Aporras1 Week 6
Discovery Questions
1. Choose two genes from Figure 4.6b (PDF of figures on Brightspace) and draw a graph to represent the change in transcription over time. Create your plot in Excel (or other program that can do plots) and display the image up on your wiki page. Alternately, you can do it by hand, scan or take a photo of the plot, and display the image on your wiki page.
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.)
Given the ratio of red experimental mRNA to green experimental mRNA, repression or induction would be determined by which is greatest. If red is greater, resulting in a value > 1.0, it is an induction (red). If green is greater, resulting in a value < 1.0, it is a repression (green). If they are equal, in which the ratio is 1:1, the color would be black. Below is a table of the genes and their respective colors from Figure 4.7.
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?
From Figure 4.7b, genes X and Y were transcribed similarly because of their similar induction in hour 3 (ratios 2.2 & 4.5), close to equal ratio of red:green in hour 5 (ratios 1.0 & 0.95), and repression during hour 9 (ratios 0.15 & 0.05)
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?
At the first time point, most of the spots would be yellow because there hasn't been any change either towards induction or repression. Therefore the ratio of green to red dye in cells is relatively equal to begin. The actual reason why the spot is yellow is because when you combine red and green you get yellow. Biologically, if the experiment resulted in most spots being yellow this would infer genes were neither suppressed or induced in the experiment and genes were relatively unchanged in expression.
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).
Over the course of the experiment, TEF4 was repressed because it appears to be very light green (according to my non-colorblind friend). If the cell was responding to a reduction in available glucose, translation would be affected because glucose acts as a signaling molecule in Saccharomyces' pathways involving translation and thus, gene expression. Without glucose, S. cerevisiae wouldn't be able to use pathways which require glucose as a signaling molecule. If translation of mRNA to proteins isn't occurring, therefore the TEF4 gene will be repressed because of its involvement in translation.
6. (Question, 11, p. 120) Why would TCA cycle genes be induced if the glucose supply is running out?
TCA cycle, also known as Kreb's cycle, genes would be induced because those genes are associated with energy metabolism which are induced as glucose is consumed. On the other hand, protein production genes are repressed during consumption of glucose. The primary reason for the induction of these genes is because of the increase of acetyl-COA entering the cycle from alcohol dehydrogenase.
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 genome could use common promoters or transcription factors to ensure genes for enzymes in a common pathway are induced or repressed simultaneously.
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?
TUP1 acts as a repressor for glucose-repressed genes which are repressed by the presence of glucose. Without TUP1, these genes wouldn't be repressed by the presence of glucose and would continue transcription and thus produce red colored spots.
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?
One would expect these spots to be red because as the glucose levels are depleted, thus causing stress to the cell, Yap1p would confer resistance. In order for it to confer resistance, it would induce high levels of transcription during the time of stress.
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 perform the same experiment with glucose depletion and compare that to a normal microarray of TUP1 and Yap1p. You would specifically look at the target genes of these transcription factors and the dots would be red in the presence of glucose thus confirming TUP1 deletion. On the other hand, one would expect the dots of targets genes of Yap1p to be bright red as glucose concentration decreased to confirm its overexpression.
Acknowledgements
- Met outside of class with Eddie Bachoura to discuss any questions we had prior to meeting and throughout the process of completing the Week 6 assignment.
- I contacted Dr. Dionisio and Dr. Dalquist through email to further explain a question I had on the assignment.
While I worked with the people noted above, this individual journal entry was completed by me and not copied from another source.
Aporras1 (talk) 16:22, 8 October 2017 (PDT)
References
- Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. Studying Gene Expression and Function. Available from: https://www.ncbi.nlm.nih.gov/books/NBK26818/
- Ashe, M. P., De Long, S. K., & Sachs, A. B. (2000). Glucose Depletion Rapidly Inhibits Translation Initiation in Yeast. Molecular Biology of the Cell, 11(3), 833–848.
- Brown, P. O., & Botstein, D. (1999). Exploring the new world of the genome with DNA microarrays. Nature genetics, 21.
- Campbell, A. M., Heyer, L. J., & Laurie, J. H. (2007). Discovering genomics, proteomics, and bioinformatics (No. 04; QH447, C3 2007.).
- DeRisi, J. L., Iyer, V. R., & Brown, P. O. (1997). Exploring the metabolic and genetic control of gene expression on a genomic scale. Science, 278(5338), 680-686.
- LMU BioDB 2017. (2017). Week 5. Retrieved October 09, 2017, from https://xmlpipedb.cs.lmu.edu/biodb/fall2017/index.php/Week_6
