Alondra Vega: Week 9: Difference between revisions

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===Materials and Methods===
===Materials and Methods===
====Strains Used====
 
====Strains Used====
*The Schade lab used strains BY4743 and BSY25.  These are a result of a cross of two single-mutant strains from the American Type Culture Collection.
*The Schade lab used strains BY4743 and BSY25.  These are a result of a cross of two single-mutant strains from the American Type Culture Collection.
*W303 was also used for growth curve experiments.
*W303 was also used for growth curve experiments.

Revision as of 07:07, 23 March 2011

Vocabulary

  1. Doubling time:the time taken for a cell to complete the cell cycle. The amount of time between successful cell divisions.[1]
  2. Reverse transcription:The process of making a double stranded DNA molecule from a single stranded RNA template through the enzyme, reverse transcriptase.[2]
  3. Heat shock:The response is mediated by heat shock transcription factor (HSF), which is present in a monomeric, non-DNA binding form is unstressed cells and is activated by stress to a tromeric form which can bind to promoters of heat shock genes.[3]
  4. Glycogen:A branched polymer of glucose that is mainly produced in liver and muscle cells, and functions as secondary long-term energy storage in animal cells.[4]
  5. Trehalose:A sweet-tasting, crystalline disaccharide, C12H22O11, found in trehala and in many fungi.[5]
  6. Osmotic Stress: The osmotic stress technique is a method for measuring the effect of water on biological molecules, particularly enzymes. Just as the properties of molecules can depend on the presence of salts, pH, and temperature, they can depend significantly on the amount of water present.[6]
  7. Hyperosmotic:Of, relating to, or characterized by an increased osmotic pressure (typically higher than the physiological level).[7]
  8. Osmolarity: The molarity of an ideal solution of a nondissociating substance that exerts the same osmotic pressure as the solution being considered. [8]
  9. Dendrogram:a branching diagram representing a hierarchy of categories based on degree of similarity or number of shared characteristics especially in biological taxonomy.[9]
  10. Biogenesis:The process in which life forms arise from similar life forms.[10]

Outline: Cold Adaptation in Budding Yeast; Schade et al.

Abstract

  • The paper focused on determining the transcriptional response to a stress, specifically cold shock, in budding yeast.
  • Microarrays were used to check the transcript abundance, which clearly showed two different groups of transcriptionally modulated genes.
    • Early cold response (ECR)
    • Late cold response (LCR)
  • Comparisons were made to other stresses.
  • Trehalose and glycogen are induced in the LCR, which means that the environmental stress response (ESR) happens during the LCR.
  • Msn2p and Msn4p are involved in induction of genes and they control the stress response in the LCR.
  • Cold-specific early response is not mediated by Msn2p and Msn4p, but by a unknown regulatory mechanism.

Introduction

  • Unicellular organisms can be exposed to changes such as changes in nutrients, acidity, osmolarity, temperature and exposure to toxic agents and radiation.
  • Cells have developed a way to adapt to these changes, whether it be by protein phosphorylation and degradation or by transcriptional changes.
  • Previous research in heat shock shows that the mediated transcription factor in this process is HSF1p.
  • Genome-wide transcriptional profiling has shown that approximately 10% of the genes are induced and/or repressed in the response to a specific stress. The genes involved are called the environmental stress response (ESR).
  • Repressed ESR genes can be found in processes such as RNA metabolism, nucleotide biosynthesis, secretion and ribosomal performance.
  • The regulation of the ESR genes is determined by two distinct transcription factors, Msn2p and Msn4p.
  • Cold shock has not been studied as much as the other stresses, which is a motive for this research. Researchers know that cold causes changes in the physical and biochemical properties of the cell.
    • An example of this is a decrease in membrane fluidity, which will result in slower lateral diffusion of membrane proteins.
  • Cold shock in E. coli is described.
  • Previous research shows that in yeast, differential hybridization revealed a set of genes that were up-regulated during a cold shock: NSR1, TIP1, TIR1, TIR2 and NSR1. These genes have been fund to be part of pre-rRNA processing and ribosome biogenesis.
  • TIR1, TIP1, and TIR2 are responsible for maintaing the cell wall integrity during stress.
  • This article is focused on describing the global transcriptional analysis in the stress cold shock in yeast. From their data, they were able to compare their results to other stresses to measure the general stress response as well as the trehalose and glycogen levels.

Materials and Methods

Strains Used

  • The Schade lab used strains BY4743 and BSY25. These are a result of a cross of two single-mutant strains from the American Type Culture Collection.
  • W303 was also used for growth curve experiments.
    • Note: Knowing the how and where the Schade Lab got their strains is important when trying to replicate this experiment.

Growth Medium and Culture Condition

  • Yeast were grown in YPD medium, which contains glucose bactopeptone and yeast extract.
  • Cultures were inoculated and grown overnight at 30°C in 50mL of medium.
  • The cultures were then diluted in fresh medium, grown to have a concentration of 0.6 OD600 at 30°C and the transferred to a 10°C water bath shaker.
  • They cells were incubated for 10, 30 and 120 minutes at 170rpm before harvesting.
  • The temperature then decreased 4°C per minute.
  • Doubling time was determined to be 20.7 hours. Based on this observation, the diluted cultures for the 12hour experiment were only allowed to reach a concentration of 0.4 OD600 before they were changed to the 10°C flask.
  • The 60hour experiments were diluted to 0.05 OD600, it was not until the cells reached a concentration of 0.1OD600 that they were then moved to the 10°C flask.
  • All cultures reached a final OD600 of 0.6-0.8 before harvesting the cells at 10°C. Cell platelets were frozen in liquid nitrogen and stored at -80°C.
    • Note: The control for this experiment was harvested at 30°C.

Isolation of RNA

  • Total RNA was isolated form the cells using the hot phenol method, which can be found in the Kohrer and Domney 1991 paper, with some modifications.
  • Cells from a 500mL culture were processed by extracting phenol twice for 10 minutes apiece.
  • For the 60-hr experiment, RNA isolation was shown to be inefficient, thus to make this process easier and actually retrieve some RNA, glass beads were added.
  • Also, for their mRNA purification the Oligotex Spin-Column Protocol was used.
    • Note: The Oligotex Spin-Column Protocol is a Kit that can be purchased that come with its own protocol and materials to purify the mRNA.

RNA labeling and DNA Microarray Hybridization

  • Three micrograms of mRNA were labeled by incorporating Cy3 and Cy5 through reverse transcription.
  • The cDNA that was retrieved from the previous step was hybridized onto yeast genomic DNA microarrays.
  • Prehybridization was done 20:1:1.
  • Microarray were washed twice in 01XSSC buffer for 2min/wash at 42°C, airstream dried, and immediately hybridized.

Data Acquisition and Analysis

  • Microarrays were scanned using a lte scanner at a 1-μm resolution and the TIFF files were quantified using the QuantArray software.
  • Normalization and quality controls were all done in an Excel sheet.
  • Each DNA spot had to pass three quality controls.
    • The signal intensity had to be much greater than the background intensity.
    • The signal intensity had to be between the dynamic range of the photomultiplier tube.
    • The raw intensities of the duplicate spots had to be within 50% within each other.
  • If the spots met all three criterion, then the ratios of the intensities were normalized by the median ratio of the entire subarray. In this case the subarray consisted of 400 spots.
  • Each subarray was normalized individually, which yielded more reproducible data.
  • The log2 values for each duplicate were averaged.
  • Statistical Analysis and visualization were performed with GeneSpring software.

Experimental Design

  • Samples were taken before harvesting to determine the budding index and the glucose content in the medium.
  • On averaged there were about 70% budded cells and the medium glucose content was 16g/L.
  • The 12-h and 60-h experiment were tested for diauxic shift-inducible genes.
  • There were three time course experiments with the wild type strain that were performed with three time points, 0,2 and 12hours. There were two repeats. for the experiments in the times of 10 min, 30 min, and 6 hours, there were three repeats.
  • For each experiment performed the Cy dye was changed for the reference and experimental samples.
  • The control also had three replicates with the dye swapping.
  • From the control hybridization, data was obtained from 5559 genes and about 14 genes showed an average variation >1.5 fold.
  • Genes with twofold variation were selected.
  • Student T-test was used with a p-value of <0.03 for the experimental analysis.
  • The expression ratios were averaged.
  • A total of 43 microarrays were used.

Comparison with Other S. cerevisiae Stress Data

  • this section of the methods section of the paper describes were they got all their comparison data. The ESR genes and the study of the response to variety of stresses were obtained from different websites. The data for cold shock was obtained from the Sahara et al. 2002 database. All comparisons were done through GeneSpring, which is a program, using standard correlation.

Biochemical and Analytical Procedures

  • To find the trehalose and glycogen levels, they looked at the Parrou and Francois 1997 paper.
  • For this experiment, the cells were grown and harvested as they were in the DNA microarray analysis.
  • Glucose levels were determined by using the Glucose kit.

Results

Alondra Vega

BIOL398-01/S11:Assignments

Lab Journal

Class Journal Week 1 Shared Journal: Week 5 Shared Journal: Week 9
Class Journal Week 2 Shared Journal: Week 6 Alondra Vega: Week 10 Shared Journal: Week 13
Class Journal Week 3 Alondra Vega: Week 7 Shared Journal: Week 11 Shared Journal: Week 14
Shared Journal: Week 4 Shared Journal: Week 8 Shared Journal: Week 12 Alondra Vega:Week 15

Individual Assignments

Alondra Vega: Week 2 Alondra Vega: Week 6 Alondra Vega: Week 11
Alondra Vega: Week 3 Alondra Vega: Week 7 Alondra Vega: Week 12
Alondra Vega: Week 4 Alondra Vega: Week 8 Alondra Vega: Week 13
Alondra Vega: Week 5 Alondra Vega: Week 9 Alondra Vega: Week 14