QLanners Week 12

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Deliverable

Antonio and Quinn Journal Club Presentation

Definitions of 10 Terms

  1. anneal: two complementary nucleic acids aligning in an opposing orientation to allow the nucleotide bases of one strand to form hydrogen bonds with the nucleotide bases of the complementary strand (GenScript, n.d.)
  2. hybridization: Mating of different species (or varieties) to create a hybrid offspring (Biology-Online, 2008)
  3. putative: an inference that is based on what is commonly believed, not direct proof/evidence (Biology-Online, 2014)
  4. cyclins: a particular family of regulatory proteins that are involved in the progression of the cell cycle (BioNinja, n.d.)
  5. assay: a scientific procedure to determine the amount of a particular constituent of a mixture or of the potency of a drug (Biology-Online, 2005)
  6. oligonucleotide: chain of ~13-25 nucleotides. Designs to hydrolize to specific DNA/RNA sequences and often used for research (News Medical Life Sciences, 2014)
  7. biplot: a way to visualize data that is an enhanced scatterplot with both points and vectors to represent structure (forrest.psych.unc.edu, 1999)
  8. transcriptome: the full range of mRNA molecules expressed by an organism or produced by a specific cell/tissue (Nature, n.d.)
  9. polypropylene: a plastic polymer of propylene (which has the molecular formula (C3H5)n) (Dictionary.com, n.d.)
  10. haploid: cell that contains only one set of chromosomes. produced from meiosis (Difference Between.com, 2011)
  11. diploid: cell that contains two sets of chromosomes - one maternal, one paternal (Difference Between.com, 2011)

Outline

Background Information

  • Saccharomyces cerevisiae used as model organism for research
  • Haploid Strain FY73 used in study
  • Studies have been done at growth temperature of 37◦ C and 39◦ C.
  • Upregulation of five genes in cold shock from 30◦ C to 10◦ C have been discovered.
  • This study looked at more severe heat and hold shock.

Methods

  • Four fractions of cells grown at 30◦ C
  • The fractions each experienced a different change in temperature
    • Fraction 1: 4◦ C for 180 minutes
    • Fraction 2: 45◦ C for 15 minutes
    • Fraction 3: 37◦ C for 30 minutes
    • Fraction 4: Half at 37◦ C and other half at 45◦ C for 15 minutes
  • After specified time interval, each fraction harvested and frozen in liquid nitrogen
  • RNA extracted through the use of a Micro-Dismembrator, mixing with TRI- ZOL Reagent, and the method of Chomczynski and Sacchi
  • Probe generation was done through the following steps
    • 60 μg total RNA from each fraction was annealed to oligonucleotide dT15
      • This was used as a template to radiolabel first strand
    • First strand cDNA radio labeled with 50 μCi of [α-33P]-dCTP using SuperScript II (reaction at 43◦ C for one hour)
    • RNA hydrolysed with NaOH (at 65◦ C for 30 minutes)
    • Probe purified with isopropanol
  • Filters prehybridized in hybridization mix (5× SSC, 5× Denhardt’s solution and 0.5% SDS)
  • Probe denatured at 100◦ C for five minutes
  • Probe hybridized with arrays overnight (65◦ C)
  • Washed two times (5 minutes and 20 minutes)
  • Filter regeneration
    • Poured a boiling solution of 5 mM sodium phosphate (pH 7.5) and 0.1% SDS over the filters
  • Filters exposed to storage phosphor screen for 24 hours
  • Data from filters collected using a Phos- phorImager Scanning Instrument 425
  • Data images analyzed using Array Vision software
  • For each tested temperature environment, data from 4 hybridizations were analysed, using two different arrays and two different RNA samples
    • Two replica-spots per gene on each array
    • Total of eight replica-spots per gene were analyzed
  • Statistical analysis carried out using MATLAB
    • Significance of variance classified as:
      • High significance (met the min-max separation criteria)
      • Medium significance (met the standard deviation separation criteria)
      • Low (the rest of the data)

Flowchart

Flowchart of Experimental Methods outlined in journal article

Results

  • Figure 1: Biplot obtained using a bipole algorithm called correspondence analysis. The different lines indicate the different fraction environments. The black dots all indicate intensity signals for individual genes. The closer a gene is to a particular colored line, the more correlated that gene's expression is with that line's condition. Furthermore, the positive and negative values on the axis indicate whether the expression of the gene was up- or down-regulated.
  • Table 1: Count of the number of genes whose expression level changed in each condition tested. Only genes whose expression changed to a high or medium statistical significance were counted.
  • Figure 2: A figure depicting the upregulation or downregulation of genes of various clusters. Grene indicated repressed while red indicated induced. The response of each gene for each heat stress environment (30>37, 30>45, 30>37>45) was included. There is also a more thorough breakdown of the genes included in the two primary gene clusters C and F.
  • Figure 3: A Venn diagram that depicts the number of genes upregulated in each of the environmental heat stress categories and the overlap between the upregulation of these genes.
  • Figure 4: A clustering of all of the genes upregulated in all of the heat shock environments and the cold shock environment. The intensity of the color depicts the intensity of the upregulation. There is also a legend to the right that summarizes the genes in the cluster that have promoter sequences known to have binding factors associated with other stress responses.

Findings from Results

  • Effects of temperature shock include both activation and repression on genes
  • More downregulated genes than upregulated genes. This finding is consistent with previous research from Gasch et al. (2000).
  • More gene's are associated with the heat shock than the cold shock
    • And the number of genes affected is related to the intensity of the heat shock (higher the temperature, the more genes affected)
  • The pre-adaptation of a lower temperature of heat shock results in less upregulated genes. However, this is not the same for downregulated genes.
    • Suggesting different regulatory circuits control the upregulated vs downregulated genes
  • There was a general repression of several genes with functions related to metabolism, cell growth and division, transcription, ribosomal proteins and protein synthesis and destination. This is in agreement with previous findings from other studies (Gasch et al., 2000; Causton et al., 2001).
    • This indicates that repression is a very general response to heat, that serves to slow down/stop cell growth and division and to use energy to overcome the heat stress
    • Most apparent in cluster C of figure 2
  • They couldn't find a regulatory signal common to the different promoters that are associated with the genes found to be repressed by heat shock.
    • Suggesting a more complex response which involves several complicated mechanisms
  • 31 promotors for genes repressed in cluster C and that are involved in cell growth and division contained "consensus sequence for Xbp1p binding". Xbp1p is a transcriptional repressor and has been found to be upregulated in stressful environmental conditions (Mai and Breeden, 1997)
  • There was major up-regulation of genes related to heat-shock proteins and lipid metabolism. This is consistent with the hypothesis that found lipid modifications to be important in resistance to heat and salt tolerance (Mahua et al., 2000).
    • This is found in Cluster F on Figure 2
  • Majority of genes that upregulated at 45◦C show no change if there is a pre-adaptation to 37◦C.
  • Results support the idea of common environmental response (CER) genes, which take part in a general response to very diverse stimuli (temperature, oxidation, nutrient availability, pH and osmolarity). The idea that these CER genes provide protection from a severe environmental condition by first experiencing pre-exposure to a less-severe environment is found in the results and consistent with previous hypotheses (Causton et al., 2001)
  • Response to cold shock effects fewer genes than the response to heat shock
  • Five genes (Zhang et al., 2001) and he TIP and TIR genes (Kondo and Inouye 1991; Kowalski et al., 1995; Donzeau et al., 1996) previously found to be associated with cold shock were not significantly impacted in this experiment (which had more severe cold shock conditions).
    • This could be because the extreme cold shock worked to slow down the very repression mechanisms that slow down these cold-repressed genes
    • Or could just be due to the strategies chosen for statistical analysis
  • The majority of genes upregulated in cold shock were also upregulated in heat shock, suggesting the genes in this cluster for general stress response (not specifically cold shock).

Acknowledgements

  1. I worked in class with my teammate Antonio to read and understand the findings of our assigned journal article. I also met with Antonio outside of class on Monday, November 20 to finish up the presentation. Furthermore, we plan to meet the day of the presentation to run through it together to ensure that we give a quality presentation to the class.
  2. I checked base with my teammates Simon and Eddie in class on Thursday to make sure our team page was filled out correctly, and also that they knew were to enter their information each week on the page. I also texted Eddie to work on making the fixes to our team page.
  3. Dr. Dahlquist for answering the plethora of questions I had regarding the format of the journal club presentation.

Qlanners (talk) 21:53, 20 November 2017 (PST)

References

  1. Anneal. (n.d.). Genscript. Retrieved on November 19, 2017 from https://www.genscript.com/molecular-biology-glossary/11896/anneal
  2. Assay.(2005). Biology-Online. Retrieved on November 19, 2017 http://www.biology-online.org/dictionary/Assay
  3. Becerra, M., Lombardia, L.J., Gonzalez-Siso, M.I., Rodriguez-Belmonte, E., Hauser, N.C., & Cerdan, M.E. (2003). Genome-wide analysis of the yeast transcriptome upon heat and cold shock. Comparative and Functional Genomics, 4(4), 366-375. doi: 10.1002/cfg.301
  4. Causton HC, Ren B, Koh SS, et al. 2001. Remodelling of yeast genome expression in response to environmental changes. Mol Biol Cell, 12: 323–337.
  5. Cyclins. (n.d.). BioNinja. Retrieved on November 19, 2017 from http://ib.bioninja.com.au/standard-level/topic-1-cell-biology/16-cell-division/cyclins.html
  6. Donzeau M, Bourdineaud JP, Lauquin GJ. 1996. Regulation by low temperatures and anaerobiosis of a yeast gene specifying a putative GPI-anchored plasma membrane protein. Mol Microbiol, 20: 449–459.
  7. Gasch AP, Spellman PT, Kao CM, et al. 2000. Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell, 11: 4241–4257.
  8. HAW. (2011). Difference Between Haploid and Diploid. Difference Between.com. Retrieved on November 19, 2017 from http://www.differencebetween.com/difference-between-haploid-and-vs-diploid/
  9. Hybridization. (2008). Biology-Online. Retrieved on November 19, 2017 from http://www.biology-online.org/dictionary/Hybridization
  10. Jung US, Levin DE. 1999. Genome-wide analysis of gene expression regulated by the yeast cell wall integrity-signalling pathway. Mol Microbiol, 34: 1049–1057.
  11. Kondo K, Inouye M. 1991. Cold-induction of yeast NSR1 protein and its role in pre-rRNA processing. J Biol Chem, 267: 16 259–16 265.
  12. Kowalski LR, Kondo K, Inouye M. 1995. Cold-shock induction of a family of TIP1-related proteins associated with the membrane in Saccharomyces cerevisiae. Mol Microbiol, 15: 341 – 353.
  13. Mai B, Breeden L. 1997. Xbp1, a stress-upregulated transcrip- tional repressor of the Saccharomyces cerevisae Swi4/Mbp1 family. Mol Cell Biol, 17: 6491–6501.
  14. Oligonucleotide. (2014). News Medical Life Sciences. Retrieved on November 19, 2017 from https://www.news-medical.net/life-sciences/What-is-an-Oligonucleotide.aspx
  15. Polypropylene. (n.d.). Dictionary.com. Retrieved on November 19, 2017 from http://www.dictionary.com/browse/polypropylene
  16. Putative. (2014). Biology-Online. Retrieved on November 19, 2017 from http://www.biology-online.org/dictionary/Putative
  17. Transcriptome. (n.d.). Nature. Retrieved on November 19, 2017 from https://www.nature.com/scitable/definition/transcriptome-296
  18. Young, F.W. (1999). Principle Components. forrest.psych.unc.edu. Retrieved on November 19, 2017 from http://forrest.psych.unc.edu/research/vista-frames/help/lecturenotes/lecture13/biplot.html
  19. Zhang L, Ohta A, Horiuchi H, Takagi M, Imai R. 2001. Multiple mechanisms regulate expression of low temperature responsive (LOT) genes in Saccharomyces cerevisiae. Biochem Biophys Res Commun, 283: 531 – 535.

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