EcoR II is an unreliable enzyme for studies of CpNpG methylation in Arabidopsis thaliana

被引:1
作者
Godager, LH [1 ]
Meza, TJ [1 ]
Borud, B [1 ]
Jakobsen, KS [1 ]
Aalen, RB [1 ]
机构
[1] Univ Oslo, Div Gen Genet, N-0315 Oslo, Norway
关键词
genomic sequencing; Lea genes; low temperature-induced genes; methylation; promoters;
D O I
10.1023/A:1007428026143
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Methylation patterns from cold-inducible and embryo-specific Arabidopsis thaliana gene promoter regions were investigated. Pairs of restriction enzymes sensitive and insensitive to methylation in the same recognition sequence were used to digest genomic DNA, and the methylation status was visualized by Southern hybridization. The pair BstN I/ EcoR II should detect CpNpG methylation due to the sensitivity of EcoR II to 5-methylcytosine in the second position in the recognition sequence (5'-CC(A/T)GG-3'), The pail Msp I/Hpa II will detect both CpNpG methylation and CpG methylation, since Msp I does not digest the recognition sequence (5'-CCGG-3') when the first C residue is methylated, while Hpa II restriction is inhibited by methylation of either of the two C residues. EcoR II digestion studies suggested CpNpG methylation in all genes tested and demethylation after cold stress in all genes (including two control embryo-specific Lea genes not induced by low temperature). Control experiments indicated an unexpected pattern of methylation and low temperature demethylation in chloroplast genes;Additional control experiments, using the: methylation sensitive enzyme, Serf I (recognizing the sequence 5'-CCNGG-3'), disproved the presence of 5-methylcytosine in common sites not digested by EcoR II. (CpNpG-methylation was revealed in one Serf I site in one gene and in Msp, I/Hpa II sites in two genes. CpG methylation was not found in any gene tested.) Our study indicates that results obtained using EcoR II for DNA methylation studies should be interpreted with caution. The peculiarities of the EcoR II enzyme are further discussed.
引用
收藏
页码:19 / 32
页数:14
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