In vitro DNA methylation inhibits gene expression in transgenic tobacco

A hemimethylated chimeric gene, containing the cauliflower mosaic virus 35S promoter, the beta-glucuronidase coding region and the polyadenylation signal of nopaline synthase, was introduced into tobacco protoplasts by polyethylene glycol mediated transfection. Hemimethylation led to complete inhibition of transient gene expression. In regenerated transgenic plants the integrated gene was constitutively hypermethylated at the sequences CpG and CpNpG and this was correlated with an inactivation of beta-glucuronidase in 12 out of 18 analyzed plant lines whereas two showed slight and four strong activity. From 10 control lines transformed with nonmethylated DNA, only two were inactive; three showed slight and five strong activity. 5-aza-cytidine treatment of plant tissue from 'hypermethylated' lines led to induction of beta-glucuronidase in most cases. Shoots regenerated from azaC treated calli revealed stable enzyme restoration and demethylation of the integrated transgene.