Aporras1 Week 12

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User page: Antonio Porras

Team page: JASPAR the Friendly Ghost

Assignment page: Week 12 Assignment

Presentation Prep

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

  1. Write an outline of the article. The length should be a minimum of the equivalent of 2 pages of standard 8 1/2 by 11 inch paper (you can use the "Print Preview" option in your browser at 100% scale to see the length). Your outline can be in any form you choose, but you should utilize the wiki syntax of headers and either numbered or bulleted lists to create it. The text of the outline does not have to be complete sentences, but it should answer the questions listed below and have enough information so that others can follow it. However, your outline should be in YOUR OWN WORDS, not copied straight from the article.
    • What is the importance or significance of this work?
    • Briefly state the result shown in each of the figures and tables.
    • How do the results of this study compare to the results of previous studies (See Discussion).

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.

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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