Affymetrix GeneChip Yeast Genome S98 [YG_S98]

Global translational responses to oxidative stress impact upon multiple levels of protein synthesis

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Global restriction of protein synthesis is a hallmark of cellular stress. Using hydrogen peroxide, we monitor the transcript level and also the translation status for each RNA using cycloheximide to freeze elongating ribosomes. Polyribosome fractionation of cell extracts was used to separate highly translated and poorly translated mRNAs that were then separately analysed.

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Transcription profiling by array of Saccharomyces cerevisiae after treatment with hydrogen peroxide

Transcription profiling and translation status analysis in yeast with hydrogen peroxide and cycloheximide treatment

Global restriction of protein synthesis is a hallmark of cellular stress. Using hydrogen peroxide, we monitor the transcript level and also the translation status for each RNA using cycloheximide to freeze elongating ribosomes. Polyribosome fractionation of cell extracts was used to separate highly translated and poorly translated mRNAs that were then separately analysed.


Affymetrix:Protocol:Hybridization-EukGE-WS2v4_450

Title: Affymetrix EukGE-WS2v4_450 Hybridization. Description:

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-16287

The yeast strain CY4 (MATa ura3-52 leu2-3 leu2-112 trp1-1 his3-11 can1-100) [Wheeler GL et. al, 2003] was grown at 30 C in a synthetic minimal media with 2%(w/v) glucose, lacking non-essential but containing essential amino acids.  Mid-log phase yeast (optical density at 600nm) of 0.7 was used.

P-MEXP-16288

Hydrogegn Peroxide (0.2mM) was added to the liquid culture (OD 0.7) for 15 min.

P-MEXP-16289

Hydrogegn Peroxide (2mM) was added to the liquid culture (OD 0.7) for 15 min.

P-MEXP-8413

 Pooled fractions were collected directly into 2 volumes of Trizol (Invitrogen), mixed and  1 volume of chloroform was added with glycogen (50-150mg/ml) and sodium acetate (0.1 M pH 4.5).  After centrifugation, the aqueous phase was collected and 0.7 volumes of isopropanol added.  The RNA precipitate was pelleted, washed in 80% ethanol and resuspended in water. Equal amounts were analysed by northern blot analysis.  For microarrays the RNA samples were diluted, precipitated again by the addition of an equal volume of LiCl buffer (4 M LiCl, 20 mM Tris-HCl pH 7.5, 10 mM EDTA) and stored overnight at -20C.  The RNA pellet was washed twice with 80% ethanol and resuspended in water. 

P-MEXP-8415

Cell lysates were prepared essentially as described previously (Ashe et al. 2000).  Briefly, after incubation with cycloheximide 100mg /ml at 4C, cells were pelleted and washed twice in 50ml of ice-cold lysis buffer (Ashe et al. 2000). Cell pellets were resuspended in 500ml of lysis buffer and rapidly frozen in liquid nitrogen.  Lysates were prepared by grinding under liquid nitrogen.  The ground yeast were thawed on ice and spun at 16000g at 4C for 5 min.  The extract was cleared at 16000g for 30 min at 4C.  60 A260 units were layered onto 35 ml 15-50% sucrose gradients. The gradients were sedimented via centrifugation at 16900 rpm for 13 hours.  Gradients were collected as described previously (Ashe et al. 2000) and fractionated into 15 2.2 ml aliquots. For comparison of polysomal RNA levels, fractions 4-8 and 11-15 from the gradient were pooled together for monosomal and polysomal RNA samples respectively.  As a control all 15 fractions from a second gradient were pooled. 

P-MEXP-8418

In vitro transcription by using double-stranded cDNA as a template in the presence of biotinylated UTP and CTP, using an ENZO BioArray HighYield RNA Transcript Labeling Kit (Affymetrix, Santa Clara, CA, and ENZO Diagnostics, Farmingdale, NY) according to the manufacturer's instructions.

P-MEXP-8427

Total RNA samples were prepared according standard protocols.

Transcription profiling by array of Saccharomyces cerevisiae after treatment with hydrogen peroxide

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