Difference between revisions of "Bklein7 Week 7"

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#*''Saccharomyces cerevisiae releases chemical energy from glucose through anaerobic respiration (fermentation) in glucose-rich environments, despite it being the energetically inefficient pathway when compared to aerobic respiration. However, in glucose-limited environments, yeast cells switch to the aerobic pathway. Upon switching pathways, ethanol is converted to acetyl-CoA, which enters the TCA cycle (aerobic pathway), and glucose is converted into storage sugars. This diauxic shift is captured in the microarray experiment. In the glucose-limited experimental group, TCA cycle genes are induced to shift from fermentation to the aerobic respiration pathway. This shift happens in response to glucose depletion, because it enables the cell to produce more ATP through the more energetically productive TCA cycle and conserve glucose. Thus, the cell is essentially preparing for survival in a glucose-limited environment by freeing up higher levels of chemical energy to sustain cellular processes and storing glucose for emergency use.
 
#*''Saccharomyces cerevisiae releases chemical energy from glucose through anaerobic respiration (fermentation) in glucose-rich environments, despite it being the energetically inefficient pathway when compared to aerobic respiration. However, in glucose-limited environments, yeast cells switch to the aerobic pathway. Upon switching pathways, ethanol is converted to acetyl-CoA, which enters the TCA cycle (aerobic pathway), and glucose is converted into storage sugars. This diauxic shift is captured in the microarray experiment. In the glucose-limited experimental group, TCA cycle genes are induced to shift from fermentation to the aerobic respiration pathway. This shift happens in response to glucose depletion, because it enables the cell to produce more ATP through the more energetically productive TCA cycle and conserve glucose. Thus, the cell is essentially preparing for survival in a glucose-limited environment by freeing up higher levels of chemical energy to sustain cellular processes and storing glucose for emergency use.
 
# (Question 12, p. 120)  What mechanism could the genome use to ensure genes for enzymes in a common pathway are induced or repressed simultaneously?
 
# (Question 12, p. 120)  What mechanism could the genome use to ensure genes for enzymes in a common pathway are induced or repressed simultaneously?
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#*The genome could induce or repress genes in a common pathway simultaneously if the different genes all contained equivalent regulatory sequences that bound to the same regulatory protein. In this case, the specific regulatory protein would have to only act on genes present in that pathway (or associated processes). If so, genes involved in the same pathway could be regulated by controlling the synthesis of just one protein, regardless of their diverse positions in the genome.
+
#*The genome could induce or repress genes in a common pathway simultaneously if the different genes all contained equivalent regulatory sequences in their promotor regions that bound to the same regulatory protein. In this case, the regulatory protein would have be specific in that it only would act on genes present in that pathway (or associated processes). If so, genes involved in the same pathway could be regulated by controlling the synthesis of just one protein, regardless of their diverse positions in the genome.
 
# (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?
 
# (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?
  

Revision as of 01:33, 20 October 2015

Introduction to DNA Microarrays

Discovery Questions from Campbell & Heyer Chapter 4

Answer the following questions related to Chapter 4 of Campbell & Heyer (2003). Note that some of the questions below have been reworded from the Discovery Questions in the book:

  1. (Question 5, p. 110) Choose two genes from Figure 4.6b (PDF of figures on MyLMUConnect) 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 in hard copy and turn it in in class.
    • Bklein7 Week7 Question1.png
    • Disclaimer: I am red-green colorblind, so assessing the red-green color scale for changes in transcription (Figure 4.5) proved difficult. In correlating the colors from Figure 4.6b to values, I made my best guesses using the aid of pixel color analysis tools.
    • (note to self: improve x-axis scale?)
  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.)
    • Bklein7 Week7 Question2.png
  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?
    • The transcription of genes X and Y from Figure 4.7b exhibited similar patterns in response to the gradual loss of oxygen. The transcription of both genes was induced at the 3 hour mark (90% of normal oxygen level), showed very little or no change when compared to the control at the 5 hour mark (~62% of normal oxygen level), and finally was repressed at the 9 hour mark (10% of normal oxygen level). Despite these similar patterns of up and down-regulation, the magnitudes of the transcriptional changes did vary between the two genes. Gene Y was both more dramatically induced at the 3 hour mark and more dramatically repressed at the 9 hour mark. Additionally, gene Y was very slightly down-regulated at the 5 hour mark whereas gene X showed no change from its transcription at a normal oxygen level.
  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, the experimental group was the least glucose-limited when compared to all other time points, and therefore these experimental conditions were most similar to that of the control (glucose rich environment). Spots on a DNA microarray appear yellow when approximately equal amounts of cDNA from the control group, labelled green, and the experimental group, labelled red, bind to a specific spot. Thus, yellow spots indicate no change in the transcription rates of genes when comparing the experimental and control groups. Because the first time point was the time at which the experimental conditions best matched the control conditions (little/no glucose limitation), there consequentially was very little transcriptional change, resulting in most spots being yellow.
  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).
    • According to the Saccharomyces Genome Database, TEF4 is a transcription elongation factor that stimulates the binding of AA-tRNA to ribosomes. Thus, the translation of the TEF4 gene induces protein production. In reviewing the DNA microarray results in Figure 4.12, TEF4 is repressed by OD 0.46, transcribed at rates consistent with the control by OD 0.8, and then increasingly repressed over the remainder of the experiment. The time marked by OD 0.46 is associated with the onset of glucose limitation. It is possible that the yeast detect this initial decrease in available chemical energy (as glucose is necessary for glycolysis and consequentially the production of ATP), and they respond by down-regulating protein production genes to conserve chemical energy. In this sense, the cell would essentially be preparing for a possible energy deficit. At OD 0.8, when the glucose levels had only slowly decreased, the yeast cells may have terminated this seemingly aggressive response to potential energy deficiency. However, when the environment became more dramatically glucose-limited, the cells resumed the strategy of conserving chemical energy by decreasing protein production. By the time the environment was almost entirely devoid of glucose, TEF4 experienced over a 20 fold repression. This suggests that protein production was dramatically limited to endeavor to preserve the cell's life as long as possible before glucose levels could be restored. The trend of down-regulating protein producing genes in a glucose-limited environment is supported by Figure 4.13.
  6. (Question, 11, p. 120) Why would TCA cycle genes be induced if the glucose supply is running out?
    • Saccharomyces cerevisiae releases chemical energy from glucose through anaerobic respiration (fermentation) in glucose-rich environments, despite it being the energetically inefficient pathway when compared to aerobic respiration. However, in glucose-limited environments, yeast cells switch to the aerobic pathway. Upon switching pathways, ethanol is converted to acetyl-CoA, which enters the TCA cycle (aerobic pathway), and glucose is converted into storage sugars. This diauxic shift is captured in the microarray experiment. In the glucose-limited experimental group, TCA cycle genes are induced to shift from fermentation to the aerobic respiration pathway. This shift happens in response to glucose depletion, because it enables the cell to produce more ATP through the more energetically productive TCA cycle and conserve glucose. Thus, the cell is essentially preparing for survival in a glucose-limited environment by freeing up higher levels of chemical energy to sustain cellular processes and storing glucose for emergency use.
  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 genome could induce or repress genes in a common pathway simultaneously if the different genes all contained equivalent regulatory sequences in their promotor regions that bound to the same regulatory protein. In this case, the regulatory protein would have be specific in that it only would act on genes present in that pathway (or associated processes). If so, genes involved in the same pathway could be regulated by controlling the synthesis of just one protein, regardless of their diverse positions in the genome.
  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?
  1. (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?
  2. (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?

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