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| Homework 7 | |||
Answer Key in PDF format
Bioc471a/571a Homework 7 - Due at the start of class October 30
All 13 of the questions listed below refer to material presented in Lecture 17 "Gene Expression Lab Practicum." Choose any five of the questions and answer them on a separate page referring to them by number (2 pts each).
1. What is the difference between the yeast Two-hybrid screen and the yeast One-hybrid screen with regard to the research objective (i.e., what do you need as materials for each strategy and what is the outcome)?
2. What could the scientist do if the pancreatic library she was screening did not contain any clones corresponding to the 5' end of the ORF (the library was made with oligo dT)? Describe two approaches and the advantages/disadvantages of each.
3. Based on the observation that IBx4 reporter gene worked almost as well as the insulin reporter gene in this assay, what does this suggest about the mechanism of Igs in insulin gene regulation? Which experiment with PanC cells supports this conclusion?
4. One interpretation of the terminal deletion experiments is that protein domains required for Igs function (SLH interactions) are encoded by the deleted amino acids. What is an alternative explanation that might explain the loss of Igs activity?
5. Why is it necessary to engineer in the ATG and TAG codons into the coding sequence of the N-terminal and C-terminal deletion mutants, respectively? How would you do this?
6. What explanation for the "super-shifted" band in the EMSA assay in the presence of Dib-TC protein extract best fits the researcher's hypothesis? What is an alternative explanation and how would you distinguish between the two?
7. What do the data from the Gain of Function experiments indicate with regard to the relative "strength" of the two putative activation domains? Assuming the researcher's model is correct, what is the most likely biochemical explanation for this difference?
8. Do the gain of function experiments prove that Igs proteins interact with SLH proteins? Explain. How is it thought that "activation domains" stimulate transcriptional initiation rates?
9. What is the purpose of adding the Flag epitope sequences to the N-terminus of the Igs coding sequence? Why not just use anti-Igs antibodies for these co-immunoprecipitation studies?
10. How would you construct the internal Igs deletants IgsD31-99 and IgsD31-204 used in the co-immunoprecipitation experiments? How would you confirm unambiguously that the deletions you created did not inadvertently cause a "frame shift mutation?"
11. What are psi- packaging cells and why are they required for retroviral expression systems? Do the infected DiB-TC cells produce recombinant virus? Explain.
12. What is the source of the two bands in the Ponceau-stained nitrocellulose filter and why are these the only protein bands observed?
13. What do the results of the IgsD658 and IgsD31-204 co-immunoprecipitation results suggest about the role of activation region 2 in SLH binding?
