BIOL368/F14:Chloe Jones Week 10

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Addendum for Week 9

  • I downloaded Huang et al. (2005) Structure 2B4C into StarBiochem and adjusting the size of atoms and secondary structure, I was able to obtain a clear picture of the V3 region on the protein an then look at the amino acids that corresponded to the ones that changed in Psipred.
  • I worked specifically with the Non-trending group, which experienced two amino acids changes in the first visit, and 1 amino acid change in the second visit.
    • Initial Visit
    • Residue 49 :Threonine (polar) vs. Alanine (nonpolar)
    • Residue 56 :Aspartic acid (polar) vs. Asparagine (polar)
    • Final Visit
    • Residue 49 :Threonine (polar) vs. Alanine (nonpolar)
Figure 1.The initial visit with the amino acids highlighted that changed
Figure 2.The final visit with the amino acid highlighted that changed
Table1 . The changes that occurred in amino acid sequence in the V3 region for the non-trending group

Introduction to DNA Microarrays

Questions were answered using Chapter 4 of Campbell & Heyer (2003).

  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.
    • Figure 1. Change in transcription over time in the genes DMC1 and DIM1.
  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.)
    • To interpret fractional ratio as a fold expression must take its reciprocal (i.e. 1/.05=20)
    • Table 1: DNA microarray data given in the form of ratios. Determine color from ratios.
1 hour 3 hours 5 hours 9 hours
Gene X 1.0 black 2.2 dim red 1.0 black 0.15 dim green
Gene Y 1.0 black 4.5 dim red 0.95 dim green 0.05 bright green
Gene Z 1.0 black 1.5 dim red 2.0 dim red 2.0 dim red
  1. (Question 7, p. 110) Were any of the genes in Figure 4.7b transcribed similarly? If so, which ones were transcribed similarly?
Table 2.
DNA microarray data used to measure similarity amongst genes.Pertains to question 3.
    • To measure similarity have to find the correlation coefficient , abbreviated letter R
      • R=1, expression pattern track perfectly
      • R=1, expression pattern track perfectly in opposite direction
      • R~0, expressions patterns of two genes do not track each other at all
    • Step 1: Compute mean and standard deviation for each clone.See if in log format
      • x̄X=1.088, x̄Y=1.625 x̄Z=1.625
      • Sx~0.843, Sy~1.966, Sx~0.480
    • Step :Subtract mean value from each number in row then divide by standard deviation.(letterNORM)
      • Xnorm=-0.104,1.319,-0.104,-1.113
      • Ynorm=-0.318, 1.462, -0.343, -0.801
      • Znorm= -1.302, -0.260, 0.781, 0.781
    • Step 3: Multiply the first number in Xnorm by the first number in Y norm, repeat for the next three sets of number. Add all the numbers and then divide by the number of elements in each row, in this case (4). Repeat to test similarity between each different pair.
      • r(X,Y)=(-0.104*-0.318)+(1.319*1.462)+(-0.104*-0.343)+(-1.113*-0.801)/4=0.722
      • r(X,Z)=(-0.104*-1.302)+(1.319*-0.260)+(-0.104*0.781)+(-1.113*0.781)/4=-0.315
      • r(Y,Z)=(-0.318*-1.302)+(1.462*-0.260)+(-0.343*0.781)+(-0.801*0.781)/4=-0.215
    • No similarity between the genes.
  1. (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?
    • Yellow spots correspond to red to green ratio of 1:1, so essentially neutral. Spots would be yellow at the first time point because here wouldn’t be that much change to gene expression. The biological reason for most spots appearing yellow in an experiment would show that the quantity of gene expression in terms of reduced and induced was equal.
  2. (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 highlighted as a bright green color in its frame which indicates repression. TEF4 is involved in translation and therefore involved in the production of proteins. The reduction of glucose impacts protein production, because without it the gene can no longer participate in translation. So, it make sense as to why TEF4 is involved in repression when glucose levels are low.
  3. (Question, 11, p. 120) Why would TCA cycle genes be induced if the glucose supply is running out?
    • TCA cycle genes would be induced if glucose it running out because the TCA cycle generates energy and storage sugars and therefore would have to compensate for loss of energy that glucose provided.
  4. (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 use "guilt by association" theory which assumes that genes which are associated with or interacting with one another are more likely to share the same function. This method can then be confirmed using experimental testing, such as the presence or absence of glucose and whether it gets induced or repressed.
  5. (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?
    • I would expect the spots to remain red for the glucose-represented genes because deletion of the TUP1 is no longer repressing the glucose repressing genes(green).
  6. (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?
    • Similar to the TUP1 deletion, I would expect the over expression of Yap1p to result in red spots, because there is repression of glucose repressed genes which would wouldn't allow for repressor genes (i.e. green spots)
  7. (Question 15, p. 121) Could the loss of a repressor or the overexpression of a transcription factor result in the repression of a particular gene?
    • Yes, it could result in the repression of a particular gene if a repressor is lost or a transcription factor is overexpressed. Genes correspond to one another so the repression or overexpression of one gene can have an affect on another gene. However, based on the two studies above when the microarray was red when TUP1 was deleted and Yap1p was overexpressed it really just depends on the gene I think and their association.
  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?
    • To verify that you have deleted or overexpressed a gene you could look at the microarray chip. First, you can observe the control spots that are not affected by the repressed or overexpressed transcription factors and then second you could observe if the gene is truly repressed it will not appear on the microarray data and if overexpressed it will be shown as a bright red color (i.e. induced).

Electronic Lab Notebook

Weekly Assignments

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Chloe Jones 03:46, 15 October 2014 (EDT)Chloe Jones