Investigation Title	Transcription profiling of S. cerevisiae grown in acetate, ethanol and maltose limited conditions to investigate transcriptional regulation in controlling fluxes in central carbon metabolism	
Comment[Submitted Name]	Role of Transcriptional Regulation in Controlling Fluxes in Central Carbon Metabolism of Saccharomyces cerevisiae	
Experimental Design	unknown_experiment_design_type	transcription profiling by array	
Experimental Design Term Source REF		EFO	
Comment[ArrayExpressReleaseDate]	2008-06-16	
Comment[SecondaryAccession]	GSE8895	
Comment[AEMIAMESCORE]	3	
Comment[ArrayExpressAccession]	E-GEOD-8895	
Comment[MAGETAB TimeStamp_Version]	2010-08-09 11:42:34 Last Changed Rev: 13058	
Experimental Factor Name	
Experimental Factor Type	
Experimental Factor Term Source REF	
Person Last Name	Daran	
Person First Name	Jean-Marc	
Person Mid Initials	
Person Email	j.m.daran@tnw.tudelft.nl	
Person Phone	
Person Fax	
Person Address	Kluyver centre for genomics of industrial organisms,Department of Biotechnology,TU Delft,Julianalaan 67,Delft,2628BC,The Netherlands	
Person Affiliation	TU Delft	
Person Roles	submitter	
Person Roles Term Source REF	The MGED Ontology	
Quality Control Type	
Quality Control Term Source REF	
Replicate Type	
Replicate Term Source REF	
Normalization Type	
Normalization Term Source REF	
Date of Experiment	
Public Release Date	2008-06-16	
PubMed ID	14630934	
Publication DOI	14630934	
Publication Author List	Pascale Daran-Lapujade, Mickel L A Jansen, Jean-Marc Daran, Walter van Gulik, Johannes H de Winde, Jack T Pronk	
Publication Title	Role of transcriptional regulation in controlling fluxes in central carbon metabolism of Saccharomyces cerevisiae. A chemostat culture study.	
Publication Status	journal_article	
Publication Status Term Source REF	The MGED Ontology	
Experiment Description	In contrast to batch cultivation, chemostat cultivation allows the identification of carbon source responses without interference by carbon-catabolite repression, accumulation of toxic products, and differences in specific growth rate. This study focuses on the yeast Saccharomyces cerevisiae, grown in aerobic, carbon-limited chemostat cultures. Genome-wide transcript levels and in vivo fluxes were compared for growth on two sugars, glucose and maltose, and for two C2-compounds, ethanol and acetate. In contrast to previous reports on batch cultures, few genes (180 genes) responded to changes of the carbon source by a changed transcript level. Very few transcript levels were changed when glucose as the growth-limiting nutrient was compared with maltose (33 transcripts), or when acetate was compared with ethanol (16 transcripts). Although metabolic flux analysis using a stoichiometric model revealed major changes in the central carbon metabolism, only 117 genes exhibited a significantly different transcript level when sugars and C2-compounds were provided as the growthlimiting nutrient. Despite the extensive knowledge on carbon source regulation in yeast, many of the carbon source-responsive genes encoded proteins with unknown or incompletely characterized biological functions. In silico promoter analysis of carbon source-responsive genes confirmed the involvement of several known transcriptional regulators and suggested the involvement of additional regulators. Transcripts involved in the glyoxylate cycle and gluconeogenesis showed a good correlation with in vivo fluxes. This correlation was, however, not observed for other important pathways, including the pentose-phosphate pathway, tricarboxylic acid cycle, and, in particular, glycolysis. These results indicate that in vivo fluxes in the central carbon metabolism of S. cerevisiae grown in steadystate, carbon-limited chemostat cultures are controlled to a large extent via post-transcriptional mechanisms. Experiment Overall Design: Cultivation of microorganisms in chemostats offers numerous advantages for studying the structure and regulation of metabolic networks (11). In chemostat cultures, individual culture parameters can be changed, while keeping other relevant physical and chemical culture parameters (composition of synthetic medium, pH, temperature, aeration, etc.) constant. An especially important parameter in this respect is the specific growth rate, which, in a chemostat, is equal to the dilution rate, which can be accurately controlled. This allows the experimenter to investigate the effects of environmental changes or genetic interventions at a fixed specific growth rate, even if these changes result in different specific growth rates in batch cultures. In a chemostat, growth can be limited by a single, selected nutrient. The very low residual concentrations of this growth-limiting nutrient in chemostat cultures alleviate effects of catabolite repression and inactivation. Furthermore, these low residual substrate concentrations prevent substrate toxicity, which, for example, occurs when S. cerevisiae is grown on ethanol or acetate as the carbon source in batch cultures . Experiment Overall Design: The central goal of the present study is to assess to what extent carbon source-dependent regulation of fluxes through central carbon metabolism in S. cerevisiae is regulated at the level of transcription. To this end, we compare the transcriptome of carbon-limited, aerobic chemostat cultures grown on four different carbon sources: glucose, maltose, ethanol, and acetate. Data from the transcriptome analysis are compared with flux distribution profiles calculated with a stoichiometric metabolic network model. Questions that will be addressed are as follows: (i) does glucose-limited aerobic cultivation lead to a complete alleviation of glucose-catabolite repression; (ii) how (in)complete is our understanding of the genes involved in the transcriptional response of S. cerevisiae to four of the most common carbon sources for this yeast; and (iii) to what extent do transcriptome analyses with microarrays provide a reliable indication of flux distribution in metabolic networks?	
Protocol Name	P-G8895-14	P-G8895-6	P-G8895-2	P-G8895-10	P-G8895-13	P-G8895-5	P-G8895-17	P-G8895-1	P-G8895-9	P-G8895-16	P-G8895-8	P-G8895-19	P-G8895-4	P-G8895-12	P-G8895-15	P-G8895-7	P-G8895-18	P-G8895-3	P-G8895-11	Affymetrix:Protocol:Hybridization-EukGE-WS2v4	Affymetrix:Protocol:Hybridization-Unknown	Affymetrix:Protocol:Hybridization-Mini_Euk1	Affymetrix:Protocol:Hybridization-EukGE-WS2	P-AFFY-6	
Protocol Type	specified_biomaterial_action	specified_biomaterial_action	specified_biomaterial_action	specified_biomaterial_action	grow	grow	grow	grow	grow	nucleic_acid_extraction	nucleic_acid_extraction	nucleic_acid_extraction	nucleic_acid_extraction	nucleic_acid_extraction	labeling	labeling	labeling	labeling	labeling	hybridization	hybridization	hybridization		feature_extraction	
Protocol Description	liquid nitrogen quenching	Liquid N2 Quenching	liquid N2 Quenching	Liquid N2 quenching	Strain and Growth Conditionsâ€"Wild-type S. cerevisiae strain CEN.PK113â€"7D (MATa) (1) was grown at 30 °C in 2-liter chemostats (Applikon), with a working volume of 1.0 liter as described in Ref. 2.  Cultures were fed with a defined mineral medium that limited growth by glucose, ethanol, acetate, or maltose with all other growth requirements in excess. The dilution rate was set at 0.10 h-1. The pH was measured on-line and kept constant at 5.0 by the automatic addition of 2 M KOH with the use of an Applikon ADI 1030 biocontroller. Stirrer speed was 800 rpm, and the airflow was 0.5 liters_min-1. Dissolved oxygen tension was measured online with an Ingold model 34-100-3002 probe, and was between 60 and 75% of air saturation. The off-gas was cooled by a condenser connected to a cryostat set at 2 °C and analyzed as previously described (3). Steady-state samples were taken after 10â€"14 volume changes to avoid strain adaptation caused by long term cultivation (4). Mediaâ€"The defined mineral medium composition was based on that described by Verduyn et al. (5). The carbon source was 256 mmol of carbon/liter. 1. Pronk, J. T., Wenzel, T. J., Luttik, M. A. H., Klaassen, C. C. M., Scheffers, W. A., and van Dijken, J. P. (1994) Microbiology 140, 601â€"610 2. van den Berg, M. A., de Jong-Gubbels, P., Kortland, C. J., van Dijken, J. P., Pronk, J. T., and Steensma, H. Y. (1996) J. Biol. Chem. 271, 28953â€"28959 3. van Maris, A. J. A., Luttik, M. A. H., Winkler, A. A., van Dijken, J. P., and Pronk, J. T. (2003) Appl. Environ. Microbiol. 69, 2094â€"2099 4. Ferea, T. L., Botstein, D., Brown, P. O., and Rosenzweig, R. F. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9721â€"9726 5- Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1992) Yeast 8, 501â€"517	Chemostat Cultivation Steady-state chemostat cultures were grown in Applikon laboratory fermentors of 1-liter working volume as described in detail elsewhere [van den Berg, M. A., de Jong-Gubbels, P., Kortland, C. J., van Dijken, J. P., Pronk, J. T., and Steensma, H. Y. (1996) J. Biol. Chem. 271, 28953-28959]. In brief, the cultures were fed with a defined mineral medium containing glucose as the growth-limiting nutrient [. Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1990) Microbiol.Rev. 58, 616-630]. The dilution rate (which equals the specific growth rate) in the steady-state cultures was 0.10 h_1, the temperature was 30 °C, and the culture pH was 5.0. Aerobic conditions were maintained by sparging the cultures with air (0.5 liter_min_1). The dissolved oxygen concentration, which was continuously monitored with an Ingold model 34-100-3002 probe, remained above 80% of air saturation.	Strain and Growth Conditions Wild-type S. cerevisiae strain CEN.PK113-7D (MATa) (1) was grown at 30 °C in 2-liter chemostats (Applikon), with a working volume of 1.0 liter as described in Ref. 2.  Cultures were fed with a defined mineral medium that limited growth by glucose, ethanol, acetate, or maltose with all other growth requirements in excess. The dilution rate was set at 0.10 h-1. The pH was measured on-line and kept constant at 5.0 by the automatic addition of 2 M KOH with the use of an Applikon ADI 1030 biocontroller. Stirrer speed was 800 rpm, and the airflow was 0.5 liters_min-1. Dissolved oxygen tension was measured online with an Ingold model 34-100-3002 probe, and was between 60 and 75% of air saturation. The off-gas was cooled by a condenser connected to a cryostat set at 2 °C and analyzed as previously described (3). Steady-state samples were taken after 10-14 volume changes to avoid strain adaptation caused by long term cu ltivation (4). Media The defined mineral medium composition was based on that described by Verduyn et al. (5). The carbon source was 256 mmol of carbon/liter. 1. Pronk, J. T., Wenzel, T. J., Luttik, M. A. H., Klaassen, C. C. M., Scheffers, W. A., and van Dijken, J. P. (1994) Microbiology 140, 601-610 2. van den Berg, M. A., de Jong-Gubbels, P., Kortland, C. J., van Dijken, J. P., Pronk, J. T., and Steensma, H. Y. (1996) J. Biol. Chem. 271, 28953-28959 3. van Maris, A. J. A., Luttik, M. A. H., Winkler, A. A., van Dijken, J. P., and Pronk, J. T. (2003) Appl. Environ. Microbiol. 69, 2094-2099 4. Ferea, T. L., Botstein, D., Brown, P. O., and Rosenzweig, R. F. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9721-9726 5- Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1992) Yeast 8, 501-517	Chemostat Cultivationâ€" Steady-state chemostat cultures were grown in Applikon laboratory fermentors of 1-liter working volume as described in detail elsewhere [van den Berg, M. A., de Jong-Gubbels, P., Kortland, C. J., van Dijken, J. P., Pronk, J. T., and Steensma, H. Y. (1996) J. Biol. Chem. 271, 28953â€"28959]. In brief, the cultures were fed with a defined mineral medium containing glucose as the growth-limiting nutrient [. Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1990) Microbiol.Rev. 58, 616â€"630]. The dilution rate (which equals the specific growth rate) in the steady-state cultures was 0.10 h_1, the temperature was 30 °C, and the culture pH was 5.0. Aerobic conditions were maintained by sparging the cultures with air (0.5 liter_min_1). The dissolved oxygen concentration, which was continuously monitored with an Ingold model 34-100-3002 probe, remained above 80% of air saturation.	Chemostat Cultivation Steady-state chemostat cultures were grown in Applikon laboratory fermentors of 1-liter working volume as described in detail elsewhere [van den Berg, M. A., de Jong-Gubbels, P., Kortland, C. J., van Dijken, J. P., Pronk, J. T., and Steensma, H. Y. (1996) J. Biol. Chem. 271, 28953-28959]. In brief, the cultures were fed with a defined mineral medium containing glucose as the growth-limiting nutrient [. Verduyn, C., Postma, E., Scheffers, W. A., and van Dijken, J. P. (1990) Microbiol.Rev. 58, 616-630]. The dilution rate (which equals the specific growth rate) in the steady-state cultures was 0.10 h_1, the temperature was 30°C, and the culture pH was 5.0. Aerobic conditions were maintained by sparging the cultures with air (0.5 liter_min_1). The dissolved oxygen concentration, which was continuously monitored with an Ingold model 34-100-3002 probe, remained above 80% of air saturation.	Sampling of cells from chemostats, probe preparation, and hybridization to Affymetrix GeneChip® microarrays were performed as described previously (6) 6- Piper, M. D. W., Daran-Lapujade, P., Bro, C., Regenberg, B., Knudsen, S.,Nielsen, J., and Pronk, J. T. (2002) J. Biol. Chem. 277, 37001â€"37008	Sampling and RNA Isolation Samples from the chemostat cultures were taken as rapidly as possible to limit any potential changes in transcript profiles during the procedure. 40-60 ml of culture broth was sampled directly from the chemostat into a beaker containing 200 ml of liquid nitrogen. With vigorous stirring, the sample froze instantly. The frozen sample was then broken into small fragments and transferred to a 50-ml centrifuge tube. The sample was then thawed at room temperature, ensuring that it remained as close to zero as possible. Cells were pelleted (5000 rpm at 0 °C for 4 min), resuspended in 2 ml of ice-cold AE buffer (50 mM sodium acetate, 10 mM EDTA, pH 5.0) and aliquoted into 5 Eppendorf tubes. This corresponded to _20 mg of dry weight per tube. For each array, total RNA was extracted from a single tube using the hot-phenol method (32) or the FastRNA kit, Red (BIO 101, Inc., Vista, CA).	Sampling of cells from chemostats, probe preparation, and hybridization to Affymetrix GeneChip microarrays were performed as described previously (6) 6- Piper, M. D. W., Daran-Lapujade, P., Bro, C., Regenberg, B., Knudsen, S.,Nielsen, J., and Pronk, J. T. (2002) J. Biol. Chem. 277, 37001-37008	Sampling and RNA Isolationâ€" Samples from the chemostat cultures were taken as rapidly as possible to limit any potential changes in transcript profiles during the procedure. 40â€"60 ml of culture broth was sampled directly from the chemostat into a beaker containing 200 ml of liquid nitrogen. With vigorous stirring, the sample froze instantly. The frozen sample was then broken into small fragments and transferred to a 50-ml centrifuge tube. The sample was then thawed at room temperature, ensuring that it remained as close to zero as possible. Cells were pelleted (5000 rpm at 0 °C for 4 min), resuspended in 2 ml of ice-cold AE buffer (50 mM sodium acetate, 10 mM EDTA, pH 5.0) and aliquoted into 5 Eppendorf tubes. This corresponded to _20 mg of dry weight per tube. For each array, total RNA was extracted from a single tube using the hot-phenol method (32) or the FastRNA kit, Red (BIO 101, Inc., Vista, CA).	Probe Preparation and Hybridization to Arrays—mRNA extraction, cDNA synthesis, cRNA synthesis and labeling, as well as array hybridization were performed as described in the Affymetrix users’ manual (1). Briefly, poly(A)_ RNA was enriched from total RNA in a single round using the Qiagen Oligotex kit. Double-stranded cDNA synthesis was carried out incorporating the T7 RNA-polymerase promoter in the first round. This cDNA was then used as template for in vitro transcription (ENZO BioArray High Yield IVT kit), which amplifies the RNA pool and incorporates biotinylated ribonucleotides required for the staining procedures after hybridization. 15 mg of fragmented, biotinylated cRNA was hybridized to Affymetrix yeast S98 arrays at 45 °C for 16 h as described in the Affymetrix users’ manual (1). Washing and staining of arrays were performed using the GeneChip Fluidics Station 400 and scanning with the Affymetrix GeneArray Scanner. (1) Affymetrix (2000) Affymetrix GeneChip Expression Analysis Technical Manual, Santa Clara, CA	Sampling of cells from chemostats, probe preparation, and hybridization to Affymetrix GeneChip® microarrays were performed as described previously (6) 6- Piper, M. D. W., Daran-Lapujade, P., Bro, C., Regenberg, B., Knudsen, S.,Nielsen, J., and Pronk, J. T. (2002) J. Biol. Chem. 277, 37001â€"37008	Probe Preparation and Hybridization to Arrays—mRNA extraction, cDNA synthesis, cRNA synthesis and labeling, as well as array hybridization were performed as described in the Affymetrix users manual (1). Briefly, poly(A)_ RNA was enriched from total RNA in a single round using the Qiagen Oligotex kit. Double-stranded cDNA synthesis was carried out incorporating the T7 RNA-polymerase promoter in the first round. This cDNA was then used as template for in vitro transcription (ENZO BioArray High Yield IVT kit), which amplifies the RNA pool and incorporates biotinylated ribonucleotides required for the staining procedures after hybridization. 15 mg of fragmented, biotinylated cRNA was hybridized to Affymetrix yeast S98 arrays at 45 °C for 16 h as described in the Affymetrix users™ manual (1). Washing and staining of arrays were performed using the GeneChip Fluidics Station 400 and scanning with the Affymetrix GeneArray Scanner. (1) Affymetrix (2000) Affymetrix GeneChip Expression Analysis Technical Manual, Santa Clara, CA	Sampling of cells from chemostats, probe preparation, and hybridization to Affymetrix GeneChip microarrays were performed as described previously (6) 6- Piper, M. D. W., Daran-Lapujade, P., Bro, C., Regenberg, B., Knudsen, S.,Nielsen, J., and Pronk, J. T. (2002) J. Biol. Chem. 277, 37001-37008	Probe Preparation and Hybridization to Arraysâ€"mRNA extraction, cDNA synthesis, cRNA synthesis and labeling, as well as array hybridization were performed as described in the Affymetrix users’ manual (1). Briefly, poly(A)_ RNA was enriched from total RNA in a single round using the Qiagen Oligotex kit. Double-stranded cDNA synthesis was carried out incorporating the T7 RNA-polymerase promoter in the first round. This cDNA was then used as template for in vitro transcription (ENZO BioArray High Yield IVT kit), which amplifies the RNA pool and incorporates biotinylated ribonucleotides required for the staining procedures after hybridization. 15 mg of fragmented, biotinylated cRNA was hybridized to Affymetrix yeast S98 arrays at 45 °C for 16 h as described in the Affymetrix users’ manual (1). Washing and staining of arrays were performed using the GeneChip Fluidics Station 400 and scanning with the Affymetrix GeneArray Scanner. (1) Affymetrix (2000) Affymetrix GeneChip Expression Analysis Technical Manual, Santa Clara, CA	Probe Preparation and Hybridization to Arrays—mRNA extraction, cDNA synthesis, cRNA synthesis and labeling, as well as array hybridization were performed as described in the Affymetrix users’ manual (1). Briefly, poly(A)_ RNA was enriched from total RNA in a single round using the Qiagen Oligotex kit. Double-stranded cDNA synthesis was carried out incorporating the T7 RNA-polymerase promoter in the first round. This cDNA was then used as template for in vitro transcription (ENZO BioArray High Yield IVT kit), which amplifies the RNA pool and incorporates biotinylated ribonucleotides required for the staining procedures after hybridization. 15 mg of fragmented, biotinylated cRNA was hybridized to Affymetrix yeast S98 arrays at 45 °C for 16 h as described in the Affymetrix users’ manual (1). Washing and staining of arrays were performed using the GeneChip Fluidics Station 400 and scanning with the Affymetrix GeneArray Scanner. (1) Affymetrix (2000) Affymetrix GeneChip Expression Analysis Technical Manual, Santa Clara, CA	Title: Fluidics Station Protocol. Description: 	Title: Affymetrix Generic Hybridization. Description: 			Title: Affymetrix CEL analysis. Description: 	
Protocol Parameters	
Protocol Hardware	
Protocol Software																				MicroArraySuite 5.0	MicroArraySuite 5.0			MicroArraySuite 5.0	
Protocol Contact	
Protocol Term Source REF																					mo	
SDRF File	E-GEOD-8895.sdrf.txt	
Term Source Name	The MGED Ontology		ArrayExpress	EFO	The MGED Ontology	mo	
Term Source File	http://mged.sourceforge.net/ontologies/MGEDontology.php		http://www.ebi.ac.uk/arrayexpress	http://www.ebi.ac.uk/efo/	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://mged.sourceforge.net/ontologies/MGEDontology.php	
Term Source Version