SEQUENCE-SPECIFIC HYPOMETHYLATION OF THE TOBACCO GENOME INDUCED WITH DIHYDROXYPROPYLADENINE, ETHIONINE AND 5-AZACYTIDINE

Higher plant DNA is methylated at CG and CNG targets. In this study we have investigated the tobacco methylation system in tissue culture us ing the methylation inhibitors 5-azacytidine (5-azaC), dihydroxypropyl adenine (DHPA) and ethionine (Ethi), and methylation-sensitive restric tion endonucleases HpaII, MspI, HhaI, EcoRII, scrFI, and Fnu4HI. Surpr isingly, CAG/CTG sequences, contrary to CG doublets and CCG/CGG triple ts, appeared to be refractory to the inhibitory effect of 5-azaC. Thus 5-azaC cannot be considered a general inhibitor of DNA methylation in tobacco cells. On the other hand, DHPA, the inhibitor of S-adenosylho mocysteine (SAH) hydrolase, and Ethi caused hypomethylation of both CA G/CTG and CCG/CGG triplets but not of the CG doublets. The sensitivity of triplet-specific methylation to the inhibition of SAH hydrolase su ggests the possibility that plant-specific DNA methylation at CNG targ ets might be modulated by alterations of the SAH/S-Adenosylmethionine ratio in plant cells.