Moneil5 Week 9

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Margaret J. ONeil

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Purpose

The purpose of this week's assignment is to better understand where gene microarray data comes from and what the data means. This is so that in future weeks when we run models and calculate statistics for data obtained from microarrays, we know what the raw data means, and where it is coming from.

Workflow/Methods

  1. (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.
    File:MO.Week9.Question1.tif
  2. (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
      • Hour 1: black
      • Hour 3: medium red
      • Hour 5: black
      • Hour 9: bright green
    • Gene Y
      • Hour 1: black
      • Hour 3: bright red
      • Hour 5: dim green
      • Hour 9: bright green
    • Gene Z
      • Hour 1: black
      • Hour 3: dim red
      • Hour 5: medium red
      • Hour 9: medium red
  3. (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?
    Based on the data in figure 4.7b it does appear as though genes X and Y are transcribed despite the large differences in magnitude of expression. Gene x and gene Y follow similar trends of activation and repression over time, however gene Y is activated almost 2 fold greater than gene X at 3 hours.
  4. (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?
    The first time point of expression shows most spots as being yellow because not enough time has passed between the start of the experiment and the time point (no time has passed) meaning that there hasn't been time yet for most genes to have been activated or repressed. Most are set at their typical expression levels with no change seen until the next time point. Also on a more specific experiment, given the large number of genes in an organism such as yeast, only so many out of the 6000+ genes will be activated or repressed due to a given environmental stress.
  5. (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).
    Based on the expression data from Figure 4.12 it seems as though over the course of the experiment, TEF4 has been repressed. Based on the Saccharomyces Genome Database entry for TEF4, TEF4 plays a role in respiration and the breakdown of glucose, so in the presence of limited glucose the gene is repressed because the protein that it transcibes is not as needed in the absence of glucose.
  6. (Question, 11, p. 120) Why would TCA cycle genes be induced if the glucose supply is running out?
    TCA cycle genes would be activated if glucose supply was running out because the yeast cells would have to turn to another carbon source, in this case ethanol, in order to continue the cell cycle and be competitive with other yeast cells.
  7. (Question 12, p. 120) What mechanism could the genome use to ensure genes for enzymes in a common pathway are induced or repressed simultaneously?
    To ensure genes for enzymes in a common pathway are activated or repressed simultaneously, the genome might have a single activating or repressing transcription factor for that cluster of genes. This would allow for a single gene to be controlling the activation or repression of a common pathway.
  8. (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?
    I would expect the glucose-repressed genes to show up as red, or induced/activated over the course of the experiment. TUP1 being deleted would mean that the repressor gene is deleted, which in conjunction with the glucose decrease would mean those genes repressed by glucose are activated over time.
  9. (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?
    I would expect red spots because YAP1 codes for resistance to environmental stresses, and when YAP1 is overexpressed, it will over-activate it's target genes as well.
  10. (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?
    Using microarray data, you should expect the deletion strain of TUP1 to have a black spot where TUP1 is supposed to be since it is not present in the genome. As for YAP1, you should expect to see a very red dot at the YAP1 spot indicating over-expression of the gene.

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.

TEF4. (n.d.). Retrieved March 22, 2017, from http://www.yeastgenome.org/locus/S000001564/overview

Acknowledgments

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

Margaret J. Oneil 23:22, 22 March 2017 (EDT)