Rlegaspi Week 7

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Introduction to DNA Microarrays

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Individual Journal Assignment

Answer the following questions related to Chapter 4 of Campbell & Heyer (2003). Note that some of the questions below have been reworded from the Discovery Questions in the book:

  1. (Question 5, p. 110) Choose two genes from Figure 4.6b (PDF of figures on MyLMUConnect) 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 in hard copy and turn it in in class.
  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
      • 1 hour: 1.0 = black
      • 3 hours: 2.2 = dim red
      • 5 hours: 1.0 = black
      • 9 hours: 0.15 = medium green
    • Gene Y
      • 1 hour: 1.0 = black
      • 3 hours: 4.5 = medium red
      • 5 hours: 0.95 = dim green
      • 9 hours: 0.05 = bright green
    • Gene Z
      • 1 hour: 1.0 = black
      • 3 hours: 1.5 = dim red
      • 5 hours: 2.0 = dim red
      • 9 hours: 2.0 = dim 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?
    • Genes X and Y show a similar transcription pattern. Firstly, both start with a black color at the 1 hour mark, then show a red color (induced) at the 3 hour mark, then a black color at the 5 hour mark (repression), and finally a green color at the 9 hour mark (further repression).
  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 spots show mostly a yellow color at the first time point because cells have not reacted to the environmental change (i.e. available oxygen). More time is needed to determine if transcription was induced or repressed; therefore, no change is indicated by the yellow color.
  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).
    • Looking at Figure 4.12 and observing the TEF4 spot over the course of this experiment, I would conclude that TEF4 is being repressed since the TEF4 spot began at a yellow color that eventually turns to a bright and bright green color. TEF4 is responsible for stimulating the binding of aminoacyl-tRNA (AA-tRNA) to ribosomes' (from Saccharomyces Genome Database). With the consumption of glucose over time and the increase of cell density, I would hypothesize that the cell is conserving energy for the expression of other genes. Within the slide of OD 3.7, an observation of GLK1 shows a red color meaning that this gene is being induced; therefore, the repression of TEF4 may give way to the induction of other genes within the Saccharomyces cell.
  6. (Question, 11, p. 120) Why would TCA cycle genes be induced if the glucose supply is running out?
    • TCA cycle genes would be induced if the glucose supply is running out because the cell is in need of glucose to run other cellular activity; therefore, the TCA cycle would produce oxaloacetate that can be converted into glucose through a metabolic pathway called gluconeogenesis.
  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 induced or repressed simultaneously, the genome could use same transcription factors for all the genes within that 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 time course of glucose depletion where cells at t0 (plenty of glucose available) are labeled green and cells at later time points (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?
    • The spots of the glucose-repressed genes would be of a red color (induced) in the later time points of the experiment.
  9. (Question 14, p. 121) Consider a microarray experiment where cells that over-express 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?
    • The spots that represented Yap1p target genes would be of a red color (induced) in the later time points of the experiment.
  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 the microarray data, you could very that you truly deleted TUP1 because of the presence of black spots of the TUP1 gene (since it would be deleted, no change in transcription throughout the duration of the experiment); on the other hand, you can very the over-expressed YAP1 through the presence of bright red spots.

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Ron Legaspi
BIOL 367, Fall 2015

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