FCH 532 Homework 3
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1. Which of restriction enzymes listed in Table 5-4 is a blunt end cutter?
( http://rebase.neb.com
can be used to help you figure it out).
Which sets are isoschizomers (enzymes with the same recognition sequence but do not necessarily cleave the same sites) and which are isocaudamers (enzymes that produce identical sticky ends)
2. Translate the following sequence to a polypeptide chain in three reading frames.
Show each of the reading frames relative to the nucleotide sequence.
5’-ATGTATACGAAGATTATTGGTACTGGCAGCTATCTGCCCGAACAAGTGC-3’
3.
Reconstruct the following plasmid from the fragment sizes:
Restriction enzyme
Eco RI
Hin dIII
Fragment sizes (kb)
5.4
2.1, 1.9, 1.4
Sal I
Eco RI + Hin dIII
Eco RI + Sal I
Hin dIII + Sal I
5.4
2.1, 1.4, 1.3. 0.6
3.2, 2.2
1.9, 1.4, 1.2, 0.9
4. The plasmid pBR322 contains the amp R and tet R genes, which respectively confer resistance to the antibiotics ampicillin and tetracycline. The tet R gene contains a cleavage site for Sal I, the only such site in the entire plasmid. Describe how one can select for E. coli that had been transformed by pBR322 that contains a foreign DNA insert into its Sal I site.
5. A protein segment of sequence Phe-Cys-Gly-Val-Leu-His-Lys-Met-Glu-Thr- is encoded by the following DNA segment:
5’-TTGTGCGGAGTCCTACACAAGATGGAGACA-3’
Design an 18-base oligonucleotide that could be used to change the protein’s Leu-His segment to Ile-Pro via site-directed mutagenesis.
6. Define the following terms as they relate to biochemistry a. RFLP b. 3’ to 5’ exonuclease c. 5’ to 3’ exonuclease d. primase e. DNA polymerase I

f. DNA polymerase III g. X-gal h. IPTG i. Southern Blotting f. PCR g. clone h. colony hybridization i. genome j. transgenic k. relaxed control plasmid l. stringent control plasmid m. chromosome walking
7. Describe the lac operon and how it works. Use figures and drawings to help explain your answer. Why is an inducible system instrumental for blue-white screening?