Project description:Background: As carbon sources are exhausted, Saccharomyces cerevisiae cells exhibit reduced metabolic activity and cultures enter stationary phase. We asked whether cells in stationary-phase cultures respond to additional stress at the level of transcript abundance. Results: Microarrays were used to quantify changes in transcript abundance in cells from stationary-phase cultures. In response to oxidative stress, more than 800 mRNAs increased within 1 minute. A significant number of these mRNAs encode proteins involved in stress responses. We tested whether mRNA increases were due to new transcription, rapid poly-adenylation of message (which would not be detected by microarrays), or potential release of mature mRNA sequestered in the cell but insoluble during RNA isolation. Examination of the response to oxidative stress in an RNA polymerase II mutant, rpb1-1 suggested new transcription was not required. Quantitative RT-PCR analysis of a subset of these transcripts further suggested that essentially all isolated transcripts were polyadenylated. In contrast, over 1000 transcripts increased after protease treatment of cell-free lysates from stationary-phase but not exponentially growing cultures. We also determined that oxidative stress and temperature upshift led to the release of different transcripts, suggesting that mRNA release is stress specific. Conclusions: A large number of mRNAs are sequestered in a protease-labile, rapidly releasable form in cells in stationary-phase cultures but not exponentially growing cultures. The differences between mRNAs released by protease treatment and those observed with oxidative stress and temperature upshift, suggest different stresses cause the release of different transcripts. We hypothesize that P-bodies are involved in this sequestration. Keywords: stress response
Project description:Oxidative stress is a harmful condition in a cell, tissue, or organ, caused by an imbalnace between reactive oxygen species and other oxidants and the capacity of antioxidant defense systems to remove them. The budding yeast S. cerevisiae has been the major eukaryotic model for studies of response to oxidative stress. We used microarrays to study the genome-wide temporal response of the yeast S. cerevisiae to oxidative stress induced by cumene hydroperoxide. Keywords: time course The effects of oxidative stress induced by CHP on the transcriptional profile of S. cerevisiae was studied from a dynamical perspective. Yeast cultures were grown in controlled batch conditions, in 1 L fermentors. Three replicate cultures in mid-exponential phase were exposed to 0.19 mM CHP, while three non-treated cultures were used as controls. Samples were collected at t=0 (immediately before adding CHP) and at 3, 6, 12 and 20 min after adding the oxidant. Samples were processed for RNA extraction and profiled using Affymetrix Yeast Genome S98 arrays.
Project description:Determine if yeast cell in stationary-phase cultures respond to oxidative stress Yeast cells in stationary-phase cultures were exposed to oxidative stress with samples harvested every 30-minutes over 8 hours. All experimental samples are over an common reference. There are two replicates for each time point and six replicates of T0.
Project description:Background: Recent studies have demonstrated that antisense transcription is pervasive in budding yeasts and is conserved between Saccharomyces cerevisiae and S. paradoxus. While studies have examined antisense transcripts of S. cerevisiae for inverse transcription in stationary phase and stress conditions, there is a lack of comprehensive analysis of the conditional specific evolutionary characteristics of antisense transcription between yeasts. Here we attempt to decipher the evolutionary relationship of antisense transcription of S. cerevisiae and S. paradoxus cultured in mid log, early stationary phase, and heat shock conditions. Results: Massively parallel sequencing of sequence strand-specific cDNA library was performed from RNA isolated from S. cerevisiae and S. paradoxus cells at mid log, stationary phase and heat shock conditions. We performed this analysis using a stringent set of sense ORF transcripts and non-coding antisense transcripts that were expressed in all the three conditions, as well as in both species. We found the divergence of the condition specific anti-sense transcription levels is higher than that in condition specific sense transcription levels, suggesting that antisense transcription played a potential role in adapting to different conditions. Furthermore, 43% of sense-antisense pairs demonstrated inverse transcription in either stationary phase or heat shock conditions relative to the mid log conditions. In addition, a large part of sense-antisense pairs (67%), which demonstrated inverse transcription, were highly conserved between the two species. Our results were also concordant with known functional analyses from previous studies and with the evidence from mechanistic experiments of role of individual genes. Conclusions: This study provides a comprehensive picture of the role of antisense transcription mediating sense transcription in different conditions across yeast species. We can conclude from our findings that antisense regulation could act like an on-off switch on sense regulation in different conditions.
Project description:Determine if yeast cells in stationary-phase cultures respond to oxidative stress Yeast cells in stationary-phase cultures were exposed to oxidative stress with samples harvested every 1-minute over 35 minutes, with a final time point at 60 minutes. All experimental samples are over an common reference. There are two replicates for each time point and six replicates of T0.
Project description:Purpose: Lactococcus lactis is the primary constituent of many industrial starter cultures used in food fermentations. However, this bacterium is exposed to several stressful conditions during the industrial process. L. lactis TOMSC161 has been shown to resist better to freeze-drying and storage when cells are harvested at the late stationary compared to the early stationary phase. Methods: In this work, the physiological changes that occur during growth (early and late stationary phases) were studied using transcriptomic, proteomic and cytometric approaches in order to elucidate the cell mechanisms involved in the stress tolerance of L. lactis TOMSC161 to freeze-drying and storage processes. Results: The majority of altered abundances for genes determined by transcriptomic analysis indicated a slowdown at the late stationary phase in general metabolism and in the incomplete reduction of O2 leading to reactive oxygen species (ROS), in comparison to the early stationary phase. Furthermore, the expression of genes involved in general and oxidative stress responses was over-expressed at the late stationary phase when compared to the early stationary phase, thus explaining the better resistance to freeze-drying and the storage of cells harvested after six hours of stationary phase. Superoxide anion detection was performed by flow cytometry and related to the ROS production of starters. Superoxide anion concentration was lower for cells harvested at the late stationary phase when compared to cells harvested at the early stationary phase. Conclusions: L. lactis TOMSC161 was thus able to develop an oxidative stress “pre-adaptation” during the stationary phase making it possible to better resist freeze-drying and storage stresses and especially oxidative stress.