Aporras1 Week 12

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User page: Antonio Porras

Team page: JASPAR the Friendly Ghost

Assignment page: Week 12 Assignment

Terms, Definitions, & Citations

1. YPD media: medium used for growing yeast. Made of of yeast extract, peptone, and glucose. It is put in an autoclave to ensure it's sterility. (University of Texas, n.d.)

2. Method of Chomczynski and Sacchi: "RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h" (Chomczynski, P., & Sacchi, N., 1987) Used for large amounts of samples and to isolate RNA from a small amount of cells.

3. Correspondence analysis: "Correspondence analysis is a statistical technique that provides a graphical representation of cross tabulations (which are also known as cross tabs, or contingency tables)" (Yelland, P. M., 2010). Useful when there are two or more categories.

4. Glutathione: "A peptide C10H17N3O6S that contains one amino acid residue each of glutamic acid, cysteine, and glycine..." (Merriam-Webster.com, n.d.) Often found in tissues of animals and plants as it has an essential role as a coenzyme of oxidation-reduction reactions within those tissues.

5. Consensus sequence: "A sequence of DNA having similar structure and function in different organisms" (Oxford Dictionaries, n.d.).

6. In silico: "...an expression used to mean “performed on computer or via computer simulation”" (mpkb.org, n.d.).

7. Cluster analysis: "Statistical classification technique in which cases, data, or objects (events, people, things, etc.) are sub-divided into groups (clusters) such that the items in a cluster are very similar (but not identical) to one another and very different from the items in other clusters" (BusinessDictionary.com, n.d.). Useful to observe or analyze patterns or relationships in data.

8. Haploid: "A cell (especially gametes or germ cells) containing half of the number of homologous chromosomes in somatic cells" (Biology-Online.org, n.d.).

9. Diploid: "...describes a cell that contain two copies of each chromosome" (Nature.com, n.d.).

10. Annealing: "..process of two strands of DNA rejoining" (Study.com, 2017).

Outline of Article

Background on Article

  • Used Saccharomyces cerevisiae as model for gene expression because of availability of the microorganism's genome.
    • Strain FY73 was used.
  • DNA microarrays used to observe transcriptional changes.
  • Exposed the cells to both heat shock and cold shock.
  • Had four different temperature conditions described in the methods.
  • Used specific conditions to observe a wider array of genes that were regulated.

Methods

  • Cells were first grown in YPD media with 2% glucose.
    • Temperature: 30◦C
  • Four fractions were given the following conditions:
    • Shifted down to 4◦C for 180 minutes
    • Shifted to 45◦C for 15 minutes
    • Shifted to 37◦C for 30 minutes
      • One remained at 37◦C
      • One shifted up to 45◦C for 15 minutes
  • Previous conditions/shifts before collecting were chosen based off of information from literature.
  • The harvested cells were put in liquid nitrogen.
  • The membranes were disrupted using a Micro-Dismembrator.
    • Then, it was mixed with TRIZOL.
    • Finally, the RNA was extracted by Chomczynski and Sacchi.
  • The probe was generated through the following steps/processes:
    • 60 μg of RNA was annealed to an oligonucleotide (dT15).
      • Used as a template to radiojlabel first strand.
    • Radiolabed with 50 μCi of [α-33P]-dCTP with the use of Superscript II.
      • Reaction was allowed to proceed for 1hr at 43◦C.
    • NaOH was used to hydrolyze the RNA for 30 minutes at 65◦C.
    • Purified with isopropanol.
  • Filters, with the use of hybridization mix, were prehybridized for one hour at 65◦C.
    • Mix contained: 5x SSC, 5x Denhardt's solution & 5% SDS.
  • Probe was allowed to denature at 100◦C for 5 minutes.
  • Probe was then cooled with ice and hybridized with arrays at 65◦C.
    • Hybridization was done overnight.
  • Two washes were completed at 65◦C:
    • 5 minutes and 20 minute washes in 2x SSC 0.1% SDS.
  • Boiling solution of 5mM NaPO4 and 0.1% SDS was poured over the filters to regenerate the filters.
  • Filters were allowed to be exposed to a phosphor screen for 24 hours.
  • Data was collected using a PhosphorImager Scanning Instrument 425.
    • The signal quantification was done using Array Vision software.
  • Four hybridizations were analyzed and two different samples of RNA.
    • Overall, eight replica slots were analyzed per gene.
  • M-CHIPS software was used to perform data quality assessment, normalization, and statistical analysis.
    • Written in MATLAB.
  • Correspondence analysis was performed.
  • Additionally, additional cluster analysis was performed to find co-regulated genes using GeneCluster and TreeView software.
  • RSA tools facilities was used to perform in silicon analysis of promoters.
    • Area up to -800 bp from ATG of each gene.

Flowchart

File:900pixels

Flowchart of methods and data analysis completed by article: Genome-wide analysis of the yeast transcriptome upon heat and cold shock.

Results

  • Figure 1: Biplot created with the use of correspondence analysis of the data by the different conditions of heat shock and cold shocks. The overwhelming black dots represent the individual genes. The colored lines represent the different conditions and their respective temperatures. The more positive values represent up regulation and the more negative values represent down regulation. The medians of the hybridizations are represented by the colored circles in the biplot.
  • Table 1: The number of genes which respond to the different conditions/changes. Important to note: only genes whose expression changes were high to medium significance were included.
  • Figure 2: Microarray data of clustering of genes which are regulated by heat shock where the green represents down regulation and the red represents up regulation. The figure further breaks down the genes into their respective major clusters.
  • Figure 3: Venn diagram which puts the ORFs up regulated during heat shock. The pre-adaptation was up to 37◦C. 31 of the 34 have between 1 and 12 copies of the two consensus sequences which haven't yet been identified.
  • Figure 4: Clustering of the genes which were up regulated by heat and cold shock. The higher intensity of the color corresponds with the regulatory ratio and the box provides the number of genes in the respective cluster.

Conclusions

  • The profiles in each of the four conditions are different and specific to each shock they were put under.
  • The effect that temperature shock has on gene expression is both upregulation and downregulation. However the amount of genes which are downregulated surpasses the amount of genes which are upregulated.
    • Conclusion in agreement with previous experiment (Gasch et al. 2000).
  • More genes are related to heat shock than cold shock.
    • Specifically in the heat shock, the number of genes which are affected is related to the level of heat shock/overall stress.
  • Pre-adaptation causes the cells to show less upregulation in genes.
  • Categories associated with being affected by down regulation are often:
    • Protein synthesis
      • Ribosomal proteins
    • Protein destination
    • Cell growth
    • Cell division
  • They were not able to find a common regulatory signal to the promoters of the genes which are down regulated after heat shock.
    • Downregulation could be an indirect result by protein degradation.
  • Cluster F was found to have 237 genes whose expression was unregulated by the heat shock.
    • Some proteins but importantly, lipids.
      • Supports the hypothesis that "lipid modifications are a major contributor to the induced heat and salt tolerance of yeast cells (Mahuaet al.,2000)".
  • There is a dependence on the level of heat shock the cells underwent associated with the number of unregulated genes.
  • Additionally its possible to find genes which are associated with general stress and not just temperature changes.
    • These genes are known as "CER" or common environmental response genes.
    • These genes could be associated with tolerance to changes as the cells treated with pre-adaptation had a double in the gene expression of these CER genes which could point to their association with tolerance or changes in temperature.
  • Genes which were upregulated during cold shock were also upregulated during heat shock which could ultimately suggest that these genes are for general stress response and not just cold shock or heat shock alone.

Deliverables

Journal Club Presentation: Media:Genome-wide_analysis_of_the_yeast_transcriptome_upon_heat_and_cold_shock._QL_AP.zip

Acknowledgements

  1. Met with Quinn Lanners outside of class to work on the presentation. We also texted each other regarding checkpoints in the completion of the assignment and worked in class alongside each other during the initial parts of the assignment.
  2. Emailed Dr. Dahlquist regarding formatting of the presentation slides and received additional in-class help from both Dr. Dahlquist and Dr. Dionisio.

While I worked with the people noted above, this individual journal entry was completed by me and not copied from another source.

Aporras1 (talk) 23:39, 20 November 2017 (PST)

References

  1. 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
  2. Chomczynski, P., & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical biochemistry, 162(1), 156-159.
  3. Cluster analysis. BusinessDictionary.com. Retrieved November 19, 2017, from BusinessDictionary.com website: http://www.businessdictionary.com/definition/cluster-analysis.html
  4. Consensus sequence | Definition of consensus sequence in English by Oxford Dictionaries. (n.d.). Retrieved November 20, 2017, from https://en.oxforddictionaries.com/definition/consensus_sequence
  5. Differences between in vitro, in vivo, and in silico studies. (n.d.). Retrieved November 20, 2017, from https://mpkb.org/home/patients/assessing_literature/in_vitro_studies
  6. Glutathione. (n.d.). Retrieved November 20, 2017, from https://www.merriam-webster.com/dictionary/glutathione
  7. Haploid. (n.d.). Retrieved November 20, 2017, from http://www.biology-online.org/dictionary/Haploid
  8. LMU BioDB 2017. (2017). Week 12. Retrieved November 16, 2017, from https://xmlpipedb.cs.lmu.edu/biodb/fall2017/index.php/Week_12
  9. (n.d.). Retrieved November 19, 2017, from http://fg.cns.utexas.edu/fg/protocol%3A_YPD_plates.html
  10. (n.d.). Retrieved November 20, 2017, from https://www.nature.com/scitable/definition/diploid-310
  11. Study.com. (2017). What is Annealing? - Definition, Biology & Process | Study.com. [online] Available at: http://study.com/academy/lesson/what-is-annealing-definition-biology-process.html [Accessed 21 Nov. 2017].
  12. Yelland, P. M. (2010). An introduction to correspondence analysis. Math. J, 12, 1-23.
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