Week 6 Individual Journal

Purpose

The purpose of this assignment is understand how DNA microarray assays are performed and how to interpret the data compiled from them.

Week 6 Assignment

Questions

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. 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.

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
  • 1 Hr: YELLOW
  • 3 Hr:BRIGHT RED
  • 5 Hr: YELLOW
  • 9 Hr:DIM GREEN
• Gene Y
  • 1 Hr: YELLOW
  • 3 Hr: BRIGHT RED
  • 5 Hr: DIM GREEN
  • 9 Hr: BRIGHT GREEN
• Gene Z
  • 1 Hr: YELLOW
  • 3 Hr: MEDIUM RED
  • 5 Hr: BRIGHT RED
  • 9 Hr: DIM 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. The ratio of their transcription was the same at the first hour, peaked after the third hour, and descending with a similar negative slope in the last couple hours.

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?

The yellow coloration at the first time point occurs because the glucose deprived cells have not yet acclimated to the lack of glucose and thus are operating in a similar manner as the control, expressing similar genes. The yellow color is an indication that the genes in question are being transcribed in both transcriptomes. In other words, the cells are operating in a biologically similar manner. The yellow color comes from the presence of both the green and red fluorescent tags from both transcriptomes in each spot which merge together to create a yellow dye.

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).

The green coloration of the TEF4 spot indicates that the gene was repressed during the experiment. It could be possible that given the lack of available nutrients, the yeast cells repressed the expression of certain genes in order to minimize metabolic activity that requires the ATP produced by the oxidation of glucose.

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

The induction of the TCA cycle genes during glucose deprivation may be caused by the cells relying on other metabolic sources of carbons since their glucose supply is nearly depleted. In response to such starved conditions, cells can metabolize other biomolecules such as the hydrocarbons of fatty acids and triglycerides or the carbon backbone of amino acids into acetly-CoA to keep the TCA cyle running.

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 yeast cells can use different gene regulatory pathways in order to simultaneously control the expression of similar genes. Through the use of the negative and positive feedback loops, such cells can activate or inhibit certain transcription factors necessary to induce or repress genes near certain promoter regions.

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?

The glucose repressed genes should be red since the TUP1 gene suppresses the expression of the glucose repressed genes during glucose deprivation. If TUP1 was deleted, then those genes would be expressed and emit a red color.

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?

Since Yap1p is associated with resistance to environmental stressors, the overexpression of the Yap1p gene can be expected to cause the spots representing the target genes of Yap1p to become red since their expression will increase as well.

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?

In order to verify whether Tup1 had been deleted or whether Yap1 had been overexpressed, one can either use a microarray experiment and locate the spots associated with both genes. If the mutated Tup1 appears green, then its expression has been reduced, and if the mutated Yap1 appears red, then its expression has been induced. Similarly, one could also observe the spots associated with the target genes of the mutated Tup1 and Yap1. Since Tup1 is a repressor, it can be expected that its deletion would induce the expression of its target genes, and since Yap1 is a transcription factor, its overexpression would also be expected to induce the expression of its target genes.

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Acknowledgements

Dr. Kam Dahlquist; Professor

John Nimmers-Minor; Homework Partner

Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

Ymesfin (talk) 20:45, 9 October 2019 (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 2019. (2019). Week 6. Retrieved October 10, 2019, from https://xmlpipedb.cs.lmu.edu/biodb/fall2019/index.php/Week_6

Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

Ymesfin (talk) 18:32, 6 October 2019 (PDT)