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.

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

IV. Conclusion

V. Further Implications

Deliverable

Journal Club Week 12 Presentation