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Multiway real-time PCR gene expression profiling in yeast Saccharomyces cerevisiae reveals altered transcriptional response of ADH-genes to glucose stimuli.

Ståhlberg A, Elbing K, Andrade-Garda JM, Sjögreen B, Forootan A, Kubista M - BMC Genomics (2008)

Bottom Line: In multiway studies samples are characterized by their expression profiles to monitor changes over time, effect of treatment, drug dosage etc.The data are analyzed by matrix-augmented PCA, which is a generalization of PCA for 3-way data, and the results are confirmed by hierarchical clustering and clustering by Kohonen self-organizing map.The technique also identifies genes that show perturbed expression in specific strains.

View Article: PubMed Central - HTML - PubMed

Affiliation: TATAA Biocenter, Odinsgatan 28, 411 03 Göteborg, Sweden. anders.stahlberg@neuro.gu.se

ABSTRACT

Background: The large sensitivity, high reproducibility and essentially unlimited dynamic range of real-time PCR to measure gene expression in complex samples provides the opportunity for powerful multivariate and multiway studies of biological phenomena. In multiway studies samples are characterized by their expression profiles to monitor changes over time, effect of treatment, drug dosage etc. Here we perform a multiway study of the temporal response of four yeast Saccharomyces cerevisiae strains with different glucose uptake rates upon altered metabolic conditions.

Results: We measured the expression of 18 genes as function of time after addition of glucose to four strains of yeast grown in ethanol. The data are analyzed by matrix-augmented PCA, which is a generalization of PCA for 3-way data, and the results are confirmed by hierarchical clustering and clustering by Kohonen self-organizing map. Our approach identifies gene groups that respond similarly to the change of nutrient, and genes that behave differently in mutant strains. Of particular interest is our finding that ADH4 and ADH6 show a behavior typical of glucose-induced genes, while ADH3 and ADH5 are repressed after glucose addition.

Conclusion: Multiway real-time PCR gene expression profiling is a powerful technique which can be utilized to characterize functions of new genes by, for example, comparing their temporal response after perturbation in different genetic variants of the studied subject. The technique also identifies genes that show perturbed expression in specific strains.

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Schematic diagram of central metabolism to indicate position of relevant/studied enzymes in metabolism.
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Figure 1: Schematic diagram of central metabolism to indicate position of relevant/studied enzymes in metabolism.

Mentions: Genes known to be induced or repressed in wild-type strain, with normal glycolytic flux, were included in the study to allow for the classification of the ADH-genes (Figure 1). The established glucose induced genes were Triose-phosphate isomerase 1 (TPI1, catalyzing reaction step 5), Phosphoglycerate kinase 1 (PGK1, catalyzing reaction step 7), Pyruvate decarboxylase (PDC1, catalyzing reaction step 11) and Alcohol dehydrogenase 1 (ADH1, catalyzing reaction step 12), which are all members of the lower glycolysis [8,23]. The established glucose repressed genes involved in gluconeogenesis and the glyoxylate cycle genes were Fructose-1,6-bisphosphatase (FBP1, catalyzing the reverse reaction of step 4), Alcohol dehydrogenase 2 (ADH2, catalyzing the reverse reaction of step 12) and Malate dehydrogenase 2 (MDH2, catalyzing oxalacetate in the glyoxylate cycle) [23]. In addition, Sucrose fermentation (SUC2) was included, which is a well studied target gene of glucose repression/derepression [24-27], the Multicopy Inhibitor of GAL (MIG1), which is a central repressor of SUC2 [28], the mitochondria localized Cytocrome C (CYC1), which is regulated by MIG1 and repressed by glucose [29,30] and heat shock protein 12 (HSP12), which is known to be repressed by very low glucose concentrations and is also stress induced [31-33]. Inorganic pyrophosphatase (IPP1), Actin (ACT1), and Pyruvate dehydrogenase 1 (PDA1) were included as tentative house-keeping/reference genes. In addition to the well characterized genes, for which raw data have been partly reported before [3], we also included a series of ADH-genes (ADH3-6) that are less well understood, to investigate their responses in relation to different glycolytic rates. Throughout this paper the genes in figures and tables are color coded as follows: ADH1, PGK1, TPI1, PDC1 and MIG1 genes, which are expected to be induced by glucose, are shown in blue, FBP1, ADH2, MDH2 and SUC2, which are expected to be repressed by glucose, are shown in red, ADH3, ADH4, ADH5 and ADH6 genes, whose functions are rather unknown, are shown in yellow, HSP12 is shown in black and CYC1 in green.


Multiway real-time PCR gene expression profiling in yeast Saccharomyces cerevisiae reveals altered transcriptional response of ADH-genes to glucose stimuli.

Ståhlberg A, Elbing K, Andrade-Garda JM, Sjögreen B, Forootan A, Kubista M - BMC Genomics (2008)

Schematic diagram of central metabolism to indicate position of relevant/studied enzymes in metabolism.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2335116&req=5

Figure 1: Schematic diagram of central metabolism to indicate position of relevant/studied enzymes in metabolism.
Mentions: Genes known to be induced or repressed in wild-type strain, with normal glycolytic flux, were included in the study to allow for the classification of the ADH-genes (Figure 1). The established glucose induced genes were Triose-phosphate isomerase 1 (TPI1, catalyzing reaction step 5), Phosphoglycerate kinase 1 (PGK1, catalyzing reaction step 7), Pyruvate decarboxylase (PDC1, catalyzing reaction step 11) and Alcohol dehydrogenase 1 (ADH1, catalyzing reaction step 12), which are all members of the lower glycolysis [8,23]. The established glucose repressed genes involved in gluconeogenesis and the glyoxylate cycle genes were Fructose-1,6-bisphosphatase (FBP1, catalyzing the reverse reaction of step 4), Alcohol dehydrogenase 2 (ADH2, catalyzing the reverse reaction of step 12) and Malate dehydrogenase 2 (MDH2, catalyzing oxalacetate in the glyoxylate cycle) [23]. In addition, Sucrose fermentation (SUC2) was included, which is a well studied target gene of glucose repression/derepression [24-27], the Multicopy Inhibitor of GAL (MIG1), which is a central repressor of SUC2 [28], the mitochondria localized Cytocrome C (CYC1), which is regulated by MIG1 and repressed by glucose [29,30] and heat shock protein 12 (HSP12), which is known to be repressed by very low glucose concentrations and is also stress induced [31-33]. Inorganic pyrophosphatase (IPP1), Actin (ACT1), and Pyruvate dehydrogenase 1 (PDA1) were included as tentative house-keeping/reference genes. In addition to the well characterized genes, for which raw data have been partly reported before [3], we also included a series of ADH-genes (ADH3-6) that are less well understood, to investigate their responses in relation to different glycolytic rates. Throughout this paper the genes in figures and tables are color coded as follows: ADH1, PGK1, TPI1, PDC1 and MIG1 genes, which are expected to be induced by glucose, are shown in blue, FBP1, ADH2, MDH2 and SUC2, which are expected to be repressed by glucose, are shown in red, ADH3, ADH4, ADH5 and ADH6 genes, whose functions are rather unknown, are shown in yellow, HSP12 is shown in black and CYC1 in green.

Bottom Line: In multiway studies samples are characterized by their expression profiles to monitor changes over time, effect of treatment, drug dosage etc.The data are analyzed by matrix-augmented PCA, which is a generalization of PCA for 3-way data, and the results are confirmed by hierarchical clustering and clustering by Kohonen self-organizing map.The technique also identifies genes that show perturbed expression in specific strains.

View Article: PubMed Central - HTML - PubMed

Affiliation: TATAA Biocenter, Odinsgatan 28, 411 03 Göteborg, Sweden. anders.stahlberg@neuro.gu.se

ABSTRACT

Background: The large sensitivity, high reproducibility and essentially unlimited dynamic range of real-time PCR to measure gene expression in complex samples provides the opportunity for powerful multivariate and multiway studies of biological phenomena. In multiway studies samples are characterized by their expression profiles to monitor changes over time, effect of treatment, drug dosage etc. Here we perform a multiway study of the temporal response of four yeast Saccharomyces cerevisiae strains with different glucose uptake rates upon altered metabolic conditions.

Results: We measured the expression of 18 genes as function of time after addition of glucose to four strains of yeast grown in ethanol. The data are analyzed by matrix-augmented PCA, which is a generalization of PCA for 3-way data, and the results are confirmed by hierarchical clustering and clustering by Kohonen self-organizing map. Our approach identifies gene groups that respond similarly to the change of nutrient, and genes that behave differently in mutant strains. Of particular interest is our finding that ADH4 and ADH6 show a behavior typical of glucose-induced genes, while ADH3 and ADH5 are repressed after glucose addition.

Conclusion: Multiway real-time PCR gene expression profiling is a powerful technique which can be utilized to characterize functions of new genes by, for example, comparing their temporal response after perturbation in different genetic variants of the studied subject. The technique also identifies genes that show perturbed expression in specific strains.

Show MeSH
Related in: MedlinePlus