Great question Janet. UV is a form of mutagen analogous to hydroxyurea, and induces damage like thymidine dimers and DSBs across the entirety of the genome. As a result, proteins that are likely to be involved in the human DNA damage repair pathway would be upregulated during such conditions (assuming said DNA damage did not result in destruction of the promoter or enhancer regions of critical repair proteins).
How can you account for the PCR product in the NTC ATPVOC6 loading control lane on your validation gel? Did this preclude validation of your primer pair?
Thank you for the question Nickles. Normally, unexpected product would invalidate our primer validation results and force a replication of the experiment to try and remove such sources of contamination. However, the additional bands in our loading control were all the same size as that of our cDNA amplification product for ATPV0C6. As such, we were able to attribute our contamination to a definite source that we did not see in our GS lanes. Because of this, we decided that this contamination error was probably not significant enough to change the observation that our primers annealed to and amplified our GOI. In an ideal world, we probably would have redone the experiment, but for our purposes the fact that we can at least characterize the error allowed us to move forward with other experiments.
For your future directions, how do you think researchers should test to see if UPEC is an antagonist, or just a marker for normal DNA upkeep to rule one or the other out?
Great question Mason. To test Upec’s potential function as a damage repair antagonist, researchers could perform ChIP Seq experiments to see if Upec binds to repressor sequences of T. thermophila repair enzymes during times of basal DNA function. To test its potential as a DNA upkeep marker, researchers could KO Upec and see if this mutation results in isolated cells upregulating repair enzymes (IE, the cell would suddenly believe that it was in danger of severe damage or apoptosis). I hope this answers your question!
Because our experiments focused almost completely on the expression levels of the gene, I honestly do not know. I think it would be more interesting if Upec was an antagonist / repressor of DNA repair mechanisms though, since this would elucidate how the pathway is shutdown and a cell is returned to basal function. From my research, this ramp down seems to be less understood, despite it being just as important to cell function as the instigation of the repair pathway.
You mentioned that the differences in expression are most likely due to DNA damage, is there a method for getting a quantitative value of the differences in expression/the differences due to the damage?
During our experiments, we performed a qRT PCR, which measures quantitative expression levels of genes that you are testing in real time (as opposed to a gel, which only gives you a qualitative idea of whether expression increased or decreased). During the semester, we performed this experiment for Upec to compare the quantitative expression values for cells that were untreated or treated with hydroxyurea (Figure 6). We found that there was a definite quantitative decrease in the expression of Upec under conditions of DNA damage. Raw results demonstrated UT samples with an expression level of ~800 and HU samples with a level of about ~300. If we normalize this to our UT sample, like we did in figure 6, we can see that our HU expression is only about a third of that seen for cells that were not treated with any damaging agent.
curesymposium.org 
Which human proteins would you specifically target to see an increase expression during UV exposure in your future directions?
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Great question Janet. UV is a form of mutagen analogous to hydroxyurea, and induces damage like thymidine dimers and DSBs across the entirety of the genome. As a result, proteins that are likely to be involved in the human DNA damage repair pathway would be upregulated during such conditions (assuming said DNA damage did not result in destruction of the promoter or enhancer regions of critical repair proteins).
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How can you account for the PCR product in the NTC ATPVOC6 loading control lane on your validation gel? Did this preclude validation of your primer pair?
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Thank you for the question Nickles. Normally, unexpected product would invalidate our primer validation results and force a replication of the experiment to try and remove such sources of contamination. However, the additional bands in our loading control were all the same size as that of our cDNA amplification product for ATPV0C6. As such, we were able to attribute our contamination to a definite source that we did not see in our GS lanes. Because of this, we decided that this contamination error was probably not significant enough to change the observation that our primers annealed to and amplified our GOI. In an ideal world, we probably would have redone the experiment, but for our purposes the fact that we can at least characterize the error allowed us to move forward with other experiments.
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For your future directions, how do you think researchers should test to see if UPEC is an antagonist, or just a marker for normal DNA upkeep to rule one or the other out?
LikeLike
Great question Mason. To test Upec’s potential function as a damage repair antagonist, researchers could perform ChIP Seq experiments to see if Upec binds to repressor sequences of T. thermophila repair enzymes during times of basal DNA function. To test its potential as a DNA upkeep marker, researchers could KO Upec and see if this mutation results in isolated cells upregulating repair enzymes (IE, the cell would suddenly believe that it was in danger of severe damage or apoptosis). I hope this answers your question!
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What do you think is more likely? That UPEC is an
antagonist of DNA repair mechanisms, or a marker of normal
DNA upkeep?
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Because our experiments focused almost completely on the expression levels of the gene, I honestly do not know. I think it would be more interesting if Upec was an antagonist / repressor of DNA repair mechanisms though, since this would elucidate how the pathway is shutdown and a cell is returned to basal function. From my research, this ramp down seems to be less understood, despite it being just as important to cell function as the instigation of the repair pathway.
LikeLike
You mentioned that the differences in expression are most likely due to DNA damage, is there a method for getting a quantitative value of the differences in expression/the differences due to the damage?
LikeLike
During our experiments, we performed a qRT PCR, which measures quantitative expression levels of genes that you are testing in real time (as opposed to a gel, which only gives you a qualitative idea of whether expression increased or decreased). During the semester, we performed this experiment for Upec to compare the quantitative expression values for cells that were untreated or treated with hydroxyurea (Figure 6). We found that there was a definite quantitative decrease in the expression of Upec under conditions of DNA damage. Raw results demonstrated UT samples with an expression level of ~800 and HU samples with a level of about ~300. If we normalize this to our UT sample, like we did in figure 6, we can see that our HU expression is only about a third of that seen for cells that were not treated with any damaging agent.
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