5-azacytidine increases the activity of neomycin phosphotransferase II in transgenic Nicotiana tabacum : posttranslational mechanism might play an important role

In previous studies, tobacco protoplasts were transformed with the bacterial gene encoding neomycin phosphotransferase II (npt II). Transformed calli produced from transformed protoplast cultures lost NPT II activity after several subcultures. Activity of this enzyme, however, could be increased by treating the calli with 5-azacytidine. Present studies show that the effect of 5-azacytidine could not be blocked by the DNA replication inhibitor, hydroxyurea, nor by its analogue, cytidine. In addition, the level of mRNA of npt II was not increased by 5-azacytidine, indicating the effect of 5-azacytidine was not at the transcriptional level. Treatment with cycloheximide, a protein synthesis inhibitor, had no effect on 5-azacytidine-increased NPT II activity, indicating that current protein synthesis was not involved in increasing NPT II activity. These results suggested that the effect of 5-azacytidine might be a posttranslational one.

This posttranslational hypothesis was further supported by ELISA analysis which showed that there was no increase of NPT II protein caused by 5-azacytidine, whereas 5-azacytidine increased the activity of the enzyme. In contrast to 5- azacytidine, however, the auxin 2,4-D increased both the quantity and the activity of NPT II.

Furthermore, in vitro studies involving standard bacterial NPT II enzyme and crude extracts from untreated and 5-azacytidine- or hydroxyurea-treated calli showed that the activity of NPT II added to the untreated extracts was lower than the activity of NPT II added to the extracts from calli treated with 5-azacytidine or hydroxyurea, indicating that there was an unknown factor(s) existing in the crude extracts. This factor affected the activity of NPT II and itself was affected by 5-azacytidine and hydroxyurea treatment.

5-azacytidine is a potent DNA demethylating agent. However, present studies indicate that the effect of 5-azacytidine on increasing NPT II activity was not necessarily due to its demethylation function, and that 5-azacytidine might affect the enzyme in a post translational mechanism.

Assays for malate dehydrogenase demonstrated that the effect of 5-azacytidine and hydroxyurea on NPT II was not due to an overall effect on callus metabolism.