Msamdars Week 6

Electronic Lab Notebook

Purpose

The purpose of this assignment is for us to understand how DNA microarray experiments work, and how to analyze the data that we gather from these microarray experiments.

Questions

1. Choose two genes from Figure 4.6b 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. 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.
• Answer in table below

  	Timepoint
  Genes	1 hour	3 hour	5 hour	9 hour
  gene X	black	medium red	black	bright green
  gene Y	black	bright red	black	bright green
  gene Z	black	dim red	medium red	medium red

3. Were any of the genes in Figure 4.7b transcribed similarly? If so, which ones were transcribed similarly to which ones?
• Gene X followed a similar transcription pattern as Gene Y. Both were measured as having an early heavy spike in transcription rates at the 4 hour timepoints, returned to near baseline rates at the 5 hour timepoints, and a substantial reduction in transcription rates at the 9 hour timepoints, with gene Y being more exaggerated than gene X in displaying these patterns.
4. 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?
• Most spots would be yellow at the first time point because the cDNA with different dyes they have incorporated have thus far hybridized to the assays in an equal amount, and thus the merged image produced by the computer would show that the dye shows up in equal amounts.
5. 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).
• As the optical density of the cell culture grew, the spot for TEF4 grew brighter green, meaninng that the gene was being repressed over the course of the experiment. When a cell undergoes a lack of availability of a metabolic reactant or precursor (such as glucose) it would probably want to decrease its overall metabolic activity so as to conserve its energy and maintain homeostasis. Thus, the production of TEF4, which is involved in translation, is minimized, as it is used in a process that consumes energy.
6. Why would TCA cycle genes be induced if the glucose supply is running out?
• The TCA cycle conserves the amount of energy used to make more energy, thus decreasing the overall amount of glucose used. The cell would want to promote the use of this energy saving measure.
7. What mechanism could the genome use to ensure genes for enzymes in a common pathway are induced or repressed simultaneously?
• The genome could use shared transcription factors and portions of promotion sequences to ensure that certain specific enzymes are transcribed upon binding of those common transcription proteins.
8. 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?
9. 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 previously-glucose-repressed genes would show up in the later timepoints of the ΔYap1p strain and be colored red. Thus, the merged image would show normal production rates as compared to the the previously heavily repressed (and green) images, and would probably be yellow or light red.
10. 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?
• A black microarray dot at the TUP1 location would indicate that no DNA hybridized to that particular location on the microarray chip, thus confirming deletion of the gene. Furthermore, to test for over expression of YAP1, simply look for an increased red color on the microarray chip, as compared to a control microarray chip.

Acknowledgements

This week my partner was Christina Dominguez. We worked in class together.

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

Other Links

Links
Mihir Samdarshi User Page
Assignment Pages	Personal Journal Entries	Shared Journal Entries
Week 1	Journal Week 1	Class Journal Week 1
Week 2	Journal Week 2	Class Journal Week 2
Week 3	FAS2/YPL231W Week 3	Class Journal Week 3
Week 4	Journal Week 4	Class Journal Week 4
Week 5	Database - AmtDB	Class Journal Week 5
Week 6	Journal Week 6	Class Journal Week 6
Week 7	Journal Week 7	Class Journal Week 7
Week 8	Journal Week 8	Class Journal Week 8
Week 9	Journal Week 9	Class Journal Week 9
Assignment Pages	Personal Journal Entries
Week 10	Journal Week 10
Week 11	Journal Week 11
Week 12/13	Journal Week 12/13
Team Project Links
Skinny Genes Team Page

References

1. LMU BioDB 2019. (2019). Week 6. Retrieved October 8, 2019, from https://xmlpipedb.cs.lmu.edu/biodb/fall2019/index.php/Week_6
2. MediaWiki (2019). Category: Help. Retrieved October 8, 2019, from https://www.mediawiki.org/wiki/Category:Help