Project description:The enzyme telomerase ensures the integrity of linear chromosomes by maintaining telomere length. As a hallmark of cancer, cell immortalization and unlimited proliferation is gained by reactivation of telomerase. However, a significant fraction of cancer cells instead uses alternative telomere lengthening mechanisms to ensure telomere function, collectively known as Alternative Lengthening of Telomeres (ALT). Although the budding yeast Naumovozyma castellii (Saccharomyces castellii) has a proficient telomerase activity, we demonstrate here that telomeres in N. castellii are efficiently maintained by a novel ALT mechanism after telomerase knockout. Remarkably, telomerase-negative cells proliferate indefinitely without any major growth crisis and display wild-type colony morphology. Moreover, ALT cells maintain linear chromosomes and preserve a wild-type DNA organization at the chromosome termini, including a short stretch of terminal telomeric sequence. Notably, ALT telomeres are elongated by the addition of ?275 bp repeats containing a short telomeric sequence and the subtelomeric DNA located just internally (TelKO element). Although telomeres may be elongated by several TelKO repeats, no dramatic genome-wide amplification occurs, thus indicating that the repeat addition may be regulated. Intriguingly, a short interstitial telomeric sequence (ITS) functions as the initiation point for the addition of the TelKO element. This implies that N. castellii telomeres are structurally predisposed to efficiently switch to the ALT mechanism as a response to telomerase dysfunction.
Project description:RNAi, a gene-silencing pathway triggered by double-stranded RNA, is conserved in diverse eukaryotic species but has been lost in the model budding yeast, Saccharomyces cerevisiae. We report that RNAi is present in other budding-yeast species, including Saccharomyces castellii and Candida albicans. These species use noncanonical Dicer proteins to generate siRNAs, which mostly correspond to transposable elements and Y´ subtelomeric repeats. In S. castellii, RNAi mutants are viable but have excess Y´ mRNA levels. In S. cerevisiae, introducing Dicer and Argonaute of S. castellii restores RNAi, and the reconstituted pathway silences endogenous retrotransposons. These results identify a novel class of Dicer proteins, bring the tool of RNAi to the study of budding yeasts, and bring the tools of budding yeast to the study of RNAi. Examine mRNA abundance in two biological replicates of wild-type (DPB005) and RNAi deletion strains (DPB007, DPB009) of S. castellii.
Project description:RNAi, a gene-silencing pathway triggered by double-stranded RNA, is conserved in diverse eukaryotic species but has been lost in the model budding yeast, Saccharomyces cerevisiae. We report that RNAi is present in other budding-yeast species, including Saccharomyces castellii and Candida albicans. These species use noncanonical Dicer proteins to generate siRNAs, which mostly correspond to transposable elements and Y´ subtelomeric repeats. In S. castellii, RNAi mutants are viable but have excess Y´ mRNA levels. In S. cerevisiae, introducing Dicer and Argonaute of S. castellii restores RNAi, and the reconstituted pathway silences endogenous retrotransposons. These results identify a novel class of Dicer proteins, bring the tool of RNAi to the study of budding yeasts, and bring the tools of budding yeast to the study of RNAi. Overall design: Examine mRNA abundance in two biological replicates of wild-type (DPB005) and RNAi deletion strains (DPB007, DPB009) of S. castellii.
Project description:Phytate (myo-inositol hexakisphosphate) is the primary storage form of phosphate in seeds and legumes (Reddy et al., 1982). Phytases are phosphatases that hydrolyze phytate to less phosphorylated myo-inositol derivatives and inorganic phosphate. The crystal structure of phytase from Debaryomyces castellii has been determined at 2.3 A resolution. The crystals belonged to space group P6(5)22, with unit-cell parameters a = 121.65, c = 332.24 A. The structure was solved by molecular replacement and refined to a final R factor of 15.7% (R(free) = 20.9%). The final model consists of a dimer (with two monomers of 458 residues), five NAG molecules and 628 water molecules.
Project description:Total RNA versus genomic DNA hybridization on custom arrays designed for all Saccharomyces castellii genes Total RNA was collected in mid-log phase from Saccharomyces castellii cells grown in rich medium (abbreviated CM, in house recipe). RNA was then converted to cDNA, Cy3-labeled and hybridized competitively against Cy5 labeled genomic DNA from Saccharomyces castellii.
Project description:Total RNA versus genomic DNA hybridization on custom arrays designed for all Saccharomyces castellii genes Overall design: Total RNA was collected in mid-log phase from Saccharomyces castellii cells grown in rich medium (abbreviated CM, in house recipe). RNA was then converted to cDNA, Cy3-labeled and hybridized competitively against Cy5 labeled genomic DNA from Saccharomyces castellii.
Project description:RNA interference (RNAi), a gene-silencing pathway triggered by double-stranded RNA, is conserved in diverse eukaryotic species but has been lost in the model budding yeast Saccharomyces cerevisiae. Here, we show that RNAi is present in other budding yeast species, including Saccharomyces castellii and Candida albicans. These species use noncanonical Dicer proteins to generate small interfering RNAs, which mostly correspond to transposable elements and Y' subtelomeric repeats. In S. castellii, RNAi mutants are viable but have excess Y' messenger RNA levels. In S. cerevisiae, introducing Dicer and Argonaute of S. castellii restores RNAi, and the reconstituted pathway silences endogenous retrotransposons. These results identify a previously unknown class of Dicer proteins, bring the tool of RNAi to the study of budding yeasts, and bring the tools of budding yeast to the study of RNAi.
Project description:The yeast Candida glabrata is a major opportunistic pathogen causing mucosal and systemic infections in humans. Systemic infections caused by this yeast have high mortality rates and are difficult to treat due to this yeast's intrinsic and frequently adapting antifungal resistance. To understand and treat C. glabrata infections, it is essential to investigate the molecular basis of C. glabrata virulence and resistance. We established an RNA interference (RNAi) system in C. glabrata by expressing the Dicer and Argonaute genes from Saccharomyces castellii (a budding yeast with natural RNAi). Our experiments with reporter genes and putative virulence genes showed that the introduction of RNAi resulted in 30 and 70% gene-knockdown for the construct-types antisense and hairpin, respectively. The resulting C. glabrata RNAi strain was used for the screening of a gene library for new virulence-related genes. Phenotypic profiling with a high-resolution quantification of growth identified genes involved in the maintenance of cell integrity, antifungal drugs, and ROS resistance. The genes identified by this approach are promising targets for the treatment of C. glabrata infections.