METHYLATION OF CODING REGION ALONE INHIBITS GENE-EXPRESSION IN PLANT-PROTOPLASTS

Derivatives of the cauliflower mosaic virus 35S promoter lacking CG an d CNG methylation targets mere constructed and used to direct transcri ption of reporter gene constructs in transiently transformed protoplas ts. Such methylation-target-free (MTF) promoters, although weaker than the 35S promoter, retain significant activity despite mutation of the as-1 element. The effect of methylation on gene expression in MTF- an d 35S-promoter driven constructs was examined. Even when the promoter region was free of methylation targets, reporter gene expression was m arkedly reduced when cytosine residues in CG dinucleotides were methyl ated in vitro prior to transformation. Mosaic methylation experiments, in which only specific parts of the plasmids were methylated, reveale d that methylation of the coding region alone has a negative effect on reporter gene expression, Methylation nearer the 5' end of the coding region was more inhibitory, consistent with inhibition of transcripti on elongation.