Transgenic yeast (pAG305GPD-AAE13) vs. control yeast (pAG305GPD-ccdB)
ABSTRACT: The yeast expression vectors used to express AAE13 was constructed as follows: AAE13 (At3g16170) was amplified from the vector pENTR-AAE13. The amplified product was cloned into pCR®8⁄GW⁄TOPO® (Invitrogen). The resulting plasmid was combined with the Advanced Gateway destination vectors pAG305GPD-ccdB (Addgene, Boston, MA) in an attL X attR recombination reaction to generate the vectors pAG305GPD-AAE13. The integrating vector pAG305GPD-AAE13 was introduced into WAT11, which was also transformed empty vector pAG305GPD-ccdB as control (B1, B2, B3 and B4). The transgenic and control yeast cells were cultured in SD-drop out medium at 30 °C under shaken conditions, malonate was fed into cells at final concentration 0.2 mM when the value of OD600 reached at 0.1. The cells were harvested at mid-log phase. Two-condition experiment, Transgenic yeast (pAG305GPD-AAE13) vs. control yeast (pAG305GPD-ccdB). Biological replicates: 4 control replicates, 4 transgenic replicates.
Project description:We did transcription profiling on the effect of RCK1 over-expression. rck1 mutant strain was transformed with empty high copy vector pRS425 empty vector), or with RCK1 cloned into pRS425 (the RCK1-overexpressing strain: RCK1 cloned into pRS425). Analysis was performed with rck1 (Empty vector, pRS425) mutant and RCK1 over-expressed cells (pRS425-RCK1). Yeast cells were grown on SD-leu medium. Yeast cells were grown overnight at 30°C . The culture was refreshed to 0.2 O.D and grown at 30°C for 2h 30min. Cell wall stress was applied by addition of zymolyase to a final concentration of 5 unit/ml . Cells were collected at 2 hours of growth and processed for RNA extraction.
Project description:Virulence of Cryptococcus neoformans for mammals was proposed to emerge from evolutionary pressures on its natural environment by protozoan predators, which selected for strategies that allow survival within macrophages. In fact, Acanthamoeba castellanii ingests yeast cells, which then replicate intracellularly. In addition, most fungal factors needed to establish infection in the mammalian host are also important for survival within the amoeba. To better understand the origin of C. neoformans virulence, we compared the transcriptional profile of yeast cells internalized by amoebae and murine macrophages after 6 h of infection. Our results showed 656 and 293 genes whose expression changed at least two-fold in response to the intracellular environments of amoebae and macrophages, respectively. Among the genes common to both groups, we focused on the ORF CNAG_05662, which was potentially related to sugar transport. We constructed a mutant strain and evaluated its ability to grow on various carbon sources. The results showed that this gene, named PTP1 (Polyol Transporter Protein 1), is involved in the transport of 5- and 6-carbon polyols but its absence had no effect on virulence. Overall, our results are consistent with the hypothesis that mammalian virulence originated from fungal-protozoal interactions and provide a better understanding of how C. neoformans adapts to the mammalian host. Four conditions, pairwise-compared: cells in vegetative growth at 28C versus cells within amoebae at 28C; and cells in vegetative growth at 37C/5% CO2 versus cells within macrophages at 37C/5% CO2. Three biological replicates for each condition. One replicate per array.
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:Transcriptional profiling of four different yeast FZF1 alleles: S. cerevisiae, S. paradoxus and two reciprocal chimeras with the coding and 5' noncoding region from opposite species of S. cerevisiae and S. paradoxus, both before and 15 minutes after sulfite addition. FZF1 is a transcription factor that is known to be turned on in response to sulfite. Here we determine whether the FZF1 allele from two species leads to different transcriptional responses and the effect of the individual noncoding and coding regions on the transcriptional response. Two-color experiment, 4 Strains x 2 Timepoints each compared to a reference pool made up of all samples. 3 Biologic replicates. With dye-swaps
Project description:26972c yeast cells were transformed with either empty vector (pYES3) or pYES3:Gm:bHLHm1. Cells were grown on low ammonium concentrations to observe transcriptional changes in the yeast genome in response to the soybean bHLHm1 transcription factor. The ammonium transport mutant (26972c) was transformed with either a empty vector control (pYES3) or pYES3 containing GmbHLHm1.Independent replicates (3) for each treatment were grown for 12 hours in the presence of 1 mM ammonium and 2 % w/vol galactose to activate the Gal promoter located on pYES3. Cells were harvested and total RNA extracted. Quantified RNA was analaysed using microarray analysis using the [Yeast_2] Affymetrix Yeast Genome 2.0 Array.
Project description:Data on absolute molecule numbers are rare but will empower the modeling, understanding and comparison of cellular functions and biological systems. We quantified transcriptomes and proteomes in fission yeast during both proliferation and quiescence. This rich resource provides the first comprehensive reference for all RNA and most protein concentrations in a eukaryote under key physiological conditions. This integrated dataset will support quantitative biology and afford unique biological insights into cell regulation. While mRNAs are typically expressed in a narrow range above 1 copy/cell, most long non-coding RNAs, except for a specific subset, are strongly repressed below 1 copy/cell. Cell cycle-regulated transcription tunes mRNA numbers to phase-specific requirements but can also lead to switch-like expression. Proteins greatly exceed mRNAs in abundance and dynamic range, and their numbers scales with functional demands. Upon transition to quiescence, the proteome composition changes substantially but, in stark contrast to mRNAs, proteins do not uniformly decrease but scale with cell volume.
Project description:Transcriptome yeast cells expressing modified GFPs were analyzed to elucidate the consequences of growth defect 7 samples were analyzed. In each sample, differently modified GFP is highly expressed. One of them is a empty vector control.
Project description:HBV-transgenic mice were treated i.v. with 1x10e11 particles AAV serotype 8 vectors expressing different RNAi-triggers targeting the HBV transcripts. To determine possible toxicities caused by the different vectors (including off-target activity against endogenous genes) we performed a whole transcriptome analysis of RNA of livers harvested 15 days after injection. 4 replicates per treatment group were analyzed. As controls, one group of mice was treated with 0.9% saline (mock) and another group of animals was treated with an empty AAV8 vector (1x10e11 particles i.v.).
Project description:This study aimed at characterizing the targets of the bZIP transcription factor Yap7 in two yeast species: Saccharomyces cerevisiae and the human pathogen Candida glabrata. Transcriptome analyses were thus conducted in wild type and yap7 knock out strains using Agilent microarrays.
Project description:We did transcription profiling on the effect of KDX1 over-expression. Analysis was performed with wild type and KDX1 over-expressed cells ( pRS426-KDX1). Yeast cells were grown on SD-ura medium. Yeast cells were grown overnight at 30°C . The culture was refreshed to 0.2 O.D and grown at 30°C for 5h 30min. Cells were collected and processed for RNA extraction.