Elena Rustchenko - Academia.edu (original) (raw)

Papers by Elena Rustchenko

Revista Iberoamericana de Micología

Utilization of L-sorbose, D-arabinose or primary fluconazole resistance in Candida albicans are c... more Utilization of L-sorbose, D-arabinose or primary fluconazole resistance in Candida albicans are controlled by copy number of specific chromosomes. On the other hand, spontaneous morphological mutants have a wide range of chromosomal alterations. We have investigated the UV and X-ray sensitivity of these mutants, as well as C. albicans laboratory strains. While L-sorbose utilizing mutants had normal sensitivities, a large subclass of D-arabinose utilizing mutants was abnormally sensitive to UV. Spontaneous morphological mutants responded differently, an expected result because of the heterogeneous nature of their electrophoretic karyotypes. We suggest that the differences in UV and X-ray sensitivity are due to gene imbalance caused by some chromosomal alterations. In this respect, the radiation sensitivity is similar to other features impaired by changes in chromosomes, but is unlike the acquisition of the ability to utilize alternative nutrients or the acquisition of resistance to fluconazole. Our studies also revealed that strains of C. albicans heterozygous for the mating type loci exhibited the same X-ray sensitivity as homozygous or hemizygous strains, a finding which is in contrast to the properties of Saccharomyces cerevisiae, where heterozygous strains are more resistant. This feature of C. albicans strains may be indicative of an inefficient repair system that may be related to inefficiency of mating.

Journal of Bacteriology

Several different repetitive DNA sequences have been isolated from the pathogenic yeast Candida a... more Several different repetitive DNA sequences have been isolated from the pathogenic yeast Candida albicans. These include two families of large dispersed repeat sequences (Ca3, Ca24) and a short (23-bp) tandemly repeated element (Ca7) associated with C. albicans telomeres. In addition, a large subtelomeric repeat (WOL17) has been cloned. DNA fragments containing the telomeric repeats are highly variable among different C. albicans strains. We have shown that the Ca3 repeat is relatively more stable and is suitable for use as a species-specific and strain-specific probe for C. albicans.

Genetics, 1999

This is the first report that adaptive mutagenesis can arise by chromosomal nondisjunction, a phe... more This is the first report that adaptive mutagenesis can arise by chromosomal nondisjunction, a phenomenon previously associated exclusively with DNA alterations. We previously uncovered a novel regulatory mechanism in Candida albicans in which the assimilation of an alternative sugar, l-sorbose, was determined by copy number of chromosome 5, such that monosomic strains utilized l-sorbose, whereas disomic strains did not. We present evidence that this formation of monosomy of chromosome 5, which is apparently a result of nondisjunction, appeared with increased frequencies after a selective condition was applied, i.e., by adaptive mutagenesis. The rate of formation of l-sorbose-utilizing mutants per viable cell per day ranged from 10(-6) at the initial time of detection to 10(-2) after 4 days of incubation on the selective plate.

Journal of bacteriology, 1999

The exposure of Candida albicans to fluconazole resulted in the nondisjunction of two specific ch... more The exposure of Candida albicans to fluconazole resulted in the nondisjunction of two specific chromosomes in 17 drug-resistant mutants, each obtained by an independent mutational event. The chromosomal changes occurred at high frequencies and were related to the duration of the drug exposure. The loss of one homologue of chromosome 4 occurred after incubation on a fluconazole medium for 7 days. A second change, the gain of one copy of chromosome 3, was observed after exposure for 35 or 40 days. We found that the mRNA levels of ERG11, CDR1, CDR2, and MDR1, the candidate fluconazole resistance genes, remained either the same or were diminished. The lack of overexpression of putative drug pumps or the drug target indicated that some other mechanism(s) may be operating. The fluconazole resistance phenotype, electrophoretic karyotypes, and transcript levels of mutants were stable after growth for 112 generations in the absence of fluconazole. This is the first report to demonstrate that...

PLoS ONE, 2010

The important human pathogen Candida albicans possesses an unusual form of gene regulation, in wh... more The important human pathogen Candida albicans possesses an unusual form of gene regulation, in which the copy number of an entire specific chromosome or a large portion of a specific chromosome changes in response to a specific adverse environment, thus, insuring survival. In the absence of the adverse environment, the altered portion of the genome can be restored to its normal condition. One major question is how C. albicans copes with gene imbalance arising by transitory aneuploid states. Here, we compared transcriptomes from cells with either two copies or one copy of chromosome 5 (Ch5) in, respectively, a diploid strain 3153A and its representative derivative Sor55. Statistical analyses revealed that at least 40% of transcripts from the monosomic Ch5 are fully compensated to a disomic level, thus, indicating the existence of a genome-wide mechanism maintaining cellular homeostasis. Only approximately 15% of transcripts were diminished twofold in accordance with what would be expected for Ch5 monosomy. Another minor portion of approximately 6% of transcripts, unexpectedly, increased up to twofold and higher than the disomic level, demonstrating indirect control by monosomy. Array comparative genome hybridization revealed that only few out of approximately 500 genes on the monosomic Ch5b were duplicated, thus, not causing a global up regulation. Dosage compensation was confirmed with several representative genes from another monosomic Ch5a in the mutant Sor60. We suggest that C. albicans's unusual regulation of gene expression by the loss and gain of entire chromosomes is coupled with widespread compensation of gene dosage at the transcriptional level.

Yeast, 2005

is subject to a novel form of regulation which involves a reversible increase or decrease in the ... more is subject to a novel form of regulation which involves a reversible increase or decrease in the copy number of chromosome 5. Furthermore, the structural gene SOU1 is required for L-sorbose utilization and encodes a member of the short chain dehydrogenase family. However, the precise function of SOU1 was not known and neither was the pathway for L-sorbose utilization. We have now expressed SOU1 at a high level from a replicative plasmid having a constitutive ADH1 promoter and purified a version of Sou1p tagged with the FLAG epitope at the N-terminus. Sou1FLAGNp has a sorbose reductase activity which utilizes NADPH as a co-factor and converts L-sorbose to D-sorbitol. It can also less efficiently utilize fructose as a substrate with NADPH as a co-factor, converting fructose to mannitol. In agreement with prediction, the purified enzyme has a subunit molecular weight of 31 kDa and a pI of about 4.8. It probably consists of four identical subunits and has a pH optimum of 6.2. The L-sorbose utilization pathway in C. albicans probably converts L-sorbose to fructose-6-phosphate via D-sorbitol as an intermediate. The first step is catalysed by Sou1p. We also found that C. albicans extracts have a D-sorbitol-6phosphate dehydrogenase activity, not encoded by SOU1, which utilizes NADP as a co-factor. This activity has not been described previously in yeasts and may be involved in the conversion of phosphorylated D-sorbitol to fructose-6-phosphate or glucose-6-phosphate.

Yeast, 2004

We previously demonstrated that the pathogenic yeast Candida albicans effectively adapts to utili... more We previously demonstrated that the pathogenic yeast Candida albicans effectively adapts to utilize L-sorbose (Sou + ) by a novel mechanism based on the loss of one copy of chromosome 5, probably due to the reduction of copy number of a negative regulator located on this chromosome. We report here another negative regulator of Lsorbose utilization, an orthologue of the Saccharomyces cerevisiae BMH1 gene, which encodes the evolutionarily conserved protein 14-3-3. This essential gene is located on chromosome 1, does not have paralogues, and is supposedly a component of the regulatory network. Experiments involving disruption of one allele of BMH1 and overexpression of BMH1 revealed that BMH1 represses the transcription of SOU1, which is responsible for the utilization of L-sorbose. Although the exact mechanism of the interaction between BMH1 and SOU1 is not known, it is clear that the control is based on the ratio of gene copy number, and that BMH1 does not control the loss of chromosome 5, the major mechanism producing Sou + mutants. We propose that function of BMH1 as a negative regulator of SOU1 contributes to a general cellular homeostasis. This is a first report on the role of the C. albicans essential gene BMH1 as a negative regulator of the utilization of secondary carbon source in yeast, which further substantiates the involvement of 14-3-3 proteins in diverse functions.

Yeast, 2001

Although plasmids containing rRNA genes (rDNA) are commonly found in fungi, they have not been re... more Although plasmids containing rRNA genes (rDNA) are commonly found in fungi, they have not been reported in Candida. We discovered that the yeast opportunistic pathogen Candida albicans contains two types of rDNA plasmids which differ in their structure and number of rDNA repeats. A large circular plasmid of unknown size consists of multiple rDNA repeats, each of which includes an associated autonomously replicating sequence (ARS). In contrast, a linear plasmid, which is represented by a series of molecules with a spread of sizes ranging from 50±150 kbp, carries a limited number of rDNA units and associated ARSs, as well as telomeres. The number of linear plasmids per cell is growth cycle-dependent, accumulating in abundance in actively growing cells. We suggest that the total copy number of rDNA is better controlled when a portion of copies are on a linear extrachromosomal plasmid, thus allowing a rapid shift in the number of corresponding genes and, as a result, better adaptation to the environment. This is the ®rst report of a linear rDNA plasmid in yeast, as well as of the coexistence of circular and linear plasmids. In addition, this is a ®rst report of naturally occurring plasmids in C. albicans.

Yeast, 1994

Several recent investigations, employing restriction endonucleases that do not cleave within rDNA... more Several recent investigations, employing restriction endonucleases that do not cleave within rDNA units, revealed that a number of laboratory strains of Saccharomyces cerevisiae apparently contains a single tandem array of approximately 50 to 200 rDNA units on each chromosome XII homolog. The number of these rDNA units varies from strain to strain, among subclones of the same strain, and after different conditions of growth. In contrast, the commonly-used strain S288C and its derivatives contain two clusters on each chromosome XII homolog. Although the two clusters are stably maintained, the number of rDNA units within each cluster can vary as in strains with single clusters.

Proceedings of the National Academy of Sciences, 1998

We report the identification of the gene, SOU1, required for L-sorbose assimilation in Candida al... more We report the identification of the gene, SOU1, required for L-sorbose assimilation in Candida albicans. The level of the expression of SOU1 is determined by the copy number of chromosome III (also denoted chromosome 5), such that monosomic strains assimilate L-sorbose, whereas disomic strains do not, in spite of the fact that SOU1 is not on this chromosome. We suggest that C. albicans contains a resource of potentially beneficial genes that are activated by changes in chromosome number, and that this elaborate mechanism regulates the utilization of food supplies and possibly other important functions, thus representing a novel general means for regulating gene expression in microbes.

Mycologia, 2006

Treatment of a prototrophic laboratory strain of Candida albicans with 5-fluoro-orotic acid (5-FO... more Treatment of a prototrophic laboratory strain of Candida albicans with 5-fluoro-orotic acid (5-FOA) produced two major types of mutants with chromosomal alterations, 5-FOA-resistant (FoaR) and those remaining sensitive (FoaS). Both major types remained Ura+. FoaR mutants, produced after a long exposure, contained either a duplication of chromosome 4b or an inner enlargement of chromosome 5b. The average mutant frequency was approximately 1.0 x 10(-5). The reverse mutation of FoaR to FoaS also caused the loss of either the extra chromosome 4b or the enlarged chromosome 5b, revealing a causal relationship between the resistance and the specific chromosome constitution. The cells remained sensitive after a relatively short 24 h exposure to 5-FOA medium, but the treatment induced non-specific changes in lengths of various chromosomes. Furthermore, FoaR type mutants acquired a notable chromosomal and phenotypic instability. Our results indicate the necessity of electrokaryotyping of strains that have been exposed to 5-FOA, especially with studies of gene function and with DNA microarray assays.

Microbiology, 1997

In this study, four clinical isolates and over 100 colony morphology mutants, previously derived ... more In this study, four clinical isolates and over 100 colony morphology mutants, previously derived spontaneously from strain 31 53A during growth on glucose medium, were examined for their utilization of 21 carbon and 3 nitrogen sources at various growth temperatures. The results demonstrated extensive variability in the pattern of assimilation among the mutants and strains, including both the gain and loss of assimilating functions. The persistent alterations in assimilation patterns observed in sequentially produced subclones illustrated an extensive ability of C. albicans populations to constantly produce new combinations of assimilating functions. The variability among spontaneous mutants derived from a single strain explains the well documented variability among natural isolates. From these results we established a relationship between the previously documented broad spectrum of spontaneous chromosomal aberrations in these mutants to the expression of genes controlling the utilization of alternative carbon and nitrogen sources. The existence of cryptic genes, responsible for growth on alternative substrates, was previously deduced from the analysis of other mutants obtained as a response to the restrictive condition on media containing non-assimilating carbon sources. Thus, mutants with altered assimilation functions can arise either on glucose medium or by selection on restricted media. Extensive differences between the patterns of chromosomal aberrations and the distribution of correlated phenotypes in the two groups of mutants indicated that the same phenotypes may be produced by two different mechanisms involving the same or different genes.

Genetics, 2012

Candida albicans, a major human fungal pathogen, usually contains a diploid genome, but controls ... more Candida albicans, a major human fungal pathogen, usually contains a diploid genome, but controls adaptation to a toxic alternative carbon source L-sorbose, by the reversible loss of one chromosome 5 (Ch5). We have previously identified multiple unique regions on Ch5 that repress the growth on sorbose. In one of the regions, the CSU51 gene determining the repressive property of the region was identified. We report here the identification of the CSU53 gene from a different region on Ch5. Most importantly, we find that CSU51 and CSU53 are associated with novel regulatory elements, ASUs, which are embedded within CSUs in an antisense configuration. ASUs act opposite to CSUs by enhancing the growth on sorbose. In respect to the CSU transcripts, the ASU long antisense transcripts are in lesser amounts, are completely overlapped, and are inversely related. ASUs interact with CSUs in natural CSU/ASU cis configurations, as well as when extra copies of ASUs are placed in trans to the CSU/ASU configurations. We suggest that ASU long embedded antisense transcripts modulate CSU sense transcripts.

Gene, 2005

We have adopted a method of telomere-mediated chromosome fragmentation in order to demonstrate th... more We have adopted a method of telomere-mediated chromosome fragmentation in order to demonstrate the alignment of contigs and determination of gaps. We established the order and orientation of four contigs of Candida albicans chromosome 5 and determined the sizes of three gaps between these contigs. We confirmed this proposed alignment of contigs, as well as gap sizes, by sequencing one gap and analyzing three mega deletions of approximately 41 kbp, 58 kbp, and 77 kbp, which covered two other gaps. These gaps could be also conveniently sequenced, which is an important step in establishing a complete sequence. The combined length of contigs and gaps covered approximately 422 kbp, which is one third of chromosome 5. Telomere-mediated chromosome fragmentation, used here for the first time to align the contigs of C. albicans and determine the gaps, proved to be a reliable method. The method could be helpful in sequencing projects of other diploid organisms, in particular those in which centromeres have not been identified. In addition, our approach can be used to assign any contig to a chromosome, or to induce the loss of a specific chromosome. D

Fungal Genetics and Biology, 2007

C. albicans can adapt and grow on sorbose plates by losing one copy of Chr5. Since rad52 mutants ... more C. albicans can adapt and grow on sorbose plates by losing one copy of Chr5. Since rad52 mutants of S. cerevisiae lose chromosomes at a high rate, we have investigated the ability of C. albicans rad52 to adapt to sorbose. Carad52-ΔΔ mutants generate Sou + strains earlier than wild type but the final yield is lower, probably because they die at a higher rate in sorbose. As other strains of C. albicans, CAF2 and rad52-ΔΔ derivatives generate Sou + strains by a loss of one copy of Chr5 about 75% of the time. In addition, rad52 strains were able to produce Sou + strains by a fragmentation/ deletion event in one copy of Chr5, consisting of loss of a region adjacent to the right telomere. Finally, both CAF2 and rad52-ΔΔ produced Sou + strains with two apparent full copies of Chr5, suggesting that additional genomic changes may also regulate adaptation to sorbose.

FEMS Yeast Research, 2014

Candida albicans, a fungus that normally inhabits the digestive tract and other mucosal surfaces,... more Candida albicans, a fungus that normally inhabits the digestive tract and other mucosal surfaces, can become a pathogen in immunocompromised individuals, causing severe or even fatal infection. Mechanisms by which C. albicans can evade commonly used antifungal agents are not fully understood. We are studying a model system involving growth of C. albicans on toxic sugar sorbose, which represses synthesis of cell wall glucan and, as a result, kills fungi in a manner similar to drugs from the echinocandins class. Adaptation to sorbose occurs predominantly due to reversible loss of one homolog of chromosome 5 (Ch5), which results in upregulation of the metabolic gene SOU1 (SOrbose Utilization) on Ch4. Here, we show that growth on sorbose due to Ch5 monosomy can involve a facultative trisomy of a hybrid Ch4/7 that serves to increase copy number of the SOU1 gene. This shows that control of expression of SOU1 can involve multiple mechanisms; in this case, negative regulation and increase in gene copy number operating simultaneously in cell.

Biological & Pharmaceutical Bulletin, 2011

Yeast, 2008

Electrophoretic karyotyping of the Candida albicans revealed a different migration pattern of ChR... more Electrophoretic karyotyping of the Candida albicans revealed a different migration pattern of ChR in three different stocks of the sequencing strain SC5314. In one stock, the high instability of ChR size prevented the migration of ChR as a compact band; ChR appeared, instead, as a smear. In some stocks, ChR and/or Ch1 ploidy diminished, suggesting mixed populations of disomic and monosomic cells. Similarly, some stocks of widely used derivatives CAI4 and BWP17 contained smearing of ChR. In addition, the most manipulated strain in the lineage of SC5314, the last derivative, BWP17, acquired an increase in the size of Ch7b and revealed an unusual property. BWP17 did not tolerate a well-established procedure of telomere-mediated fragmentation of a chromosome; the remaining intact homologue always duplicated. We suggest that some stocks of SC5314 are unstable and that BWP17 may not be appropriate for general studies. Instead of BWP17 or CAI4, we recommend using for general research CAF4-2, which is a relatively stable Ura − derivative, and which has been successfully used for more than a decade in our laboratory.