Nstojan1 Week 5

Purpose

• The purpose of this lab is to learn about how microarray experiments work and how to solve problems involving microarray analysis problems as well as learn abut research ethics.

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.
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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 - 1 hour: Black, 3 hour - medium red, 5 hour - black, 9 hour - dim green
   • Gene Y - 1 hour: black, 3 hour: bright red, 5 hour: black, 9 hour: dim green
   • Gene Z - 1 hour: black, 3 hour: dim red, 5 hour - medium red, 9 hour- 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?

1. • I think that Gene X and Gene Y are transcribed similarly because the only difference is that at hour 3, Gene X is dim red and Gene Z is bright red. All of the Genes start the same with Black at hour 1 but then Gene Z remains red.
2. (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?
   • Some of the spots are yellow because the microarrays have a color scale that runs from red to green. When they are merged together they appear yellow because the system reads it as one color since its hard to separate, therefore, the yellow spots are created.
3. (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 TEF4 gene is green which indicates that it is being repressed. The change in expression that's seen in TEF4 is due to the reduction because TEF4 is involved in elongation. This alters the genes response mechanism and as a result it can’t adequately produce glucose which results in a reliance on stored energy.
4. (Question, 11, p. 120) Why would TCA cycle genes be induced if the glucose supply is running out?
   • TCA genes would be induced if the glucose supply is running out because the genes need to find another energy source in order to produce ATP and other cellular functions. When TCA genes are induced it allows them to use different substrates to produce energy which ensures they are able to survive on a low glucose supply.
5. (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 gene could use is known as the ‘guilt by association’. This derives the function of genes based on their expression patterns. This method assumes that genes with similar expression profiles share similar promoters and vice versa. Using this method, it can allow researchers to see the functions and mechanisms of a gene.
6. (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?
   • There would be spots changing from green to red over time to show that the glucose is being depleted over time.
7. (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?
   • The spots would be also red at later points of the experiment with the Yap1p gene. This is because the Yap1p gene is a transcription factor and it activates inc response to different stressors such as glucose depletion. In this case, as the glucose is depleted, Yap1p activity increases resulting in high intensity red meaning that the gene is overexpressed.
8. (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?
   • I could verify that the TUP1 gene is truly deleted by doing an analysis to see how the changes of gene expression throughout the experiment. If there were changes, the sports would represent these changes. Similarly, for the Yap1p gene, I could do an analysis to see the changes in gene expression. They could be compared to controls and if the gene is overexpressed there would be a lot more red spots.

Acknowledgments

• I worked with User:Asandle1, we spoke over text about some of the problems.
• I consulted with User:kdahlquist about accessing the work and troubleshooted issues with my Wiki.
• "Except for what is noted above, this individual journal entry was completed by me and not copied from another source"Nstojan1-new (talk) 15:36, 16 February 2024 (PST)

References

• LMU BioDB 2024. (2024). Week 5. Retrieved February 16, 2024. https://xmlpipedb.cs.lmu.edu/biodb/spring2024/index.php/Week_5
• 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.