Question

In pBR 322 plasmid the tetracycline antibiotic resistance gene has the recognition site for which of the following restriction enzymes.
I. PVU I
II. Sal I
III. BamH I
IV. Pst I

• A. I and IV
• B. I and II
• C. II and IV
• D. II and III

Hint:

Memory aid for pBR322:
Tetracycline (\(tet^{r}\)) = BamH I & Sal I (Think: "T-B-S" $\rightarrow$ Tetracycline, Bam, Sal).
Ampicillin (\(amp^{r}\)) = Pst I & Pvu I (Think: "A-P-P" $\rightarrow$ Ampicillin, Pst, Pvu).

Answer 1

The Correct Option is D

Step 1: Recall the two marker genes on pBR322.
This cloning vector carries an ampicillin resistance gene and a tetracycline resistance gene, each used as a selectable marker.
Step 2: Recall what happens when you cut inside a marker gene.
Inserting foreign DNA at a restriction site located inside the tetracycline gene disrupts that gene, so cells with the insert lose tetracycline resistance while keeping ampicillin resistance, letting us screen for recombinants.
Step 3: Recall which enzymes cut inside that particular gene.
The unique sites for this kind of insertional inactivation within the tetracycline resistance gene belong to Sal I and BamH I.
Final answer: Option 4, II and III (Sal I and BamH I).