Hohn T, Corsten S, Rieke S, Müller M, Rothnie H
Friedrich Miescher Institute, Basel, Switzerland.
Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8334-9. doi: 10.1073/pnas.93.16.8334.
Derivatives of the cauliflower mosaic virus 35S promoter lacking CG and CNG methylation targets were constructed and used to direct transcription of reporter gene constructs in transiently transformed protoplasts. 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- and 35S-promoter driven constructs was examined. Even when the promoter region was free of methylation targets, reporter gene expression was markedly reduced when cytosine residues in CG dinucleotides were methylated in vitro prior to transformation. Mosaic methylation experiments, in which only specific parts of the plasmids were methylated, revealed 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 transcription elongation.
构建了缺乏CG和CNG甲基化靶点的花椰菜花叶病毒35S启动子的衍生物，并用于在瞬时转化的原生质体中指导报告基因构建体的转录。这种无甲基化靶点（MTF）的启动子虽然比35S启动子弱，但尽管as-1元件发生了突变，仍保留了显著的活性。研究了甲基化对MTF和35S启动子驱动构建体中基因表达的影响。即使启动子区域没有甲基化靶点，在转化前体外将CG二核苷酸中的胞嘧啶残基甲基化时，报告基因的表达也会显著降低。镶嵌甲基化实验（其中仅质粒的特定部分被甲基化）表明，仅编码区的甲基化对报告基因的表达有负面影响。编码区5'端附近的甲基化抑制作用更强，这与转录延伸的抑制作用一致。
