Emmatyrnauer Week 12

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Definitions

  1. consortium: a two- (or more) membered bacterial culture (or natural assemblage) in which each organism benefits from the other (http://www.biology-online.org/dictionary/Consortium)
  2. desaturase: an enzyme that converts an unsaturated fatty acid chain to one that includes at least one carbon-carbon double bond (https://medical-dictionary.thefreedictionary.com/desaturase)
  3. glycosyl phosphatidyl inositol: a lipid anchor for many cell-surface proteins; represents a posttranslational modification of proteins with a glycolipid (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4689333/)
  4. mannoproteins: glycoproteins that contain 15 to 90% mannose by weight (https://link.springer.com/chapter/10.1007%2F978-3-642-68234-6_18)
  5. hybridization: act of binding (https://www.genome.gov/10000533/dna-microarray-technology/)
  6. Subcellular localization: the act of proteins moving to a particular position in a cell (e.g. how anchor proteins end up in particular locations) (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2845201/)
  7. thioredoxin peroxidases: a member of a newly discovered family of proteins that are conserved from yeast to mammals and to which natural killer enhancing factor belongs; these proteins are antioxidants that function as peroxidases only when coupled to a sulfhydryl reducing system. (https://www.ncbi.nlm.nih.gov/pubmed/9388194)
  8. glutathione: a peptide C10H17N3O6S that contains one amino acid residue each of glutamic acid, cysteine, and glycine, that occurs widely in plant and animal tissues, and that plays an important role in biological oxidation-reduction processes and as a coenzyme (https://www.merriam-webster.com/dictionary/glutathione)
  9. ubiquitious: present everywhere (https://www.medicinenet.com/script/main/art.asp?articlekey=26111)
  10. virulence: the degree or ability of a pathogenic organism to cause disease (http://www.biology-online.org/dictionary/Virulence)

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 how does this compare to the cellular responses of yeast which have already adapted to/able to survive at 4C; significant in understanding and carrying out preservation of samples

Materials and Methods

Experimental design flow chart
  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
      • RNA extracted and mRNA purified
      • Labeled with Cy3-UTP
    • Other group removed once growth reached mid log phase and transferred to 0C, and cooled until 4C was reached
      • Cells harvested 6, 12, 24, and 48 hours after cold shock by centrifugation
      • Cells flash frozen and stored at -80C
      • RNA was extracted and poly A mRNA was purified
      • labeled with Cy5-UTP
  2. Probe prepared and labeled
  3. Microarray hybridized and scanned
    • S. cerevisiae 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 at least 3/5 experiments = upregulated
      • Genes with less than 0.5-fold in at least 3/5 experiments = downregulated
  4. Hierarchal cluster analysis performed with GeneSpring

This experimental design is represented in the flow chart to the right.

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 hierarchical 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. For the most part, these genes were induced (trehalose and glycogen synthesis and break down genes, glycolysis and gluconeogenesis genes, and aryl-alcohol dehydrogenase genes)
  5. Table 3: Genes involved in metabolism were induced. These include genes part of the phospholipid synthesis pathway as well as the methionine biosynthesis pathway.
  6. Table 4 is representative of the genes involved in cell rescue, defense, and virulence after exposure to 4C. These genes were induced (cold shock-inducible proteins, seripauperin proteins, heat shock proteins, and proteins involved in the detoxification of active oxygen species).
  7. Table 5 represents the genes that were repressed after exposure to 4C. Many of these are involved in protein synthesis. For example, ribosomal protein large subunit genes, ribosomal protein small subunit genes, etc.

Discussion

  1. Yeast cells were able to maintain colony forming ability for more that one year after being stored at 4C
    • Therefore, preservation via low temperatures is a viable option
  2. Transcription patterns differed between cells continuously grown in 4C versus cells shifted to 4C
  3. At low temperatures, membrane fluidity and permeability decrease so induction of genes (e.g OLE1) involved with fatty acid desaturase help to prevent these drastic changes (Aguilar et al. 1999; Inaba et al. 2003; Nakagawa et al. 2002). However, this study did not observe this large induction.
    • Northern blot demonstrated by Nakagawa et al. 2002 showed activation of OLE1 at 10C so it is possible that induction occurred before the 6 hour mark
  4. Murata et al. 2003 found that phospholipid synthesis and methionine synthesis are integrated mechanisms. This relates to this study as both molecules were induced
  5. Trehalose and glycogen have been found to accumulate during exposure to stressful environments (Lillie and Pringle 1980)
  6. Mannoproteins have been identified with regulating cell membrane permeability (Zlotnik et al. 1984)
    • Similar to OLE1 they most likely are required for cold shock survival
  7. In accordance with results from this experiment, heat shock proteins's have been found to be induced at 0C (Kandror et al. 2004)
    • This is because both heat shock and cold shock are acute stresses and interfere with proper protein folding
  8. Homma et al. 2003 found that these heat shock proteins are also expressed when yeast cells are cultured at 4C for months.
  9. Methionine synthesis genes induced after cold shock in this study. Some of these genes were found to allow for sulfur assimilation (Thomas and SurdinKerjan1997).
    • These capabilities may be required for glutathione synthesis after cold shock.

Powerpoint Presentation

Media:Genome-wide_expression_analysis_of_yeast_response_during_exposure_to_4_degrees_C.pdf

Electronic Notebook

This week I worked on my journal club presentation with Katie. I outlined the paper, searched definitions that I did not understand, and also worked on my data analyses from week 8 and week 10 (more detailed description of this can be found in my executive summary on the Lights, Camera, InterACTION! page). Once the presentation was completed, it was uploaded to the wiki and linked to this page. Katie and I plan to meet up before our presentation to practice.

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 work on 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. Murata, Y., Homma, T., Kitagawa, E., Momose, Y., Sato, M. S., Odani, M., ... & Fujita, K. (2006). Genome-wide expression analysis of yeast response during exposure to 4 C. Extremophiles, 10(2), 117-128.
  3. Consortium. Retrieved November 20, 2017, from http://www.biology-online.org/dictionary/Consortium
  4. Desaturase. Retrieved November 20, 2017, from https://medical-dictionary.thefreedictionary.com/desaturase
  5. Glycosyl phosphatidyl inositol. Retrieved November 20, 2017, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4689333/
  6. Mannoproteins. Retrieved November 20, 2017, from https://link.springer.com/chapter/10.1007%2F978-3-642-68234-6_18
  7. Hybridization. Retrieved November 20, 2017, from https://www.genome.gov/10000533/dna-microarray-technology/
  8. Subcellular localization. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2845201/
  9. Thioredoxin peroxidases. Retrieved November 20, 2017, from https://www.ncbi.nlm.nih.gov/pubmed/9388194
  10. Glutathione. Retrieved November 20, 2017, from https://www.merriam-webster.com/dictionary/glutathione
  11. Ubiquitious. Retrieved November 20, 2017, from https://www.medicinenet.com/script/main/art.asp?articlekey=26111
  12. Virulence. Retrieved November 20, 2017, from http://www.biology-online.org/dictionary/Virulence

Links

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