy number typically have devastating developmental consequences in mammals, fungal cells such as Saccharomyces cerevisiae seem to tolerate trisomies without obvious impairment of growth. Here, we demonstrate that two commonly used laboratory strains of the yeast Candida albicans, CAI-4 and SGY-243, can carry three copies of chromosome 1. Although the trisomic strains grow well in the laboratory, Ura+ derivatives of CAI-4, carrying three copies of chromosome 1, are avirulent in the intravenously inoculated mouse model, unlike closely related strains carrying two copies of chromosome 1. Furthermore, changes in chromosome copy number occur during growth in an animal host and during growth in the presence of growth-inhibiting drugs. These results suggest that chromosome copy number variation provides a mechanism for genetic variation in this asexual organism.

Candida albicans, the single most frequently isolated human fungal pathogen, was thought to be asexual until the recent discovery of the mating-type-like locus (MTL). Homozygous MTL strains were constructed and shown to mate. Furthermore, it has been demonstrated that opaque-phase cells are more efficient in mating than white-phase cells. The similarity of the genes involved in the mating pathway in Saccharomyces cerevisiae and C. albicans includes at least one gene (KEX2) that is involved in the processing of the alpha mating pheromone in the two yeasts. Taking into account this similarity, we searched the C. albicans genome for sequences that would encode the alpha pheromone gene. Here we report the isolation and characterization of the gene MFalpha1, which codes for the precursor of the alpha mating pheromone in C. albicans. Two active alpha-peptides, 13 and 14 amino acids long, would be generated after the precursor molecule is processed in C. albicans. To examine the role of this gene in mating, we constructed an mfalpha1 null mutant of C. albicans. The mfalpha1 null mutant fails to mate as MTLalpha, while MTLa mfalpha1 cells are still mating competent. Experiments performed with the synthetic alpha-peptides show that they are capable of inducing growth arrest, as demonstrated by halo tests, and also induce shmooing in MTLa cells of C. albicans. These peptides are also able to complement the mating defect of an MTLalpha kex2 mutant strain when added exogenously, thereby confirming their roles as alpha mating pheromones.

Candida albicans is a diploid yeast with a dimorphic life history. It exists commensally in many healthy humans but becomes a potent pathogen in immunocompromised hosts. The underlying genetic mechanisms by which C. albicans switches from a commensal to a pathogenic form in the host are not well understood. To study the evolution of virulence in mammalian hosts, we used GAL1 as selectable marker system that allows for both positive and negative selection in selective media. We show that the deletion of one or both copies of GAL1 in the C. albicans genome does not change virulence in a systemic mouse model. We obtained estimates for the frequency of mitotic recombination at the GAL1 locus during systemic infection. Our observations suggest that genetic changes such as mitotic recombination and gene conversion occur at a high enough frequency to be important in the transition of C. albicans from a commensal to a pathogenic organism.

Candida albicans is the single, most frequently isolated human fungal pathogen. As with most fungal pathogens, the factors which contribute to pathogenesis in C. albicans are not known, despite more than a decade of molecular genetic analysis. Candida albicans was thought to be asexual until the discovery of the MTL loci homologous to the mating type (MAT) loci in Saccharomyces cerevisiae led to the demonstration that mating is possible. Using Candida albicans mutants in genes likely to be involved in mating, we analysed the process to determine its similarity to mating in Saccharomyces cerevisiae. We examined disruptions of three of the genes in the MAPK pathway which is involved in filamentous growth in both S. cerevisiae and C. albicans and is known to control pheromone response in the former fungus. Disruptions in HST7 and CPH1 blocked mating in both MTLa and MTL(alpha) strains, whereas disruptions in STE20 had no effect. A disruption in KEX2, a gene involved in processing the S. cerevisiae pheromone Mf(alpha), prevented mating in MTL(alpha) but not MTLa cells, whereas a disruption in HST6, the orthologue of the STE6 gene which encodes an ABC transporter responsible for secretion of the Mfa pheromone, prevented mating in MTLa but not in MTL(alpha) cells. Disruption of two cell wall genes, ALS1 and INT1, had no effect on mating, even though ALS1 was identified by similarity to the S. cerevisiae sexual agglutinin, SAG1. The results reveal that these two diverged yeasts show a surprising similarity in their mating processes.

Variation of the electrophoretic karyotype is common among clinical strains of Candida albicans and chromosome translocation is considered one of the causes of karyotypic variation. Such chromosome translocations may be a mechanism to confer phenotypic diversity on the imperfect fungus C. albicans. A clinical strain, TCH23, from a vaginal candidiasis patient shows distinct carbohydrate assimilation profile, serotype B, no chlamydospore formation and an atypical karyotype (Asakura et al., 1991). To examine the taxonomic relationship among C. albicans, Candida dubliniensis and this strain, we sequenced the internal transcribed spacer 1 (ITS1) of nuclear ribosomal DNA. The ITS1 sequence of TCH23 was identical with that of C. albicans but not of C. dubliniensis. Thus, strain TCH23 was classified as a variant of C. albicans with an atypical phenotype. The chromosomal DNAs of this strain were resolved into 13 bands on pulse-field gel electrophoresis (PFGE). Using DNA probes located at or near both ends of each chromosome of C. albicans, we investigated the chromosome organization of this strain. Referring to the SfiI map of C. albicans 1006 (Chu et al., 1993), we found that seven chromosomal DNA bands in strain TCH23 were reciprocal chromosome translocations. One homologue from chromosomes 1, 2 and 6 and both homologues from chromosomes 4 and 7 participated in these events. One translocation product was composed of three SfiI fragments, one each from chromosomes 2, 4 and 7. We deduced the breakpoints of chromosome translocation from the physical map of this strain; between 1J and 1J1, between 2A and 2U, both ends of 4F2, between 6C and 6O and both ends of 7F., (Copyright 2001 John Wiley & Sons, Ltd.)

The pBAC 108L and pFos 1 vectors were developed as stable propagation vectors which, due to their extremely low copy number, facilitate the cloning of a large-sized insert containing repeated DNA. However, the low copy number requires laborious end-DNA preparation for end sequencing and chromosome walking. Here we describe efficient methods for end-DNA isolation. The entire process, including small-scale DNA preparation, restriction digestion, self-ligation, and PCR with vector-based primers, is carried out in 96-well formats. Using a Fosmid library of genomic DNA of Candida albicans, PCR products ranging in size from 0.1 to 8 kbp were generated from 118 end sequences in 140 reactions from 70 Fosmid clones. A single or a prominent band was found in 101 of these reactions. Twenty-six of these bands were tested for walking and all of them proved to be specific. Thus, the system overcomes the disadvantage caused by low copy number. This system allows rapid physical mapping of genomes, and is adaptable for several other vectors including BAC (bacterial artificial chromosome), PAC (P1-derived artificial chromosome), and YAC (yeast artificial chromosome).

Fungal diseases have become increasingly important in the past few years. Because few fungi are professional pathogens, fungal pathogenic mechanisms tend to be highly complex, arising in large part from adaptations of preexisting characteristics of the organisms' nonparasitic lifestyles. In the past few years, genetic approaches have elucidated many fungal virulence factors, and increasing knowledge of host reactions has also clarified much about fungal diseases. The literature on fungal pathogenesis has grown correspondingly; this review, therefore, will not attempt to provide comprehensive coverage of fungal disease but focuses on properties of the infecting fungus and interactions with the host. These topics have been chosen to make the review most useful to two kinds of readers: fungal geneticists and molecular biologists who are interested in learning about the biological problems posed by infectious diseases, and physicians who want to know the kinds of basic approaches available to study fungal virulence.

Parasexual genetic analysis of Candida albicans utilized the dominant selectable marker that conferred resistance to mycophenolic acid. We cloned and sequenced the IMH3(r) gene from C. albicans strain 1006, which was previously identified as resistant to mycophenolic acid (MPA) (A. K. Goshorn and S. Scherer, Genetics 123:213-218, 1989). MPA is an inhibitor of IMP dehydrogenase, an enzyme necessary for the de novo biosynthesis of GMP. G. A. Kohler et al. (J. Bacteriol. 179:2331-2338, 1997) have shown that the wild-type IMH3 gene, when expressed in high copy number, will confer resistance to this antibiotic. We demonstrate that the IMH3(r) gene from strain 1006 has three amino acid changes, two of which are nonconservative, and demonstrate that at least two of the three mutations are required to confer resistance to MPA. We used this gene as a dominant selectable marker in clinical isolates of C. albicans and Candida tropicalis. We also identified the presence of autonomously replicating sequence elements that permit autonomous replication in the promoter region of this gene. Finally, we found the excision of a phi-type long terminal repeat element outside the IMH3 open reading frame of the gene in some strains. We used the IMH3(r) allele to disrupt one allele of ARG4 in two clinical isolates, WO-1 and FC18, thus demonstrating that a single ectopic integration of this dominant selectable marker is sufficient to confer resistance to MPA.

It has been suggested that Candida albicans, a diploid asexual fungus, achieves genetic diversity by genomic rearrangement. This important human pathogen may provide a system in which to analyze alternate routes to genomic diversity. C. albicans has a highly variable karyotype; its chromosomes contain a middle repeated DNA sequence called the Major Repeat Sequence (MRS), composed of subrepeats HOK, RPS, and RB2. RPS is tandemly repeated while the other subrepeats occur once in each MRS. Chromosome 7, the smallest of the eight chromosomes, has been previously mapped. The complete physical map of this chromosome was used to analyze chromosome 7 diversity in six strains, including two well-characterized laboratory strains (1006 and WO-1) and four clinical ones. We found four types of events to explain the genomic diversity: 1) Chromosome length polymorphism (CLP) results from expansion and contraction of the RPS; 2) reciprocal translocation occurs at the MRS loci; 3) chromosomal deletion; and (4) trisomy of individual chromosomes. These four phenomena play an important role in generating genomic diversity in C. albicans.

Although the diploid fungus Candida albicans, a human pathogen, has been thought to have no sexual cycle, it normally possesses mating-type-like orthologs (MTL) of both of the Saccharomyces cerevisiae mating-type genes (MAT) a and alpha. When strains containing only MTLa or MTLalpha were constructed by the loss of one homolog of chromosome 5, the site of the MTL loci, MTLa and MTLalpha strains mated, but like mating types did not. Evidence for mating included formation of stable prototrophs from strains with complementing auxotrophic markers; these contained both MTL alleles and molecular markers from both parents and were tetraploid in DNA content and mononucleate.

Significant occurrence of high-ploidy cells is commonly observed among many Candida albicans strains. We isolated two isogenic strains, STN21 and STN22, each from a half sector of a colony obtained after mild UV-irradiation of a Arg(-) derivative of CBS5736. The two strains were different from each other in ploidy states and chromosome organization. Although cells of STN22 were homogeneous in size and had a single nucleus, high-ploidy cells, with either a single large nucleus or several nuclei, were present together with apparently normal cells with a single nucleus in the cell population of STN21. Flow cytometry showed that STN22 was a stable diploid; however, STN21 seemed to be the mixture of different ploidy states, including diploid and tetraploid. The phenotype of STN21 containing high-ploidy cells is referred to here as the Sps(-) phenotype (suppressor of ploidy shift). STN22 showed a typical electrophoretic karyotype similar to strain 1006 in C. albicans. However, an extra chromosomal band appeared in some clones of STN21 at high frequency. By assignment of several DNA probes, this extra chromosome was shown to be a translocation of the 7F-7G portion of chromosome 7 with the 470 kb DNA segment containing H SfiI fragment from chromosome 4. Thus, this extra chromosome is a hybrid of 4H and 7F-7G. Since the isogenic Sps(+) strain STN22 exhibited no extra chromosome bands, a correlation is suggested between the Sps(-) phenotype and the occurrence of chromosome translocations., (Copyright 2000 John Wiley & Sons, Ltd.)

The frequency of opportunistic infections caused by the fungus Candida albicans is very high and is expected to continue to increase as the number of immunocompromised patients rises. Research initiatives to study the biology of this organism and elucidate its pathogenic determinants have therefore expanded significantly during the last 5-10 years. The past few years have also brought continuous improvement in the techniques to study gene function by gene inactivation and by regulated gene expression and to study gene expression and protein localization by using gene reporter systems. As steadily more genomic sequence information from this human fungal pathogen becomes available, we are entering a new era in antimicrobial research. However, many of the currently available molecular genetics tools are poorly adapted to a genome-wide functional analysis in C. albicans, and further development of these tools is hampered by the asexual and diploid nature of this organism. This review outlines recent advances in the development of molecular tools for functional analysis in C. albicans and summarizes current knowledge about the genomic and genetic variability of this important human fungal pathogen.

As part of the ongoing Candida albicans Genome Project, we have constructed a complete sequence-tagged site contig map of chromosome 7, using a library of 3840 clones made in fosmids to promote the stability of repeated DNA. The map was constructed by hybridizing markers to the library, to a blot of the electrophoretic karyotype, and to a blot of the pulsed-field separation of the SfiI restriction fragments of the genome. The map includes 149 fosmids and was constructed using 79 markers, of which 34 were shown to be genes via determination of function or comparison of the DNA sequence to the public databases. Twenty-five of these genes were identified for the first time. The absolute position of several markers was determined using random breakage mapping. Each of the homologues of chromosome 7 is approximately 1 Mb long; the two differ by about 20 kb. Each contains two major repeat sequences, oriented so that they form an inverted repeat separated by 370 kb of unique DNA. The repeated sequence CARE2/Rel2 is a subtelomeric repeat on chromosome 7 and possibly on the other chromosomes as well. Genes located on chromosome 7 in Candida are found on 12 different chromosomes in Saccharomyces cerevisiae.

Recent studies are beginning to delineate those pathways by which the important pathogen Candida albicans switches from one growth form to another; at the same time, insights are being gained into the importance of growth form in pathogenesis.

Candida albicans strain WO-2 was isolated as a spontaneous derivative of the white-opaque switching strain WO-1. The electrophoretic karyotype of WO-2 lacks two bands which are found in the parent. These bands correspond to one homologue of chromosome 7 and to a translocation product containing parts of chromosomes 6 and 5. Probing a blot of the karyotype demonstrated that the genetic material in these bands had been lost, yielding an aneuploid strain. UV-irradiation experiments showed that auxotrophs due to mutation in genes located in this region predominated, supporting the conclusion that WO-2 is partially haploid. WO-2 contained about 10% of its genome in the haploid state, and it grew with a doubling time of about twice that of its parent. However, it was able to undergo both the yeast-to-hyphal transition and the white-opaque transition. Hence, these processes do not require perfect diploidy.

The karotype of Candida albicans 1001, a pathogenic isolate, presents two additional chromosome bands when compared with C. albicans 1006 strain. These two bands were a 2600 kb chromosome located between chromosome group 1-R and chromosome 2 (named chromosome 2*) and a 710 kb small chromosome, called snc due to its similarity in size to the supernumerary chromosome in strain WO-1. A comparison of karyotypes of strains 1001, 1006 and WO-1 has enabled us to conclude that chromosomes 2 and 7 are involved in such a reorganization giving rise to the new chromosome bands of strain 1001. We describe a tentative physical map of C. albicans 1001 based on the previously outlined map of C. albicans strain 1006.

Candida albicans produces large amounts of the acyclic pentitol D-arabitol in culture and in infected animals and humans, and most strains also grow on minimal D-arabitol medium. An earlier study showed that the major metabolic precursor of D-arabitol in C. albicans was D-ribulose-5-PO4 from the pentose pathway, that C. albicans contained an NAD-dependent D-arabitol dehydrogenase (ArDH), and that the ArDH structural gene (ARD) encoded a 31-kDa short-chain dehydrogenase that catalyzed the reaction D-arabitol + NAD <=> D-ribulose + NADH. In the present study, we disrupted both ARD chromosomal alleles in C. albicans and analyzed the resulting mutants. The ard null mutation was verified by Southern hybridization, and the null mutant's inability to produce ArDH was verified by Western immunoblotting. The ard null mutant grew well on minimal glucose medium, but it was unable to grow on minimal D-arabitol or D-arabinose medium. Thus, ArDH catalyzes the first step in D-arabitol utilization and a necessary intermediate step in D-arabinose utilization. Unexpectedly, the ard null mutant synthesized D-arabitol from glucose. Moreover, 13C nuclear magnetic resonance studies showed that the ard null mutant and its wild-type parent synthesized D-arabitol via the same pathway. These results imply that C. albicans synthesizes and utilizes D-arabitol via separate metabolic pathways, which was not previously suspected for fungi.

In order to clarify the relationship between polyploidization and the capability of phenotypic switching in the imperfect yeast Candida albicans, two types of variants were isolated as segregants from a fusant, which produced a proportion of the cell population with a higher ploidy than the rest, either in a temperature-dependent or -independent manner, when incubated at low (28 degrees C) and high (37 degrees C) temperatures. In the case of the temperature-dependent type of variants, high-ploidy cells appeared at 37 degrees C but rarely at 28 degrees C. This phenotype was named Pldts (temperature-sensitive polyploidization), and the temperature-independent phenotype was called Pld-. The appearance of high-ploidy cells in the culture of the Pldts strain at 37 degrees C was accompanied by a significant increase in the frequency of auxotrophic variants; these variants probably occur as a result of segregation of auxotrophic markers from the heterozygous to the homozygous state. Both Pldts and Pld- phenotypes were recessive in a fusion with a Pld+ parent. An adenine auxotrophic marker (ade1) was introduced into a Pldts strain in a heterozygous state, and the individual high-ploidy cells of this strain, grown at 37 degrees C, were micromanipulated to form colonies, which consisted of red and white sectors appearing at high frequency on a pink background. When the ade1 auxotrophy was introduced into Pld- strains, frequently sectored colonies were produced. These results suggested an increased level of chromosome missegregation in both types of Pld mutants. Analyses by pulsed-field gel electrophoresis of Ade-segregants, derived from a micromanipulated high-ploidy cell of a Pld(ts) strain, suggested the occurrence of nonreciprocal recombination, some of which includes chromosome loss.

Candida albicans and the closely related species Candida stellatoidea are medically important diploid asexual yeasts. Clinical isolates frequently show variant electrophoretic karyotypes, apparently due largely to chromosomal translocations. These translocations seem to occur at hot spots characterized by the repeated DNA sequence RSP1. A programmed karyotypic rearrangement occurs in C. stellatoidea. Karyotypic rearrangement may serve as a source of genetic variation in these asexual yeasts.

The opportunistic fungal pathogen, Candida albicans, is diploid as usually isolated and has no apparent sexual cycle. Genetic analysis has therefore been very difficult. Molecular genetics has yielded important information in the past few years, but it too is hampered by the lack of a good genetic map. Using the well-characterized strain 1006 and strain WO-1, which undergoes the white-opaque phenotypic transition, we have developed a genomic restriction map of C. albicans with the enzyme SfiI. There are approximately 34 SfiI restriction sites in the C. albicans genome. Restriction fragments were separated by pulsed-field electrophoresis and were assigned to chromosomes by hybridization of complete and partial digests with known chromosome-specific probes as well as by digestion of isolated chromosomes. Telomeric fragments were identified by hybridization with a telomere-specific probe (C. Sadhu, M.J. McEachern, E.P. Rustchenko-Bulgac, J. Schmid, D.R. Soll, and J.B. Hicks, J. Bacteriol. 173:842-850, 1991). WO-1 differs from 1006 in that it has undergone three reciprocal chromosomal translocations. Analysis of the translocation products indicates that each translocation has occurred at or near an SfiI site; thus, the SfiI fragments from the two strains are similar or identical. The tendency for translocation to occur at or near SfiI sites may be related to the repeated sequence RPS 1, which contains four such sites and could provide homology for ectopic pairing and crossing over. The genome size of both strains is about 16 to 17 megabases, in good agreement with previous determinations.

Objective: The purpose of this study was to assess in the clinical setting the reliability and long-term stability of Raman spectroscopy as implemented in the RASCAL multiple gas analyzer, and to describe/analyze failure modes that manifest in regular use., Methods: Twenty-one RASCAL analyzers were tested for accuracy and precision. Without any prior external calibration or alignment, all gas analyzers were systematically tested over a consecutive 36-hour period with standard gas mixtures. Data were analyzed by evaluating the difference between the predicted value and observed value (bias residual) as reported by each RASCAL: All service data (29 months) also were analyzed for information on durability and failure modes., Results: The RASCAL exhibited a significant tendency to overread high concentrations of agent (isoflurane/enflurane); 4 of 16 instruments misread an agent by more than +/- 0.30%. Four of 16 instruments could not properly identify volatile agents in low concentrations (0.31 vol%). Inventory records show that water contamination led to the replacement of gas sample sets an average of 1.5 +/- 1.2 times per case over the 29-month period. Although many instruments had not been externally calibrated for over 63 days, linearity proved acceptable for CO2, O2, N2O, and N2. A rationale for instrument behavior and major failure modes, based on the instrument design, was developed., Conclusions: The manufacturer's suggested calibration intervals (30 days) were found to be more than adequate for reliable gas analysis using Raman spectroscopy. Without the benefit of frequent calibrations and as time passes, volatile agent quantification can be expected to drift significantly upward.

The secreted aspartyl proteinase activity from Candida albicans is thought to be a potential virulence factor. Four laboratories have cloned a gene from C. albicans encoding this enzyme. When two of these genes sharing 77% homology at the DNA level are hybridized under conditions of high stringency to contour-clamped homogeneous electric field chromosome separations of four different strains, they label different chromosomes: chromosome 6 for SAP1 and chromosome R for SAP2. The existence of different genes for the two sequences was confirmed by polymerase chain reaction. Genomic Southern blots probed with the genes and washed at low stringency revealed several cross-hybridizing bands. Contour-clamped homogeneous electric field chromosome separations probed at low stringency indicated that there was a cross-hybridizing sequence on chromosome 3 in addition to those on chromosomes R and 6. The genes for the secreted aspartyl proteinase activity in C. albicans thus constitute a gene family which we have called the SAP family.

A number of genes have been shown to be transcribed specifically during sporulation in Saccharomyces cerevisiae, yet their developmental function is unknown. The SPR1 gene is transcribed during only the late stages of sporulation. We have sequenced the SPR1 gene and found that it has extensive DNA and protein sequence homology to the S. cerevisiae EXG1 gene which encodes an exo-1,3-beta-glucanase expressed during vegetative growth (C. R. Vasquez de Aldana, J. Correa, P. San Segundo, A. Bueno, A. R. Nebrada, E. Mendez, and F. del Ray, Gene 97:173-182, 1991). We show that spr1 mutant cells do not hydrolyze p-nitrophenyl-beta-D-glucoside or laminarin in a whole-cell assay for exo-1,3-beta-glucanases. In addition to the absence of this enzymatic activity, spr1 mutant spores exhibit reduced thermoresistance relative to isogenic wild-type spores. These observations are consistent with the notion that SPR1 encodes a sporulation-specific exo-1,3-beta-glucanase.