Emmatyrnauer Week 12

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Definitions

Journal Outline

Introduction

  • Ambient temperature affects gene regulation
  • Has led to the evolution of biochemical and physiological properties
  • Known cellular activities in response to:
    • heat shock: heat shock proteins expressed more
    • cold shock: decreased permeability of cell membrane, decreased protein synthesis and folding, and decreased enzymatic activity
  • Currently there aren't any studies that examine the effects of cold shock below 10 deg C on gene expression in yeast
  • Interested in this because preservation of organisms often occurs at less than 10 deg C (i.e. 4 deg C)
  • This study exposed yeast to temperature of 4 deg C and examines changes in gene expression using cDNA microarray
    • Cluster analysis performed
      • Results include gene expression between 6 and 48 hours after 4C cold shock differs from culture kept at 35C or 4C. After cold shock, expression of certain genes was induced (e.g. energy preservation genes, phospholipid synthesis genes, and cell rescue genes)
  • Now the question is what are the cellular responses of yeast which have already adapted to/able to survive at 4C

Materials and Methods

  1. Strain and growth conditions
    • Saccharomyces cerevisia S288c grown in YPD medium at 25C in a shaker.
    • One group was left until growth reached late log phase--control group
    • Other group removed once growth reached mid log phase and transferred to 0C.
      • Upon reaching 4C, yeast was flash frozen and stored at -80C
  2. RNA was extracted and poly A mRNA was purified
  3. Probe prepared and labeled
  4. Microarray hybridized and scanned
    • S. cerevisia cDNA microarrays used (targets for 5,952 genes)
    • 5 independent culture microarray experiments carried out for each experiment
      • Genes with more than twofold increase in 3/5 experiments = upregulated
      • Genes with less than 0.5-fold in 3/5 experiments = downregulated
  5. Hierarchal cluster analysis performed with GeneSpring

Results

  1. Figure 1 is a graph representing the growth of the yeast cells at 25C and following exposure to 4C. Growth was monitored over a 60 hour period and cold exposure occurred at 20 hours. Growth occurred at a faster rate in the control group (which was left at 25C at mid log growth) compared to the group that was cooled and stored at 4C. However growth of the control group leveled out at around 30 hours.
  2. Table 1 represents changes in expression of 20 genes which demonstrated greater that two-fold up-regulation and 20 genes which demonstrated less that 0.5-fold down regulation at 6, 12, 24, and 48 hours. In general, genes that were involved in cell rescue, defense, virulence, energy and metabolism were up-regulated, whereas genes responsible for protein synthesis, binding functions/co-factor requirements, activity regulation, and fate were down-regulated.
  3. Figure 2 is an image of the heierarchical cluster analysis between 6 and 48 hours following 4C exposure, culture at 4C, and culture at 35C. Red=levels of expression greater than 2 fold in 4C group compared to control; Blue=levels of expression less than 0.5 fold after cold shock compared to control; Yellow=no significant difference between cold shock group and control group.
  4. Table 2 is representative of genes involved in energy. Induced genes include trehalose and glycogen synthesis genes, and some genes in glycolysis/glyconeogenesis that are required for use of trehalose and glycogen. Glycogen degradation genes were also induced.
  5. Table 3 is representative of genes involved in metabolism. Genes part of the phospholipid synthesis pathway as well as the methionine biosynthesis pathway were induced.
  6. Table 4 is representative of the genes involved in cell rescue, defense, and virulence after exposure to 4C. Cold shock-inducible proteins, seripauperin protein, heat shock proteins and proteins involved in the detoxification of active oxygen species were induced.
  7. Table 5 represents the genes that were repressed after exposure to 4C. Many of these are involved in protein synthesis.

Discussion

Acknowledgements

  1. I worked with my group project partner Katie during class and over text to work on the presentation
  2. I worked with Katie, Zach, and Blair, to construct the our group page.
  3. While I worked with the people noted above, this individual journal entry was completed by me and not copied from another source.

Emmatyrnauer (talk) 22:23, 19 November 2017 (PST)

References

  1. LMU BioDB 2017. (2017). Week 12. Retrieved November 19, 2017, from https://xmlpipedb.cs.lmu.edu/biodb/fall2017/index.php/Week_12
  2. Homma, T., Iwahashi, H., & Komatsu, Y. (2003). Yeast gene expression during growth at low temperature. Cryobiology, 46(3), 230-237. Retrieved November 19, 2017 from http://sq4ya5rf2q.search.serialssolutions.com/?V=1.0&sid=PubMed:LinkOut&pmid=12818212

Links

  1. My User Page
  2. List of Assignments
  3. List of Journal Entries
  4. List of Shared Journal Entries
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