Kasey E. O'Connor Week 11 Journal

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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

Tables

  • Table 1: Shows the biomass yield and residual nutrients in the nitrogen and glucose limiting chemostats at 12°C and 30°C. It gives proof that there is little effect on the growth efficiency with the changes in growth temperature.
  • Table 2: Shows the protein and storage carbohydrate contents of the yeast biomass in the chemostat at each nutrient and temperature level. Specifically, it shows that trehalose and glycogen contents are significantly lower at 12°C than 30°C.
  • Table 3:
    • (A): Shows the significantly overrepresented cis-regulatory binding motifs in 5' upstream regions in low-temperature up and down regulated genes for both limited chemostats.
    • (B): Shows the significantly overrepresented promoter elements that bing to known transcription factors.
    • These results are consistent with the induction of glycogen and trehalose biosynthesis genes due to the stress of going from optimal temperature to low temperature.

Figures

  • Figure 1: This Venn diagram shows the overlap of the 235 genes with significant transcriptional changes in both limited cultures, as well at the 806 genes that changed in nitrogen limited and the 494 that significantly changed in glucose limited chemostats.
  • Figure 2: This is the map that shows the transcription level ratio of the 1065 significantly expressed genes in the chemostat at both temperatures. This specifically shows that trehalose and glycogen was lower at 12 than at 30°C in the ammonia limited culture, whereas in the glucose-limited chemostats, trehalose contents were lower at 12°C, but glycogen content was higher at 30°C.
  • Figure 3: Shows the genes that are significantly expressed in batch cultures during adaptation. It also shows the heat map of the 259 genes that were commonly found among the 3 studies to have a significant transcriptional change.
  • Figure 4: Compares the transcript ratio of the common 259 genes in the batch cultures and the chemostat at the low temperatures. The Venn diagrams show that there were only 13 genes commonly upgraded and 16 commonly downgraded among all of the studies.
  • Figure 5: Uses Venn diagrams to compare the number of genes in the batch cultures and the chemostat that were commonly up or down regulated during acclimation and adaptation
  • Figure 6: Compares the ESR genes with the batch cultures and the chemostat in this study to find the number of genes that were commonly up or down regulated during adaptation and acclimation

Figure Presentation

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