Mavila9 Week 6
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Purpose
This investigation serves to further understanding of DNA microarrays and their use in research.
Questions
(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.
(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 | gene Y | gene Z | |
|---|---|---|---|
| hour 1 | black | black | black |
| hour 3 | medium red | bright red | dim red |
| hour 5 | black | dim green | medium red |
| hour 9 | medium green | bright green | 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 gene Y were transcribed similarly.
(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 initially show up yellow because the associated genes are neither strongly repressed or strongly expressed.
(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 because as glucose became scarce the cell reduced expression of this gene to not waste energy.
(Question, 11, p. 120) Why would TCA cycle genes be induced if the glucose supply is running out?
- TCA cycle genes are induced to promote the breakdown of acetly-coenzyme A to produce energy.
(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 genes could be put close together after the same promoter sequence so that induction or repression for the different genes are the same and simultaneous.
(Question 13, p. 121) Consider a microarray experiment where cells deleted for the repressor TUP1 were subjected to the same experiment of a time course of glucose depletion where cells at t0 (plenty of glucose available) are labeled green and cells at later time points (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 spots that represent glucose-repressed genes would be red in the later time points because glucose would be depleted, therefore no glucose would repress the genes.
(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 time points (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 that represent Yap1p gene would be red because it is overexpressed and would therefore be expressed despite glucose depletion.
(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?
- To confirm that TUP1 was deleted the spots representing it should be green with or without presence of glucose and to confirm overexpression of YAP1 the spots representing it should be red with or without sufficient glucose.
Acknowledgments
I would like to thank DeLisa Madere for helping me with this assignment. I would also like to thank Dr. Dahlquist for providing the materials along with helping me complete the assignmnet. I would also like to thank Campbell and Heyer for their work on Basic Research with DNA Microarrays.
References
LMU BioDB 2019. (2019). Week 6. Retrieved October 8, 2019, from https://xmlpipedb.cs.lmu.edu/biodb/fall2019/index.php/Week_6
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.
