Nicolekalcic Week 6

Journal Week 6 - Nicole Kalcic

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Introduction to DNA Microarrays

1. (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. 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.
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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: hour 1 is black, hour 3 is dim red, hour 5 is black, hour 9 is bright green,
   • Gene Y: hour 1 is black, hour 3 is dim red, hour 5 is dim green, hour 9 is bright green,
   • Gene Z: hour 1 is black, hour 3 is medium red, hour 5 is dim red, hour 9 is medium 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?
   • Gene X and gene Y were transcribed similarly because they both began a similar induction in hour 3, reduced back to a near equal ratio during hour 5, then reduced further.
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?
   • Spots are yellow at the first point because the sets have the same promoters. These spots contain both red and green (mixed together: yellow). If the experiment resulted in most spots being yellow, it must be because the genes have not been in the environment long enough to be affected by the changes that can be brought on by the changes in the environment.
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).
   • TEF4 was repressed throughout this experiment because glucose is needed for the translation process, and it did not have access to high amounts of glucose in this environment.
6. (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 to use as energy in order to preserve remaining glucose.
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 same transcription factor could be used for all of the genes in a common pathway so that enzymes 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?
   • Gene repression would not occur, because of the lack of TUP1. They would be present during the whole experiment. Glucose depleted cells are labeled red, meaning these cells would be red.
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?
   • YAP1 acts on the genes resisting environmental stresses. The depletion of glucose would not affect the cell at the same level that YAP1 affects the cell when it is over-expressed. I believe the spots would stay green because of the aid of YAP1 (despite glucose depletion).
10. (Question 16, p. 121) Using the microarray data, how could you verify that you had truly deleted TUP1 or over expressed YAP1 in the experiments described in questions 8 and 9?
    • To verify, you could use a control cell with normal levels of TUP1 and YAP1 to make comparisons. Spots on the TUP1 genes should be red when there is glucose if the TUP1 is deleted. Spots on the YAP1 genes would be brighter red as the glucose diminishes if the YAP1 is over expressed.

Acknowledgements

• I communicated with my partner Katie Wright about the assignment, for any questions that I had.

While I worked with the people noted above, this individual journal entry was completed by me and not copied from another source.

Nicolekalcic (talk) 13:41, 9 October 2017 (PDT)

References

• Alberts et al. (2002) Molecular Biology of the Cell, Ch. 8: Microarrays
• Brown, P.O. & Botstein, D. (1999) Exploring the new world of the genome with DNA microarrays Nature Genetics 21: 33-37.
• 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.
• DeRisi, J.L., Iyer, V.R., and Brown, P.O. (1997) Exploring the Metabolic and Genetic Control of Gene Expression on a Genomic Scale. Science 278: 680-686.
• LMU BioDB 2017. (2017). Week 6. Retrieved October 9, 2017, from https://xmlpipedb.cs.lmu.edu/biodb/fall2017/index.php/Week_6