and a 3rd idea that fits as well:
For question 5, the main thing is that you understand what you are looking at. We presented a very similar figure in lecture, going over it in quite some detail and also giving you study questions to think about. Check out related SQs going forward and the old exams, which serve as good practice.
5A. Twist expression causes β-catenin to move (relocalize) from the cell surface to the nuclei, as seen in the micrographs; in the vector control, no nuclear staining is seen (the DAPI stain – a DNA dye – tells us what nuclear localization looks like) while in the Twist cells, we now see obvious nuclear staining. No change in β-cat total protein levels (as seen by the blots) occurs, consistent with a relocalization effect.
5B. Twist expression causes LEF-1 to be expressed; as seen in both the immunolocalization data and the blot, the vector control cells have no detectable LEF-1 protein while in the Twist cells, we see LEF-1 protein present (blot) - and localized primarily to the nuclei.
5C. Actin serves as a load control. We expect that actin, as a housekeeping protein, will have a relatively constant expression level and thus it can serve as a comparison for the other proteins of interest, confirming that there are equivalent amounts of total protein in each sample and thus that observed differences -/+Twist are real and not experimental artifacts. Many students will say “load control” but not really understand it. If you are not sure, just ask!
5D. You must understand these design principles for reporters and transgenes. You will see these throughout the quarter.
Regulatory region: must be from a gene that will be expressed in these cells at a high level; okay to say “constitutively active” gene regulatory region or ”housekeeping” gene regulatory region.
Coding region: must be the cDNA of Twist. Okay to have a GFP (or other) tag cDNA fused in frame to Twist.
By the way, how confident are you that Twist was being expressed in the cells (see blot - compare Twist Ab on cell lysates of control v + Twist transgene).
Question 6 probes your knowledge of proto-oncogenes and viruses, leading to an understanding of the Berkeley Experiment. You need to understand the table – that the tsZSV is a way to turn the transforming activity off and on.
6A. The most likely single residue change would be mutant 2 (m2).
Explanation: The data indicate that the m2 mutation is temperature sensitive – at the permissive temperature, the ZSVm2 causes transformation similar to wt virus while at the restrictive temperature, it loses transforming ability. A ts mutant is almost always a point mutation; the temperature shift causes a slight change in conformation of the m2 protein, causing it to lose function. The ZSVm1 cannot transform at either temperature, suggesting a true loss of function (null) in the transforming gene.
6B. The answer is B and C.
Here the emphasis is on the Proto-Oncogene Hypothesis and the idea that the virus has picked up a
slightly mutated version of a cellular gene that normally promotes proliferation.
6C. The answer does not have to be very involved – the key point is to propose the Berkeley Experiment using the tsZSV (m2):
wt ZSV at any temp infects & transforms (control)
m2 ZSV at permissive temp infects & transforms à what happens when we the shift to the restrictive temperature, essentially shutting off the ZSV transforming ability?
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