Altius FL #2 BS section #3

[akimhaneul]

For this question, why can't the answer be A? Is it because passage says that cancer cells exhibit normal transcription?

I think I'm not understanding why the explanation says "Because transcription is blocked, answers A and B can be eliminated"

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[desperadoflakes]

Ok, I think I can answer this.

These researchers found that normal human cells have this protein, but cancer cells are deficient in it. So they want to know why, and they hypothesized that the mRNA produced by cancer cells is less stable than in normal cells. By less stable, they mean that it degrades more quickly (has a shorter half life). They think that it's because of a mutation they found on the 3'UTR. This one mutation on this UTR is the only difference on the mRNA between the cell types. To test this, they did two experiments. I think this is at least close to being right.

In the second experiment, they tested if the mutated 3'UTRs from the cancer cells had an effect on transcription compared to the WT. They put the 3'UTR from each of the cell lines on a luciferase reporter, and then measured bioluminescence 48 hours later. Transcription of mRNAs carrying the 3'UTRs from both cancer cell lines was way higher than transcription of the normal 3'UTR. This tells us that the protein deficiency is not because the mRNA is less transcriptionally active. But we conducted this experiment in Hela cells, could it be because the cancer cells themselves are less transcriptionally active? No, because the passage says that transcription of the gene proceeds normally. Lol. Also, just in general it's safe to assume that cancer cells are overall highly transcriptionally active. So it's not due to transcription.

So if the cancer cells are still making good levels of mRNA, why are they deficient in the protein? Could be that the mRNA is unstable and quickly gets degraded. So how do they measure this? From your findings in the luciferase experiment, you could predict that just doing straight up RT-qPCR would show you the same levels of transcript across all the cells. In fact, I bet that's exactly what they did to "verify that transcription proceeds normally."

So the solution is to block all transcription, wait 12 hours, and then do RT-QPCR. These cells haven't made any new transcripts in the last 12 hours. If we still see the same levels of this mRNA, that means that it's not really degrading, which means its stable. If we see a decrease in this mRNA, that means the mRNA is getting degraded and it's unstable.

This would have been a lot more straightforward if they had showed you a figure with two panels, one before the ActD and one 12 hours later. But they would like you to prove that you are worthy of a minty white coat through deductive guessing and decoding of intentionally badly written experimental design

 

[aldol16]

In the second experiment, they tested if the mutated 3'UTRs from the cancer cells had an effect on transcription compared to the WT. They put the 3'UTR from each of the cell lines on a luciferase reporter, and then measured bioluminescence 48 hours later. Transcription of mRNAs carrying the 3'UTRs from both cancer cell lines was way higher than transcription of the normal 3'UTR. This tells us that the protein deficiency is not because the mRNA is less transcriptionally active. But we conducted this experiment in Hela cells, could it be because the cancer cells themselves are less transcriptionally active? No, because the passage says that transcription of the gene proceeds normally. Lol. Also, just in general it's safe to assume that cancer cells are overall highly transcriptionally active. So it's not due to transcription.

The reason why they do this experiment has nothing to do with transcription. As they say in the passage, it's transcriptionally active - they say this without needing proof for it. The whole idea of doing the transfection of the UTR into the four cell lines is to assess whether it has an effect on translation. This bit should be fairly obvious since they're measuring luciferase activity, which is a phenotypic effect from a protein. So the experiment is simple - transfect the UTR into the cell lines and see if it down-regulates translation of the luciferase gene. As they show, it down-regulates the gene in all of the cell lines they test, showing that the UTR is responsible for translational suppression of the relevant gene in the cancer lines. The thing that makes this part most confusing is that they don't show the normalized-to-100% control - the vertical axis title only mentions that all the bars in Fig. 3 are compared to a 100% control which they don't show.

 

[desperadoflakes]

The reason why they do this experiment has nothing to do with transcription. As they say in the passage, it's transcriptionally active - they say this without needing proof for it. The whole idea of doing the transfection of the UTR into the four cell lines is to assess whether it has an effect on translation. This bit should be fairly obvious since they're measuring luciferase activity, which is a phenotypic effect from a protein. So the experiment is simple - transfect the UTR into the cell lines and see if it down-regulates translation of the luciferase gene. As they show, it down-regulates the gene in all of the cell lines they test, showing that the UTR is responsible for translational suppression of the relevant gene in the cancer lines. The thing that makes this part most confusing is that they don't show the normalized-to-100% control - the vertical axis title only mentions that all the bars in Fig. 3 are compared to a 100% control which they don't show.

They show the control in the next figure. Right, makes sense with the luciferase, thanks.

 

[aldol16]

They show the control in the next figure. Right, makes sense with the luciferase, thanks.

That control is only for Hela cells and if you look closely, the UTR transfected ones in that figure actually have a little less luciferase activity than in the preceding figure. I don't know what to make of that. But I also think it's important to note that I think they have controls for each cell line - it doesn't make any sense to compare all the cell lines to Hela. You want to compare cancer cells to cancer cells - give them luciferase with and without the UTR. This is because the cells would likely have different basal levels of luciferase expression to begin with.

 

[desperadoflakes]

Yeah, I agree, but the luciferase reporter experiment on both figures was done in Hela cells, the experimental variable they changed was each 3'UTR was taken from a different cell line. I'm pretty sure the reporter plasmid used in the first figure only has the portion of the 3'UTR with the variable site they found? And I believe that the one they used on that second figure has the full 3'UTR from the WT gene I guess

 

[aldol16]

Yeah, I agree, but the luciferase reporter experiment on both figures was done in Hela cells, the experimental variable they changed was each 3'UTR was taken from a different cell line. I'm pretty sure the reporter plasmid used in the first figure only has the portion of the 3'UTR with the variable site they found? And I believe that the one they used on that second figure has the full 3'UTR from the WT gene I guess

No. The 3'UTR is from the original KLF6 region. They're making a plasmid from that and using that same plasmid to transfect four different cell lines.

 

[desperadoflakes]

No. The 3'UTR is from the original KLF6 region. They're making a plasmid from that and using that same plasmid to transfect four different cell lines.

Wow not sure how I missed that. Thanks!

 

[aldol16]

By the way guys, the idiot test prep company posted the actual paper they took this from here: Altius FL #2 BS section #2

Earlier, we had talked about how Fig. 4 seems redundant and weird because "luciferase gene expression" is quite vague - it could refer to an assay on the protein (the physical manifestation of expression) or the mRNA. If you look at the paper, Fig. 4 is actually showing the level of mRNA and not protein. Fig. 3 is a protein assay (by assessing luminescence from the activity of that protein). So that makes sense - they're showing that this UTR is a transcriptional regulator and not a translational one. The company just decided to omit this key bit of information.

 

[Altius Premier Tutor]

The reason answers A and B cannot be correct is because Actinomycin D inhibits transcription (mentioned once in the passage and in the Figure 2 caption). The question asks the examinee to draw a conclusion about cancer cells "based on" the levels of mRNA present AFTER treatment with actinomycin D. Think about that for a moment...one cannot make an inference about transcription in cancer cells vs normal cells in an experiment in which you just BLOCKED transcription in all the cell lines.

What you can do is look at what happens to the KLF6 mRNA that's already present. The correct answer states what Figure 2 shows in its most simple sense: it shows that over the same time period, the mRNA degrades faster in the cancer cells; thus the correct answer is that cancer cells exhibit "enhanced degradation of KLF6 mRNA."

 

[desperadoflakes]

The reason answers A and B cannot be correct is because Actinomycin D inhibits transcription (mentioned once in the passage and in the Figure 2 caption). The question asks the examinee to draw a conclusion about cancer cells "based on" the levels of mRNA present AFTER treatment with actinomycin D. Think about that for a moment...one cannot make an inference about transcription in cancer cells vs normal cells in an experiment in which you just BLOCKED transcription in all the cell lines.

What you can do is look at what happens to the KLF6 mRNA that's already present. The correct answer states what Figure 2 shows in its most simple sense: it shows that over the same time period, the mRNA degrades faster in the cancer cells; thus the correct answer is that cancer cells exhibit "enhanced degradation of KLF6 mRNA."

Yes, but to assess the levels of mRNA after treatment with actinomycin D, it should be clear to us what the levels are before treatment with antinomycin D. You expected us to draw the conclusion that the levels before treatment were the same across all cell lines from vague context clues.

 

[Altius Premier Tutor]

The y-axis of Figure 2 is "fold increase vs. untreated," which makes the graph a way of comparing the "rate" of fold-increase or fold-decrease across all cell lines, whatever their baseline KLF6 mRNA levels might have been.

 

[desperadoflakes]

The y-axis of Figure 2 is "fold increase vs. untreated," which makes the graph a way of comparing the "rate" of fold-increase or fold-decrease across all cell lines, whatever their baseline KLF6 mRNA levels might have been.

Ok, I didn't see that, I should've. Thank you!