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PubMed1990

The purine-cytosine permease gene of Saccharomyces cerevisiae: primary structure and deduced protein sequence of the FCY2 gene product.

A 2.1 kb DNA segment carrying the purine-cytosine permease gene (FCY2) of Saccharomyces cerevisiae was sequenced, the primary structure of the protein (533 amino acids) deduced and a folding pattern in the membrane is proposed for the permease protein. Expression of the FCY2 gene product requires a functional secretory pathway and is reduced in mnn9, a mutant strain deficient in outer chain glycosylation. The FCY2 gene was mapped on the right arm of chromosome V close to the HIS1 gene.

Amino Acid Sequence↗

PubMed1992

In vivo and in vitro studies of the purine-cytosine permease of Saccharomyces cerevisiae. Functional analysis of a mutant with an altered apparent transport constant of uptake.

The FCY2 gene of the purine-cytosine permease (PCP) of Saccharomyces cerevisiae and the allele fcy2-21 have been cloned on the yeast multicopy plasmid pJDB207. The corresponding plasmids were introduced into a S. cerevisiae strain carrying a chromosomal deletion at the FCY2 locus. The resulting strains were designated pAB4 and pAB25 respectively. The pAB25 strain, which carries the fcy2-21 allele, contains four amino acid changes in the open reading frame of the PCP (Weber et al., 1989). The influence of these mutations was studied on cells by determination of the uptake constants of purine bases and cytosine [apparent Michaelis constant of transport (Ktapp) and Vmax] and on plasma-membrane preparations, by measurements of binding parameters at equilibrium [(Kd and maximum amount of binding sites/Bmax)]. For strain pAB4, the Ktapp and Vmax of uptake were almost similar for all solutes considered [1.8-2.6 microM and 8.5-10.2 nmol.min-1.(10(7) cells)-1]. The main effect of the mutations in strain pAB25 was based on a large increase in Ktapp for all ligands except adenine. Plasma membranes of each strain displayed one class of specific binding sites. Variations in Kd of 0.4-1 microM were observed for pAB4. These slight variations had no effect on the Ktapp of uptake measured for the corresponding solutes. In contrast, using pAB25 membranes, Kd increased dramatically; 2.6 microM, 40 microM and 96 microM for adenine, cytosine and hypoxanthine, respectively. These increments were correlated to variations in Ktapp of the uptake for cytosine and hypoxanthine. Therefore, we conclude that modification in the Ktapp of uptake in the strain carrying fcy2-21 allele is merely due to a modification of the binding ability of the permease for its ligands.

Biological Transport↗

PubMed1992

Purine-cytosine permease of Saccharomyces cerevisiae. Effect of external pH on nucleobase uptake and binding.

The cloned FCY2 gene (strain pAB4) of the purine-cytosine permease (PCP) of Saccharomyces cerevisiae and the cloned allele fcy2-21 (strain pAB25) introduced into an S. cerevisiae strain carrying a chromosomal deletion at the FCY2 locus [Weber, E., Rodriguez, C., Chevallier, M. R. & Jund, R. (1990) Mol. Microbiol. 4, 585-596] were studied. The influence of external pH (varying over 3.5-6) has been analysed on the uptake of adenine, hypoxanthine and cytosine (Ktapp, apparent Michaelis constant and Vm) and on the binding constants of these three solutes (Kdapp, apparent half-saturation constant and Bmax, total binding sites) determined on plasma membranes. For pAB4, the variations of Ktapp and Vm were the same for the three bases, i.e. an increase in Ktapp when the pH increased and a maximum Vm around pH 5. For pAB25, Ktapp values varied in the same way and were significantly higher for the three bases than those found in pAB4. There was almost no variation of Vm for adenine, and there was a continuous decrease when the pH increased in the Vm of hypoxanthine and cytosine. Equilibrium binding measurements were performed for the three bases with plasma membrane isolated from pAB4 and pAB25. One single class of binding sites was detected. For pAB4, the affinity increased when the pH decreased for the three bases. The affinity of PCP for adenine was always greater than for cytosine or hypoxanthine. For pAB25, the same phenomenon was observed. However, the curves showing the variation of Kdapp as a function of pH were shifted towards more acidic pH values. A model was used to fit the experimental binding data obtained with hypoxanthine for the calculation of the dissociation constants of its binding to PCP and to determine the ionization constants of an amino acid involved in ligand binding. For pAB4, at acid pH, the dissociation constant was 1.7 +/- 0.4 microM. An amino acid displaying a pK of 3.8 was determined; this value was shifted to pK 4.8 when hypoxanthine was bound. For pAB25, the main effects of the mutation were a large decrease in the affinity of PCP for hypoxanthine (Kd of 14.4 +/- 4.3 microM) and a shift in the pK of the amino acid towards a more acidic pH (about 2.9). The pK of this group remained similar to the value obtained with pAB4 when hypoxanthine was bound. From these data, it is proposed that the binding of hypoxanthine and H+ is a random process.

Adenine↗

PubMed2026

Resistance gene mutations and phylogenetic relationships in Candidozyma auris isolates from Russia.

INTRODUCTION: Candidozyma auris is an emerging healthcare-associated fungal pathogen with a high propensity for nosocomial transmission and development of antifungal resistance. This study aimed to identify resistance-associated genomic variants and characterize the phylogenetic structure of clinical C. auris isolates circulating in Russia. METHODS: We analyzed 82 isolates collected between 2017 and 2023 from 18 hospitals in the Northwestern and Central Federal Districts of the Russian Federation. Antifungal susceptibility testing was combined with whole-genome sequencing, targeted FCY2 sequencing, and comparative phylogenomic analysis using publicly available international genomes. RESULTS: All isolates analyzed in this study belonged to clade I and showed a highly conserved profile of elevated azole MICs. The consistent detection of ERG11 (K143R), TAC1B (A640V), and CDR1 (V704L) suggests that reduced azole susceptibility in this population is associated with both target-gene alteration and efflux-mediated mechanisms. All isolates remained susceptible to echinocandins in vitro, and no resistance-conferring mutations were detected in FKS1, consistent with the absence of an echinocandin-resistant phenotype. Decreased susceptibility to flucytosine was mainly associated with the FCY2 (L383*) nonsense mutation, which was confirmed by targeted Sanger sequencing in additional isolates. Phylogenomic reconstruction showed that the Russian isolates represented a restricted segment of global clade I diversity and revealed two major geographically structured lineages corresponding to two large metropolitan areas in European Russia. DISCUSSION: The distribution of closely related isolates across hospitals supports local persistence and inter-hospital dissemination of genetically related strains. These findings provide important insights into the molecular epidemiology, antifungal resistance mechanisms, and transmission dynamics of C. auris in Russia.

Phylogeny↗

PubMed1987

The genetic basis of resistance to 5-fluorocytosine in Candida species and Cryptococcus neoformans.

In terms of genetically determined susceptibility to the clinical antifungal agent 5-fluorocytosine (5-FC), Candida albicans may be homozygous sensitive (FCY/FCY), homozygous resistant (fcy/fcy), or heterozygous (fcy/FCY). Although heterozygotes are only slightly resistant, they occur at significant frequency among clinical strains and carry preexisting resistance determinants which may be responsible, following homozygosis, for treatment failures. There are two resistance genes (FCY1 and FCY2) known. Resistance in fcy1/fcy1 strains was associated with decreased UMP pyrophosphorylase activity, whereas resistance in fcy2/fcy2 strains was associated with decreased cytosine deaminase activity. These results were confirmed and extended in a 19F nuclear magnetic resonance study of 5-FC uptake and metabolism in genetically defined strains. By means of hybridization via spheroplast fusion, a complementation test was devised to test allelism of resistance determinants. Resistance to 5-FC was employed as a useful genetic marker in basic studies. In tetraploid hybrids which bore appropriate fcy markers, it was possible to select for reduction in ploidy by selecting for increased resistance to 5-FC; a novel parasexual system was thus generated (2n x 2n----4n----2n). In linkage studies, the gene FCY1 was shown to be linked to the gene HIS. Reciprocal mitotic recombination was demonstrated repeatedly with fcy1 and his alleles in cis and in trans configurations and evidence for nonreciprocal recombination (mitotic gene conversion) was also obtained. In Cryptococcus neoformans, mutation in either of two genes (FCY1, FCY2) is sufficient to confer resistance. These genes behave as simple Mendelian determinants which recombine freely. Diploid C. neoformans heterozygous for resistance (FCY/fcy) provided useful strains in which to develop genetic mapping methodology based on mitotic recombination.

Candida↗

PubMed1988

Evolutionary relationship and secondary structure predictions in four transport proteins of Saccharomyces cerevisiae.

The comparison of the amino acid sequences of four yeast transport proteins indicates that there is a questionable relatedness between the uracil permease (FUR4) and the purine-cytosine permease (FCY2), whereas the arginine permease (CAN1) and the histidine permease (HIP1) clearly originated from a common molecular ancestor. The analysis of the primary structure of these transport proteins by two methods of secondary structure predictions suggests the presence of 9-12 membrane-spanning alpha-helices in each polypeptide chain. These results are concordant in that 90% of the alpha-helices were determined by both methods to be at the same positions. In the aligned sequences HIP1 and CAN1, the postulated membrane-spanning alpha-helices often start at corresponding sites, even though the overall sequence similarity of the two proteins is only 30%. In the aligned DNA coding sequences of CAN1 and HIP1, synonymous nucleotide substitutions occur with very similar frequencies in regions where the replacement substitution (changing the amino acids) frequencies are widely different. Moreover, our data suggest that the replacement substitutions can be considered as neutral in the N-terminal segment, whereas the other regions are subject to a conservative selective pressure because, if compared to a random drift, the replacement substitutions are underrepresented.

Amino Acid Sequence↗

PubMed2025

Whole genome sequencing analysis of Candida glabrata isolates collected from patients with selected drug-resistant candidiasis hospitalized in Eastern Poland.

The epidemiology data for candidiasis indicate an increase in Candida glabrata infections. Moreover, several reports have shown an increasing number of drug-resistant cases of these infections. The source of drug resistance can often be traced to genetic mutations in genes related to a drug's mechanism of action. Therefore, we conducted whole genome sequencing of several drug-resistant isolates of Candida glabrata collected from patients hospitalized in Eastern Poland to assess whether mutations in selected genes correlated with susceptibility analysis results. The fungal species from patient samples were identified, and the isolated Candida glabrata were subjected to antifungal drug susceptibility testing. The results were interpreted according to the EUCAST and CLSI recommendations. Susceptibility to 5-flucytosine was assessed using the ATB FUNGUS kit. Libraries were prepared according to the NEXTERA XT DNA Library Prep and subsequently sequenced. The outcomes indicated common resistance to two of the three analyzed echinocandins, as well as two cases of simultaneous resistance to echinocandins and selected azole-based drugs. We detected several previously reported mutations in selected resistance-related genes, as well as five that are first described here: ERG5 (M267I), ERG6 (R57K), PDH1 (K438Q, V434I, F600V, V1192S), FCY1 (M129T), and FCY2 (I384F). Neither of the identified nonsynonymous mutations was correlated with the drug resistance demonstrated in the susceptibility testing. Furthermore, we can exclude the possibility of acquired drug resistance, thereby raising questions about the possibility of unknown mechanisms of resistance to azole-based and echinocandin drugs.

Candida glabrata↗