Dbashour Week 12

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.

List of 10 Unknown Words and Their Definitions

1. hypoxia - low oxygen levels in the blood (Ratini, 2016)
2. ubiquitin - plays a role in the heat-shock response, is involved in quality control of nascent proteins, membrane trafficking, cell signalling, cell cycle control, X chromosome inactivation and the maintenance of chromosome structure (Lackie, 2013)
3. Cytosolic - contents of the fluid in the cytoplasm of a cell (King, et al., 2014).
4. diauxic shift - the two-phase growth response seen in a culture of microorganisms making a phenotypic adaptation to the addition of a second substrate (AccessScience, 2015)
5. methyl methanesulfonate - A DNA damaging agent to induce mutagenesis and in recombination experiments (Lundin, 2005).
6. peptidyl-prolyl cis/trans- isomerases - catalyse the cis–trans isomerisation of peptide bonds N-terminal to proline residues in polypeptide chains, play a role in the folding of newly synthesised proteins, and assist in cell cycle control (Shaw, 2002)
7. trehalose - a sugar thought to be implicated in anhydrobiosis - the ability of plants and animals to withstand prolonged periods of desiccation (National Center for Biotechnology Information, 2016)
8. de novo - New; not present previously; just beginning (Honee, 2009)
9. midlgarithmic - cell numbers increase in a logarithmic fashion but have not reached their full reproduction rate (Rogers & Kadner, 2017)
10. 2-fold - method of reporting statistics using log2 that results in more reproducible gene lists than do the ordinary and modified t-statistics (Witten & Tibshirani, 2007)

Outline of Article

Background Information on Purpose of Experiment

• Low temperatures are known to have several effects on biochemical and physiological properties in various cells
• Cold shock proteins are induced when cells are exposed to low temperatures in order to cope with the drastic change in environment
• In yeast, the NSR1, TIP1, and OLE1 genes have been identified as important cold-inducible genes through past research
• low temperature-dependent gene expression and low temperature response are still unclear
• The purpose of this experiment is to analyze global gene expression in low temperature-exposed yeast cells using a yeast cDNA microarray to obtain fundamental information on low temperature response and low temperature-dependent gene expression in yeast cells

Cold shock and microarray procedures of yeast samples

• S. cerevisiae YPH500 was used for all the analyses
• Cultured aerobically in YPD medium (yeast extract, peptone, and glucose) at 30°C and shaken at 100 rpm
• 50 ml of the culture were collected for a reference time of 0
• Cells flash-frozen in liquid nitrogen
• Stored at -80°C in preparation for RNA
• The remaining cells were cold shocked at 10°C then cultured at the same temperature
• Cells collected at 0.25, 0.5, 2, 4, and 8 hours after the cold shock
• Cells flash-frozen in liquid nitrogen
• Stored at -80°C in preparation for RNA
• Cy3-dUTP and Cy5-dUTP were used as cDNA probes
• Labeled with fluorophore in order to carry out microarray hybridization
• Microarray hybridization performed based on the manual for S. cerevisiae cDNA microarray
• Microarrays were scanned by laser microscope
• Process repeated twice
• Averaged the expression ratios of the separate experiments for final data
• Images analyzed by computer program
• Data analyzed by analysis software
• Fluorescence intensities were normalized
• Data was clustered and referred to the Munich Information Center for Protein Sequences functional database
• Functional relationships among the genes in each cluster was determined

Results and Discussion

Global Expression Analysis of Low Temperature Response in Yeast Cells Using a cDNA Microarray

• Changes in expression of genes in yeast after cold shock was analyzed using cDNA of 5,803 genes in a yeast genome
• Ratio of fluorescent intensities was 2 fold for a mainly all the cDNA spots on the data
• Roughly 25% of yeast genes' expression levels were affected by cold shock
• Increase in number of genes up-regulated
• Increase in number of genes down-regulated
• Significantly up or down-regulated genes were classified according to the MIPS functional database
• Up-regulated and down-regulated genes increased in almost all categories when exposed to low temperatures
  • Suggests that changes in gene expression are due to the introduction of low temperatures in order to adapt to their environment
  • Other cells have demonstrated this ^^ when exposed to other environmental stresses

Clustering Analysis of Global Expression Data

• Genes close together were placed into clusters
• Clusters showed:
  • Similar functions
  • Cooperative regulation
• Clusters:
  • 1A: Unclassified proteins
  • 1B: Amino acid biosynthesis and metabolism
  • 1C: RNA polym. I & RNA processing
  • 1D: Ribosomal proteins
  • 1E: Not defined
• Up regulated genes were divided into three clusters depending on their expression profiles
  • IC: up-regulated after within 30 mins after cold shock
  • ID: high up-regulation at 2hr and at 4-8 hr
  • IE: high up-regulation at 2hr and at 4-8 hr

Genes related to transcription

• Further classification of genes:
  • 2A: RNA polymerase I & RNA processing - up regulated, then down regulated in later phase
  • 2B: rRNA processing - up regulated, then down regulated in later phase
  • 2C mRNA transcription - up-regulated in mid phase
  • 2D: mRNA transcription - continuously down regulated
• These findings suggest that...
  • The mechanisms for transcription are up regulated when exposed to low temperatures in the early phase
    • Up-regulated mRNAs = essential for basic transcriptional/translational functions, like encoding regulatory proteins for amino acid production
  • The genes for transcriptional regulation and mRNA synthesis made diverse responses in the late phase
    • Down-regulated mRNAs = not essential for survival in cold shock, like genes encoding heat shock transcription factor or a transcription factor for drug resistant genes

Ribosomal Protein Genes

• Cluster Classification:
  • 3A & 3B: cytosolic ribosome - up regulated first, then down regulated in later phase
  • 3C: translational control factors - continuously up regulated
  • 3D: tRNA synthetases - continuously down regulated
• These findings suggest that...
  • Low temperature impairs translational ability
  • Yeast genes up-regulate to compensate

Cell Rescue, Defense, Death, and Aging

• Cluster Classification:
  • 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
• These findings suggest that...
  • Heat shock protein genes down-regulated (EXCEPT for HSP12 and HSP26)
  • Protein folding genes up-regulated

Metabolism and Energy Production

• Cluster Classification:
  • 5A: nucleotide metabolism - up-regulated early-mid phase, then down-regulated
  • 5B & 5E: not specified
  • 5C & 5D: C-compound and carbohydrate metabolism - continuously up-regulated
  • 5F & 5H: amino acid metabolism - continuously down-regulated
  • 5G: C-compound and carbohydrate utilization - continuously down-regulated
• These findings suggest that...
  • Glycogen and trehalose production genes showed a lot of cooperative up-regulation
  • Trehalose may help protect cellular membrane, which may help to keep yeast cells intact at low temperatures

Signal Transduction Components

• Cluster Classification:
  • 6A: signal transduction - down-regulated
  • 6B: signal transduction - up regulated in early phase, then down-regulation
  • 6C: signal transduction - down-regulated in early phase, then up-regulation in late
• These findings suggest that...
  • Genes related to cAMP-PKA pathway and Msn2p/4p were up-regulated
  • Increase in PKA signaling has been known to be response to stresses
  • Increase in...
    • Signaling
    • Metabolism control
    • Stress resistance

Conclusion

• Gene expression changed to maintain transcription and translation and to adapt a tolerance to the colder temperature
• Transcriptional genes up-regulated first to help transcription and translation
• Ribosomal proteins up-regulated in the middle phase to further assist maintenance of translation
• Stress response induced genes up-regulated in the late phase
• Maintaining translation is priority to yeast in cold shock
• Cells need to make proteins to help maintain integrity and basic functions of cells
• Yeast cells can adapt to environment once ^^^ obtained to gain tolerance to low temperature
• Other organisms show similar responses in gene expression when exposed to environmental stresses

Deliverable

Journal Club Week 12 Presentation