Neomycin Phosphotransferase Gene - npt, Transgenic Plants, Polyacrylamide Gel Electrophoresis (PAGE), Phosphorylation

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	Neomycin Phosphotransferase Gene - npt - This gene is used both as a selectable and a scoreable marker in experiments involving transfer of genes leading to the production of transgenic plants. It imparts kanamycin resistance, so that the transformed tissue can be selected on kanamycin. An assay for NPT II enzyme is also used to detect its presence in transformed tissue or transgenic plants. The gene for NPT II enzyme is often used with nos promoter, which drives its synthesis.
In some cases, npt II gene had adverse effect on the expression of the desirable gene introduced (e.g. bt2 gene for insect resistance; see next chapter), so that alternative approaches for improving its expression had to be used. For an enzyme assay of NPT II (Reiss et al., 1984; Gene 30; 217-230), the enzyme is first fractionated using non denaturing polyacrylamide gel electrophoresis (PAGE). Since the enzyme detoxifies kanamycin by phosphorylation, radioactively labelled A TP (p32) is used with kanamycin in an agar layer, which is used to cover the gel containing the enzyme. The whole set is incubated at 35°C and the phosphorylation leading -to incorporation of 32p in kanamycin can be detected by autoradiography. The filter with dot blots is incubated with the substrates and is then subjected to autoradiography to detect the presence NPT II enzyme.