Sarah Carratt: Week 9: Difference between revisions
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*glucose levels determined using Glucose kit | *glucose levels determined using Glucose kit | ||
==== | ====Results==== | ||
* Briefly state the result shown in each of the figures and tables. | |||
** Figure 1: Overall look at the transcriptional response to cold for wild-type experiment. A: cluster analysis of microarray data for 634 genes that were determined to be significant. Green: down-regulated, red: induced. Early Cold Response genes shown in sections labeled D and E. A, B, and C show Late Cold Response genes. B and C classify the classification and amount of such classified genes for ECR in B and LCR in C. | |||
** Figure 2: Comparison of data with that collected by Gasch et. al (2000). A correlation between transcriptional response of ECR from 30 to 10 degrees is found in the shift from 37 deg to 25 deg C as in the Gasch experiment. Not all genes were analogous however, only the ones labeled a and b on the diagram were found to be in correlation. | |||
** Figure 3: Early Cold Response compared with Late Cold Response, and the genes used in both are compared in Venn Diagrams with the Environmental Stress Response. It is found that the ECR only shares 2 induced genes and 2 repressed genes with the ESR whereas the LCR and the ESR share 87 induced genes and 111 repressed genes. Responses to various drugs are also investigated. | |||
** Figure 4: Comparison of wild type gene expression for 2 hour and 12 hour time points vs the delta msn2 delta msn4 strains. This data suggests that the ECR expression profile is cold-specific whereas the LCR genes are similar to that of ESR. | |||
** Figure 5: Investigation of amounts of glycogen (top) and trehalose (bottom) in regards to the wild type and the mutant strain. The mutant strain did not produce a significant amount of trehalose at any time point and produced less glycogen than the wild type at all time points. The mutant strain was less able to accumulate reserve carbohydrates during cold treatment. | |||
** Figure 6: Comparison of transcriptional response in wild type yeast with the Sahara (2002) study. A common cluster of genes during the LCR was observed, this list includes several general stress-response genes. The data for the ECR varied drastically, however between the two data sets. The 2002 Sahara paper indicated that the ribosomal genes would be induced whereas the Schade paper noticed a decrease in transcript abundance. This could be explained by difference in yest strain or in the phase that the yeast was grown in. | |||
* Closing Remarks | |||
**Cold response happens in 2 phases: early and late cold response. In this experiment, the late cold response began sometime after 2 hours. | |||
** Early cold response is fairly unique to cold | |||
** Late cold response is similar to environmental stress response | |||
** More data must be collected in order to fully understand this phenomenon. Part of the response seems to also depend on what growth phase the yeast is in. | |||
{{Template:SarahCarratt}} | {{Template:SarahCarratt}} | ||
Revision as of 23:42, 23 March 2011
Instructions
Formatting
- Store this journal entry as "username Week 9" (i.e., this is the text to place between the square brackets when you link to this page).
- Create the following set of links. (HINT: you can do all of this easily by adding them to your template and then using the template on your pages.)
- Link to your journal entry from your user page.
- Link back from your journal entry to your user page.
- Link to this assignment from your journal entry.
- Don't forget to add the "BIOL398-01/S11" category to the end of your wiki page.
Things to Read
Article
- Schade et al. (2004) Cold Adaptation in Budding Yeast. Molecular Biology of the Cell 15: 5492–5502.
- Link to PDF version of article: use this one for printing/reading.
- Link to full text in HTML: use this one to get the figures for your PowerPoint.
Background
- Thieringer, H.A., Jones, P.G., and Inouye, M. (1998) Cold shock and adaptation. BioEssays 20: 49–57.
- Aguilera, J., Randez-Gil, F., and Prieto, J.A. (2007) Cold Response in Saccharomyces cerevisiae: New Functions for Old Mechanisms. FEMS Microbiological Reviews 31: 327–341.
- Al-Fageeh, M.B. and Smales, C.M. (2006) Control and Regulation of the Cellular Responses to Cold Shock: the Responses in Yeast and Mammalian Systems. Biochemical Journal 397: 247–259.
Preparation for Journal Club 2
- Make a list of at least 10 biological terms for which you did not know the definitions when you first read the article. Define each of the terms. List the citation(s) for the dictionary(s) you use, providing a URL to the page is fine.
- Write an outline of the article and include the following questions:
- What is the main result presented in this paper?
- What is the importance or significance of this work?
- How did they treat the cells (what experiment were they doing?)
- What strain(s) of yeast did they use? Was the strain haploid or diploid?
- What media did they grow them in? What temperature? What type of incubator? For how long?
- What controls did they use?
- How many replicates did they perform per timepoint?
- What mathematical/statistical method did they use to analyze the data?
- What transcription factors did they talk about?
- Briefly state the result shown in each of the figures and tables.
- Each group of students will be assigned one section of the paper. The group will be responsible for preparing that portion of the PowerPoint presentation and explaining the assigned section and figures in detail to the class.
- Introduction, Figures 1, 2: Sarah, Carmen
- Methods, Figures 3, 4: Alondra, Nick
- Discussion, Figures 5, 6: James
Online Sources
Student Response
Biological Terms
- transient changes: "short-lived; passing; not permanent; said of a disease or an attack" [1]
- MIPS: "MIPS Mammalian Protein-Protein Interaction Database is a collection of manually curated high-quality PPI data collected from the scientific literature by expert curators" [2]
- isoform: "any of the proteins with the same function and similar amino acid sequence, encoded by different genes (or by RNA transcript)" [3]
- aggregation: "massing of materials together as in clumping" [4]
- menadione: "a fat soluble vitamin that plays an important role in blood clotting" [5]
- expression profile: "the measurement of the activity (the expression) of thousands of genes at once, to create a global picture of cellular function" [6]
- Msn2p/Msn4p: "transciption factor that could control stress-induced expression" [7]
- hyperosmotic shock: "shock caused or characterized by an increased osmotic pressure (typically higher than the physiological level)" [8]
- trehalose: "also known as mycose or tremalose, is a natural alpha-linked disaccharide formed by an α,α-1,1-glucoside bond between two α-glucose units" [9]
- glutathione/glutaredoxin system: "part of a complex regulatory network controlling defense against oxidative stress" [10]
Outline
Abstract
- determined transcriptional response of the budding yeast S. cerevisiae to cold
- use temperatures of 10 degrees Celsius
- different lengths of times (ECR and LCR recorded)
- microarrays used to identify induced and repressed genes
- important conclusions of the paper
- environmental stress response (ESR) genes and late cold response (LCR) genes overlap
- ESR occurs in LCR
- no evidence of ESR in ECR
- Msn2p and Msn4p observed
- they control stress response pattern in LCR
- ECR is independent of these so there must be different regulator
- environmental stress response (ESR) genes and late cold response (LCR) genes overlap
Introduction
- Unicellular organisms feel the effects of extreme conditions
- nutrients, acidity, osmolarity, temperature, toxins, and radiation
- No random responses, programed into the cell
- protein generation and breakdown
- transcriptional changes lead to altered cell states
- Not much is known about mechanisms for survival in cold
- Cold causes changes in physical and biochemical properties
- Adaptability is determined by set of regulatory mechanisms
- Description of transcriptional analysis of cold response
- Comparison of cold-responses to other stress stimuli and measure key markers
Materials and Methods
Strains
- BY4743
- diploid
- wild type
- BSY25
- diploid
- much like first gene
- derived from a cross of the two single-mutant strains
- W303
- haploid
- growth curve experiments
Growth Medium and Culture Conditions
- new cultures with each experiment
- grown in 30 degrees Celsius
- 50 ml of YDP medium in 250 flask
- shaken at 170 rpm
- transfered to 10 degrees Celsius
- incubated and shaken at 170 rpm
- 10, 30, 120 minutes
- decrease of 4 degrees Celsius per minute
- doubling rate determined for 10 degrees Celsius
- 12 hour grown to .4 OD600then to cold
- 60 hour diluted to .05 OD600in new medium then grown to .4 OD600 and diluted again to .05 OD600
Isolation of RNA
- modified hot-phenol method
RNA Labeling and DNA Microarray Hybridization
- labeled by directly mixing in Cy3 and Cy5 through reverse transcription
- protocols available online for microarrays[11]
Data Acquisition and Analysis
- ScanArray lite scanner
- QuantArray software
- three quality controls in Microsoft Excel
- ratios normalized by median ratio of 400 spots
- log2 values of the ratio averaged
- GeneSpring software for statistical analysis and visualization
- hierarchical clustering based on matrix of standard correlation
Experimental Design
- ensure each culture was in same physiological state by taking samples
- time-course experiments with wild-type strain were performed with time points of:
- 0,2,12 hours (two independent biological repeats)
- 10,30 minutes & 60 hours (three independent biological repeats)
- 43 microarrays used
Comparison with Other S. cerevisiae Stress Data
- Gasch and Sahara comparison using GeneSpring
Biochemical and Analytical Procedures
- determination of glycogen and trehalose
- glucose levels determined using Glucose kit
Results
- Briefly state the result shown in each of the figures and tables.
- Figure 1: Overall look at the transcriptional response to cold for wild-type experiment. A: cluster analysis of microarray data for 634 genes that were determined to be significant. Green: down-regulated, red: induced. Early Cold Response genes shown in sections labeled D and E. A, B, and C show Late Cold Response genes. B and C classify the classification and amount of such classified genes for ECR in B and LCR in C.
- Figure 2: Comparison of data with that collected by Gasch et. al (2000). A correlation between transcriptional response of ECR from 30 to 10 degrees is found in the shift from 37 deg to 25 deg C as in the Gasch experiment. Not all genes were analogous however, only the ones labeled a and b on the diagram were found to be in correlation.
- Figure 3: Early Cold Response compared with Late Cold Response, and the genes used in both are compared in Venn Diagrams with the Environmental Stress Response. It is found that the ECR only shares 2 induced genes and 2 repressed genes with the ESR whereas the LCR and the ESR share 87 induced genes and 111 repressed genes. Responses to various drugs are also investigated.
- Figure 4: Comparison of wild type gene expression for 2 hour and 12 hour time points vs the delta msn2 delta msn4 strains. This data suggests that the ECR expression profile is cold-specific whereas the LCR genes are similar to that of ESR.
- Figure 5: Investigation of amounts of glycogen (top) and trehalose (bottom) in regards to the wild type and the mutant strain. The mutant strain did not produce a significant amount of trehalose at any time point and produced less glycogen than the wild type at all time points. The mutant strain was less able to accumulate reserve carbohydrates during cold treatment.
- Figure 6: Comparison of transcriptional response in wild type yeast with the Sahara (2002) study. A common cluster of genes during the LCR was observed, this list includes several general stress-response genes. The data for the ECR varied drastically, however between the two data sets. The 2002 Sahara paper indicated that the ribosomal genes would be induced whereas the Schade paper noticed a decrease in transcript abundance. This could be explained by difference in yest strain or in the phase that the yeast was grown in.
- Closing Remarks
- Cold response happens in 2 phases: early and late cold response. In this experiment, the late cold response began sometime after 2 hours.
- Early cold response is fairly unique to cold
- Late cold response is similar to environmental stress response
- More data must be collected in order to fully understand this phenomenon. Part of the response seems to also depend on what growth phase the yeast is in.
Individual Assignments
Class Assignments
Class Notes
Internal Links
| BIOL398-01/S11:Assignments | BIOL398-01/S11:People | BIOL398-01/S11:Sarah Carratt |
