Kasey E. O'Connor Week 11 Journal: Difference between revisions
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*Seasonal and daily temperature changes are inevitable in the lives of microorganisms living in natural environments. | *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. | **There are many different cellular responses among yeast cells. | ||
**Temperatures below the optimal environment (25- | **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 | *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 | *Two distinct phases of cold shock response have been noticed through other studies | ||
Line 32: | Line 32: | ||
'''Materials and Methods''' | '''Materials and Methods''' | ||
*The haploid strain CEN.PK113-7D was used | *The haploid strain CEN.PK113-7D was used | ||
**Grown at 12 or | **Grown at 12 or 30°C in a 1 liter chemostat | ||
**Grown in anaerobic conditions at a fixed growth rate of 0.03 h<sup>-1</sup> | **Grown in anaerobic conditions at a fixed growth rate of 0.03 h<sup>-1</sup> | ||
**pH remained constant at 5.0 | **pH remained constant at 5.0 | ||
**The transcription was analyzed in both glucose and ammonium limited chemostat | **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 | **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. | *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. | *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 | |||
Briefly state the result shown in each of the figures and tables. | Briefly state the result shown in each of the figures and tables. | ||
===Figure Presentation=== | ===Figure Presentation=== | ||
===Useful Links=== | ===Useful Links=== | ||
{{Kasey E. O'Connor}} | {{Kasey E. O'Connor}} |
Revision as of 22:35, 3 April 2013
Journal Club Assignment
Read
Vocabulary
- In vivo - within a living organism [1]
- Diurnal - occurring during the day [2]
- Mannoproteins - highly antigenic yeast cell wall proteins with large numbers of mannose groups attached [3]
- Cuvette - a transparent container with precisely-measured dimensions for holding liquid samples to be put into a spectrophotometer [4]
- Transcriptome - the complement of mature messenger RNAs produced in a given cell in a given moment of its life [|5]
- Prototropic - strains that have the same nutritional requirements as the wild-type strain [6]
- sphingolipids - structural lipid of which the parent structure is sphingosine rather than glycerol synthesised in the Golgi complex [7]
- Orthologues - genes that can be found in two or more different species that can be traced back to the same common ancestor [8]
- Oleate - A salt of oleic acid, some oleates, as the oleate of mercury, are used in medicine by way of inunction [9]
- 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
- SFK1, YPC1, and YEL073C are all involved in lipid metabolism
- PHO84, FUI1, AHA1, FCY2, and YLR413W were down-regulated
- PIR3, SFK1, YPC1, YEL073C, YNL024C, and YLR225C were up-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
Briefly state the result shown in each of the figures and tables.
Figure Presentation
Useful Links
- BIOL398-03/S13 Homepage
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