Nika Vafadari Week 9

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Electronic Lab Notebook Week 9

Purpose

To understand the purpose and function of DNA microarrays and learn how to analyze and interpret the data.

Answers to Discovery Questions from Chapter 4

  • (Question 5, p. 110) Choose two genes from Figure 4.6b (PDF of figures on Brightspace) and draw a graph to represent the change in transcription over time. You can either create your plot in Excel and put the image up on your wiki page or you can do it by hand and upload a picture or scan.
  • Title: mRNA Expression Level Over Time
  • Fig. 1 The figure above compares mRNA production of genes DMC1 and SPS1 over time, where expression level is expressed by the plotting fold induction (a ratio that compares experimental activity to control activity) over time.
  • (Question 6b, p. 110) Look at Figure 4.7, which depicts the loss of oxygen over time and the transcriptional response of three genes. These data are the ratios of transcription for genes X, Y, and Z during the depletion of oxygen. Using the color scale from Figure 4.6, determine the color for each ratio in Figure 4.7b. (Use the nomenclature "bright green", "medium green", "dim green", "black", "dim red", "medium red", or "bright red" for your answers.)
  • Gene X
    • 1 hour= black
    • 3 hours= dim red
    • 5 hours= black
    • 9 hours= medium green
  • Gene Y
    • 1 hour= black
    • 3 hours= medium red
    • 5 hours= dim green
    • 9 hours= bright green
  • Gene Z
    • 1 hour= black
    • 3 hours=dim red
    • 5 hours= dim red
    • 9 hours= dim red


  • (Question 7, p. 110) Were any of the genes in Figure 4.7b transcribed similarly? If so, which ones were transcribed similarly to which ones?
  • Yes, X and Y look like they were transcribed similarly since they start at black, indicating no change, but then move to red and then green.


  • (Question 9, p. 118) Why would most spots be yellow at the first time point? I.e., what is the technical reason that spots show up as yellow - where does the yellow color come from? And, what would be the biological reason that the experiment resulted in most spots being yellow?
  • Yellow spots indicate which genes are transcribed in both conditions (by both transcriptomes). The yellow color itself comes from the overlap of the green and red spots, since green and red create the color yellow when mixed. Many of these yellow spots may appear at the first time point since it takes time for cells to adapt to various environmental conditions and change their gene expression in response. Therefore, an experiment would result in mostly yellow spots if the cell has not yet adjusted to the environment and has not changed its gene expression in response.


  • (Question 10, p. 118) Go to the Saccharomyces Genome Database and search for the gene TEF4; you will see it is involved in translation. Look at the time point labeled OD 3.7 in Figure 4.12, and find the TEF4 spot. Over the course of this experiment, was TEF4 induced or repressed? Hypothesize why TEF4’s change in expression was part of the cell’s response to a reduction in available glucose (i.e., the only available food).

  • The TEF4 spot at time point OD 3.7 is green indicated that the gene has been repressed over the course of the experiment. One reason for this change in expression could be the fact that TEF4 is involved in protein synthesis, which requires energy. Therefore, as glucose, the energy source for the cell is reduced, the cell may repress genes, such as TEF4, that are involved in energy costing activities, such as protein synthesis in order preserve energy.


*(Question, 11, p. 120) Why would TCA cycle genes be induced if the glucose supply is running out?

  • With a reduction in glucose, there is a loss of a source of energy, since the breakdown of glucose leads to the production of ATP. Therefore, the cell needs other methods of producing energy, such as the citric acid cycle. Therefore, the cell can utilize a secondary source of carbon in the place of glucose, such as ethanol, to create acetyl coA, which can be used in the TCA cycle. This cycle then leads to the production of NADH and FADH2, which are used to make ATP through oxidative phosphorylation as well as the production of GTP, which can also be converted into ATP. Therefore, TCA cycle genes may be induced in order to ensure that the TCA cycle produces these products.


*(Question 12, p. 120) What mechanism could the genome use to ensure genes for enzymes in a common pathway are induced or repressed simultaneously?

  • The genes could be clustered in a way that they are all regulated by the same transcription factors that either activate or repress gene expression.


*(Question 13, p. 121) Consider a microarray experiment where cells deleted for the repressor TUP1 were subjected to the same experiment of a timecourse of glucose depletion where cells at t0 (plenty of glucose available) are labeled green and cells at later timepoints (glucose depleted) are labeled red. What color would you expect the spots that represented glucose-repressed genes to be in the later time points of this experiment?

  • Since the repressor TUP1 plays a role in the repression of glucose repressed genes, if it is deleted I would expect to see red spots representing the glucose-repressed genes, since their expression will mostly likely be induced because they are not longer being repressed by the TUP1 repressor as they are in cells still containing the repressor.


*(Question 14, p. 121) Consider a microarray experiment where cells that overexpress the transcription factor Yap1p were subjected to the same experiment of a timecourse of glucose depletion where cells at t0 (plenty of glucose available) are labeled green and cells at later timepoints (glucose depleted) are labeled red. What color would you expect the spots that represented Yap1p target genes to be in the later time points of this experiment?

  • Since, Yap1p plays a role in cell resistance to environmental stresses, in the case of glucose depletion, I would expect to see red spots when Yap1p is over expressed since it would most likely induce various genes in order to resist/ respond to the environmental stress.


*(Question 16, p. 121) Using the microarray data, how could you verify that you had truly deleted TUP1 or overexpressed YAP1 in the experiments described in questions 8 and 9?

  • You would have to check the colors of the spots on the microarray data. For example if TUP1 was deleted the spot would be black, indicating that the gene has not been expressed, and if YAP1 was over expressed the spot would be red.

Acknowledgments

  • Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

References

  • Campbell, A.M. and Heyer, L.J. (2003), “Chapter 4: Basic Research with DNA Microarrays”, in Discovering Genomics, Proteomics, and Bioinformatics, Cold Spring Harbor Laboratory Press, pp. 107-124.
  • Dahlquist, Kam D. (2017) BIOL398-05/S17:Week 9. Retrieved from http://www.openwetware.org/wiki/BIOL398-05/S17:Week_9 on 20 March 2017.


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