Sarah Carratt: Week 9

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

1. 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.
2. Write an outline of the article and include the following questions:
   1. What is the main result presented in this paper?
   2. What is the importance or significance of this work?
   3. How did they treat the cells (what experiment were they doing?)
   4. What strain(s) of yeast did they use? Was the strain haploid or diploid?
   5. What media did they grow them in? What temperature? What type of incubator? For how long?
   6. What controls did they use?
   7. How many replicates did they perform per timepoint?
   8. What mathematical/statistical method did they use to analyze the data?
   9. What transcription factors did they talk about?
   10. Briefly state the result shown in each of the figures and tables.
3. 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.
   1. Introduction, Figures 1, 2: Sarah, Carmen
   2. Methods, Figures 3, 4: Alondra, Nick
   3. Discussion, Figures 5, 6: James

Online Sources

• Biology Dictionary
• Gene Ontology
• NCI Dictionary of Cancer Terms

Student Response

Biological Terms

1. transient changes: "short-lived; passing; not permanent; said of a disease or an attack" [1]
2. 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]
3. isoform: "any of the proteins with the same function and similar amino acid sequence, encoded by different genes (or by RNA transcript)" [3]
4. aggregation: "massing of materials together as in clumping" [4]
5. menadione: "a fat soluble vitamin that plays an important role in blood clotting" [5]
6. expression profile: "the measurement of the activity (the expression) of thousands of genes at once, to create a global picture of cellular function" [6]
7. Msn2p/Msn4p: "transciption factor that could control stress-induced expression" [7]
8. hyperosmotic shock: "shock caused or characterized by an increased osmotic pressure (typically higher than the physiological level)" [8]
9. 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]
10. 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

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

Transcription Factors

• Msn2p/Msn4p
  • control stress-induced expression
• Spt23p, Mga2p
  • regulate lipid metabolism and membrane fluidity
• HAP5 and TYE7
  • carbohydrate metabolism
  • induced in LCR

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

Figure 1

• Color Coding System
  • down-regulated genes = green
  • up-regulated genes = red
• Clustering
  • 3 induced, 2 repressed
  • A,B,C,D,E,F (D/E = ECR and A-C = LCR)
• ECR involved in transcription
• general increase in "non-yellow" expression in LCR
• functional categories according to MIPS

Figure 2

• Compares Gasch study (37 to 25) and the 2 hour results of Schade (30 to 10)
• Testament to need for constancy, results shown strong differences in expression

Figure 3

• ECR expression contrast with other stressors
• Clusters show repressed and induced during heat shock
• Venn diagrams show similarities between LCR and ESR, repressed genes and ESR, ESR and ECR
• LCR involves the ESR
• ECR gives a "cold-specific transcriptional response

Figure 4

• 120 genes affected by cold
• Msn2p and Msn4p activate 99 LCR genes (ESR)
• Most LCR genes were not affected when Msn2p and Msn4 are gone
• Msn2p and Msn4p are independent of ECR genes

Figure 5

• No glycogen or trehalose in first two hours
• Increased glycogen and trehalose after twelve hours
• Mutant shows little change in LCR for glycogen and trehalose

Figure 6

• Sahara data presented for comparison
• Primary differences in A/B
• Problem of consistent methods

Navigation Guide

Individual Assignments

Sarah Carratt: Week 2	Sarah Carratt: Week 6	Sarah Carratt: Week 11
Sarah Carratt: Week 3	Sarah Carratt: Week 7	Sarah Carratt: Week 12
Sarah Carratt: Week 4	Sarah Carratt: Week 8	Sarah Carratt: Week 13
Sarah Carratt: Week 5	Sarah Carratt: Week 9	Sarah Carratt: Week 14

Class Assignments

Shared Journal: Week 1	Shared Journal: Week 6	Shared Journal: Week 11
Shared Journal: Week 2	Shared Journal: Week 7	Shared Journal: Week 12
Shared Journal: Week 3	Shared Journal: Week 8	Shared Journal: Week 13
Shared Journal: Week 4	Shared Journal: Week 9	Shared Journal: Week 14
Shared Journal: Week 5	Shared Journal: Week 10

Class Notes

Sarah Carratt_1.18.11	Sarah Carratt_2.3.11	Sarah Carratt_2.22.11
Sarah Carratt_1.20.11	Sarah Carratt_2.8.11	Sarah Carratt_2.24.11
Sarah Carratt_1.25.11	Sarah Carratt_2.10.11	Sarah Carratt_3.1.11
Sarah Carratt_1.27.11	Sarah Carratt_2.15.11	Sarah Carratt_3.3.11
Sarah Carratt_2.1.11	Sarah Carratt_2.17.11	Sarah Carratt_3.8.11

Internal Links

BIOL398-01/S11:Assignments

BIOL398-01/S11:People

BIOL398-01/S11:Sarah Carratt