Matthew E. Jurek Week 11

Matthew E. Jurek BIOL398-03/S13

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Biological Terms

1. trehalose- also known as mycose or tremalose, is a natural alpha-linked disaccharide formed by an α,α-1,1-glucoside bond between two α-glucose units.
2. mannoprotein- component of yeast cell walls; protein covalently linked to polymers of mannose.
3. chromatography- the separation of mixtures into their constituents by preferential adsorption by a solid, as a column of silica (column chromatography) or a strip of filter paper (paper chromatography) or by a gel.
4. immunoprecipitation- the separation of an antigen from a solution by the formation of a large complex with its specific antibody.
5. catabolite- a product of catabolic action.
6. kinetics- the branch of mechanics that deals with the actions of forces in producing or changing the motion of masses.
7. ceramides- ceramides are a family of lipid molecules. A ceramide is composed of sphingosine and a fatty acid.
8. orthologue- one of two or more homologous gene sequences found in different species.
9. ergosterol- a compound present in ergot and many other fungi. A steroid alcohol, it is converted to vitamin D2 when irradiated with ultraviolet light.
10. transcriptome- the transcriptome is the set of all RNA molecules, including mRNA, rRNA, tRNA, and non-coding RNA produced in one or a population of cells.

via Biology Online Dictionary

Acclimation of Saccharomyces cerevisiae to Low Temperature: A Chemostat-based Transcriptome Analyis

Tai et al. (2007) Acclimation of Saccharomyces cerevisiae to Low Temperature: A Chemostat-based Transcriptome Analysis. Molecular Biology of the Cell 18: 5100–5112.

1. Introduction
   • On the cellular level, yeast responds in a number of ways to temperature changes (temps outside the 25-30 degree C optimum).
   • Temperatures below the optimum range slow cellular processes.
   • Effects of low temperature depend on exposure time
   • Sudden exposure results in adaptaion
   • Prolonged exposure results in acclimation
   • Previous studies have focused on cold shock (sudden exposure) and found 2 phases of cold-shock response
   • Current low-temp transcriptome databases contain major discrepancies
   • Previous studies have focused on batch culutures: hard to distinguish temp effects on transcription from effects of specific growth rate
   • Chemostat cultures allow for control of specific growth rate independent of culture conditions such as metabolites, pH, and oxygen availability.
   • Overall Goal: Focus on genome-wide transcriptional regulation by exploring steady-state acclimatized growth of yeast at low temps
2. Materials and Methods
   • Haploid strain CEN.PK113-7D was grown at both 12 and 30 degrees C, anaerobically, in a chemostat
   • The chemstat contained a dilution rate of .03/h, pH of 5.0 and stirrer speed of 600rpm.
   • The cultures were contained in a synthetic medium that limited either carbon or nitrogen. Every other requirement was in excess.
   • Each growth condition consisted of 3 independent replicates.
   • Microarray data was analyzed using Fisher's exact test with a Bonferroni correction in order to acheive a p value threshold of .01.
   • This data was then compared to previously published low-temperature transcriptome datasets.
3. Results
   • Biomass and fermentation rates did not differ significantly between cultures grown at 12 and 30 degrees C in carbon and nitrogen limited environments.
   • Glucose-limited cultures: 494 genes yielded a significant difference amongst transcript levels at the two temps
   • Nitrogen-limited cultures: 806 genes exhibited this type of behavior
   • 235 genes showed a change in regulation when both were limited
   • Hextose transporter genes HXT2, HXT3, and HXT4 exhibited increased transcript levels at 12 degrees C
   • HXT5 and HXT16 showed reduced transcription at this temp
   • There are 3 genes that encode ammonia permeases. In the ammonium-limited cultures at 12 degrees C, 2 were reduced and one showed increased transcription.
   • Important to note the specific growth rate of .03/h in this experiment is close to the maximum specific growth rate at 12 degrees versus 30 degrees
   • Trehalose accumulation is a sign of low-temp adaptation
   • However, there was not an increase in transcription of genes that metabolize trehalose and glycogen in this experiment
   • Thus, the accumulation of trehalose and glycogen is not necessary for acclimation at low temps in yeast
   • To date, the exact relevance of storage carbohydrate metabolism during adaptation remains a mystery.
   • There was a noted up-regulation of translational machinery at low temps, especially 12 degrees C.
   • Increased protein content could be a compensation mechanism for reduced enzyme kinetics at low temps.
   • Three pre-existing datasets show 259 genes that responded to temp downshifts.
   • Genes within these datasets were observed for up-regulation or down-regulation in the given conditions.
   • Three genes consistently up-regulated at low temps were involved in lipid metabolism while three genes consistently down-regulated were involved in encoding transporters.
   • Multiple stimuli are often responsible for changes in gene regulation.
   • Batch culture results are unreliable as specific growth rate fluctuates with temperature and oxygen solubility is impacted by temperature.
   • Genes that appear to signal cold shock in batch cultures may exhibit different behaviors in chemostat cultures.
   • An ESR mechanism has been proposed in the regulation of yeast genes that exhibit transcriptional changes in response to multiple environmental stresses.
   • In this study, it was found that ESR is NOT a response to growth at low temperature. ESR occurs following sudden exposure to suboptimal temps.
4. Discussion
   • Transcriptome analysis requires a pair-wise comparison in batch cultures of two conditions in which one acts as the reference.
   • In the chemostat study, only studying glucose-limited or ammonium-limited cultures would have resulted in secondary effects. Thus, the experimental design was focused on both.
   • A properly designed, combinatorial experiment can account for context while batch experiments cannot.
   • Context is important in truly identifying genes impacted by low temperature.
   • Fixed-specific growth rate impacts transcription and chemostat experiments favor this.
   • Acclimated growth at low-temperature does not involve a Msn2/Msn4-complex (as seen in batch), but rather some other mechanism.
   • In fact, ESR is impacted by reduced specific growth rate and not low temps.
   • Ribosomal protein-encoding genes are upregulated at low temps to compensate for translational issues at low temps.
   • Incorrectly-folded proteins occur during rapid temp decreases, but are not a problem in acclimated yeast cultures.
   • Low temperature and low specific growth rate are parameters that are linked in batch cultures.
   • These parameters can be separated in chemostat cultures. It's been found that low-temperature acclimation does not simply involve reprogramming on the transcriptional level within yeast.
   • Intracellular metabolite levels are also important for acclimation.
5. Figures
   • Table 1-Illustrates biomass yields and fermentation rates did not differ greatly amongst carbon and nitrogen limited cultures at both temps. Growth efficiency was not impacted by temperature.
   • Figure 1- Compares differences in gene expression based on the various conditions, with the middle representing both conditions combined.
   • Figure 2- A heat map expressing the transcript level of various genes within the 4 different growing conditions.
   • Table 2- Expresses the storage carbohydrate content amongst the various conditions tested within the chemostat.
   • Table 3- Illustrates over-expressed binding motifs as well as a number of transcription factors and their overrepresentation.
   • Figure 3- A comparison of differently expressed genes seen in the previous studies as well as a heat map illustrating their expression.
   • Figure 4- A comparison of expression ratios of genes up regulated or down regulated in batch and chemostat cultures.
   • Figure 5- A comparison of gene expression changes as seen in previous studies as well as this study.
   • Figure 6- A comparison of genes up regulated or down regulated in the previous studies (adaptation) and also a comparison on previous studies and this study (acclimation).