Practice question

Hygromycin B blocks translation elongation through interference with ribosomal translocation step on 80S plant ribosomes. Bacterial gene hpt from Escherichia coli encodes 341 amino acid phosphotransferase that uses ATP to phosphorylate C-4 hydroxyl of destomic acid moiety of antibiotic, creating inactive phospho derivative that cannot occupy decoding site. Expression cassette includes CaMV 35S promoter and terminator for constitutive high expression in plant cytosol. Enzyme kinetics show high affinity for hygromycin but not kanamycin or gentamicin, providing specific resistance profile. Selection scheme uses 25 to 50 mg per liter in solid or liquid medium, causing necrosis of untransformed tissue within 10 days. Stable integration confirmed by PCR and Southern hybridization. Unlike genes conferring ampicillin resistance that function only in bacteria, hpt functions efficiently in plant cells, making it standard selectable marker for hairy root and embryogenic callus transformation experiments requiring clean antibiotic selection. Mechanism of detoxification involves ATP dependent phosphorylation preventing antibiotic binding to decoding center of ribosome, thus preserving translation fidelity. Enzyme purified shows Km of 0.1 mM for hygromycin. In plants, expression localized to cytoplasm, stable under field conditions without fitness cost. Co-transformation frequency with rol genes high due to simultaneous integration, enabling efficient selection of transformed