Malverso Week 7

Introduction to DNA Microarrays

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.
   • I estimated the values based on figure 4.6b and then created a graph using Excel. The chnage in transcription is found by computing the ratio of fold repressed:fold induced. (See graph above)
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.)
   • I revisited the textbook to see that a number above 1 was tinted red, and below was tinted green.
   • GENE X: 1 hour - black; 3 hour - dim red; 5 hour - black; 9 hour - medium green;
   • GENE Y: 1 hour - black; 3 hour - medium red; 5 hour - black; 9 hour - bright green;
   • GENE Z: 1 hour - black; 3 hour - dim red; 5 hour - dim red; 9 hour - 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?
   • Gene x and gene y were transcribed similarly. This is apparent because their colors (and therefore ratios) were similar.
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 technical reason is that there is an equal ratio of green dye to red dye, which when mixed equally create a yellow color.
   • The biological reason that the experiment resulted in most genes being yellow is that not many of the genes changed (either induced or repressed) because of the conditions that the experiment imposed on the DNA slides.
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).
   • I went to the database and saw that TEF4 is a "translational elongation factor" and also "stimulates the binding of aminoacyl-tRNA (AA-tRNA) to ribosomes by releasing eEF1A (Tef1p/Tef2p) from the ribosomal complex."
   • Over the course of the experiment, TEF4 was repressed (transcribed less) because the dot on the figure corresponding to TEF4 is a bright green.
   • This may be because the reduction in available glucose limited the amount of energy the yeast cells had and as the textbook noted, the cells sensed that they were beginning to starve. A reduction in this gene signifies the cell going into somewhat of a "low-power mode" where the cell did as little activity as possible in order to conserve energy. This is supported by the textbook when it says that "genes involved in protein synthesis were coordinately repressed"(118).
6. (Question, 11, p. 120) Why would TCA cycle genes be induced if the glucose supply is running out?
   • TCA cycle genes are induced because while the glucose is running out, the yeast cells are trying to figure out where else they can get energy from and also how they can store their energy for later. The cells are preparing for the depletion of their energy source.
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?
   • The genome could induce a transcription factor. That transcription factor would then induce a few genes simultaneously. So by inducing the transcription factor, the genome is basically inducing related genes.
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?
   • TUP1 is responsible for the repression of glucose-repressed genes , according the the textbook on page 121.Therefore I would expect the spots to be red, because there would be no glucose and no TUP1 to repress those genes anymore.
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?
   • Yap1p is a "transcription factor known to confer resistance to environmental stresses" (121). This factor would most likely cause the Yap1 to be induced because the reduction in glucose is an environmental stress. Therefore I would expect the color to be red.
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?
    • I would have to compute the red:green ratio by dividing red dye / green dye, and the amount of red vs green dye would be found by using photography and computers to quantify the amount of light. Of course, if TUP1 was deleted totally, no light would show up at all.

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