Bartee L, Bender J
Department of Biochemistry and Molecular Biology, Johns Hopkins University School of Public Health, 615 North Wolfe Street, Baltimore, MD 21205, USA.
Nucleic Acids Res. 2001 May 15;29(10):2127-34. doi: 10.1093/nar/29.10.2127.
In Arabidopsis a SWI2/SNF2 chromatin remodeling factor-related protein DDM1 and a cytosine methyltransferase MET1 are required for maintenance of genomic cytosine methylation. Mutations in either gene cause global demethylation. In this work we have assessed the effects of these mutations on the PAI tryptophan biosynthetic gene family, which consists of four densely methylated genes arranged as a tail-to-tail inverted repeat plus two unlinked singlet genes. The methylation mutations caused only partial demethylation of the PAI loci: ddm1 had a strong effect on the singlet genes but a weaker effect on the inverted repeat, whereas met1 had a stronger effect on the inverted repeat than on the singlet genes. The double ddm1 met1 mutant also displayed partial demethylation of the PAI genes, with a pattern similar to the ddm1 single mutant. To determine the relationship between partial methylation and expression for the singlet PAI2 gene we constructed a novel reporter strain of Arabidopsis in which PAI2 silencing could be monitored by a blue fluorescent plant phenotype diagnostic of tryptophan pathway defects. This reporter strain revealed that intermediate levels of methylation correlate with intermediate suppression of the fluorescent phenotype.
在拟南芥中，一种与SWI2/SNF2染色质重塑因子相关的蛋白质DDM1和一种胞嘧啶甲基转移酶MET1是维持基因组胞嘧啶甲基化所必需的。任一基因发生突变都会导致全基因组去甲基化。在这项研究中，我们评估了这些突变对PAI色氨酸生物合成基因家族的影响，该家族由四个紧密甲基化的基因组成，它们以尾对尾反向重复排列，外加两个不连锁的单拷贝基因。甲基化突变仅导致PAI基因座部分去甲基化：ddm1对单拷贝基因有强烈影响，但对反向重复序列影响较弱，而met1对反向重复序列的影响比对单拷贝基因的影响更强。双突变体ddm1 met1也表现出PAI基因的部分去甲基化，其模式与ddm1单突变体相似。为了确定单拷贝PAI2基因的部分甲基化与表达之间的关系，我们构建了一种新型的拟南芥报告菌株，其中PAI2的沉默可以通过诊断色氨酸途径缺陷的蓝色荧光植物表型来监测。该报告菌株显示，甲基化的中间水平与荧光表型的中间抑制相关。
