Weber H, Ziechmann C, Graessmann A
Freie Universität Berlin, Institut für Molekularbiologie und Biochemie, FRG.
EMBO J. 1990 Dec;9(13):4409-15. doi: 10.1002/j.1460-2075.1990.tb07891.x.
A hemimethylated chimeric gene, containing the cauliflower mosaic virus 35S promoter, the beta-glucuronidase coding region and the polyadenylation signal of nopaline synthase, was introduced into tobacco protoplasts by polyethylene glycol mediated transfection. Hemimethylation led to complete inhibition of transient gene expression. In regenerated transgenic plants the integrated gene was constitutively hypermethylated at the sequences CpG and CpNpG and this was correlated with an inactivation of beta-glucuronidase in 12 out of 18 analyzed plant lines whereas two showed slight and four strong activity. From 10 control lines transformed with nonmethylated DNA, only two were inactive; three showed slight and five strong activity. 5-aza-cytidine treatment of plant tissue from 'hypermethylated' lines led to induction of beta-glucuronidase in most cases. Shoots regenerated from azaC treated calli revealed stable enzyme restoration and demethylation of the integrated transgene.
一个包含花椰菜花叶病毒35S启动子、β-葡萄糖醛酸酶编码区和胭脂碱合酶聚腺苷酸化信号的半甲基化嵌合基因，通过聚乙二醇介导的转染被导入烟草原生质体。半甲基化导致瞬时基因表达完全受到抑制。在再生的转基因植物中，整合的基因在CpG和CpNpG序列上组成性地高度甲基化，这与18个分析的植物株系中有12个株系的β-葡萄糖醛酸酶失活相关，而有两个株系表现出轻微活性，四个株系表现出强活性。在用未甲基化DNA转化的10个对照株系中，只有两个无活性；三个表现出轻微活性，五个表现出强活性。对来自“高度甲基化”株系的植物组织进行5-氮杂胞苷处理，在大多数情况下会诱导β-葡萄糖醛酸酶的表达。从经氮杂胞苷处理的愈伤组织再生的芽显示出稳定的酶活性恢复和整合转基因的去甲基化。
