ANTIBIOTICS INDUCE GENOME-WIDE HYPERMETHYLATION IN CULTURED NICOTIANA-TABACUM PLANTS

Plant genomic DNA methylation was analyzed by an improved SssI methylt ransferase assay and by genomic sequencing with sodium bisulfite. Kana mycin, hygromycin, and cefotaxime (also called Claforan) are commonly used as selective agents for the production of transgenic plants. Thes e antibiotics caused DNA hypermethylation in tobacco plants grown in v itro, which was both time- and dose-dependent. An exposure of the plan tlets to 500 mg/liter cefotaxime for 1 month caused the de novo methyl ation of 3 x 10(7) CpG sites/haploid genome of 3.5 x 10(9) base pairs. It occurred in high, moderate, and low repetitive DNA and was not rev ersible upon the removal of the antibiotics. Reversion was only observ ed in progeny grown in the absence of drugs. Analysis of the promoter regions of two single-copy genes, an auxin-binding protein gene and th e class I chitinase gene, showed the hypermethylation to be heterogene ous but biased toward CpGs. The hypermethylation of the class I chitin ase and the auxin-binding protein promoters was not a consequence of a drug-induced gene amplification.