 |
 |
||
| Homework 2 | |||
Answer Key in PDF format
Bioc471a/571a Homework 2 - Due at the start of class September 4
1. (2 pts) Explain the rationale behind comparing the amino acid sequences of trypsin genes from humans and Drosophila, to that of the Drosophila chymotrypsin gene, in order to design an oligonucleotide probe for screening a mosquito cDNA library. This would be an alternative approach to that of using a Southern blot to identify a DNA fragment to use as a probe as outlined in the Lab Practicum we discussed in class.
2. (2 pts.) Using the known cDNA sequence of the mosquito trypsin gene shown in the notes for lecture 3, write the sequence of the BEST possible Drosophila-based oligonucleotide probe of 24 nucleotides that could have been designed (hindsight is always 20-20). Write the sequence in the conventional 5' to 3' direction.
3. (2 pts.) Explain why the E. coli K-12 strain used in recombinant DNA methods is not a dangerous pathogen, even though it is a close cousin of the highly virulent E. coli strain 0157:H7.
4. (2 pts.) The E. coli lacZ gene is often used in molecular genetic applications as a marker of cells that are able to activate a cell-specific gene promoter, e.g., the Nodal gene promoter-lacZ fusion gene is expressed in a limited set of mouse heart muscle cells as shown in lecture 4. What protein function is encoded by the lacZ gene and what is it about this protein that makes it a good cell marker in higher eukaryotes (hint: the Drosophila trypsin gene would NOT make a good marker gene for the opposite reason).
5. (2 pts.) The enzyme alkaline phosphatase removes the phosphate group from the 5' end of DNA strands. The enzyme ligase can only ligate together DNA strands that contain a 3' OH group and a 5' phosphate group. Explain how dephosphorylation of enzyme digested plasmid vector sequences with alkaline phosphatase reduces the number of colonies containing plasmids without inserts, but does not eliminate the recovery of recombinant plasmids? (hint: something happens inside the E. coli cell using host enzymes).
