Kasey E. O'Connor Week 11 Journal

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Contents

Journal Club Assignment

Read

Vocabulary

  1. In vivo - within a living organism [1]
  2. Diurnal - occurring during the day [2]
  3. Mannoproteins - highly antigenic yeast cell wall proteins with large numbers of mannose groups attached [3]
  4. Cuvette - a transparent container with precisely-measured dimensions for holding liquid samples to be put into a spectrophotometer [4]
  5. Transcriptome - the complement of mature messenger RNAs produced in a given cell in a given moment of its life [|5]
  6. Prototropic - strains that have the same nutritional requirements as the wild-type strain [6]
  7. sphingolipids - structural lipid of which the parent structure is sphingosine rather than glycerol synthesised in the Golgi complex [7]
  8. Orthologues - genes that can be found in two or more different species that can be traced back to the same common ancestor [8]
  9. Oleate - A salt of oleic acid, some oleates, as the oleate of mercury, are used in medicine by way of inunction [9]
  10. Assay - The determination of the amount of a particular constituent of a mixture or of the biological or pharmacological potency of a drug [10]

Outline

Introduction

  • Seasonal and daily temperature changes are inevitable in the lives of microorganisms living in natural environments.
    • There are many different cellular responses among yeast cells.
    • Temperatures below the optimal environment (25-35°C) affect enzyme kinetics, cellular growth, respiration, and lipid composition of membranes.
  • Sudden exposure to cold triggers stress response while longer exposure leads to acclimation and eventually adaptation
  • Two distinct phases of cold shock response have been noticed through other studies
    • Early Cold Response (ECR) occurs within the first 12 hours
    • Late Cold Response (LCR) occurs after 12 hours of exposure
  • Genes associated with cold shock have been consistently observed
    • TPS1, TPS2, HSP12, HSP26, HSP42, HSP104, YRO2, and SSE2
  • Already published data about low-temperature transcriptomes have shown inconsistencies among expressed genes
  • Most published low-temperature studies on yeast have been performed in batch cultures
    • This method is poorly adapted for the study of prolonged cold exposure because the growth rate is strongly affected by the temperature
  • Chemostat cultures enable control of the growth rate
    • They provide a reproducible environment for study on gene expression in fully controlled cultures
  • The goal of the study is to investigate S. cerevisiae at suboptimal temperatures and look at the entire genome transcriptional regulation
    • The results of this analysis were compared to previous studies in the batch cultures

Materials and Methods

  • The haploid strain CEN.PK113-7D was used
    • Grown at 12 or 30°C in a 1 liter chemostat
    • Grown in anaerobic conditions at a fixed growth rate of 0.03 h-1
    • pH remained constant at 5.0
    • The transcription was analyzed in both glucose and ammonium limited chemostat
    • Biomass dry weight, metabolites, dissolved oxygen, and gas profiles were constant for three volume changes before sampling
  • Samples came from three independent culture replicates for each condition (ex. 12°C and glucose limited)
  • Microsoft Excel was used to run the statistical significance of the microarrys
  • Fisher's exact test was used with the Bonferroni correction and a p-value of 0.01.

Results

  • Biomass yields and fermentation rates were similar in all of the different chemostats, which indicates that growth efficiency was not very affected by growth temperature.
  • DNA microarray analysis was used to analyze the data
    • In the glucose limited cultures, 494 genes yielded significantly different levels of transcription at the two temperature
    • 806 genes had a significant change in transcription levels in the ammonia limited cultures
    • Total, 1065 (16%) genes had a significant transcription level change
    • Only 235 genes showed significant change in transcription levels in both the glucose and ammonia limited cultures
  • Transcriptional induction of genes involved in the metabolism of carbs (trehalose) is consistently observed after cold shock
    • In a steady-state chemostat at 12°C there was no upregulation in trehalose and glycogen metabolism was not observed
    • TPS1, TPS2, TSL1, NTH1, GSY1, GSY2, GAC1, PIG2, PCL10, and PCL7 showed decreased transcript levels in 12 degrees compared to 30°C
    • Shows that the induction of genes involved in the synthesis of the compounds for glycogen and trehalose accumulation is not a prerequisite for acclimation to cold temperature
  • 16 genes associated with ribosome assembly showed higher transcript levels at 12°C than at 30°C in both cultures
  • In the ammonia limited culture, an additional 80 genes showed increases in transcription levels at 12°C
  • There was an increase of the rRNA content at low temperature in the ammonium-limited cultures
    • Supported by increased transcript levels of genes that encode subunits of polymerase I (RPA12, RPA49, RPA135, and RPC40) and 33 genes involved in rRNA processing
  • Comparison of the data with three other data sets (Sahara et al., 2002; Schade et al., 2004:Murata et al. 2006) only had 259 genes that responded to temperature in all three studies
    • Only 91 were consistently up-regulated and 48 consistently down-regulated
  • There were only 11 genes that showed a consistent pattern of regulation in all four chemostat environments
    • PIR3, SFK1, YPC1, YEL073C, YNL024C, and YLR225C were up-regulated
      • SFK1, YPC1, and YEL073C are all involved in lipid metabolism
        • Temperature affect the fluidity of the lipid bilayer, so there is a need for modification
    • PHO84, FUI1, AHA1, FCY2, and YLR413W were down-regulated
  • The biggest difference between batch cultures and chemostats is the specific growth rate
    • In batch cultures a temperature decrease causes a decrease of the specific growth rate
  • Also, in batch cultures exposed to air the dissolved oxygen concentration is temperature dependent

Discussion

  • The only group of genes that was consistently regulated among both the batch and chemostat cultures were the genes involved in lipid metabolism
    • This adaptation is necessary because the membrane composition is essential for growth
  • The chemostat data showed up-regulation of translational machinery
    • This was consistent with some batch studies but inconsistent with others.
  • Batch culture studies showed up-regulation of chaperone-encoding genes such as HSP26 and HSP42 **However, in the chemostat, these genes were down-regulated
  • This study shows that transcriptional responses to low temperature and low specific growth rate found in the batch cultures can be distinguished through running a chemostat with a constant specific growth rate

Briefly state the result shown in each of the figures and tables.

Figure Presentation

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