Jkuroda Week 7

Discovery Questions

(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.

(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.)

(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, from what I have gathered, it looks like genes X and Y were transcribed similarly, because of the fact that their behavior follows a similar pattern over the time intervals.
(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 would show most spots as yellow because that color signifies that there has been no overall change in transcription. The yellow color is a result of a computer merging two scans; one from a green laser and the other from a red laser. The biological reason why most spots ended up as yellow is because the experiment did not test conditions that caused a major change in transcription, therefore resulting in many spots showing a net zero change.
(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 all of the scans at the various time points, it looks like TEF4 was repressed. I came to this conclusion because of the fact that the TEF4 spot gradually turned green over time, which could possibly be explained by the fact that glucose is slowly becoming less available. This would cause translation to slow or halt, in correspondence with the lack of energy available.
(Question, 11, p. 120) Why would TCA cycle genes be induced if the glucose supply is running out?
- According to Wikipedia, a TCA cycle is a "series of chemical reactions used by all aerobic organisms to generate energy through the oxidation of acetate derived from carbohydrates, fats and proteins into carbon dioxide and chemical energy in the form of adenosine triphosphate (ATP)." It looks like this cycle is implemented because of the fact that the cells detect the decreasing glucose supply, thus requiring them to start to store energy to survive the lack of food in the foreseeable future.
(Question 12, p. 120) What mechanism could the genome use to ensure genes for enzymes in a common pathway are induced or repressed simultaneously?
- It looks like genes can be clustered together in order to have a shared transcription factor, which would cause them to be induced or repressed simultaneously.
(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?
- According to the reading, TUP1 is "one protein responsible for the repression of glucose-repressed genes. Thus, a cell lacking TUP1 would not act any differently when glucose is taken away. Therefore, I would expect to see red spots since the cell would run out of glucose (glucose depleted).
(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?
- According to the reading, Yap1p "is a transcription factor known to confer resistance to environmental stresses such as hydrogen peroxide, heavy metals and osmotic shock." I would then expect the spots that represented Yap1p target genes to be red in the later time points because of the fact that Yap1p would be overexpressed. This would mean that the cell would have a greater resistance to the faltering conditions, and a glucose depleted state would be observed.
(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?
- If you look at the microarray and fail to find a spot that is red or green, then you have deleted the TUP1 and/or YAP1 in the experiments, since a yellow spot represents a net-zero change in expression.