Schmitt F, Oakeley E J, Jost J P
Friedrich Miescher Institute, P. O. Box 2543, CH-4002 Basel, Switzerland.
J Biol Chem. 1997 Jan 17;272(3):1534-40. doi: 10.1074/jbc.272.3.1534.
Plant genomic DNA methylation was analyzed by an improved SssI methyltransferase assay and by genomic sequencing with sodium bisulfite. Kanamycin, hygromycin, and cefotaxime (also called Claforan) are commonly used as selective agents for the production of transgenic plants. These antibiotics caused DNA hypermethylation in tobacco plants grown in vitro, which was both time- and dose-dependent. An exposure of the plantlets to 500 mg/liter cefotaxime for 1 month caused the de novo methylation 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 reversible upon the removal of the antibiotics. Reversion was only observed in progeny grown in the absence of drugs. Analysis of the promoter regions of two single-copy genes, an auxin-binding protein gene and the class I chitinase gene, showed the hypermethylation to be heterogeneous but biased toward CpGs. The hypermethylation of the class I chitinase and the auxin-binding protein promoters was not a consequence of a drug-induced gene amplification.
通过改进的SssI甲基转移酶测定法和亚硫酸氢钠基因组测序分析植物基因组DNA甲基化。卡那霉素、潮霉素和头孢噻肟（也称为头孢曲松）通常用作生产转基因植物的选择剂。这些抗生素导致体外培养的烟草植株DNA发生超甲基化，且具有时间和剂量依赖性。将幼苗暴露于500毫克/升头孢噻肟1个月会导致单倍体基因组（3.5×10⁹碱基对）中3×10⁷个CpG位点发生从头甲基化。这种情况发生在高度、中度和低度重复DNA中，并且在去除抗生素后不可逆。仅在无药物条件下生长的后代中观察到甲基化逆转。对两个单拷贝基因（生长素结合蛋白基因和I类几丁质酶基因）的启动子区域进行分析表明，超甲基化是异质性的，但偏向于CpG。I类几丁质酶启动子和生长素结合蛋白启动子的超甲基化不是药物诱导基因扩增的结果。
