Introduction to Microarrays

Questions to Answer

The following questions are related to Chapter 4 of Campbell & Heyer (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.)
(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 had a similar transcription pattern, but they were not identical. Both genes showed induction in their earlier time points and repression at later time points, but the hour and magnitude of this change differed. Gene X indicated an induction at hour 3, and repression at hour 9. Gene 9 indicated an induction at hour 4, but with a higher magnitude than gene X, and repression at both hour 5 and hour 9, where the repression at hour 9 was of greater magnitude than that of gene X.
(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?
- A yellow spot on a merged microarray microarray image indicates a red:green (activation:repression) ratio of 1:1. This means that there was no difference between the gene's transcription in control and experimental conditions. Most spots in the first time point are yellow because at this point, there has been no change between the gene transcription in control and experimental conditions. The biological reason that this experiment resulted in most spots being yellow is because the conditions invoked onto the yeast only affected a small umber of genes in terms of their levels of transcription. All other genes that maintained their same level of transcription appeared as yellow dots.
(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).
- TEF4 is a translation elongation factor that specifically helps AA-tRNA bind to the ribosome. Over the course of this experiment, TEF4 was repressed, as indicated it's spot appearing greenish in Figure 4.12. This change in expression may have been part of the cell's response to a reduction in available glucose because glucose is broken down to be used as energy by the cell, and generally, cellular processes, including protein synthesis, require energy. With a reduction of energy, there may have been less protein synthesis, and therefore less translation requiring the cell to use less TEF4, and therefore transcribe less TEF4.
(Question, 11, p. 120) Why would TCA cycle genes be induced if the glucose supply is running out?
- TCA would be induced if the glucose supply is running out because the TCA cycle is part of cellular respiration, and through the process of cellular respiration, glucose is converted into ATP which acts as chemical energy for the cell. The TCA cycle takes the acetyl-CoA from glycolysis and uses it to create some ATP but more NADH which is used as an electron carrier in oxidative phosphorylation, which creates the most ATP out of every process in cellular respiration. The TCA cycle is induced in order to allow oxidative phosphorylation to create more ATP for the body in situations of glucose shortage to allow the body to continue it's processes for a while longer until glucose levels are repleted.
(Question 12, p. 120) What mechanism could the genome use to ensure genes for enzymes in a common pathway are induced or repressed simultaneously?
- If all genes that encode for enzymes in a common pathway were dependent on the same promoters, the genome could either increase or decrease the abundance of that promoter in order to induce or repress the transcription of the genes that ultimately influence the pathway via the enzyme they create 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?
- TUP1 is among other genes that are responsible for repression of glucose-repressed genes. Therefore, with the deletion of TUP1, the spots that represented glucose-repressed genes would be red at later time points because there would be no glucose at later time points to repress the genes, nor would there be any TUP1 to repress the glucose-repressed genes.
(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 that grants resistance to environmental stressors. An example of this environmental stress could be glucose depletion. At later times when the glucose has been depleted, the environmental stress in the cell increases, so overexpression of YAP1p occurs. This is an activation response, so the spots that represent YAP1p would be red at later time points.
(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?
- We can verify that TUP1 has been deleted by confirming that the spots that represent TUP1 are completely green. This indicates complete repression in the experimental conditions, which makes sense because complete repression is comparable to deleting the gene altogether. YAP1p would be red because this indicates induction in the experimental condition.

Anuvarsh Week 7

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