Inhibition of Cytosine Methylation Allows Efficient Cloning of T-DNA Tagged Plant DNA, of Arabidopsis thaliana by Plasmid Rescue

Plasmid rescue can provide an efficient way of cloning T-DNA-tagged genomic DNA of plants. However, rescue has often been hampered by extensive rearrangements in the cloned DNA. We have demonstrated using a transgenic line of Arabidopsis thaliana that the plant DNA flanking the T-DNA tag was heavily cytosine methylated. This methylation could be completely inhibited by growing the plants in the presence of azacytidine. Rescue of the T-DNA tag together with the flanking plant genomic DNA sequences from nontreated control plants into an modified cytosine restriction (mcr) proficient strain of Escherichia coli resulted in rearrangements of the majority of the rescued plasmids. These rearrangements could be avoided if the methylation was inhibited in the transgenic plants by azacytidine treatment or by cloning into an mcr-deficient strain of E. coli. The results indicate that cytosine methylation of the DNA in the transgenic plants is the main cause of the DNA rearrangements observed during plasmid rescue and suggest efficient strategies to eliminate such artifacts.