William A. C. Gendron Week 10: Difference between revisions
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#*Cis-regulatory: This is a sequence near the gene being referenced, which acts as a binding site for transcription factors. It may be in or near the gene. Source:http://www.bio.brandeis.edu/haberlab/jehsite/chIP.html | #*Cis-regulatory: This is a sequence near the gene being referenced, which acts as a binding site for transcription factors. It may be in or near the gene. Source:http://www.bio.brandeis.edu/haberlab/jehsite/chIP.html | ||
#*Chromatin immunoprecipitation: Chromatin immunoprecipitation, or ChIP, refers to a procedure used to determine whether a given protein binds to or is localized to a specific DNA sequence in vivo. The diagram below illustrates the basic steps of this procedure. Source: http://www.nature.com/scitable/definition/cis-regulatory-element-cis-regulatory-element-75 | #*Chromatin immunoprecipitation: Chromatin immunoprecipitation, or ChIP, refers to a procedure used to determine whether a given protein binds to or is localized to a specific DNA sequence in vivo. The diagram below illustrates the basic steps of this procedure. Source: http://www.nature.com/scitable/definition/cis-regulatory-element-cis-regulatory-element-75 | ||
==Outline of "Acclimation of Saccharomyces cerevisiae to Low Temperature: A Chemostat-based Transcriptome Analysis" | |||
*Introduction | |||
**This research used Saccharomyces cerevisiae as the model organism. | |||
**Focused on discovering the true genes associated with cold shock reaction. | |||
**Batch based experiments create a unique situation that is not just cold shock stress. | |||
**Prior studies show discrepancies in ribosomal protein expression. Why is that? | |||
**Discrepancies between carbohydrates and trehalose usage. | |||
**Further explore the Msn2p/Msn4p complex. | |||
**Evaluate adaptation and acclimation studies. | |||
**A chemostat is a unique way to study yeast: it supplies a stable environment that can be constantly growing. Changes growth rate and other features. | |||
*Materials and Methods | |||
**Saccharomyces cerevisiae was was grown at 12 and 30 degrees Celsius. Growth rate for the 12 degrees was .03h^-1 with a volume of 1.01 liters. Set ups of nitrogen and carbon limited chemostats. | |||
**Basic Analysis: | |||
***Supernatants were collected to do liquid chromatography and other tests. This evaluated the level of glucose and metabolites. | |||
***Ammonium concentration was evaluated with a similar chromatography method. | |||
***Glucose creation from glycogen and trehalose was evaluated by a Roche kit. | |||
**Microarray analysis: | |||
***Measured three times and used Microsoft Excel to run the statistics. | |||
***Used two-fold difference as a threshold and calculated a median false rate at 1%. | |||
***Evaluated overrepresentation with DAVID(Database for Annotation, Visualization and Integrated Discovery) along with a Fisher's exact test, a Bonferroni correction and a p-test. | |||
***Compared data with data from Beltran et al., Murata et al., Sahara et al., Schade et al., Gasch et al. and the Stanford Yeast Stress Database. | |||
*Results | |||
**Low-Temperature Chemostat Cultivation Results in Altered Uptake Kinetics for the Limiting Nutrient and Enhanced Catabolite Repression | |||
***More residual nutrients in 12 degree than 30. Reduced transport and metabolism is the root of this. | |||
***Hexose transport genes increased transcription, in theory to account for the decreased rate of transport in the 12 degree. | |||
***HXT5 and 16 increased in 30 degree. | |||
***In nitrogen limited cultures, permeases that targeted ammonia changed. MEP3 increased for 12 degree while high affinity MEP1 and 2 were reduced. | |||
**Acclimation to Nonfreezing Low Temperature Does Not Require a High Storage Carbohydrate Content | |||
***Transcription was not changed in genes for trehalose and glycogen metabolism. | |||
***Carbohydrate synthesis decreased at lower temperatures. | |||
**Up-Regulation of the Translation Machinery at Low Temperature | |||
***Higher amounts of transcription seen in 16 of the ribosome biogenesis genes at 12 degrees(both nitrogen and carbon limited). | |||
***12 degrees nitrogen and carbon limited showed evidence of much higher rRNA and protein than 30. | |||
**Transcriptional Responses to Low Temperature: Adaptation versus Acclimation | |||
***Compared to previous studies | |||
***Found that our of 259 genes, 139 matched up or down regulation across cold shock genes. | |||
***Specific genes were identified to be highly consistent. | |||
**Context Dependency of Temperature Response | |||
***They describe why this has better controls than other experiments. | |||
***Better O2 control than flask batches | |||
***Found genes that did not decrease with temperature but use to be linked to other experiments that suggested that they were. | |||
**Environmental Stress Response, a Low-Temperature Adaptation-specific Response | |||
***Compared to database of genes that react in environmentally stressful situations | |||
***About 1/3 of the genes from this study were found to be linked to ESR. | |||
*Discussion | |||
**Chemostat-based Low-Temperature Transcriptomics: Experimental Design | |||
***Explains that there are too many variables to control for and multiple experiments are required to test the limits. | |||
***Requires multiple tests to get a full picture of the genes. | |||
**Specific Growth Rate and ESR | |||
***Growth rates between temperature groups were quite different. | |||
***ESR genes were found to make up a portion of the genes activated. | |||
**Transcriptional Responses to Low Temperature: Acclimation versus Adaptation | |||
***Confusing terms but it focused on the timing of different genes. I would have preferred another word instead of adaptation, because I think of it as an evolutionary term. | |||
***It wrapped up saying that that this alternative system can be used to parse out genes that are caused by the batch system. | |||
[Media:Journal Club 2WilliamGendronLaurenMageeKarinaAlvarezAlyssa.pptx] |
Revision as of 00:20, 24 March 2015
Paper Analysis- Acclimation of Saccharomyces cerevisiae to Low Temperature: A Chemostat-based Transcriptome Analysis
- Definitions
- Trehalose(also called mycose): This is "a non reducing disaccharide" which in this context it is being used as a cryoprotectant as it is brought to near freezing conditions. It can also act as a preservative and an anti-oxidant. TPS1 and TPS2 are responsible for its biosynthesis in yeast. Source: http://www.oxfordreference.com/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-19924?rskey=ZqLfPq&result=1
- Mannoproteins: These are proteins "that contain 15 to 90% mannose by weight" Sources: http://link.springer.com/chapter/10.1007%2F978-3-642-68234-6_18
- Desaturase: An enzyme that catalyzes desaturation. Desaturation is when "two hydrogen atoms or a hydrogen and a hydroxyl group are removed from adjacent atoms." Source: http://www.oxfordreference.com/view/10.1093/oi/authority.20110803095712459 http://www.oxfordreference.com/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-4993?rskey=k9Vxrx&result=1
- Permease: A membrane transport protein in or out of the cell. There really should not be two names for this. Source: http://www.oxfordreference.com/view/10.1093/oi/authority.20110803100318301
- Zero trans-influx transport assay: I could not find an exact definition to how they do this but it measures the rates of glucose uptake in this context. None of the sources I found really defined how it was done even when I did a wider google search. Source: http://link.springer.com/article/10.1007/s00253-002-1085-6
- Homeoviscous: A cell will alter the number of long or saturated fatty acids in its cell membrane to adjust for the temperature. Source: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC388039/
- DEAD Box Family: This refers to 4 amino acids that form in this order: -D-E-A-D- (Asp-Glu-Ala-Asp) and this acts as an ATP binding site. Source: http://www.oxfordreference.com/view/10.1093/oi/authority.20110803095704770?rskey=nZKYQA&result=2
- First-Order Kinetics: This is a chemical reaction that is only dependent on one of the reactants. Source: http://www.biology-online.org/dictionary/First-order_kinetics
- Cis-regulatory: This is a sequence near the gene being referenced, which acts as a binding site for transcription factors. It may be in or near the gene. Source:http://www.bio.brandeis.edu/haberlab/jehsite/chIP.html
- Chromatin immunoprecipitation: Chromatin immunoprecipitation, or ChIP, refers to a procedure used to determine whether a given protein binds to or is localized to a specific DNA sequence in vivo. The diagram below illustrates the basic steps of this procedure. Source: http://www.nature.com/scitable/definition/cis-regulatory-element-cis-regulatory-element-75
==Outline of "Acclimation of Saccharomyces cerevisiae to Low Temperature: A Chemostat-based Transcriptome Analysis"
- Introduction
- This research used Saccharomyces cerevisiae as the model organism.
- Focused on discovering the true genes associated with cold shock reaction.
- Batch based experiments create a unique situation that is not just cold shock stress.
- Prior studies show discrepancies in ribosomal protein expression. Why is that?
- Discrepancies between carbohydrates and trehalose usage.
- Further explore the Msn2p/Msn4p complex.
- Evaluate adaptation and acclimation studies.
- A chemostat is a unique way to study yeast: it supplies a stable environment that can be constantly growing. Changes growth rate and other features.
- Materials and Methods
- Saccharomyces cerevisiae was was grown at 12 and 30 degrees Celsius. Growth rate for the 12 degrees was .03h^-1 with a volume of 1.01 liters. Set ups of nitrogen and carbon limited chemostats.
- Basic Analysis:
- Supernatants were collected to do liquid chromatography and other tests. This evaluated the level of glucose and metabolites.
- Ammonium concentration was evaluated with a similar chromatography method.
- Glucose creation from glycogen and trehalose was evaluated by a Roche kit.
- Microarray analysis:
- Measured three times and used Microsoft Excel to run the statistics.
- Used two-fold difference as a threshold and calculated a median false rate at 1%.
- Evaluated overrepresentation with DAVID(Database for Annotation, Visualization and Integrated Discovery) along with a Fisher's exact test, a Bonferroni correction and a p-test.
- Compared data with data from Beltran et al., Murata et al., Sahara et al., Schade et al., Gasch et al. and the Stanford Yeast Stress Database.
- Results
- Low-Temperature Chemostat Cultivation Results in Altered Uptake Kinetics for the Limiting Nutrient and Enhanced Catabolite Repression
- More residual nutrients in 12 degree than 30. Reduced transport and metabolism is the root of this.
- Hexose transport genes increased transcription, in theory to account for the decreased rate of transport in the 12 degree.
- HXT5 and 16 increased in 30 degree.
- In nitrogen limited cultures, permeases that targeted ammonia changed. MEP3 increased for 12 degree while high affinity MEP1 and 2 were reduced.
- Acclimation to Nonfreezing Low Temperature Does Not Require a High Storage Carbohydrate Content
- Transcription was not changed in genes for trehalose and glycogen metabolism.
- Carbohydrate synthesis decreased at lower temperatures.
- Up-Regulation of the Translation Machinery at Low Temperature
- Higher amounts of transcription seen in 16 of the ribosome biogenesis genes at 12 degrees(both nitrogen and carbon limited).
- 12 degrees nitrogen and carbon limited showed evidence of much higher rRNA and protein than 30.
- Transcriptional Responses to Low Temperature: Adaptation versus Acclimation
- Compared to previous studies
- Found that our of 259 genes, 139 matched up or down regulation across cold shock genes.
- Specific genes were identified to be highly consistent.
- Context Dependency of Temperature Response
- They describe why this has better controls than other experiments.
- Better O2 control than flask batches
- Found genes that did not decrease with temperature but use to be linked to other experiments that suggested that they were.
- Environmental Stress Response, a Low-Temperature Adaptation-specific Response
- Compared to database of genes that react in environmentally stressful situations
- About 1/3 of the genes from this study were found to be linked to ESR.
- Low-Temperature Chemostat Cultivation Results in Altered Uptake Kinetics for the Limiting Nutrient and Enhanced Catabolite Repression
- Discussion
- Chemostat-based Low-Temperature Transcriptomics: Experimental Design
- Explains that there are too many variables to control for and multiple experiments are required to test the limits.
- Requires multiple tests to get a full picture of the genes.
- Specific Growth Rate and ESR
- Growth rates between temperature groups were quite different.
- ESR genes were found to make up a portion of the genes activated.
- Transcriptional Responses to Low Temperature: Acclimation versus Adaptation
- Confusing terms but it focused on the timing of different genes. I would have preferred another word instead of adaptation, because I think of it as an evolutionary term.
- It wrapped up saying that that this alternative system can be used to parse out genes that are caused by the batch system.
- Chemostat-based Low-Temperature Transcriptomics: Experimental Design
[Media:Journal Club 2WilliamGendronLaurenMageeKarinaAlvarezAlyssa.pptx]