Leanne Kuwahara-Week 3

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Purpose

To prepare for Thursday's journal club and to understand the genes involved with cold adaptation-as well as other stressors-in yeast (S. cerevisiae).

Unfamiliar Terms

  1. Differential Hybridization: A method for comparison of different organs, or physiological or disease states by the genes that are uniquely expressed in them. The mRNAs of two types of cell to be compared, e.g. normal and cancer cells, are isolated, differently labelled for detection and hybridized to an appropriate cDNA library, preferably a normalized library. The mRNAs are labeled with two different fluorophores, e.g. red emission for cancer cells and green emission for normal cells, so that when they are hybridized to the cDNA library that is arrayed in a relative excess on a single glass surface, the hybridization of both fluorophores to a single cDNA fragment will appear yellow, while the hybridization of only one, e.g. the normal cell mRNA, will appear green. (GeneScript)
  2. Trehalose: Also known as mycose, trehalose is a 1-alpha (disaccharide) sugar found extensively but not abundantly in nature. It is thought to be implicated in anhydrobiosis - the ability of plants and animals to withstand prolonged periods of desiccation. The sugar is thought to form a gel phase as cells dehydrate, which prevents disruption of internal cell organelles by effectively splinting them in position. Rehydration then allows normal cellular activity to be resumed without the major, generally lethal damage that would normally follow a dehydration/reyhdration cycle. Trehalose is a non-reducing sugar formed from two glucose units joined by a 1-1 alpha bond giving it the name of alpha-D-glucopyranoglucopyranosyl-1, 1-alpha-D-glucopyranoside. The bonding makes trehalose very resistant to acid hydrolysis, and therefore stable in solution at high temperatures even under acidic conditions. The bonding also keeps non-reducing sugars in closed-ring form, such that the aldehyde or ketone end-groups do not bind to the lysine or arginine residues of proteins (a process called glycation. (National Center for Biotechnology Information)
  3. Diauxic Shift: The switch from rapid fermentative growth in the presence of a rich carbon source to slower exponential growth by aerobic respiration using ethanol once the preferred carbon source has been exhausted. (Saccharomyces Gene Database)
  4. Growth Curve: A curve in graph form that shows the change in the number of cells (or single-celled organisms) in an experimental culture at different times. The classic growth curve, as exemplified by a newly established bacterial colony, is divided into four phases, in order of their appearance: (1) lag phase; (2) log (logarithmic), or exponential, phase; (3) stationary phase; and (4) death, or decline, phase. (Britannica, 2016)
  5. Global Transcriptional Profile: Also known as expression profiling, transcriptional profiling involves the quantification of gene expression of many genes in cells or tissue samples at the transcription (RNA) level. The quantification can be done by collecting biological samples and extracting RNA (in most cases, total RNA) following a treatment or at fixed time-points in a time-series, thereby creating "snap-shots" of expression patterns. (European Bioinformatics Institute)
  6. Two-Dimensional Heirarchial Clustering: The most frequently used mathematical technique. Attempts to group genes into small clusters and to group clusters into higher-level systems. The resulting hierarchical tree is easily viewed as a dendrogram. (Augen, 2004)
  7. Dendrogram: A dendrogram is a type of tree diagram showing hierarchical clustering — relationships between similar sets of data. They are frequently used in biology to show clustering between genes or samples, but they can represent any type of grouped data. (Stephanie, 2016)
  8. log Phase: Also called the logarithmic phase. It is the period of growth of a population of cells in a culture medium during which numbers increase exponentially and which is represented by a part of the growth curve that is a straight line segment if the logarithm of numbers is plotted against time. (Merriam-Webster)
  9. Protein Kinase A Pathway: Protein kinase A (PKA) signaling is a widely used intracellular pathway and the major route for channeling the second messenger cAMP signal. PKA cross-talks or integrates with other signaling pathways. The PKA signaling pathway has been implicated in many cellular and physiological processes, from metabolism and reproduction, to cardiac function, memory and learning. (RGD)
  10. Protein Kinase C Pathway: Protein kinase C (PKC) family members regulate numerous cellular responses including gene expression, protein secretion, cell proliferation, and the inflammatory response. (Cell Signaling Technology)

Outline of Article

Introduction & Background Information
  • Unicellular organisms subjected to drastic changes to environment
    • Develop stress responses
      • Short term: alter protein phosphorylation and degratation
      • Long term: alter transcription
  • Environmental stress response (ESR) genes: common set of genes whose transcription is changed in response to environmental stressors
    • ~10% of genome
    • Induced ESR genes involved in:
      • Protein folding/degradation
      • Transport
      • Carbohydrate metabolism
    • Repressed ESR genes involved in:
      • RNA metabolism
      • Nucleotide biosynthesis
      • Secretion
      • Ribosomal performance
    • ESR regulated by Msn2p and Msn4p
      • Bind to stress response elements (STREs) in promoter of target gene
  • Little is known about response to cold-shock
    • Cold decreases membrane fluidity
    • Yeast upregulate certain genes in response to cold-shock
      • NSR1: nucleolin-like protein (pre-RNA processing and ribosome biogenesis)
      • TIP1/TIR1/TIR2: encode Ser & Ala-rich cell wall proteins (may be involved in maintaining cell wall integrity)
      • OLE1: encode fatty acid desaturases
        • Common in other organisms-suggest membrane fluidity adaptation is ubiquitous response to cold
  • GOAL
    • Describe transcriptional response to to cold in WT (control) 'S. cerevisiae' and msn2-msn4- mutants (experimental unit)
    • Compare cold response to responses to other stressors by measuring trehalose and glycogen acculmulation
Methods
  • Experimental Design
    • WT-took time points at:
      • 0, 2, and 12hr (two replicates)
      • 10min, 30min, and 60hr (three replicates)
  • Strains
    • BY4743
    • BSY25
    • W303
      • All strains were diploid
  • Growth Medium and Culture Conditions
    • Medium used: YPD (50mL)
    • Grown overnight at 30C in a shaking incubator at 170rpm
    • Decreased temperature 4C/min
    • Harvested cells during early log phase at either 10C (experimental unit) or 30C (control)
  • Isolation of RNA
    • Used hot-phenol method
      • Modifications
        • Extract with phenol 2x for 10min each
        • Improved extraction of 60hr expt by adding glass beads
    • RNA purified using Oligotex Spin-Column Protocol
  • RNA Labeling and DNA Microarray Hybridization
    • DNA microarrays used for global transcriptional profiling to determine possible changes in gene expression due to cold-shock treatment
    • Labeled directly by incorporating Cy3- and Cy5-dCTP through reverse transcription
    • Labeled cDNA hybridized onto yeast genome microarray
      • Pre-hybridization done in 20:1:1 DigEasyHyb soln, yeast tRNA, and sonicated salmon sperm for 2hr at 42C
      • Microarrays washed 2x in 0.1xSSC buffer for 2min at 42C and airstream dried-immediately hybridized
  • Data Acquisition and Analysis
    • Three quality control tests for DNA spot:
  1. Signal intensity had to be significantly greater than background
  2. Signal intensity had to be within dynamic range of photomultiplier tube
  3. Raw intensities of duplicate spots for each gene had to be within 50% of one another
  • Performed hierarchial clustering and statistical analysis with GeneSpring software
Results
  • Exposure to decreased temperatures (10C) reduced yeast growth rate
    • Did not alter growth curve
    • Alter gene expression in response to temperature shift (30C-->10C)
  • There exists and early cold response (ECR) and a late cold response (LCR)
    • ECR: Subset of cold response genes that were induced or repressed during the first 2hr of cold treatment
    • Induced genes
      • RNA helicase genes: DED1 & DBP2
      • RNA processing genes: NSR1, HRP1, NRD1, STP4, NOG2, & HUL5
      • RNA polymerase subunit gene: RPA49
      • Lipid metabolism genes: UFD1, MGA2 (induction did not exceed 2X), & NPL4 (induction did not exceed 2X)
        • All 3 genes work to activate OLE1 (fatty acid desaturase)
    • Repressed genes
      • Heat shock protein genes: Hsp104p, Hsp82p, Hsp60p, & Hsp10p
        • Important for protein folding and stress response
    • LCR: Subset of cold response genes that were induced or repressed after 12 and 60hr
    • Induced genes
      • Glycolysis genes: GLK1, HXK1, PYK2, & GPD1
      • Glycogen metabolism genes: GLC3, PGM2, GPH1, GDB1, GYS1, & GYS2
      • Trehalose metabolism genes: TPS1, TPS2, & TSL1
      • Carbohydrate regulatory factors: HAP5 & TYE7
      • Heat shock protein genes: HSP12, HSP26, HSP42, HSP104, YRO2, & SSE2
        • Involved in stress response-prevent protein aggregation and facilitate protein degradation/refolding
      • Oxidative stress response genes: GTT2, HYR1, GPX1, TTR1, & PRX1
    • Repressed genes
      • Protein synthesis/ribosomal protein genes
      • Nucleotide biosynthesis genes
      • Protein modification genes
      • Vesicle transport genes
        • Repression of protein synthesis contributes to cold adaptation
  • LCR genes common to ESR
  • Many ECR genes showed opposing transcriptional regulation compared to other ESR
    • LCR involves the ESR, whereas ECR demonstrates a "cold-specific" response
  • 78% LCR genes were not affected by the deletion of the ESR regulatory transcription factor, Msn2/Msn4
    • Likely other transcriptional regulators for LCR gene expression
  • Transcriptional regulation of ESR is independent of Msn2/Msn4
    • Supports claim of "cold-specific" response
  • ECR does not contribute to the accumulation of reserve carbohydrates, glycogen and trehalose
  • LCR does contribute to this accumulation of carbohydrates
    • Supports microarray data
Figures
  • Figure 1a. 2D hierarchical cluster dendrogram
    • X-axis: Similarities in gene expression and R:Gr ratio of transcript abundance
    • Y-axis: Different times of exposure to cold
    • Measurements were made using the microarray data and clustering was done using the GeneSpring software
    • Trends/Conclusions drawn: Groups D and E represent ECR genes, while groups A, B, and C represent LCR genes (not sure what group F represented- never stated in paper)
  • Figure 1b/c. Gene classification of (B) ECR and (C) LCR
    • X-axis: Gene functional categories
    • Y-axis: Number of genes either induced or repressed
    • Measurements were made using the microarray data and clustering was done using the GeneSpring software
    • Trends/Conclusions drawn: Early response upregulates genes mainly concerning transport, transcription, and metabolism (also down-regulates mostly metabolism-related genes) Late response upregulates genes mainly concerning metabolism stress response (downregulates genes concerning metabolism and protein synthesis)
  • Figure 2. Transcriptional profile of ECR genes during cold-shock
    • X-axis: Time and temperature downshift of comparison data (Gasch et al., 2000)
    • Y-axis: R:Gr ratio of transcript abundance
    • Measurements were made using the microarray data at 2hr and systematic comparison to similar data obtained from Gasch et al. (2000)
    • Trends/Conclusions drawn: Certain clusters of genes (a & b) were consistent with previous data, however genes outside these clusters showed an opposing trend
  • Figure 3. Comparison of transcriptional responses to cold and other environmental stressors of (A) ECR genes and (B) LCR genes
    • X-axis: Time and type of stressor
    • Y-axis: R:Gr ratio of transcript abundance
    • Measurements were made using the microarray data at 2hr and 12hr and systematic comparison to similar data obtained from Gasch et al. (2000)
    • Trends/Conclusions drawn: ECR genes have a reciprocal transcriptional response compared to other stressors, while LCR genes respond similarly to ESR
  • Figure 4. Regulation of gene expression during cold treatment
    • X-axis: Time and either WT or -msn2-msn4 mutant
    • Y-axis: R:Gr ratio of transcript abundance
    • Measurements were made using the microarray data at 0hr (30C), 2hr (10C), and 12hr (10C)
    • Trends/Conclusions drawn: Overall less change in transcriptional regulation in mutants compared to WT. Regarding ESR, LCR genes were induced in the WT and repressed in the mutant, demonstrating the need for these reg. transcription factors in the ESR
  • Figure 5. Accumulation of reserve carbohydrates during cold-shock
    • X-axis: Time (hr)
    • Y-axis: Concentration of either glycogen or trehalose (ug glucose/10^7 cells)-categorized by WT or mutant
    • Measurements were made using cultures from microarray data and glucose was measured using the Sigma-Aldrich Glucose kit
    • Trends/Conclusions drawn: Most carbohydrate reserves were produced during the late cold response, and mutant produced less reserve carbohydrate that WT
  • Figure 6. Comparison of transcriptional response to cold with Sahara et al. (2002)
    • X-axis: Time- categorized into Sahara study and current study
    • Y-axis: R:Gr ratio of transcript abundance
    • Measurements were made using the microarray data and clustered using GeneSpring software. Systematic comparison used to compare data with Sahara et al. (2002)
    • Trends/Conclusions drawn: There is a common cluster of genes that include general response to stress in the LCR, however, the ECR ribosomal genes had contradicting transcriptional profiles compared to Sahara et al. (2002)
Discussion
  • Significance of study:
    • Showed that yeast alter gene expression in response to cold-shock
    • Demonstrated that yeast have an early and late response to cold shock
      • Early response in "cold-specific" and reciprocal of ESR
      • Late response is consistent with the response to other environmental stressors
  • There exists other transcription factors/mechanisms that regulate the expression of a majority of LCR and ECR genes
  • Results seen in this study are consistent with those done in bacteria
    • Previous studies demonstrated that genes involved in the stabilization of mRNA secondary structures and membrane fluidity and upregulated
    • Different form prokaryotic studies is the discovery of an early and late response to cold-shock
  • Future Directions:
  1. Determine regulatory mechanisms involved in response to cold-shock
  2. Study why the early response is opposite of the response to other environmental stressors
  3. Study the effects these transcriptional changes have on membrane integrity
Critical Evaluation
The study presented in the paper supplied sufficient evidence to the claims they made, however I saw a need for more statistical evidence to be presented (either in the article itself or supplementary materials). Throughout the article, I saw the significance values presented only one or two times, which could bring into question the validation of their study. The figures were also very difficult to interpret without reading the rest of the article. Particulary figure one had a group (F) that was labeled in the figure, but never mentioned in the paper.

Acknowledgements

-Texted once to clarify aspects of the assignment and go over figure assigned
  • Copied syntax from the BIOL388/S19 Week 3 assignment page to link references
Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

References

Links

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