Ntesfaio Week 6
Electronic Lab Notebook
Purpose
The purpose of this lab is to review DNA microarrays and analyze data points plotted as a result of DNA microarray methodology.
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.
File:Ntesfaio Week 6 Gene Comparison.pdf
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 Hours: Bright Red 5 Hours: Black 9 Hours: Bright Green
Gene Y: 1 Hour: Black 3 Hours: Bright Red 5 Hours: Black 9 Hours: Bright Green
Gene Z: 1 Hour: Black 3 Hours:Dim Red 5 Hours:Bright Red 9 Hours:Bright 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?
Genes X and Y were the most similar to one another. Genes X, Y, and Z were all black at 1 hour. After the one hour mark the genes differed.
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 spots may appear yellow because the spots are merged together. The reason spots show up as yellow is because there were merged microarray spots from red and green.
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 is repressed since it appears green. It's expression was a part of the cell's response to a reduction in available glucose because of the limited amount of resources. The cells repressed because of the inability to use glucose for ATP production.
6. (Question, 11, p. 120) Why would TCA cycle genes be induced if the glucose supply is running out?
The TCA cycle's total energy gained comes from a breakdown of a glucose molecule. TCA cycle genes would be induced when glucose levels are low because it will need to find another source of glucose. Phosphoenolpyruvate carboxykinase and fructose 1,6-biphosphatase are both induced. These two proteins control irreversible steps in glycolysis. This can come from stored glucose in the body or stimulating glycogen to turn into glucose. Cells can also respond to avoid starvation.
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 guilt by association method when an unknown gene is regulated the same way. The function of the unknown gene can be guessed by comparing to the known gene. This is done using microassays.
8. (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 kpoints (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?
Glucose-repressed genes would be red because the green cells have plenty of glucose available. Glucose-repressed genes increase and in later points appear more red.
9. (Question 14, p. 121) Consider a microarray experiment where cells that over express 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?
Yap1p is a conscription factor that is known to confer resistance to environmental stresses. It would repress more overtime, making a brighter green colored spot.
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?
Analyze the changes in gene expression based on spots. This can be done by looking at the color of TUP1 and Yap1p. Use the database explained under Discovery Questions and take note of the amount of variation in the replicated experiment. Determine if there is a significant impact on the cell when one repressor is deleted.
Ntesfaio (talk) 18:12, 9 October 2019 (PDT)
Acknowledgements
My homework partner this week was Jonar User:Jcowan4. Jonar and I exchanged numbers to keep in contact throughout this assignment.
Dr. Dahlquist for leading the instructions to this assignment
For the references section I copy and pasted all the readings that needed to be completed for this assignment. The readings were copied from the Week 6 page.
I also copy and pasted the questions that needed to be answered from the Week 6 assignment page.
"Except for what is noted above, this individual journal entry was completed by me and not copied from another source."
Ntesfaio (talk) 10:36, 7 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 7, 2019, from https://xmlpipedb.cs.lmu.edu/biodb/fall2019/index.php/Week_6
