Alex J. George Week 11

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Terms

1. Trehalose- disaccharide that is involved in the ability of plants/animals to withstand desiccation due to its hight water retention
2. fatty acid desaturase- an enzyme that removes two hydrogens from a fatty acid, resulting in a carbon-carbon double bond
3. specific growth rate- increase in cell mass per unit cell mass per unit time-- unites = reciprocal time
4. chemostat culture- a culture method in which fresh medium is continuously added and culture liquid is removed to keep the culture at a constant volume; allows for the control of the organism's growth rate
5. batch culture- a large-scale closed culture in which cells are grown in a fixed volume of nutrient medium under specific environmental conditions
6. dilution rate- the rate of flow of medium divided by the volume of the culture; when the culture is steady, the specific growth rate= dilution rate
7. prototrophic- having the same metabolic capabilities and nutritional requirements as the wild type
8. liquid chromatography- form of column chromatography used to separate compounds based on polarities and interactions with the column phase
9. Fisher's exact test- significance test used in analysis of tables where the sample sizes are small
10. cis-regulatory motif- a motif is a nucleotide/ amino acid sequence that is widespread and is believed to have biological significance

Introduction

• Main result: Yeast have a significant difference in gene response to long-term cold exposure versus rapid cold-shock exposure
• Cold temperatures have been shown to affect enzymatic activity, growth, respiration, membrane composition and trehalose content in yeast
• Sudden exposure results in rapid responses; long exposure leads to acclimation or even mutation
• 2 phases of cold-shock response: early (within 12 hours) and late (longer than 12 hours)
• Msn/Msn4 binding factors believed to coordinate regulation of low-temp. responses
• Trehalose only necessary for near freezing conditions
• Limitations of previous studies: Specific growth rate has strong impact on transcript profiles which isn't accounted for in batch cultures-- chemostat cultures used instead to control specific growth rate

Materials/Methods

   * How many replicates did they perform of each type?
o Biological replicates are made from entirely different biological samples.
o Technical replicates are made when one biological sample is split at a particular stage in the procedure and then carried through to the end of the procedure.
* What do they say about how they performed each of the steps listed in the Overview of Microarray Data Analysis section above?


Growth Conditions

• yeast (Saccharomyces cervisiae)grown at constant specific growth rate of .03/hr at 12C and 30C
• Medium was limited by carbon or nitrogen and constant pH of 5.0

Analytical Methods

• Rapid sampling method in Mashego et al.
• Liquid chromatography used to analyze glucose concentrations
• Trehalose determined by triplicates, glycogen by duplicates

Microarray Analysis

• Used Microsoft Excel to compare pair-wise data
• Results of each growth condition were compared against 3 independent strains of control groups
• Analyzed 12 different single strain chips (6 glucose-limited cultures--three 12C and three 30C; 6 nitrogen-limited cultures-- three 12C and three 30C)
• For this study, they used in affymetrix chip, therefore, rather than pairing up the two samples, they ran one sample at a time and computed the total number of pixels expressed, rather than a proportion of green to red
• The color stain is irrelevant because of the single sample analysis

Comparison

• Downloaded batch datasets from internet in order to compare results

Results

• Biomass and fermentation rates similar at both 12 and 30C--> Growth efficiency not severely affected by growth temperature
• Table 1: Similar values of each culture at different temperatures indicate little effect on biomass; Also shows that residual glucose and ammonia were higher at 12C than 30C
• More genes in the nitrogen-limiting culture (~800) showed significant difference than the carbon-limited (~500)
• Figure 1: Venn diagram indicates the number of genes up/down regulated for the nitrogen-limited, carbon-limited and both
• Figure 2: Analyzed temperature-responsive genes (1065) for specific functional categories and cis-regulatory motifs
• Kinetics involving uptake of growth-limiting nutrients were altered--> low affinity glucose genes were expressed more in 12C glucose plates than 30C glucose plates, but high affinity glucose genes showed no significant difference
• High affinity ammonia permeases were expressed less in 12C sample and the low affinity was expressed more in 12C sample
• Protein synthesis genes were more expressed at 12C than 30C, especially for N-limiting cultures
• May be result that the specific growth rate of .03/hr is closer to the specific g.r. maximum of 12C and not 30C
• Increased concentration of limiting nutrients resulted in catabolite repression
• Trehalose levels (usually accumulated in response to cold-shock) didn't change between temps in glucose-limited cultures
• Table 2: Trehalose and glycogen levels were significantly lower at 12C (except for glycogen in glucose-limited culture)
• Table 3: Analysis of the promoters showed more stress response elements upstream of genes that were reduced in transcript levels
• More PAC cis-regulatory motifs which is involved in regulation of ribosomal protein-encoding genes
• Result: once cells become adapted to cold temp, the stress response and up-regulation of carb storage recedes
• Figure 3: Comparison of 3 studies showed 259 genes that responed to low temp in all 3 studies; only 91 consistently up-regulated
• Batch cultures are temperature-dependent, but changes in temp. with chemostat cultures does not affect growth rate
• Figure 4: 29 genes were regulated during both adaptation and acclimation; only 11 were consistent
• Figure 5: 25% of down-regulated genes and 10% of up-regulated are likely to be only related to specific growth rate. Overlap is negligible.
• Exposure to oxygen is likely in batch cultures; N gas used in this study to ensure anaerobicity
• Figure 6: Indicates a significant overlap between consistent up/down regulated genes in batch cultures to those genes that responded to multiple stimuli

Discussion

• Environmental factors are closely linked, therefore it is impossible to change only one parameter without altering another
• As a result of keeping growth rate steady in this experiment, glucose concentration was higher in the 12C culture
• Running both ammonium and glucose limiting cultures, error was minimized
• Evidence indicates specific growth rate has effect on genome-wide transcription
• Chemostat cultures didn't see increase in regulation of storing carbohydrates
• Genes in lipid metabolism were the only genes commonly regulated between batch and chemostat cultures; correlates to importance of membrane
• Chaperone-encoding genes conflicted between batch and chemostat
• Low-temp acclimation is not only transcriptional