Lenaolufson Week 7

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.
   • Will turn hard copy in at 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: black
     • 2 hour: dim red
     • 5 hour: black
     • medium green
   • Gene Y:
     • 1 hour: black
     • 3 hour: medium red
     • 5 hour: dim green
     • 9 hour: bright green
   • Gene Z:
     • 1 hour: black
     • 3 hour: dim red
     • 5 hour: dim red
     • 7 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?
   • The genes X and Y were transcribed similarly to one another as they both first experienced no change in their transcriptions during the first hour, but then displayed changes during the third hour. Afterwards, during the fifth hour both genes X and Y underwent a reduction in transcription followed by a further reduction in the ninth hour.
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 yellowness that appears on most spots initially is due to the red and green molecules that occur in equal amounts on the same dot. This is possibly a signal that the cells have not yet adjusted to the surrounding environmental conditions thus far. When the yellow color appears, it means that there is no change in gene expression at that particular spot for that particular gene.
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).
   • Upon finding the TEF4 spot, it appeared a green color, signaling that the gene had been repressed throughout the length of the experiment. From knowing that a TEF4 is a subunit of an elongation factor, which contributes to the ribosomal complex and consequently to protein synthesis, a hypothesis can be formed. If the amount of glucose that is readily available to a cell is reduced, then the protein synthesis genes would be repressed as well. When performing protein synthesis, the cell expends a great deal of energy and thus the cell would want to repress genes that use energy unnecessarily if the reliability of energy is unclear.
6. (Question, 11, p. 120) Why would TCA cycle genes be induced if the glucose supply is running out?
   • The TCA cycle genes are responsible for controlling the operation of the TCA cycle. If the glucose supply of a cell is running low, these TCA cycle genes would be turned on in order to make pyruvate shift to be a part of the TCA cycle as well as induce two key proteins to take control of glycolysis. By activating these genes, the efficiency of the TCA cycle can be maximized in generating useable energy for the cell during times of high stress such as low glucose levels.
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 mechanism that could be used by the genome to ensure that the genes for enzymes in a common pathway are induced or repressed simultaneously is the "Guilt by Association" method. By using this method, the genes might be in close proximity to one another and thus have similar promoters or transcription factors. Because of this, the genes that are expressed by similar means are able to activate other genes that are nearby (that also possess similar functions) to be expressed as well.
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?
   • From the given information, you would expect the spots that represented glucose-repressed genes to be red during later times of the experiment due to increased expression. If the repressor were to be deleted then the glucose-repressed genes would not be expected to be repressed as well, even if there is a lack of glucose present.
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?
   • From this information, you would expect the Yap1p target genes to be red at later points of the experiment due to the resistance to environmental stress that Yap1p provides. Even if the glucose levels decrease during the experiment, these target genes are still expected to be expressed if they are over expressed.
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?
    • You could verify the over expression of YAP1 in the above experiments if the dots on the YAP1 target genes appeared red, thus demonstrating that the genes were expressed even in the absence of glucose. You could verify the deletion of TUP1 in the above experiments if the spots appeared black and there was an absence of glucose, thus demonstrating that no change had occurred. Instances where glucose is absent but TUP1 is deleted, you would expect to see an increased expression of glucose-repressed genes, but if TUP1 is still present then these genes would be repressed.

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