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. 2010 Feb;61(4):558–569. doi: 10.1111/j.1365-313X.2009.04079.x

Figure 3.

Figure 3

The Dim1B gene from Arabidopsis thaliana can complement site specific methylation defects in E. coli. The coding sequence of the Dim1B gene from Arabidopsis thaliana was cloned into vectors driving transcription in E. coli from either a lacZ or a T7 promoter as indicated in the scheme and introduced into the E. coli strains JM101 or BL21(DE3) lacking the ksgA gene. The specific adenosine dimethylation in the 3′ terminal stem–loop structure of the bacterial 16S rRNA is depicted on the left. Primer extension in the presence of ddATP confirms the absence of this methylation in the ksgA− mutant bacteria and yields no signal at the adenosine doublet but terminates primer extension at the next uridine in the 16S rRNA sequence. Introduction of a functional ksgA gene or either of the expression constructs of the Dim1B gene from Arabidopsis restores the methylation signal at the adenosine dinucleotide. The left part of the gel image shows the corresponding RNA sequence reactions for orientation of the ddATP termination signals.