Kzebrows Week 7

Questions from Campbell & Heyer, Chapter 4 (2003)

(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.)
- 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
(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 Y were transcribed similarly. They both experienced no change in transcription (along with Gene Z) in the first hour, but Genes X and Y were induced in the third hour (although Gene Y to a greater extent). Then, both experienced a reduction in transcription in the 5th hour and a greater reduction in the 9th hour. In contrast, Gene Z was continuously induced.
(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?
- Most spots are yellow at first because initially there are red and green molecules that occur in the same amounts on the same dot, meaning that perhaps the cells haven't adjusted to environmental conditions yet. The yellow color indicates that there is no change in gene expression at that spot/for that gene.
(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).
- The TEF4 spot is green, indicating that the gene was repressed over the course of the experiment. TEF4 is a subunit of an elongation factor that contributes to the ribosomal complex and therefore to protein synthesis; therefore, if the amount of glucose available to the cell is reduced, it makes sense that protein synthesis genes would be repressed. Protein synthesis is an energy-expensive process so the cell would want to repress genes that spend energy unnecessarily when it's unclear if more will be available soon.
(Question, 11, p. 120) Why would TCA cycle genes be induced if the glucose supply is running out?
- If glucose supply is running out, TCA cycle genes would be induced to cause pyruvate to shift into the TCA cycle and induce two proteins (phosphoenolpyruvate carboxykinase and fructose 1,6-biphosphatase) to control glycolysis. Inducing these genes will maximize how efficient the TCA cycle can be in providing usable energy for the cell in times of stress, which in this case is low glucose.
(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 use the mechanism known as the "Guilt by Association" method. In this situation, the genes might be clustered together and have similar promoters or transcription factors. That way, genes that are expressed in similar ways can induce nearby genes with similar functions to be expressed as well.
(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?
- You would expect to see the glucose-repressed genes to be red in later time points of the experiment, indicating increased expression. If the repressor is deleted then glucose-repressed genes would not be repressed even if there is no glucose.
(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 confers resistance to environmental stress, so at later time points of the experiment target genes of Yap1p would still be red. if these target genes are overexpressed they will still keep being expressed even if there is less glucose.
(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 that TUP1 was truly deleted if the spots were black and there was no glucose, which would indicate that no change had occurred. In situations of no glucose but deleted TUP1 there should be increased expression of glucose-repressed genes, but if there is still TUP1 then those genes would be repressed.
- For YAP1, you could verify that it was overexpressed if the dots for the YAP1 target genes were red, showing that they were expressed even if there was no glucose left (stress response).