Kwrigh35 Week 12

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Electronic Notebook

Link to Presentation PDF

Genome-wide expression analysis of yeast response during exposure to 4 degrees C

New Terms

Make a list of at least 10 terms for which you did not know the definitions when you first read the article. Define each of the terms and provide a citation for each term.

  1. Trehalose
    • A non-reducing dissacharide found in many organisms in each kingdom. It is often used in stress response and as a transport suger, among other uses.
    • Lunn, J. E. (Apr 2016). Sucrose Metabolism [Abstract]. eLS. John Wiley & Sons Ltd, Chichester. Retrieved from http://www.els.net [doi: 10.1002/9780470015902.a0021259.pub2]
  2. DEPC-treated water

Article Outline

Importance and significance of this work

  • Cold shock response in yeast has been researched quite a bit, but the authors claim that this work is the first to study the effects at 4°C. The Authors believe that their work is the first to study cold-shock response in yeast at a temperature below 10°C.
  • It is important to study organisms at low temperatures because many organisms are exposed to freezing or near-freezing temperatures in the natural world. Some organisms (such as E. coli) are quite often exposed to these low temperatures during experimental preparation.
  • It is also important to study the differences between organisms that have never been exposed to cold temperatures and organisms that have been grown in these cold conditions. This will allow us to determine if/how well yeast can adapt to the cold.

Methods and Materials

Preparation of yeast and probes
  • Strain S288c was grown in the following conditions:
    • Grown at 25°C until mid-log phase
    • Cooled to 4°C and harvested after 6, 12, 24 & 48 h
    • 2 Controls grown continuously in 4°C & 35°C environments.
  • RNA was extracted by the hot-phenol method.
  • mRNA was purified with an mRNA purification kit.
  • The probes were prepared by mixing 1-2μg of mRNA with two primers and a dNTP nucleotide mix.
    • Reverse transcribed cDNA
    • Probes were purified using a G-50 column
Experimental design of the microarray data
  • The cDNA microarrays contained targets for 5,952 genes
    • cDNA microarray chip comes from "DNA Chip Research, Kanagawa, Japan"
  • 20μL of cDNA was mixed with 40μL of DEPC-treated water and 60μL of 2× hybridization solution. This solution was heated to 90°C for three minutes.
    • Hybridization solution comes from "CyScribe first strand cDNA labeling kit"
  • The solution was then placed onto the array and incubated overnight at 65°C to allow hybridization to occur.
  • The microarray was scanned with a Scanarray 4000 scanner
  • The spots were located on the chip by GenePix4000 software.
  • Corrections were made to account for the variations in quality of the spots and "cut off was done at an average background 118 value of +2SD."
  • 20% of the data was used to calculate the Lowess fit at each point of a log-intensity vs log-ratio plot. The resulting curve was used to adjust the control value for each measurement.
  • Multiple DNA microarray trial were run, and at least 5 independent cultures were used.
  • Genes with hypridization ratios > 2.0 and < 0.5 in at least 3 out of 5 experiments were considered up-regulated or down-regulate (respectively).

Results

  • Growth in yeast cells slowed once they were moved from 25°C to 4°C.
  • Hierarchical cluster analysis can be used to determine which sets of data are most similar to each other. The process begins by grouping the two most similar data sets together into a "cluster." Data sets are then added one-by-one to the initial cluster in order of similarity, until all data is categorized into one cluster. This technique revealed which time-points have the greatest similarity in gene expression.
    • Hours 12 and 24 have the most similar gene-expression profile, and therefore are clustered together first.
    • The next closest to this cluster is 48 hours, which can then be added to the 12/24h cluster form another, bigger cluster.
    • Hour 6 is the next closest to this cluster.
    • Then the expression profile of the yeast constantly exposed to 4°C
    • And finally, with the least similar gene expression, is the yeast constantly exposed to 35°C.
  • Up and down-regulation of genes occurred according to what functions were needed for cell viability
    • Genes involved in cell rescue, defense and virulence, and energy and metabolism were up-regulated
    • Genes involved in protein synthesis, binding functions, activity regulation and fate were down-regulated
  • Transcription of some genes go into overdrive after cold shock, others don’t
    • Cold-shock causes up-regulation of genes that lead to
      • Energy preservation and cold tolerance
      • Membrane maintenance and permeability increases
      • Detoxification of active oxygen species
      • Revitalization of enzyme activity
    • Causes down-regulation of genes that lead to growth
      • Allows yeast to adapt to new environment

Results of this study in comparison to the results of previous studies

The results of this study looked very similar to those of previous studies. Here are a couple of examples:

  • Genes encoding HSPs were induced by 4°C exposure. These genes were also observed to be induced at 0°C during a 2004 study of near-freezing response by Kang et al.
  • Trehalose synthesis was also shown to be important at low temperatures during a 2004 study by Kandror et al.

There are also some instances where the results of this study are different from previous ones. For example:

  • TPS1 and TPS2 genes were found to have a smaller fold induction in this study compared to a previous study by Kandror et al in 2004.
    • This discrepancy may be explained by use of different carbon sources in the two studies.

Emma and Katie's personal ideas on further research

  • It was somewhat frustrating that this paper made so many references to previous papers, and yet did not compare its results to very many.
  • The paper also did not indicate what the authors wanted to pursue as far as future research
  • Because of the previous two points, Emma and I decided to suggest the following two ideas for future research:
    • How different types of stresses lead to similar or different changes in gene expression (e.g. salinity, pressure, chemicals, etc)
    • If there is significant change in gene expression depending on the temperature used for cold shock (ie cold shock at 2°C, 4°C and 6°C and 8°C).

Acknowledgements

  • I'd like to thank the team Data Analyst/my co-presenter Emma Tyrnauer for working with me on the assignment this week. We talked during class, over text, as well as during our meet-up on Monday afternoon.
  • I'd also like to thank my other team partners Blair Hamilton and Zach Van Ysseldyke for continuing to work diligently on their portion of the project. We communicated during class and via text.

While I worked with the people noted above, this individual journal entry was completed by me and not copied from another source.

Kwrigh35 (talk) 20:19, 20 November 2017 (PST)

Referneces

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