Anthony J. Wavrin Week 11

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Contents

Definitions

  • Trehalose - Trehalose is found in nearly every organism living in extreme cold environments and is found in human organs such as the kidney, liver, and blood plasma (organs that can withstand extended freezing times). Citation
  • Mannoproteins - Yeast cell wall components that are proteins with large numbers of mannose groups attached; highly antigenic. Citation
  • Desaturase - Any of several enzymes that put double bonds into the hydrocarbon areasof fatty acids. Citation
  • Liquid Chromatography - A type of chromatography in which both the stationary phase and the mobile phase are liquid. Citation
  • Allantoin - Any of many nitrogen-containing organic compounds which are found in some plants and in the urine of some mammals. Allantoin is used to treat wounds and ulcers. [1]
  • Morphogenesis - The development of form; the overall consequence of determination, differentiation, and growth. Citation
  • Batch culture - A large-scale closed system culture in which cells are grown in a fixed volume of nutrient culture medium under specific environmental conditions (e.g. nutrient type, temperature, pressure, aeration, etc.) up to a certain density in a tank or airlift fermentor, harvested and processed as a batch, especially before all nutrients are used up. Citation
  • Sphingolipid - Structural lipid of which the parent structure is sphingosine rather than glycerol. Synthesised in the Golgi complex. Citation
  • Northern analysis - A procedure similar to the southern blot analysis, used mostly to separate and identify RNA fragments; typically via transferring RNA fragments from an agarose gel to a nitrocellulose filter followed by detection with a suitable probe. Citation
  • Fishers exact test - A statistical test of independence much used in medical research. It tests the independence of rows and columns in a 2 X 2 contingency table (with 2 horizontal rows crossing 2 vertical columns creating 4 places for data) based on the exact sampling distribution of the observed frequencies. Hence it is an exact test. Devised by the British geneticist and biostatistician R. A. Fisher (1890-1962). Citation

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 and each condition was repeated three times.
  • 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 the transcription factor changes that were present in both nitrogen and glucose limited yeast, there was only down regulation.
  • In nitrogen and glucose limited yeast, Hsf1p is down regulated.
    • Hsf1p is a trimeric heat shock transcription factor.
    • Other transcription factors down regulated were Aft2p, Nrg1p, Phd1p, Rcs1p, Rox1p, Sok2p, Nrg1-Aft1, Phd1-Nrg1, Rox1-Phd1, and Sok2-Nrg1.

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 up-regulated in all studies.
  • only 5 of the 16 overlapping genes between Murata, Sahara, and Schade, and this study were consistently down-regulated in all studies.

Figure 5

  • Overall, there is little overlap between genes that were regulated between batch studies and this study with the growth rate studies.
  • This indicates the lack on consistency between data and may suggest other factors affecting the regulation besides solely cold shock.

Figure 6

  • While there is small overlap between the ESR study and the Batch studies, there is consistency between down-regulated genes and up-regulated genes.
  • This is more overlap between the ESR study and this study but, there is a very obvious conflict in which genes that are down-regulated in the ESR study and up-regulated in this study and vice versa.

Conclusion

  • There is a difference in regulation to cold shock depending on if it is due to adaptation (Murata, Sahara, and Schade) or acclimation(this study).
  • There is variability across the transcriptome regulation that is likely due to other conditions besides cold shock.
  • Overall, this study found 235 genes that were regulated the same during cold shock in both glucose and nitrogen limitation, and they were involved in carbohydrate metabolism, response to stimulus, transportation,nuclear export, ribosome biogenesis and assembly, and rRNA processing.


Week 11 Assignment


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