Project description:We examined the mating response of W303 bar1delta a-type cells to six alpha-factor concentrations (0.06, 0.2, 0.6, 6, 60 and 600 nM). In each alpha factor concentration between five to seven time points were collected. The time points in all experiments (except for concentration 600 nM were time point 15 min was omitted) were 5, 15, 30, 60, and 90 min. In some of the concentrations also time points 120 and 150 min were considered. The expression of cells before and after addition of alpha-factor were compared using S. cerevisiae cDNA microarrays, in all experiments the same sample before adding alpha factor was used as a control. Keywords: yeast mating response, comparison among alpha factor concentrations
Project description:We examined the mating response of three yeast species, by subjecting a-type cells from each of them to alpha-factor (6uM). The expression of cells before and after (60min) addition of alpha-factor were compared using S. cerevisiae cDNA microarrays. In addition, we verified our results with a true mating experiment: the expression of a-type and alpha-type cells from S. cerevisiae were compared before and after mixing them for 90 minutes (i.e. allowing them to mate). Keywords: yeast mating response, comparison among species
Project description:Haploid budding yeast has two mating types, defined by the alleles of the MAT locus, MATa and MATM-NM-1. Mating occurs when two haploid cells of opposite mating types signal to each other using reciprocal pheromones and receptors, polarize and grow towards each other, and eventually fuse to form a single diploid cell. The pheromones and receptors are necessary and sufficient to define a mating type, but other mating type-specific proteins make mating more efficient. We examined the role of these proteins by genetically engineering M-bM-^@M-^\transvestiteM-bM-^@M-^] cells that swap the pheromone, pheromone receptor, and pheromone processing factors of one mating type for another. These cells can mate with each other, but their mating is inefficient. By characterizing their mating defects and examining their transcriptomes, we found Afb1 (a-factor barrier), a novel MATM-NM-1-specific protein that interferes with a-factor, the pheromone secreted by MATa cells. We show that strong pheromone secretion is essential for efficient mating and that the weak mating of transvestites can be improved by boosting their pheromone production. Using synthetic biology, it is possible to characterize the factors that control efficiency in biological processes. In the case of budding yeast mating, selection for increased mating efficiency is likely to have continually boosted pheromone levels and the ability to discriminate between partners who make more (potentially fitter) and less (potentially less fit) pheromones. This sensitivity to which partner makes more pheromone comes at a cost: it means mating is not robust in situations where all potential partners make less pheromone. 4 conditions were analysed, each with 3 biological replicates. The conditions were unstimulated MATa cells in YPD. Stimulated MATa cells in YPD+10nM M-NM-1-factor. Unstimulated MATM-NM-1 cells in YPD. Stimulated MATM-NM-1 cells in YPD+10nM M-NM-1-factor.
Project description:We examined the mating response of three yeast species, by subjecting a-type cells from each of them to alpha-factor (6uM). The expression of cells before and after (60min) addition of alpha-factor were compared using S. cerevisiae cDNA microarrays. In addition, we verified our results with a true mating experiment: the expression of a-type and alpha-type cells from S. cerevisiae were compared before and after mixing them for 90 minutes (i.e. allowing them to mate). Keywords: yeast mating response, comparison among species Three (or four) samples were processed in parallel for each species (biological replicates). All samples were measured by hybridization to S. cerevisiae cDNA microarray
Project description:Identification of differentially expressed genes in a deletion strain of α-domain mating-type transcription factor MAT1-1-1 compared to wild type P2niaD18 Analysis of total RNA from P. chrysogenum strains grown for 36, 60, and 96 h on solid M322 medium under light conditions at 27°C
Project description:We measured the response of S. cerevisiae to arrest in the presence of alpha factor. These were collected in support of a related DNaseI-sequencing study. Keywords: Alpha-factor arrest S.cerevisiae R276 (MATa ura3Δ0 leu2Δ0 his3Δ1 met15Δ0 bar1Δ::KanMX) (C. Boone, University of Toronto; S288c background derived from BY4741), was cultured overnight in 50 ml rich medium (YPD) at 30°C, diluted into 500 ml fresh YPD to an OD660 of ~0.8, and treated with yeast α-factor (Sigma-Aldrich) at a final concentration of 50 ng / ml. This culture was incubated at 30°C with shaking for 3 hours (final OD660 ~1). After this treatment, approximately 90% of the cells had formed mating projections when checked by light microscopy. Total RNA from these cells was isolated using hot acidic phenol. 50 μg of total RNA was treated with Turbo Dnase (Ambion), and checked for integrity using a Bioanalyzer 2100 (Agilent). Total RNA was labeled according to the manufacturer’s protocol and applied to Affymetrix Yeast 2.0 arrays. Data were analyzed using the “affy” package from Bioconductor.
Project description:Haploid budding yeast has two mating types, defined by the alleles of the MAT locus, MATa and MATα. Mating occurs when two haploid cells of opposite mating types signal to each other using reciprocal pheromones and receptors, polarize and grow towards each other, and eventually fuse to form a single diploid cell. The pheromones and receptors are necessary and sufficient to define a mating type, but other mating type-specific proteins make mating more efficient. We examined the role of these proteins by genetically engineering “transvestite” cells that swap the pheromone, pheromone receptor, and pheromone processing factors of one mating type for another. These cells can mate with each other, but their mating is inefficient. By characterizing their mating defects and examining their transcriptomes, we found Afb1 (a-factor barrier), a novel MATα-specific protein that interferes with a-factor, the pheromone secreted by MATa cells. We show that strong pheromone secretion is essential for efficient mating and that the weak mating of transvestites can be improved by boosting their pheromone production. Using synthetic biology, it is possible to characterize the factors that control efficiency in biological processes. In the case of budding yeast mating, selection for increased mating efficiency is likely to have continually boosted pheromone levels and the ability to discriminate between partners who make more (potentially fitter) and less (potentially less fit) pheromones. This sensitivity to which partner makes more pheromone comes at a cost: it means mating is not robust in situations where all potential partners make less pheromone.
Project description:RpoN was shown to have a pleiotropic role in different microorganisms. This study was performed to elucidate role in B. cereus. In this study we compared the transcriptomic profiles of the WT, rpoN mutant and the complemented strain in aerated and static growth conditions. Matrix is compatible with the Agilent-017343 array design. Aerated cultures were sampled at two time points, upon reaching OD (600 nm) values of 0.2 (t1) and 1.0 (t2), which corresponded to mid-exponential and transition growth phases, respectively. Statically grown cultures were sampled at OD=0.2 (600 nm) corresponding to exponential growth (exp/static). Two independent biological replicates were hybridized on the arrays with either 2 (WT) or 3 (Δrpon and ΔrpoN-comp) technical replicates for each biological replicate.