Anthony J. Wavrin Week 11

Outline

Introduction

• Microorganisms are susceptible to temperature changes due to their inability to move, thus they must have physiological means to adapt to these changing environments.
• The temperature situation tested in this study was the effect of cold temperatures, termed cold shock.
• Recent studies have also looked at cold shock in yeast but, used a method of growing them in batch cultures.
• In this study, yeast were grown in chemostats to keep the conditions more constant.
• The yeast's adaptation to cold shock is determined by looking at changes in the transcriptome.

Materials and Methods

• The yeast are grown in four different conditions:
  • 12C with glucose as a limiting nutrient (experimental)
  • 30C with glucose as a limiting nutrient (control)
  • 12C with ammonium as a limiting nutrient (experimental)
  • 30C with ammonium as a limiting nutrient (control)
    • Two different limiting nutrients were used to censor the nutrient dependent changes in the transcriptome.
• The chemostat had a 1.0 liter culture of defined synthetic media with either glucose or ammonia as a limiting nutrient.
• The yeast strain used was CEN.PK113-7D (MATa) and was in a haploid state.
• The growth rate of the yeast was constant at 0.03 h-1, which is 75% max growth rate at 12C and 10% max growth rate at 30C.
• The study used a pair-wise analysis of the DNA microarray data for transcription levels and used Fisher's exact test with a Bonferroni correction for overrepresentation of transcription-factor binding sites.

Results

Table 1

• Table 1 shows that the chemostat conditions were either ammonia or glucose limiting by reporting the residual ammonia or glucose, respectively.
• Growth efficiency between the yeast at 12C and 30C was also relatively constant.
• Utilization of glucose and production of ethanol and carbon dioxide are not severely different between 12ºC and 30ºC.

Figure 1

• Figure 1 shows the overlap between significant changes in the transcriptome by gene of the yeast limited by either glucose or ammonia.
• In total, 1065 genes were significantly "regulated" but, only 235 genes were consistent in both conditions.
• This is a visual representation of genes that may be nutrient limiting specifically regulated.

Figure 2

• Genes that were down regulated at 12C in both nutrient limiting conditions are involved with carbohydrate metabolism, response to stimulus, and transportation.
• Genes that were up regulated at 12C in both nutrient limiting conditions are involved with nuclear export, ribosome biogenesis and assembly, and rRNA processing.

Table 2

• There were no indications of a build up trehalose or glycogen in the yeast at 12C in either condition.
• Ammonium limiting yeast had a significant more amount of protein per dry weight at 12C compared to 30C.

Table 3

Table 3A

• In nitrogen limited yeast, the motif STRE which regulates Msn2/Msn4 and the motif GATAA which codes for Gln3/Gat1/Dal80/Gzf3 is down regulated
  • Msn2/Msn4 is a transcriptional regulator that is activated in stress conditions.
  • Gln3/Gat1/Dal80/Gzf3 are associated with nitrogen catabolism.
• In nitrogen and glucose limited yeast,the motif PAC is up regulated.

Table 3B

• In nitrogen and glucose limited yeast, Hsf1p is down regulated.
  • Hsf1p is a trimeric heat shock transcription factor.
• In the transcription factor changes that were present in both nitrogen and glucose limited yeast, there was only down regulation.

Figure 3

• Only 139 of the 256 genes that were reported by Murata, Sahara, and Schade were consistent in either up regulation or down regulation.
  • 48 genes were down regulated.
  • 91 genes were up regulated.

Figure 4

• only 6 of the 13 overlapping genes between Murata, Sahara, and Schade, and this study were consistently upregulated in all studies.
• only 5 of the 16 overlapping genes between Murata, Sahara, and Schade, and this study were consistently downregulated in all studies.