Question

In a cloning experiment using E.coli, a DNA fragment was inserted in an EcoRI restriction site of a plasmid vector that contained the $ka{n}^R$ and $spe{c}^R$ genes for resistance to the antibiotics kanamycin and spectinomycin, respectively. It was observed that all the positive clones (containing the DNA fragment of interest) grew on medium with kanamycin but not on media containing spectinomycin. Colonies, which grew on media with both antibiotics, did not contain the fragment of interest. In the absence of any other confounding factors, the statement(s) which could independently explain these observations is/are

• A. The $spe{c}^R$ gene contains a mutation which generates a stop codon within the gene
• B. The EcoRI site was located within the $spe{c}^R$ gene
• C. The cloned DNA fragment was lethal to the cell
• D. The clone fragment of interest produces a protein which binds and inactivates the protein produced by the $spe{c}^R$ gene

Answer 1

Answer: (B), (D)

The EcoRI site was located within the $spe{c}^R$ gene
The clone fragment of interest produces a protein which binds and inactivates the protein produced by the $spe{c}^R$ gene

The positive clones with DNA fragment of interest grew on medium with kanamycin and not in spectinomycin. This shows that the fragment was inserted in the region of the gene of spec${}^R$. This means that EcoRI site was located in this region.

The cloned fragment inactivates the spec${}^R$ gene by producing a protein which can bind to the protein of spec${}^R$ gene. This binding of the two proteins will cause inactivation of the protein of spec${}^R$ gene.

Thus, the correct answers are options B and D.