Mutation of the CgPDR16 gene attenuates azole tolerance and biofilm production in pathogenic Candida glabrata.

The PDR16 gene encodes the homologue of Sec14p, participating in protein secretion, regulation of lipid synthesis and turnover in vivo and acting as a phosphatidylinositol transfer protein in vitro. This gene is also involved in the regulation of multidrug resistance in Saccharomyces cerevisiae and pathogenic yeasts. Here we report the results of functional analysis of the CgPDR16 gene, whose mutation has been previously shown to enhance fluconazole sensitivity in Candida glabrata mutant cells. We have cloned the CgPDR16 gene, which was able to complement the pdr16Δ mutation in both C. glabrata and S. cerevisiae. Along with fluconazole, the pdr16Δ mutation resulted in increased susceptibility of mutant cells to several azole antifungals without changes in sensitivity to polyene antibiotics, cycloheximide, NQO, 5-fluorocytosine and oxidants inducing the intracellular formation of reactive oxygen species. The susceptibility of the pdr16Δ mutant strain to itraconazole and 5-fluorocytosine was enhanced by CTBT [7-chlorotetrazolo(5,1-c)benzo(1,2,4)triazine] inducing oxidative stress. The pdr16Δ mutation increased the accumulation of rhodamine 6G in mutant cells, decreased the level of itraconazole resistance caused by gain-of-function mutations in the CgPDR1 gene, and reduced cell surface hydrophobicity and biofilm production. These results point to the pleiotropic phenotype of the pdr16Δ mutant and support the role of the CgPDR16 gene in the control of drug susceptibility and virulence in the pathogenic C. glabrata.

Overexpression of the YAP1, PDE2, and STB3 genes enhances the tolerance of yeast to oxidative stress induced by 7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine.

7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine (CTBT) is an antifungal agent that induces oxidative stress and enhances the activity of other antifungals with different modes of action. A genome-wide screening of Saccharomyces cerevisiae genomic library in the high-copy-number plasmid revealed three genes, YAP1, PDE2, and STB3, which increased the CTBT tolerance of the parental strain. The YAP1 gene is known to activate many genes in response to oxidants. The PDE2 and STB3 genes encode the high-affinity cAMP phosphodiesterase and the transcription factor recognizing the ribosomal RNA processing element in promoter sequences, respectively. The protective effects of their overexpression against CTBT toxicity was observed in the absence of certain proteins involved in stress responses, cell wall integrity signaling, and chromatin remodeling. The enhanced CTBT tolerance of the YAP1, PDE2, and STB3 transformants was a consequence of their high antioxidant enzyme activities at the beginning of CTBT treatment in comparison with that of the parental strain, for that they inactivated the CTBT-induced reactive oxygen species. These results point to the complex interplay among the oxidant sensing, cAMP-protein kinase A signaling, and transcription reprogramming of yeast cells, leading to their better adaptation to the stress imposed by CTBT.

A yeast cell-based system for screening Candida glabrata multidrug resistance reversal agents and selection of loss-of-function pdr1 mutants.

In the pathogenic yeast Candida glabrata, multidrug resistance is associated with the overexpression of drug efflux pumps caused by gain-of-function mutations in the CgPDR1 gene. CgPdr1p transcription factor, which activates the expression of several drug efflux transporter genes, is considered to be a promising target for compounds sensitizing the multidrug-resistant yeast cells. Here, we describe a cell-based screening system for detecting the inhibitory activity of compounds interfering with the CgPdr1p function in a heterologous genetic background of the hypersensitive Saccharomyces cerevisiae mutant strain. The screening is based on the ability to abrogate the growth defect of cells suffering from the galactose-induced and CgPdr1p-driven overexpression of a dominant lethal pma1(D378N) allele placed under the control of the ScPDR5 promoter. The system allows rapid identification of multidrug resistance reversal agents inhibiting the CgPdr1p activity or loss-of-function Cgpdr1 mutations, and is amenable to high-throughput screening on solid or liquid media.

Chemogenomic and transcriptome analysis identifies mode of action of the chemosensitizing agent CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine).

BACKGROUND: CTBT (7-chlorotetrazolo [5,1-c]benzo[1,2,4]triazine) increases efficacy of commonly used antifungal agents by an unknown mechanism. It increases the susceptibility of Saccharomyces cerevisiae, Candida albicans and Candida glabrata cells to cycloheximide, 5-fluorocytosine and azole antimycotic drugs. Here we elucidate CTBT mode of action with a combination of systematic genetic and transcriptome analysis. RESULTS: To identify the cellular processes affected by CTBT, we screened the systematic haploid deletion mutant collection for CTBT sensitive mutants. We identified 169 hypersensitive deletion mutants. The deleted genes encode proteins mainly involved in mitochondrial functions, DNA repair, transcription and chromatin remodeling, and oxidative stress response. We found that the susceptibility of yeast cells to CTBT depends on molecular oxygen. Transcriptome analysis of the immediate early response to CTBT revealed rapid induction of oxidant and stress response defense genes. Many of these genes depend on the transcription factors Yap1 and Cin5. Yap1 accumulates rapidly in the nucleus in CTBT treated cells suggesting acute oxidative stress. Moreover, molecular calculations supported a superoxide generating activity of CTBT. Superoxide production in vivo by CTBT was found associated to mitochondria as indicated by oxidation of MitoSOX Red. CONCLUSION: We conclude that CTBT causes intracellular superoxide production and oxidative stress in fungal cells and is thus enhancing antimycotic drug effects by a secondary stress.

Site-directed mutagenesis of Asp853 in Pdr3p transcriptional activator from Saccharomyces cerevisiae.

The PDR3 gene encodes one of the main transcriptional activators involved in the control of multidrug resistance in the yeast Saccharomyces cerevisiae. Recently, it has been demonstrated that a specific D853Y mutation results in the loss of transactivation activity of Pdr3p and its conversion to multicopy suppressor of multidrug resistance. In this study, the Asp853 in Pdr3p was replaced by eight different amino acids and the function of mutated proteins was analysed. Different levels of complementation of cycloheximide hypersensitivity and expression of autoregulated PDR3 and its PDR5 target in the pdr1Deltapdr3Delta mutant strain, ranging from that of the wild-type to loss-of-function alleles, were observed in pdr3 mutants containing Pro, Glu, Arg, Asn, Ser, Leu, Phe, Ile or Tyr instead of Asp853 in Pdr3p. The introduction of the D853Y mutation into gain-of-function Pdr3p suppressed the transcription of the PDR3 and PDR5 genes and reduced both the rhodamine 6G efflux rate and the drug resistance level in corresponding double mutants. The results indicate that, while Pdr3p can tolerate several substitutions of Asp853, the occurrence of a hydrophobic amino acid at this position has an adverse effect on its function.

Molecular analysis of Candida glabrata clinical isolates.

Candida glabrata is an important human pathogen, and an understanding of the genetic relatedness of its clinical isolates is essential for the prevention and control of fungal infections. In this study, we determined the relatedness of 38 Candida glabrata clinical isolates originating from two teaching hospitals in Slovakia. The 14 different genotypes were found by using microsatellite marker analysis (RPM2, MTI and Cg6) and DNA sequencing for analysis of the entire ERG11 gene. Subsequent sequencing of amplified DNA fragments of the PDR1, NMT1, TRP1 and URA3 loci in ten selected clinical isolates revealed identical DNA sequence profiles in five of them. They displayed the same microsatellite marker sizes and contained the same H576Y amino acid substitution recently described in the Pdr1p multidrug resistance transcription factor responsible for azole resistance. These results demonstrate the genetic diversity of C. glabrata clinical isolates in our hospitals and indicate a common clonal origin of some drug resistant ones.

Chemosensitisation of drug-resistant and drug-sensitive yeast cells to antifungals.

Multidrug resistance in yeast results from overexpression of genes encoding drug efflux transporters owing to gain-of-function mutations in transcription factors regulating their expression. We have screened a library of synthetic compounds for modulators of drug resistance using the multidrug-resistant Saccharomyces cerevisiae pdr3-9 mutant strain. One of the compounds, 7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine (CTBT), displayed weak antifungal activity and strongly inhibited the growth of yeast cells in combination with subinhibitory concentrations of other antifungals with a different mode of action. Biological activity of CTBT was demonstrated in Saccharomyces, Kluyveromyces and Candida yeast species grown on solid and in liquid media. The chemosensitising effect of CTBT, manifested as increased antifungal activity of fluconazole, was demonstrated in yeast mutant strains with deleted genes encoding the major multidrug resistance transcription factors Yap1p, Pdr1p and Pdr3p as well as the drug efflux pumps Pdr5p and Snq2p in S. cerevisiae or their counterparts in Candida albicans and Candida glabrata, named Cdr1p and Mdr1p, respectively. Importantly, CTBT also increased the sensitivity to fluconazole in multidrug-resistant cells overexpressing the efflux pumps. Yeast cells grown in the presence of subinhibitory concentrations of CTBT exhibited an altered sterol composition and a slightly enhanced accumulation of Rhodamine 6G, which suggests that the plasma membrane plays a role in sensitisation. This novel chemosensitisation by CTBT that can overcome multidrug resistance in yeast may prove useful in combined treatment of infections caused by drug-resistant fungal pathogens.

Resistance mechanisms in fluconazole-resistant Candida albicans isolates from vaginal candidiasis.

Candida albicans is the most frequently identified yeast species causing mycotic vaginitis. A significant number of vaginal yeast isolates are resistant to azole antifungal agents in vitro. Here we investigated the molecular mechanisms of resistance in 22 randomly selected fluconazole-resistant vaginal C. albicans isolates. Twelve isolates in this collection were found to be cross-resistant to itraconazole and 15 to voriconazole. Most of them also displayed decreased susceptibility to terbinafine. Northern blot analyses revealed overexpression of the MDR1 gene in all isolates, which in some isolates was accompanied by elevated levels of CDR1/CDR2 and ERG11 expression. Sequence analysis of the polymerase chain reaction-amplified ERG11 gene of selected azole-resistant isolates identified D116E and V488I amino acid alterations in Erg11p that are known to be conserved in fluconazole-resistant strains. The results demonstrate that decreased susceptibilities of vaginal yeast isolates to clinically used azole derivatives are the result of a combination of several molecular mechanisms involving drug efflux and alterations in the structure or cellular amount of 14-alpha-lanosterol demethylase.

Stress response and expression of fluconazole resistance associated genes in the pathogenic yeast Candida glabrata deleted in the CgPDR16 gene.

In yeasts, the PDR16 gene encodes a phosphatidylinositol transfer protein which belongs to the Sec14 homologue (SFH) family and localizes to lipid droplets, microsomes and at the cell periphery. The loss of its function alters the lipid droplet metabolism and plasma membrane properties, and renders yeast cells more sensitive to azole antimycotics. In this study, the entire chromosomal CgPDR16 ORF was replaced by the ScURA3 gene both in azole sensitive and azole resistant strains of Candida glabrata bearing a gain-of-function mutation in the CgPDR1 gene, and their responses to different stresses were assessed. The CgPDR16 deletion was found to sensitize the mutant strains to azole antifungals without changes in their osmo- and halotolerance. Fluconazole treated pdr16Δ mutant strains displayed a reduced expression of several genes involved in azole tolerance. The gain-of-function CgPDR1 allele as well as the cycloheximide and hydrogen peroxide treatments of cells enhanced the expression of the CgPDR16 gene. The results indicate that CgPDR16 belongs to genes whose expression is induced by chemical and oxidative stresses. The loss of its function can attenuate the expression of drug efflux pump encoding genes that might also contribute to the decreased azole tolerance in pdr16Δ mutant cells.

Screening for effectors that modify multidrug resistance in yeast.

The yeast transcription factors Pdr1p and Pdr3p regulate the expression of several genes that encode energy-dependent efflux pumps involved in multidrug resistance. They recognize specific pleiotropic drug resistance elements in the promoters of the target gene such as PDR5 coding for a major multidrug transporter. Gain-of-function mutations in Pdr1p/Pdr3p result in over-expression of transporter genes and establishment of multidrug resistance. We developed a novel yeast-based screening procedure designed to detect compounds that specifically modify multidrug resistance due to an interference with the expression of drug efflux transporter genes. The screening is based on the ability to abrogate the growth defect of cells suffering from the galactose induced Pdr3p driven over-expression of a dominant-lethal allele of the PMA1 gene placed under the control of the PDR5 promoter. Validation of the assay was achieved by showing that growth inhibition was relieved by mutant Pdr3p devoid of activation domain. This screening system may also be used to select the loss-of-function pdr3 (or pdr1) mutants and to identify specific gene(s) whose over-expression or deletion will suppress the expression of multidrug transporters and increase the susceptibility of yeast cells to antifungals.

Yeast strains designed for screening of reversal agents and genetic suppressors of multidrug resistance.

Multidrug resistance in yeast results from over-expression of drug efflux transporter genes due to gain-of-function mutations in transcription factors. To suppress multidrug resistance at the level of gene expression, we have developed a yeast-based screening system for the detection of compounds down-regulating the major multidrug ABC transporter Pdr5p expressed under the control of Pdr3p transcription factor. Here, we report the construction and properties of the improved set of yeast strains designed along with such screening also for a global analysis of genetic suppressors of multidrug resistance. The basic components of this system, the P(GAL1)-PDR3 and P(PDR5)-pma1(D378N) fusion genes, were individually or simultaneously integrated into corresponding chromosomes of a hypersensitive S. cerevisiae strain deleted in the PDR1 and PDR3 genes. This resulted in increased mitotic stability of a set of new test strains compared with the original prototrophic strain ZK11-1 developed previously. In addition, some of the strains designed are auxotrophic for leucine, uracil and histidine allowing them to be used in genetic screens for positive selection of multicopy or loss-of-function genetic suppressors of multidrug resistance.

Antibacterial activity of CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine) generating reactive oxygen species.

CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine) is an antifungal and chemosensitizing agent that induces oxidative stress in yeast and filamentous fungi and enhances the cytotoxic activity of 5-fluorocytosine and azole antimycotics. This study reports the effect of CTBT on bacterial cells. CTBT inhibited the growth of both Gram-positive and Gram-negative bacterial species. The action of CTBT was bactericidal. In Escherichia coli, CTBT induced an increased formation of reactive oxygen species (ROS), as determined with a ROS specific probe 2',7'-dichlorodihydrofluorescein diacetate. In zone inhibition assays, bacterial cells were more sensitive to CTBT compared with paraquat, menadione and hydrogen peroxide. The deletion of oxidative stress related genes resulted in increased susceptibility of E. coli mutant strains to CTBT treatment. Exogenous antioxidants such as ascorbic acid, cysteine and glutathione exhibited a protective effect against the growth inhibition induced by CTBT. CTBT may be a useful tool in the studies of ROS generation, oxidant sensing and oxidative stress response in different bacterial species.

CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine) producing ROS affects growth and viability of filamentous fungi.

CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine) causes intracellular superoxide production and oxidative stress and enhances the susceptibility of Saccharomyces cerevisiae, Candida albicans, and C. glabrata cells to cycloheximide, 5-fluorocytosine, and azole antimycotic drugs. Here, we demonstrate the antifungal activity of CTBT against 14 tested filamentous fungi. CTBT prevented spore germination and mycelial proliferation of Aspergillus niger and the pathogenic Aspergillus fumigatus. The action of CTBT is fungicidal. CTBT increased the formation of reactive oxygen species in fungal mycelium as detected by 2',7'-dichlorodihydrofluorescein diacetate and reduced the radial growth of colonies in a dose-dependent manner. Co-application of CTBT and itraconazole led to complete inhibition of fungal growth at dosages lower than the chemicals alone. Antifungal and chemosensitizing activities of CTBT in filamentous fungi may be useful in combination treatments of infections caused by drug-resistant fungal pathogens.

Mutations in the CgPDR1 and CgERG11 genes in azole-resistant Candida glabrata clinical isolates from Slovakia.

Candida glabrata is an important human pathogen that is naturally less susceptible to antimycotics compared with Candida albicans. Ten unmatched C. glabrata clinical isolates were selected from a collection of isolates exhibiting decreased susceptibilities to azole antifungals. Overexpression of the CgPDR1 gene, encoding the main multidrug resistance transcription factor, and its target genes CgCDR1 and CgCDR2, coding for drug efflux transporters, was observed in six fluconazole-resistant isolates. Sequence analysis of the polymerase chain reaction (PCR)-amplified DNA fragments of each isolate's CgPDR1 gene was used to identify two novel L347F and H576Y mutations in CgPdr1p. These proved to be responsible for fluconazole resistance in transformants of the C. glabrata pdr1Delta mutant strain. Five isolates harbouring the H576Y mutation also contained the mutation E502V in CgErg11p 14C-lanosterol-demethylase. Heterologous expression of the CgERG11 mutant allele did not provide evidence for its involvement in azole resistance. In four fluconazole-sensitive isolates that were itraconazole-resistant, slightly enhanced CgCDR2 expression was observed. No upregulation of the CgERG11 gene was observed in any of the ten isolates. The results demonstrate that decreased susceptibilities of C. glabrata clinical isolates to azole antifungals mainly results from gain-of-function mutations in the gene encoding the CgPdr1p transcription factor.

RPD3 and ROM2 are required for multidrug resistance in Saccharomyces cerevisiae.

The PDR5 gene encodes the major multidrug resistance efflux pump in Saccharomyces cerevisiae. In drug-resistant cells, the hyperactive Pdr1p or Pdr3p transcriptional activators are responsible for the PDR5 upregulation. In this work, it is shown that the RPD3 gene encoding the histone deacetylase that functions as a transcriptional corepressor at many promoters and the ROM2 gene coding for the GDP/GTP exchange protein for Rho1p and Rho2p participating in signal transduction pathways are required for PDR5 transcription under cycloheximide-induced and noninduced conditions. Transposon insertion mutations in ROM2, RPD3 and some other genes encoding specific subunits of the large Rpd3L protein complex resulted in enhanced susceptibility of mutant cells to antifungals. In the rpd3 Delta and rom2 Delta mutants, the level of PDR5 mRNA and the rate of rhodamine 6G efflux were reduced. Unlike rpd3 Delta, in rom2 Delta mutant cells the drug hypersensitivity and the defect in PDR5 expression were suppressed by PDR1 or PDR3 overexpressed from heterologous promoters and by the hyperactive pdr3-9 mutant allele. The results indicate that Rpd3p histone deacetylase participating in chromatin remodeling and Rom2p participating in the cell integrity pathway are involved in the control of PDR5 expression and modulation of multidrug resistance in yeast.

Functional characterization of the CgPGS1 gene reveals a link between mitochondrial phospholipid homeostasis and drug resistance in Candida glabrata.

Cardiolipin and its precursor phosphatidylglycerol are two anionic phospholipids that are essential for the biogenesis of functional mitochondria. To assess their role in mitochondrial and cellular functions in the pathogenic yeast Candida glabrata, a functional characterization of the CgPGS1 gene encoding the phosphatidylglycerolphosphate synthase has been carried out. Transposon insertion mutation in CgPGS1 resulted in the loss of phosphatidylglycerolphosphate synthase activity and in deficiency of both phosphatidylglycerol and cardiolipin. The Cgpgs1 Delta mutant cells displayed reduced amounts of cytochrome b and cytochrome a, and had impaired growth on minimal media containing non-fermentable carbon and energy sources. They did not grow at elevated temperatures and failed to form colonies after induction of mitochondrial DNA deletions. The mutant cells also displayed a decreased susceptibility to fluconazole, ketoconazole, clotrimazole, voriconazole and posaconazole. In the Cgpgs1 Delta mutant, a quantitative real time PCR revealed enhanced mRNA levels for multidrug resistance associated genes such as CgPDR1 encoding transcriptional activator and CgCDR1, CgPDH1 and CgSNQ2 coding for drug efflux transporters. These results indicate that CgPGS1 and anionic phospholipids are required for optimal mitochondrial functions and maintenance of yeast susceptibility to azole antifungals.

Loss-of-function pdr3 mutations convert the Pdr3p transcription activator to a protein suppressing multidrug resistance in Saccharomyces cerevisiae.

The PDR1 and PDR3 genes encode the main transcription activators involved in the control of multidrug resistance in Saccharomyces cerevisiae. To identify the amino acids essential for Pdr3p function, the loss-of-function pdr3 mutants were isolated and characterized. Two plasmid-borne pdr3 alleles, pdr3-E902Ter and pdr3-D853Y, which failed to complement drug hypersensitivity in the Deltapdr1Deltapdr3 mutant strain, were isolated. The E902Ter mutation resulted in a truncated protein lacking the C-terminal activation domain. The D853Y mutation allowed the expression of entire Pdr3p, but its transactivation function was lost. When overexpressed from the P(GAL1) promoter, the two mutant alleles increased the sensitivity of wild-type cells to cycloheximide and fluconazole and suppressed drug resistance in gain-of-function pdr1 and pdr3 mutant strains. The drug-sensitizing effect of overexpressed loss-of-function pdr3 mutant alleles correlated with their ability to suppress PDR5 transcription and rhodamine 6G accumulation in transformants of the wild-type and Deltapdr1 mutant strains. These results demonstrate that amino acid residue Asp853 is essential for Pdr3p function, and indicate that specific loss-of-function pdr3 mutations can convert the Pdr3p transcription activator to a multicopy suppressor of multidrug resistance.

YAP1-mediated KNQ1 expression in Kluyveromyces lactis.

The b-Zip transcription factor Yap1p plays an important role in oxidative stress response and multidrug resistance in Saccharomyces cerevisiae. We have previously demonstrated that the KNQ1 gene, encoding a multidrug transporter of the major facilitator superfamily in Kluyveromyces lactis and containing two potential Yap1p response elements in its promoter, is a putative transcriptional target of KlYap1p, the structural and functional homologue of ScYap1p. In this work, we provide evidence that KlYAP1 controls the expression of the KNQ1 gene. Using a P(KNQ1)-gusA fusion construct we showed that the expression of KNQ1 is induced upon cell treatment with the oxidizing agents H2O2 and menadione and that this induction is mediated by KlYap1p. These results were confirmed by Northern-blot analysis showing that the expression of KNQ1 is responsive to hydrogen peroxide and dependent on the presence of KlYap1p. The role of KlYAP1 in the control of KNQ1 expression was further demonstrated by EMSA experiments and drug resistance assays. These results clearly demonstrate the involvement of the KlYap1p transcription factor in the control of KNQ1 gene expression.

Fluconazole and itraconazole susceptibility of vaginal yeast isolates from Slovakia.

Vulvovaginal candidiasis is a common mucosal infection caused by opportunistic yeasts of the Candida genus. In this study, we isolated and identified the yeast species in the vagina of patients treated in the gynecology clinic and tested in vitro activities of fluconazole and itraconazole against 227 clinical yeast isolates by the NCCLS microdilution method. C. albicans (87.6%) was the most frequently identified species followed by C. glabrata (6.2%) and C. krusei (2.2%). Almost thirteen percent of yeast strains were resistant to fluconazole and 18.5% were resistant to itraconazole. Cross-resistance analyses of C. albicans isolates revealed that fluconazole resistance and itraconazole resistance were also associated with decreased susceptibilities to other azole derivatives mainly to ketoconazole and miconazole. At the same time no cross-resistance to polyene antibiotics amphotericin B and nystatin was observed. These results support the notion that antifungal agents used to treat vaginitis may be contributing to the drug resistance problem by promoting cross-resistance to a range of clinically used antifungals.

Isolation, heterological cloning and sequencing of the RPL28 gene in Kluyveromyces lactis.

By virtue of heterologous functional complementation of the Saccharomyces cerevisiae Delta pdr5 mutant strain, using a Kluyveromyces lactis genomic library, three different K. lactis chromosomal inserts were obtained. Transformation of the S. cerevisiae Delta pdr1 Delta pdr3 mutant strain, hypersensitive to drugs, with isolated plasmids resulted in resistance to cycloheximide and fluconazole. Transformation of K. lactis host strains, using the cloned chromosomal fragments, led to an increased level of resistance to some mitochondrial inhibitors and azole antifungals. The nucleotide sequence of the cloned inserts revealed that two of them contain the drug efflux transporter gene Kl-PDR5 and the third contains a DNA segment homologous to chromosome VII of S. cerevisiae. Along with three novel ORFs, encoding two proteins of unknown molecular function and one putative hexose transporter, this segment also contained the Kl-RPL28 gene, found to be responsible for the cycloheximide resistance of heterologous transformants. This gene codes for the large subunit ribosomal protein (149 amino acids) that shares 89.9% identity with its S. cerevisiae counterpart. The coding region of Kl-RPL28 was found to be interrupted with one intron near the 5' end. The nucleotide sequence data reported in this paper were submitted to GenBank and assigned the accession number AF493565.