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Article

Sahara, T., Goda, T., & Ohgiya, S. (2002). Comprehensive expression analysis of time-dependent genetic responses in yeast cells to low temperature. Journal of Biological Chemistry, 277(51), 50015-50021.

Flow Chart

Outline

Experiment design and procedures

• Purchased cDNA microarray of Saccharomyces cervisiae from DNA Chip Research Inc.
• Used S. cervisiaeYPH500
• Grew yeast cells aerobically in YPD medium at 30 degrees celsius and shaken at 100rpm.
• YPD is made up of:
  • 1% yeast extract
  • 2% peptone
  • 2% glucose
• Yeast cells were grown to "mid-log phase" where they were still maturing, but not fully reproducing
• 50mL of culture was taken and centrifuged to collect the cells and be used as a time 0 reference for the rest of the experiment
• The time 0 reference cells were stored at -80 degrees celsius
• The rest of culture was used for the experimental samples and was cold shocked at 10 degrees celsius
• Samples were collected at various times: 0.25, 0.5, 2, 4, and 8h
• Acidic phenol method and RNeasy Mini Kit were used to prepare the RNA
• The RNA was used to prepare fluorophore-labeled cDNA probes
• These probes marked the cells for array hybridization
• The microarrays were scanned with a laser microscope and were analyzed
• Repeated process twice
• Averaged the expression ratios of the separate experiments for final data

Results & discussion

• Analyzed the microarray of cDNAs of 5,803 genes in yeast genome
• There was a diauxic shift in cells that experienced cold shock
  • Down-shift of some diauxic shift-inducible genes in late phase of cold shock
• Low temperature affects expression of ~25% of the yeast genes
• Number of up-regulated genes increased from 41 at 0.25h to 536 at 8h
• Number of down-regulated genes also increased from 4 at 0.25h to 488 at 8h
• Gene expression changes significantly in both ways (up & down-regulation) to adapt cells to colder environment, similar to reactions to other stresses, like heat, salinity, hydrogen peroxide, and osmotic stresses
• Table 1 shows the number of genes that significantly changed expression (2-fold or more), up-regulating or down-regulating

Clustering analysis

• Clustering of genes (genes that were close together) were analyzed in the ways they responded
• Genes separated into 5 different clusters (Fig. 1):
  • 1A: Unclassified proteins
  • 1B: Amino acid biosynthesis and metabolism
  • 1C: RNA Polymerase I & RNA processing
  • 1D: Ribosomal proteins
  • 1E: Not labeled
• Shows cooperative regulation
  • 1C: up-regulated in early phase (0-0.5h), then down-regulated in late phase (4-8h)
  • 1D & 1E: up-regulated in mid-late phase (2h & 4-8h)

Transcription related genes

• Looked at clusters of genes relating to RNA polymerase I & RNA processing
  • All up-regulated in early phase
• Cooperative regulation of genes involved in transcription (Fig. 2)
• 2 clusters of these genes:
  • Down-regulating (2A, 2B, & 2D)
    • 2A (RNA polymerase I & RNA processing) & 2B (rRNA processing) up-regulated in early phase, then down regulated in late phase
    • 2D: mRNA transcription
  • Up-regulating (2C)
    • 2C: mRNA transcription
    • High up-regulating in mid phase
• Up-regulated genes that had to do with basic transcriptional functions, like genes encoding for regulatory proteins for amino acid production
• Down-regulated genes were not essential for basic life, like genes encoding heat shock transcription factor or a transcription factor for drug resistant genes
• Factors essential to transcription & processing of rRNAs were up-regulated
• Genes for synthesis and transcription regulation of mRNAs had mix responses

Ribosomal protein related genes

• Genes separated into four clusters (Fig. 3):
  • 3A & 3B: cytosolic ribosome
  • 3C: translational control factors
  • 3D: tRNA syntheatases
• 3A up-regulated in early-mid phase, then down-regulated in late phase
• 3B up-regulated in early-mid phase, and slightly down-regulated in the late phase
• 3C continuously up-regulated starting in the early phase
• 3D overall down-regulated
• Genes encoding ribosomal proteins = most of up-regulated genes at 2h
• Almost all up-regulating ribosomal proteins had similar expressions, showing cooperative regulation
• Low temperature impairs translational ability
• When genes related to ribosomal proteins and rRNA processing don't work, there is cold sensitivity
• Yeast up-regulates genes encoding ribosomal proteins, so compensates for less efficient/productive translation to overcome cold sensitivity

Cell rescue, defense, death, and aging genes

• Genes separated into four clusters (Fig.. 4):
  • 4A: not labeled
  • 4B & 4C: stress response - high up-regulation in mid-late phase
  • 4D: stress response and chaperone - high down-regulation in mid-late phase
• Heat shock protein genes (HSPs) were down-regulated in low temperatures, except HSP12 and HSP26
• This may mean HSP12 and HSP26 have different transcription regulation than other HSPs
• Graumann, et al., 1996 did similar experiment with Bacillus subtilis and found peptidyl prolyl cis/trans isomerases were induced in low temperatures, so protein folding genes were up-regulated

Metabolism and energy production genes

• 8 clusters (Fig. 5):
  • 5A: nucleotide metabolism
  • 5B & 5E: not annotated
  • 5C & 5D: C-compound and carbohydrate metabolism
  • 5F & 5H: amino acid metabolism
  • 5G: C-compound and carbohydrate utilization

Terms

1. Nucleolin: a protein associated with nucleolar in growing eukaryotic cells (NCBI, 2017).
2. Hypoxia: low levels of oxygen in body tissues (Hine & Martin, 2015).
3. Ubiquitin: a protein that marks which proteins are going to be broken down (Hine & Martin, 2015).
4. "Mid-log" or "lag" phase: bacterial cells are maturing, but they have not yet reached their maximum reproduction rate (Hine & Martin, 2015).
5. Diauxic shift: the switch of a microorganism from using one type of sugar to using another (King, et al., 2014).
6. Cytosolic: contents of the fluid in the cytoplasm of a cell (King, et al., 2014).
7. Peptidyl prolyl cis/trans isomerases: Enzymes that changes conformation of prolyl bonds to cis or trans, chaning the tertiary structure of a protein (Lackie, 2013).
8. cAMP-PKA pathway: the activation of PKA by cAMP (Lackie, 2013).
9. GTPase: enzymes that hydrolyze GTP (Lackie, 2013).
10. Phosphatase: an enzyme that helps remove a phosphate group from an organic compound (Hine & Martin, 2015).

Acknowledgments

1. I met with Dina outside of class, and we worked on our presentation together.

While I worked with the people noted above, this individual journal entry was completed by me and not copied from another source. Cwong34 (talk) 17:28, 20 November 2017 (PST)

References

1. Graumann, P., Schröder, K., Schmid, R., & Marahiel, M.A. (1996). Cold shock stress-induced proteins in Bacillus subtilis. Journal of Bacteriology, 178(15), 4611-4619. doi: 10.1128/jb.178.15.4611-4619.1996
2. Hine, R. & Martin, E. (Eds.). (2015). A Dictionary of Biology. In Oxford Reference. Retrieved from http://www.oxfordreference.com/view/10.1093/acref/9780198714378.001.0001/acref-9780198714378
3. King, R.C., Mulligan, P.K., & Stansfield, W.D. (Eds.). (2014). A Dictionary of Genetics. In Oxford Reference. Retrieved from http://www.oxfordreference.com/view/10.1093/acref/9780199766444.001.0001/acref-9780199766444
4. Lackie, J.L. (2013). The Dictionary of Cell and Molecular Biology. In Science Direct. Retrieved from http://www.sciencedirect.com/science/book/9780123849311
5. LMU BioDB 2017. (2017). Week 12. Retrieved November 14, 2017, from https://xmlpipedb.cs.lmu.edu/biodb/fall2017/index.php/Week_12
6. NCBI. (2017). Nucleolin. Retrieved November 20, 2017, from http://www.uniprot.org/uniprot/P19338
7. Sahara, T., Goda, T., & Ohgiya, S. (2002). Comprehensive expression analysis of time-dependent genetic responses in yeast cells to low temperature. Journal of Biological Chemistry, 277(51), 50015-50021.

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