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

<p>Abstract</p> <p>Background</p> <p>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 <it>Saccharomyces cerevisiae </it>strains with different glucose uptake rates upon altered metabolic conditions.</p> <p>Results</p> <p>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 <it>ADH4 </it>and <it>ADH6 </it>show a behavior typical of glucose-induced genes, while <it>ADH3 </it>and <it>ADH5 </it>are repressed after glucose addition.</p> <p>Conclusion</p> <p>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.</p

The Role of Hexose Transport on Glycolytic Flux and Glucose Induced Responses in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, glucose is primarily fermented rather than respired. Only in cultivation modes such as chemostats or fed-batches, where the external glucose is kept low, a fully respiratory catabolism is observed. We have constructed a series of yeast strains starting from a mutant (the hxt Null strain) that is unable to take up glucose. In these strains glucose uptake is governed by chimeras between the two hexose transporters Hxt1 and Hxt7. These strains display a broad range of glucose uptake capacities. They have been used to show that glucose uptake has a high degree of control on the glycolytic flux only when the uptake rate is significantly reduced. One of the strains, with a low uptake capacity, presented a completely Crabtree negative phenotype. This shows that a switch between fermentative and respiratory metabolism can be achieved by changing only a single metabolic step. The transferability of the phenotype has been confirmed by integration of the chimeric construct into a hxt1-hxt7 haploid wine strain. Strains with different glycolytic rates have also allowed quantitative studies on glucose-induced responses and dissection of different signaling mechanisms. A strong correlation between glycolytic rate and expression of glycolytic genes was observed. mRNA profiles of several groups of glucose-regulated genes suggest that sensing is primarily intracellular. Contrary to what is presently believed, SUC2 expression did not always correlate to high phosphorylation levels of Snf1 and Mig1, key regulators of glucose-controlled gene expression. These results suggest regulation either through additional pathway/s or independent regulation of the Snf1/Mig1 targeted kinases and phosphatases

The Role of Hexose Transport on Glycolytic Flux and Glucose Induced Responses in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, glucose is primarily fermented rather than respired. Only in cultivation modes such as chemostats or fed-batches, where the external glucose is kept low, a fully respiratory catabolism is observed. We have constructed a series of yeast strains starting from a mutant (the hxt Null strain) that is unable to take up glucose. In these strains glucose uptake is governed by chimeras between the two hexose transporters Hxt1 and Hxt7. These strains display a broad range of glucose uptake capacities. They have been used to show that glucose uptake has a high degree of control on the glycolytic flux only when the uptake rate is significantly reduced. One of the strains, with a low uptake capacity, presented a completely Crabtree negative phenotype. This shows that a switch between fermentative and respiratory metabolism can be achieved by changing only a single metabolic step. The transferability of the phenotype has been confirmed by integration of the chimeric construct into a hxt1-hxt7 haploid wine strain. Strains with different glycolytic rates have also allowed quantitative studies on glucose-induced responses and dissection of different signaling mechanisms. A strong correlation between glycolytic rate and expression of glycolytic genes was observed. mRNA profiles of several groups of glucose-regulated genes suggest that sensing is primarily intracellular. Contrary to what is presently believed, SUC2 expression did not always correlate to high phosphorylation levels of Snf1 and Mig1, key regulators of glucose-controlled gene expression. These results suggest regulation either through additional pathway/s or independent regulation of the Snf1/Mig1 targeted kinases and phosphatases

Structure of the Superantigen Staphylococcal Enterotoxin B in Complex with TCR and Peptide-MHC Demonstrates Absence of TCR-Peptide Contacts.

Superantigens are immune-stimulatory toxins produced by Staphylococcus aureus, which are able to interact with host immune receptors to induce a massive release of cytokines, causing toxic shock syndrome and possibly death. In this article, we present the x-ray structure of staphylococcal enterotoxin B (SEB) in complex with its receptors, the TCR and MHC class II, forming a ternary complex. The structure, in combination with functional analyses, clearly shows how SEB adopts a wedge-like position when binding to the β-chain of TCR, allowing for an interaction between the α-chain of TCR and MHC. Furthermore, the binding mode also circumvents contact between TCR and the peptide presented by MHC, which enables SEB to initiate a peptide-independent activation of T cells

Expression and purification of rat glucose transporter 1 in Pichia pastoris

Large amounts of pure and homogenous protein are a prerequisite for several biochemical and biophysical analyses, and in particular if aiming at resolving the three-dimensional protein structure. Here we describe the production of the rat glucose transporter 1 (GLUT1), a membrane protein facilitating the transport of glucose in cells. The protein is recombinantly expressed in the yeast Pichia pastoris. It is easily maintained and large-scale protein production in shaker flasks, as commonly performed in academic research laboratories, results in relatively high yields of membrane protein. The purification protocol describes all steps needed to obtain a pure and homogenous GLUT1 protein solution, including cell growth, membrane isolation, and chromatographic purification methods

Glucose transport machinery reconstituted in cell models.

Here we demonstrate the production of a functioning cell model by formation of giant vesicles reconstituted with the GLUT1 glucose transporter and a glucose oxidase and hydrogen peroxidase linked fluorescent reporter internally. Hence, a simplified artificial cell is formed that is able to take up glucose and process it

NMR quantification of diffusional exchange in cell suspensions with relaxation rate differences between intra and extracellular compartments.

Water transport across cell membranes can be measured non-invasively with diffusion NMR. We present a method to quantify the intracellular lifetime of water in cell suspensions with short transverse relaxation times, T2, and also circumvent the confounding effect of different T2 values in the intra- and extracellular compartments. Filter exchange spectroscopy (FEXSY) is specifically sensitive to exchange between compartments with different apparent diffusivities. Our investigation shows that FEXSY could yield significantly biased results if differences in T2 are not accounted for. To mitigate this problem, we propose combining FEXSY with diffusion-relaxation correlation experiment, which can quantify differences in T2 values in compartments with different diffusivities. Our analysis uses a joint constrained fitting of the two datasets and considers the effects of diffusion, relaxation and exchange in both experiments. The method is demonstrated on yeast cells with and without human aquaporins