Cameron M. Rehmani Seraji Week 9

Purpose

• The purpose of the week 9 assignment is to read the literature and understand the basics of DNA microarrys. The information from the readings will be used to answer the following questions related to Chapter 4 of Campbell & Heyer (2003).

Discovery Question Answers

(Question 5, p. 110) Choose two genes from Figure 4.6b (PDF of figures on Brightspace) 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 by hand and upload a picture or scan.

• The image of the graph can be found here

(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.)

Time    1 hour     3 hour	    5 hour	    9 hour
Gene X	 Black	 Medium Red	  Black	        Bright Green
Gene Y	 Black	 Bright Red	  Dim Greeen    Bright Green
Gene Z	 Black	 Dim Red	  Medium Red	 Medium 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. If you look at the table I generated in the previous question, you can see that they had the same colors at the different times. The one exception is at hour 3. At hour 3, transcription was induced slightly more for gene X than it was for gene Y.

(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 would be yellow at the first time point because the genes are not being overly induced or overly repressed. The yellow color suggests that the gene was evenly induced and repressed. The biological reason that the experiment resulted in most spots being yellow because the cells have not adjusted to adjust to the changing environment.

(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 color of the TEF4 gene is a dark green. This means that TEF4 was slightly repressed. As a result of there being less glucose available, the cell repressed the production of TEF4 to limit the amount of genes that break down glucose.

(Question, 11, p. 120) Why would TCA cycle genes be induced if the glucose supply is running out?

• The TCA cycle genes would be induced if the glucose supply is running out because the cell would use ethanol as a second source of carbon to continue to reproduce and compete with other cells.

(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 the genome could use similar repressor and activator transcription factors to sense the conditions of the external environment to help regulate the rate which genes are being repressed or induced.

(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?

• Compared to the mutant lacking glucose based repressor, I would expect there to be several more red spots showing that there is increased activity of the gene.

(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?

• At later time points of the experiments the color of the spots that represent Yap1p should be red because Yap1p codes for the resistance of environmental stress. This means that Yap1p would allow cells to continue expression at the same level or an increased rate even though there is a decrease in the amount of glucose available.

(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?

• Using microarray data, the deletion of TUP1 or overexpressed YAP1 in the experiments could be verified by making sure that the dot that represents TUP1 is black because the gene was deleted and should not be repressed or induced. For the YAP1 gene, the dot that represents it should be bright red because it is being induced.

Acknowledgments

• I worked with Lauren M. Kelly in Seaver 120 on Wednesday, March 22, 2017.
• Except for what is noted above, this individual journal entry was completed by me and not copied from another source
• Cameron M. Rehmani Seraji 20:26, 22 March 2017 (EDT):

References

• Campbell, A.M. and Heyer, L.J. (2003), “Chapter 4: Basic Research with DNA Microarrays”, in Discovering Genomics, Proteomics, and Bioinformatics, Cold Spring Harbor Laboratory Press, pp. 107-124.

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