Individual Journal Week 11: Difference between revisions
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*Lower temperatures, typically result in the slower cellular processes: growth phase, respiration, lipid composition of membranes, and trehalose content. | *Lower temperatures, typically result in the slower cellular processes: growth phase, respiration, lipid composition of membranes, and trehalose content. | ||
*Time scale of exposure to cold-shock relevant: | *Time scale of exposure to cold-shock relevant: | ||
**Sudden exposure => adaptation | |||
**Prolonged exposure=> acclimation | |||
**Long term exposure => evolutionary adaptation | |||
*Two distinct phases during cold-shock response | *Two distinct phases during cold-shock response | ||
#First 12 hours, early cold shock (ECS) | #First 12 hours, early cold shock (ECS) | ||
Line 28: | Line 28: | ||
*Growth rate an important factor on transcription | *Growth rate an important factor on transcription | ||
*Research seeks to control specific growth rate and other culture conditions, through use of chemostat, in order to establish steady-state and, thus, investigate transcription regulation influenced by suboptimal temperatures | *Research seeks to control specific growth rate and other culture conditions, through use of chemostat, in order to establish steady-state and, thus, investigate transcription regulation influenced by suboptimal temperatures | ||
===Materials & Methods=== | |||
*Yeast was grown in one chemostat with temperature 12C as well as a second chemostat set at 30C, both set at a dilution rate of 0.03h-1 with a working volume of 1.0 L | |||
*Carbon or nitrogen limitations, other media/ nutrients in excess | |||
*pH constant at 5.0 by addition of 2 M KOH | |||
*Anaerobic growth | |||
* Stirrer set at constant 600 rpm | |||
*Grown in conditions in which ammonia was limiting at 12C, ammonia was limiting at 30C, glucose was limiting at 12C, and glucose was limiting at 30C | |||
*Cultures analyzed through microarray and statistical analysis | |||
*Microarray analysis: | |||
**Determined RNA quality | |||
**growth condition derived from three independently cultured replicates | |||
**Northern analysis used | |||
*Statistical Analysis | |||
**Excel used to run significance of microarray add-in, through "pair-wise comparisons" | |||
**Data displayed in Venn diagrams and heat-maps | |||
**Promoters analyzed through use of web-based software | |||
**p-value calculated | |||
**Outside transcriptome datasets used for comparison | |||
===Results=== | |||
*Table 1 | |||
**At both 12C and 30C biomass yields and fermentation rates similar in both carbon and nitrogen limited chemostats | |||
**Therefore, growth efficiency not significantly affected by growth temperature | |||
*Figure 1 | |||
**Ven diagram displaying: Glucose limited cultures, 494 genes yielded significantly different transcript levels at both 12C and 30C | |||
** 806 genes yield significant transcription in nitrogen-limited culture | |||
**16% of S. cervisiae genome temperature-responsive genes | |||
**Under both nutrient limitations, 235 genes demonstrated up or down regulation | |||
*Figure 2 | |||
**Heat map of 1065 different genes in anaerobic glucose and ammonia limited chemostat cultures at 12C and 30C | |||
**Change in transcript levels of genes involved in uptake of growth-limiting nutrients was reflected by changes in transport kinetics | |||
**Higher limiting nutrients at 12C resulted in higher catabolite repression | |||
*Table 2 | |||
**Displays protein and storage carbohydrates grown in ammonium and glucose limited anaerobic chemostat | |||
**In ammonium-limited chemostat, trehalose and glycogen significantly lower at lower temperature | |||
*Table 3(A) | |||
**Msn2/Msn4 via STREs in their promoters regulate key genes relevant to storage carbohydrate synthesis | |||
**Overrpresentation of STRE elements in upstream regions of genes that showed a reduced transcript level at 12C in nitrogen limited chemostats | |||
**Consistent with stress induced glycogen and tehalose genes at low temps | |||
*Table 3 (B) | |||
**Enrichment of PAC cis-regulatory motifs 5' upstream of sequences, increased transcript level at 12C | |||
**Transcription Factors: Mbp1p, Hap2-Hap1, Hap3-Hap1, Fhl1p, Sfp1p, Gln3p, Gln3-Dal82, Hap2-dal82, Aft2p, Hsflp, Nrg1p, Phd1p, Rsc1p, Sok2p, Nrg1-Aft2, Phd1-Nrg1, Rox1-Phd1, Sok2-Nrg1 | |||
*Figure 3 | |||
**Displays genes that are common to the three batch-culture studies on transcriptional adaptations in relation to low temperatures | |||
**Heat map displays transcript ratio of 259 genes found in common in the three batch-culture low temp transcriptome data sets. | |||
*Figure 4 | |||
**235 genes that displayed transcription changes resulting from acclimation were compared to 259 low-temperature adaptation genes. | |||
**In total there were 11 genes that displayed a pattern of regulation in all datasets. | |||
*Figure 5 | |||
**Through the use of Venn diagrams looking at adaptation and acclimation, up or down regulated genes are compared. | |||
**Overlap was considered "negligible" between growth-rate-responsive genes in comparison to temperature-responsive genes. | |||
*Figure 6 | |||
**Compared to environmental stress, 233 genes show an opposite transcriptional response to low temperature | |||
**ESR is, thus, a response that occurs during sudden exposure to suboptimal temperatures |
Latest revision as of 23:19, 3 April 2013
Terms
- 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. [1]
- Catabolite: product of catabolism, the breakdown of complex molecules into simpler ones. [2]
- Gene Regulatory Protein: Any protein that interacts with dna sequences of a gene and controls its transcription. [3]
- Up-regulation:process that increases ligand/receptor interactions due to an increase in the number of available receptors. [4]
- Down-regluation: the process that decreases ligand and receptor interactions or reduces the responsiveness of a cell to a stimulus following first exposure.This is often accompanied by an initial decrease in affinity of receptors for the agent and a subsequent reduction in the number of available receptors expressed on the surface which can result from internalisation of the ligand:receptor complex or from decreased expression of the receptor. [5]
- Motif: The smallest group of atoms in a polymer that, when under the influence of a rotation-translation operator, will assemble the rest of the atoms in the chain. [6]
- Desaturase: Any of several enzymes that putdouble bonds into the hydrocarbon areasof fatty acids. [7]
- Biogenesis: The process in which life forms arise from similar life forms. [8]
- Trehalose: a crystalline disaccharide C12H22O11 that is found in various organisms (as fungi and insects), is about half as sweet as sucrose, and is sometimes used as a sweetener in commercially prepared foods [9]
- Transcriptome: the complement of mature messenger RNAs produced in a given cell in a given moment of its life. [10]
Outline
Introduction
- The study observes the effects of suboptimal temperatures on transcriptional regulation in yeast.
- Transcriptional responses were observed at temperatures 12C and 30C.
- Lower temperatures, typically result in the slower cellular processes: growth phase, respiration, lipid composition of membranes, and trehalose content.
- Time scale of exposure to cold-shock relevant:
- Sudden exposure => adaptation
- Prolonged exposure=> acclimation
- Long term exposure => evolutionary adaptation
- Two distinct phases during cold-shock response
- First 12 hours, early cold shock (ECS)
- After first 12 hours, Late cold response (LCR)
- TPS1 and TPS2, commonly observed trehalose-biosynthetic transcriptional induction genes- observed in cold and heat shock conditions
- Genes involved in cold-shock response typically involved in other stress response
- Growth rate an important factor on transcription
- Research seeks to control specific growth rate and other culture conditions, through use of chemostat, in order to establish steady-state and, thus, investigate transcription regulation influenced by suboptimal temperatures
Materials & Methods
- Yeast was grown in one chemostat with temperature 12C as well as a second chemostat set at 30C, both set at a dilution rate of 0.03h-1 with a working volume of 1.0 L
- Carbon or nitrogen limitations, other media/ nutrients in excess
- pH constant at 5.0 by addition of 2 M KOH
- Anaerobic growth
- Stirrer set at constant 600 rpm
- Grown in conditions in which ammonia was limiting at 12C, ammonia was limiting at 30C, glucose was limiting at 12C, and glucose was limiting at 30C
- Cultures analyzed through microarray and statistical analysis
- Microarray analysis:
- Determined RNA quality
- growth condition derived from three independently cultured replicates
- Northern analysis used
- Statistical Analysis
- Excel used to run significance of microarray add-in, through "pair-wise comparisons"
- Data displayed in Venn diagrams and heat-maps
- Promoters analyzed through use of web-based software
- p-value calculated
- Outside transcriptome datasets used for comparison
Results
- Table 1
- At both 12C and 30C biomass yields and fermentation rates similar in both carbon and nitrogen limited chemostats
- Therefore, growth efficiency not significantly affected by growth temperature
- Figure 1
- Ven diagram displaying: Glucose limited cultures, 494 genes yielded significantly different transcript levels at both 12C and 30C
- 806 genes yield significant transcription in nitrogen-limited culture
- 16% of S. cervisiae genome temperature-responsive genes
- Under both nutrient limitations, 235 genes demonstrated up or down regulation
- Figure 2
- Heat map of 1065 different genes in anaerobic glucose and ammonia limited chemostat cultures at 12C and 30C
- Change in transcript levels of genes involved in uptake of growth-limiting nutrients was reflected by changes in transport kinetics
- Higher limiting nutrients at 12C resulted in higher catabolite repression
- Table 2
- Displays protein and storage carbohydrates grown in ammonium and glucose limited anaerobic chemostat
- In ammonium-limited chemostat, trehalose and glycogen significantly lower at lower temperature
- Table 3(A)
- Msn2/Msn4 via STREs in their promoters regulate key genes relevant to storage carbohydrate synthesis
- Overrpresentation of STRE elements in upstream regions of genes that showed a reduced transcript level at 12C in nitrogen limited chemostats
- Consistent with stress induced glycogen and tehalose genes at low temps
- Table 3 (B)
- Enrichment of PAC cis-regulatory motifs 5' upstream of sequences, increased transcript level at 12C
- Transcription Factors: Mbp1p, Hap2-Hap1, Hap3-Hap1, Fhl1p, Sfp1p, Gln3p, Gln3-Dal82, Hap2-dal82, Aft2p, Hsflp, Nrg1p, Phd1p, Rsc1p, Sok2p, Nrg1-Aft2, Phd1-Nrg1, Rox1-Phd1, Sok2-Nrg1
- Figure 3
- Displays genes that are common to the three batch-culture studies on transcriptional adaptations in relation to low temperatures
- Heat map displays transcript ratio of 259 genes found in common in the three batch-culture low temp transcriptome data sets.
- Figure 4
- 235 genes that displayed transcription changes resulting from acclimation were compared to 259 low-temperature adaptation genes.
- In total there were 11 genes that displayed a pattern of regulation in all datasets.
- Figure 5
- Through the use of Venn diagrams looking at adaptation and acclimation, up or down regulated genes are compared.
- Overlap was considered "negligible" between growth-rate-responsive genes in comparison to temperature-responsive genes.
- Figure 6
- Compared to environmental stress, 233 genes show an opposite transcriptional response to low temperature
- ESR is, thus, a response that occurs during sudden exposure to suboptimal temperatures