1. How should you define the following words:
- Expression libraries
- Transcription factors
- Subtracted libraries
- Foldback cDNA
- House-keeping genes
- Screenable marker gene
2. How does RNAse H help us get nearly full-length cDNA that could not be obtained otherwise?
3. Why isn’t the product full-length?
4. What genetic sequences can be found in genomic libraries that cannot be found in cDNA libraries?
5. What is the purpose for building an expression library?
6. Why aren’t the genes in fold-back libraries good enough to use in an expression library?
7. Why did the snapdragon group need to prime 2nd strand cDNA as they did, rather than by using RNAse H? In more general terms, when would we need to prime 2nd strand cDNA that way instead of by treatment with RNAse H?
8. If you wanted to look for genes associated specifically with hairiness, would you make the library from normal cats or hairless cats?
9. If you wanted to look for as many genes as you can find associated with hairiness, would you probe that library with radioactive cDNA from normal cats, or hairless cats or neither or both?
10. In order to find these genes, would you have to probe 1 by 1 with every clone in your library, or would you make a “multi-gene” probe? [Hint: I am trying to get you to review how a differential screen is done. Review that method and you will find the answer.]
11. Would those probes be made from liver, ear, skin, whole animal, or hare (look up the meaning of this word as spelled here) RNA?
12. How do eukaryotic transposons resemble and differ from prokaryotic ones?
13. if we needed to make cDNA libraries to find a gene by complementation, would it be wiser to prime second-strand synthesis using fold-back techniques, or instead use RNAse H?
14. What determines the abundance of a given gene in a genomic library?
15. What determines the abundance of a given gene in a cDNA library?
16. How would you isolate the transposon that caused the defA– mutation?
17. List the 3 ways we can transform animal cells.
18. List the one way we prefer to transform animal eggs.
19. Why, (and this question requires an answer that draws from your knowledge of development) do you think, this latter method is preferred over the former ones when we transform eggs.
20. What happens to micro-injected DNA that that fails to integrate?
21. What mechanism allows micro-injected DNA to integrate?
22. What causes chimeric animals to arise?
23. What is the difference in meaning between a chimeric animal and a heterozygous animal?
24. If we have a chimera, how could you get a uniformly transformed animal from it?
25. Would this uniformly transformed animal be homozygous or heterozygous?
26. Would breeding 2 chimeras from different transformations with the same gene be able to produce homozygous offspring?
27. Describe the changes you would need to make to convert a bacterial npt gene into a selectable marker for human tissue culture cells.
28. What sequence directs the poly-A polymerase to add a poly-A tail?
29. What is the average size of a polyA tail?
30. What is a wings-clipped transposon?
31. True or false: In eukaryotes, constitutively expressed genes use a single enhancer that is functional in all cells all the time. Justify your answer very briefly.
32. True or false: Enhancers are DNA sequences that have a direct (=work without the need of any intermediate), stimulatory effect on RNA polymerase II. Justify your answer very briefly.
33. True or false: Eukaryotic transcription terminates at defined sequences. Justify your answer very briefly.
34. True or false: npt protects cells against all antibiotics. Justify your answer very briefly.
35. What is meant by the phrase: “natural and engineered proteins are modular”?
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