Transcription profiling of yeast treated with rapamycin
Ontology highlight
ABSTRACT: A diploid yeast strain was grown at 30C in YPD medium. At an OD of 0.6 (600nm) rapamycin was added to a final concentration of 0.20 ug/ml and the incubation continued for 90 min. A control culture was treated with an equal amount of DMSO vehicle. Cells were harvested at an OD of 0.9 rapamycin and 1.1 control.
Project description:Target of rapamycin complex 1 (TORC1) is implicated in growth control and aging from yeast to humans. Fission yeast is emerging as a popular model organism to study TOR signaling, although rapamycin has been thought to not affect cell growth in this organism. Here we analyzed the effects of rapamycin and caffeine, singly and combined, on multiple cellular processes in fission yeast. The two drugs led to diverse and specific phenotypes that depended on TORC1 signaling pathway inhibition, including prolonged chronological lifespan, inhibition of global translation, inhibition of cell growth and division, and reprogramming of global gene expression mimicking nitrogen starvation. Rapamycin and caffeine differentially affected these various TORC1-dependent processes. Combined drug treatment augmented most phenotypes and effectively blocked cell growth. Although rapamycin showed a much more subtle effect on global translation than did caffeine, rapamycin was more effective in prolonging chronological lifespan. Rapamycin prolonged the lifespan of non-growing cells only when applied during the growth phase but not when applied after cells had stopped proliferation. The doses of rapamycin and caffeine strongly correlated with growth inhibition and with lifespan extension. This comprehensive analysis will inform future studies into TORC1 function and cellular aging in fission yeast and beyond.
Project description:Comparison between Lp Paris wt and rpoN mutant in exponential phase (OD 3.3). We investigated the role of FleQ, FleR, RpoN, and FliA on the regulation of the expression of transmissive traits in L. pneumophila strain Paris at different time points (replicative phase, early transmissive phase, late transmissive phase).
Project description:To comparatively analyze the transcriptional responses of yeast cells to the presence of rapamycin or caffeine the homozygous hoΔ/hoΔ strain of S. cerevisiae BY4743 were grown in fully controlled fermenters in the presence of 200 nM rapamycin or 5 mM caffeine. Cells were cultured overnight in YPD medium (2% [w/v] D-glucose, 2% [w/v] peptone, 1% [w/v] yeast extract) at 30°C in an orbital shaker at 180 rpm prior to the fermentations. For the cultivations in the fermenters defined synthetic medium was used. The batch cultivations were carried out in duplicates in 2L B-Braun Biostat B Plus fermenters with 1.5L working volume kept at 30°C with the rate of agitation at 800rpm. pH was controlled at 5.5 with 0.5M NaOH and HCl. Sampling was carried out at the mid-exponential phase of growth at an OD range of 0.6-0.8. Samples harvested for the transcriptome analysis were immediately frozen in liquid nitrogen and were stored at -80oC until RNA isolation.
Project description:Nuclear depletion of the essential transcription termination factor Nrd1 in Saccharomyces cerevisiae was studied using a combination of RNA-Seq, ChIP-Seq of Pol II and PAR-CLIP of Nrd1. The drug rapamycin induces the formation of a ternary complex between a protein of interest, the drug and the small subunit of the ribosome (both proteins are genetically engineered). The small ribosome subunit is transported out of the nucleus. therefore the protein of interest can be depleted from nucleus upon treatment with rapamycin.
Project description:We are interested in the study of the already reported sensitivity of yeast cells lacking the type 1 protein phosphatase Ptc1 to the drug rapamycin. In order to carry out our studies, we analyzed the changes in the transcription profile that a short-term incubation with rapamycin (200 ng/mL) has in both, wild type and ptc1 cells. It has been shown that inhibition of the TOR pathway by treatment with rapamycin has profound effects on the global transcriptional profile. We confirmed the previously described transcription changes induced by the drug in wild type cells. Under our working conditions rapamycin increased, in wild type cells, at least 2-fold the expression of 667 genes, whereas it decreased the mRNA level of 721 genes (13.8% and 14.9% of genes with measurable expression level, respectively). Gene ontology analysis shows that, as previously documented, many induced genes falls into the NCR, Msn2/Msn4 regulated stress response or Retrograde response categories, whereas genes encoding cytoplasmic, but not mitochondrial, ribosomal proteins and the so called RIBI regulon where largely repressed. Deletion of PTC1 decreases the number of genes induced or repressed at least 2-fold by rapamycin treatment. Remarkably, lack of Ptc1 seems to lead to a general attenuation of changes triggered by rapamycin. The wt and the ptc1 mutant strains were analyzed in this series. We compared the expression profile of each strain treated with Rapamycin (200 ng/ml for 1h) with that of the same strain mock-treated (90% ethanol, 10% Tween-20). A Dye-swap was carried out for each RNA sample. Total number of chips analyzed: 4
Project description:Transcript level changes in transcription factor mutants after rapamycin treatment, compared to the wild-type strain. This data set accompanies the study "Widespread Misinterpretable ChIP-seq Bias in Yeast" (GSE51251) Four separate wild-type cultures of yeast were grown, and each culture was split into two. Then, one was treated with DMSO and the other one was treated with rapamycin. For transcription factor mutants, two separate cultures were grown, and rapamycin was treated in the same way as treated to the wild type. Total RNA was recovered from each culture, then labeled oligo was prepared following NimbleGene standard protocol. Each chip measures the expression levels of 5,777 genes from yeast.
Project description:Colletotrichum orbiculare Whi2, yeast stress response Whi2 homolog, is involved in switch from biotrophic to necrotrophic stage. To elucidate downstream genes regulated by Co Whi2, we have conducted DNA microarray. About 3100 genes were up or down regulated in the Co whi2Î mutant compared with the wild-type. In particularly, 44 genes among up-regulated 58 genes in the Co whi2Î mutant are ribosomal protein related gene. Eukaryote is widely conserved TOR (Target Of Rapamycin) which is known to regulator of ribosomal gene expression. To elucidate whether up-regulated ribosomal genes in the Co whi2Î mutant are regulated by TOR activity, we have conducted DNA microarray in the Co whi2Î mutant treated with rapamycin inhibiting TOR activity. The enormous ribosomal gene expression in the Co whi2Î mutant treated with the rapamycin is lower than that without rapamycin treatment. In gene expression of the Co whi2Î mutant, the wild-type and the Co whi2Î mutant infecting on cucumber cotyledons were assessed at 4 hours post-inoculation. In gene expression of the Co whi2Î mutant with rapamycin treatment, Co whi2Î mutant treated with 100nM rapamycin and Co whi2Î mutant without rapamycin treatment infecting on cucumber cotyledons were assesed at 4 hours post-inoculation. Four replication were performed for each experiments.
Project description:The first aim was to identify genes whose transcription is induced by rapamycin feeding in Drosophila larvae. Secondly, the goal was to find out which contribution the transcription factor REPTOR (=CG13624) has to the observed changes in expression. We thus compared gene epxression between rapamycin fed and control fed larvae in wild type larvae and in REPTOR KO larvae. 3 biological replicates from 4 conditions: control larvae plus/miuns rapamycin, KO larvae plus/minus rapamycin; overall 12 samples