James P. McDonald Week 11
- Trehalose: "A crystalline disaccharide that is found in various organisms, is about half as sweet as sucrose, and is sometimes used as a sweetener in commercially prepared foods." []
- Batch Culture: "A large-scale closed system culture in which cells are grown in a fixed volume of nutrient culture medium under specific environmental conditions." []
- Transcriptome: "The complete set of RNA transcripts produced by the genome at any one time." []
- Ergosterol: "Crystalline steroid alcohol that occurs especially in yeast, molds, and ergot and is converted by ultraviolet irradiation ultimately into vitamin D2" []
- Cryostat: "An apparatus for maintaining a constant low temperature especially below 0°C" []
- Cuvette: "A transparent or translucent box-shaped container with precisely-measured dimensions for holding liquid samples to be put into a spectrophotometer." []
- Desaterase: "Any of several enzymes that putdouble bonds into the hydrocarbon areasof fatty acids." []
- Mannoproteins: "Yeast cell wall components that are proteins with large numbers of mannose groups attached; highly antigenic." []
- Prototrophic: "Strain's that have the same nutritional requirements as the wild-type strain." []
- Supernatant: "The soluble liquid fraction of a sample after centrifugation or precipitation of insoluble solids." []
- Transcriptional regulation was studied in Saccharomyces cerevisiae in response to low temperatures.
- This study focused on long-term low-temperature acclimation rather than rapid transitions to low temperature or "cold shock."
- This experiment was performed in a chemostat, in contrast to most literature, which used batch cultures.
- Transcription levels of various genes were measured at low temperatures.
- The main result of the study revealed that there are large differences in transcription levels in many of the genes between the literature data, that looked at for rapid transitions to cold temperatures, and this study, that looked at long-term low-temperature acclimation.
- The effect of cold temperatures on transcription in yeast has been studied greatly.
- The studies has always been done in batch cultures.
- This study uses a steady-state chemostat model to eliminate the effects of specific growth rates.
- Other studies focused on a rapid transition to low temperatures, a cold shock.
- This study focuses looks at the effects on yeast transcription with a slow transition to low-temperatures.
- This allowed the yeast to acclimate rather than quickly adapt.
- These different measures were taken in this experiment so that they could compare their results with the literature data.
- The results showed big differences between this study and the literature data.
Materials and Methods
- The yeast used was phototropic, haploid, S. cerevisiae strain: CEN.PK113-7D (MATa)
- The yeast was grown in steady-state chemostats in anaerobic conditions.
- Chemostats allowed for the control of the specific growth rates.
- The chemostats were 2.0 l with a working volume of 1.0 l.
- The dilution rate was 0.03h-1.
- The pH was kept constant at 5.0.
- The stirrer speed was set constant at 600 rpm.
- The yeast were grown at temperatures of 12oC and 30oC.
- The growth medium was a deﬁned synthetic medium, limited by carbon or by nitrogen. All other growth requirements were present in excess.
- For each chemostat, triplicate trehalose measurements and duplicate glycogen measurements were made.
- The yeast were grown in four different conditions:
- 12oC, glucose limiting.
- 30oC, glucose limiting.
- 12oC, ammonia limiting.
- 30oC, ammonia limiting.
- There were three independently cultured replicates for each of the four conditions.
- The data was analyzed using microarray analysis and statistical assessment.
- RNA quality was determined using the microarray analysis.
- Microsoft Excel was used for significance analysis.
- Data was visualized using venn diagrams and heat maps on specific software.
- Web-based software was used for the promoter analysis.
- Statistical assessment was done for overrepresentation of GO biological processes and overrepresentation of transcription-factor binding sites.
- The data results from this study were compared with datasets from other studies.
- Transcription factors: Mbp1p, Hap2-Hap1, Hap3-Hap1, Fhl1p, Sfp1p, Gln3p, Gln3-Dal82, Hap2-Dal82, Aft2p, Hsf1p, Nrg1p, Phd1p, Rcs1p, Rox1p, Sok2p, Nrg1-Aft2, Phd1-Nrg1, Rox1-Phd1, Sok2-Nrg1
- Table displaying the physiological characteristics of the yeast grown in all four conditions.
- Shows that the biomass yields and fermentation rates were similar at 12oC and 30oC, proving that the growth efficiency of the yeast was not greatly affected by the temperature.
- Venn Diagram showing the number of genes that had significantly different transcript levels at the two temperatures, 12oC and 30oC.
- 494 genes had significant changes in glucose limitation, while 806 had significant changes in nitrogen limitation.
- Heat map showing the transcription levels of 1065 genes grown in the four conditions.
- There was a change in transcription of genes that involve the uptake of growth limiting nutrients, showing a change in transport kinetics.
- Eight target genes for glucose catabolite repression were down regulated indicating a high degree of catabolic repression at 12oC.
- Table displaying protein and storage carbohydrates in the yeast.
- Showed that the cellular protein and nitrogen contents were much higher at 12oC than at 30oC in the ammonium limited chemostat but not in the glucose limited chemostat.
- Two part table displaying overrepresented binding motifs and overrepresented promoter elements.
- The reduced transcription in the yeast at 12oC in the nitrogen limited chemostat indicated there was an over expression of STRE elements.
- There was an enrichment of PAC cis-regulatory motifs shown in the analysis of 5 upstream sequences.
- The venn diagram shows all the genes that are common to the three previous experiments and the heat map shows the transcription ratios of the 259 common genes.
- There is a group of 259 common to three other studies that all responded to the decreased temperatures, but the responses differed.
- Venn diagrams comparing the genes that were either up or down regulated in both the acclimation study and adaptation study.
- There was very little overlap observed in the growth-rate-responsive genes when the temperature responsive gene datasets of Castrillo and Regenberg were compared with the 139 genes that showed a consistent response to low temperatures.
- Venn diagrams of the genes that are specifically up and down regulated in low temperatures comparing them between this study (acclimation) and past studies (adaptation), where ESR genes are identified.
- A great overlap was observed when ESR genes were compared with the batch culture genes that were consistently up and down regulated at low temperatures.
- James P. McDonald
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