5-AZACYTIDINE INCREASES THE ACTIVITY OF NEOMYCIN PHOSPHOTRANSFERASE-II IN TRANSGENIC NICOTIANA-TABACUM - A POSTTRANSLATIONAL MECHANISM MAY PLAY A ROLE

In previous studies, tobacco protoplasts were transformed with the bac terial gene encoding neomycin phosphotransferase II (NPT II). Transfor med calluses lost neomycin phosphotransferase II activity after severa l subcultures. Treatment of calluses with 5-azacytidine, a demethylati ng agent, restored enzyme activity, suggesting that methylation of npt II sequences might be responsible for loss of NPT II activity. Studie s presented here were designed to test that hypothesis. Results indica ted that the effect of 5-azacytidine could not be blocked by the DNA r eplication inhibitor, hydroxyurea, nor by the 5-azacytidine analogue, cytidine as would be expected with a DNA demethylation mechanism. The level of NPT II mRNA was not increased by 5-azacytidine. Treatment wit h cycloheximide, a protein synthesis inhibitor, had no effect on 5-aza cytidine-increased NPT II activity. There was no increase of NPT II pr otein caused by 5-azacytidine, whereas 5-azacytidine increased activit y of NPT II. In contrast, the auxin 2,4-D increased both the NPT II pr otein and activity. Assays for malate dehydrogenase demonstrated that the effect of 5-azacytidine and hydroxyurea on NPT II was not due to a n overall effect on callus metabolism. In vitro studies involving stan dard bacterial NPT II enzyme and crude extracts from untreated and 5-a zacytidine- or hydroxyurea-treated calluses showed that the activity o f NPT II added to the untreated extracts was lower than the activity o f NPT II added to the extracts from calluses treated with 5-azacytidin e or hydroxyurea, indicating that there was an unknown factor (or fact ors) in callus extracts which affected the activity of NPT II and itse lf was affected by 5-azacytidine and hydroxyurea treatment. These resu lts suggested that one effect of 5-azacytidine in increasing NPT II ac tivity was posttranslational.