William A. C. Gendron Week 10

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

  1. Definitions

Outline of "Acclimation of Saccharomyces cerevisiae to Low Temperature: A Chemostat-based Transcriptome Analysis

  • Introduction
    • This research used Saccharomyces cerevisiae as the model organism.
    • Focused on discovering the true genes associated with cold shock reaction.
    • Batch based experiments create a unique situation that is not just cold shock stress.
    • Prior studies show discrepancies in ribosomal protein expression. Why is that?
    • Discrepancies between carbohydrates and trehalose usage.
    • Further explore the Msn2p/Msn4p complex.
    • Evaluate adaptation and acclimation studies.
    • A chemostat is a unique way to study yeast: it supplies a stable environment that can be constantly growing. Changes growth rate and other features.
  • Materials and Methods
    • Saccharomyces cerevisiae was was grown at 12 and 30 degrees Celsius. Growth rate for the 12 degrees was .03h^-1 with a volume of 1.01 liters. Set ups of nitrogen and carbon limited chemostats.
    • Basic Analysis:
      • Supernatants were collected to do liquid chromatography and other tests. This evaluated the level of glucose and metabolites.
      • Ammonium concentration was evaluated with a similar chromatography method.
      • Glucose creation from glycogen and trehalose was evaluated by a Roche kit.
    • Microarray analysis:
      • Measured three times and used Microsoft Excel to run the statistics.
      • Used two-fold difference as a threshold and calculated a median false rate at 1%.
      • Evaluated overrepresentation with DAVID(Database for Annotation, Visualization and Integrated Discovery) along with a Fisher's exact test, a Bonferroni correction and a p-test.
      • Compared data with data from Beltran et al., Murata et al., Sahara et al., Schade et al., Gasch et al. and the Stanford Yeast Stress Database.
  • Results
    • Low-Temperature Chemostat Cultivation Results in Altered Uptake Kinetics for the Limiting Nutrient and Enhanced Catabolite Repression
      • More residual nutrients in 12 degree than 30. Reduced transport and metabolism is the root of this.
      • Hexose transport genes increased transcription, in theory to account for the decreased rate of transport in the 12 degree.
      • HXT5 and 16 increased in 30 degree.
      • In nitrogen limited cultures, permeases that targeted ammonia changed. MEP3 increased for 12 degree while high affinity MEP1 and 2 were reduced.
    • Acclimation to Nonfreezing Low Temperature Does Not Require a High Storage Carbohydrate Content
      • Transcription was not changed in genes for trehalose and glycogen metabolism.
      • Carbohydrate synthesis decreased at lower temperatures.
    • Up-Regulation of the Translation Machinery at Low Temperature
      • Higher amounts of transcription seen in 16 of the ribosome biogenesis genes at 12 degrees(both nitrogen and carbon limited).
      • 12 degrees nitrogen and carbon limited showed evidence of much higher rRNA and protein than 30.
    • Transcriptional Responses to Low Temperature: Adaptation versus Acclimation
      • Compared to previous studies
      • Found that our of 259 genes, 139 matched up or down regulation across cold shock genes.
      • Specific genes were identified to be highly consistent.
    • Context Dependency of Temperature Response
      • They describe why this has better controls than other experiments.
      • Better O2 control than flask batches
      • Found genes that did not decrease with temperature but use to be linked to other experiments that suggested that they were.
    • Environmental Stress Response, a Low-Temperature Adaptation-specific Response
      • Compared to database of genes that react in environmentally stressful situations
      • About 1/3 of the genes from this study were found to be linked to ESR.
  • Discussion
    • Chemostat-based Low-Temperature Transcriptomics: Experimental Design
      • Explains that there are too many variables to control for and multiple experiments are required to test the limits.
      • Requires multiple tests to get a full picture of the genes.
    • Specific Growth Rate and ESR
      • Growth rates between temperature groups were quite different.
      • ESR genes were found to make up a portion of the genes activated.
    • Transcriptional Responses to Low Temperature: Acclimation versus Adaptation
      • Confusing terms but it focused on the timing of different genes. I would have preferred another word instead of adaptation, because I think of it as an evolutionary term.
      • It wrapped up saying that that this alternative system can be used to parse out genes that are caused by the batch system.