Affymetrix GeneChip Yeast Genome 2.0 Array [Yeast_2]

Reconstruction of the yeast Snf1 kinase regulatory network reveals its role as a global energy regulator.

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The protrotophic laboratory strain CEN.PK113-7D (MAT a) and three knock-out strains snf1, snf4 and snf1snf4 were grown in laboratory fermentors with a working volume of 1 litre at dilution rate (D) of 0.10 per hour (in triplicate for each strain). At steady state, samples from each of the 12 continuous cultures were taken and cooled below 2 degree C within ten seconds by mixing 40% sample and 60% crushed ice.

genotype

Transcription profiling by array of S. cerevisiae wild type, SNF1, SNF4, and double knock outs to investigate SNF1/4 dependent gene expression

The protrotophic laboratory strain CEN.PK113-7D (MAT a) and three knock-out strains snf1, snf4 and snf1snf4 were grown in laboratory fermentors with a working volume of 1 litre at dilution rate (D) of 0.10 per hour (in triplicate for each strain). At steady state, samples from each of the 12 continuous cultures were taken and cooled below 2 degree C within ten seconds by mixing 40% sample and 60% crushed ice.


Affymetrix:Protocol:Hybridization-Mini_Euk2v2

Title: Affymetrix Mini_Euk2v2 Hybridization. Description:

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

Chemostat Cultivation-- Steady-state chemostat Aerobic steady state chemostat cultures were obtained in well-controlled 2-litre jacketed bioreactors (B. Braun Biotech, Melsungen, Germany) with a constant working volume of 1.0 litre and a dilution rate of 0.10 h-1 (which equals the specific growth rate). Cultivations were carried out at 30°C with a stirrer speed of 700 rpm and were aerated with air (1 liter×min-1). pH was kept at 5.0 by automatic addition of 4 M KOH. The bioreactors were fitted with cooled condensers, and the off-gas was led to a gas analyser (INNOVA, Denmark) to measure the content of CO2. Steady state was assumed when at least 5 residence times had passed after switching to continuous cultivation. Steady state was verified by constant (± 2 %) biomass concentration, metabolite concentrations and carbon dioxide evolution rate at 3 measurements taken over 5 residence times. The cultures were fed with a defined medium, which contained per litre: 7.5 g glucose; 5 g (NH4)2SO4; 3.0 g KH2PO4; 0.5 g MgSO4 × 7H2O; and trace metals and vitamins as described by Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1990) Microbiol. Rev. 58, 616-630. 300 ml/L antifoam (Sigma A-8436) was added to avoid foaming. Glucose was autoclaved separately from the mineral medium and afterwards added to the medium together with a sterile filtrated solution containing the vitamins. Analysis of Extracellular Metabolites—Culture samples for determination of glucose, ethanol, glycerol, pyruvate and acetate concentrations were filtered through a 0.45-µm cellulose acetate filter (Osmonics, Minnetonka, MN) immediately after sampling, and the filtrate was frozen at –20 °C until further analysis. The concentrations of the metabolites were determined by high pressure liquid chromatography on an Aminex HPX-87Hm column (Bio-Rad) kept at 65 °C and eluted at 0.6 ml/min with 5 mM H2SO4. Acetate was detected spectrophotometrically by a Waters 486 turnable absorbance detector at 210 nm. Glucose, ethanol, glycerol, and pyruvate were detected refractometrically by a Waters 410 differential refractometer. Ammonium concentration was analyzed by ion exchange HPLC, using a Waters 474 scanning fluorescence detector

P-MEXP-125058

Samples for RNA isolation were taken from the aerobic chemostat cultivation by rapidly sampling 20 ml of culture into a tube with 30 ml of crushed ice. Hereby the temperature decreased to below 2 °C in less than 10 s. Cells were quickly pelleted (4500 rpm at 0 °C for 1 min), instantly frozen in liquid nitrogen, and thereafter stored at –80 °C. Total RNA was extracted by using the FastRNA kit, Pro (BIO 101, Inc., Vista, CA) after thawing the samples on ice.

Five µg of total-RNA were used to generate cDNA using the One-Cycle cDNA Synthesis Kit (Affymetrix, P/N 900431). cDNA synthesis with a T7-Oligo(dT) Primer was performed according to the GeneChip manual (Affymetrix, 701021 Rev. 5) chapter ‘One-Cycle cDNA Synthesis’ (step 2 & step 3), and subsequently extracted using Sample Cleanup Module (Affymetrix, P/N 900371) and according to the GeneChip manual chapter ‘Cleanup of Double-Stranded cDNA for Both the One-Cycle and Two-Cycle Target Labeling Assays’. In vitro transcription was done with double-stranded cDNA as a template in the presence of biotinylated nucleotide analog/ribonucleotide mix, using an IVT Labeling Kit (Affymetrix, P/N 900449) according to the instructions given in the GeneChip manual chapter ‘Synthesis of Biotin-Labeled cRNA for Both the One-Cycle and Two-Cycle Target Labeling Assays’.

/nfs/ma/ma-exp/EXPERIMENTS/MEXP/E-MEXP-1407

P-MEXP-125091

DAT and .CEL files were generated with MAS 5.0 software from Affymetrix

Transcription profiling by array of S. cerevisiae wild type, SNF1, SNF4, and double knock outs to investigate SNF1/4 dependent gene expression

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