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Six independent transformation experiments of tobacco leaf explants were carried out using two different constructs - (1) construct I containing only a Hygromycin-resistance gene $(HYG^R)$ as a selection marker and (2) construct II with the $(HYG^R)$ gene as a selection marker and a passenger gene encoding a dephosphorylase enzyme $(DEP)$ that inhibits phosphorylation in cells in which it is expressed. The marker as well as passenger genes were placed under transcriptional control of a constitutive promoter $(CaMV35S)$, which expresses in all cells/tissues. The results obtained are tabulated below.

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Explain how The $HYG^R$ gene used in construct II lost its functionality probably due to mutation(s) either in the gene or in the promoter.

My approach: There is a probability that Expression of the $DEP$ gene is lethal to the transformed cells.

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Having a toxic product for the cell in an expression vector is a not uncommon problem when overexpressing proteins.

The other possibility is that the selection marker itself is the problem. Here you can have mutations either in the promoter of the gene as well as in the gene itself resulting in a protein which is not capable of protecting the cells from the selection. Both possibilities (no expression and mutated protein) leave to the death of the transfected cells, since there is no protection of them by the resistance protein.

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