Kovarík A, Koukalová B, Lim K Y, Matyásek R, Lichtenstein C P, Leitch A R, Bezdek M
Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno.
Chromosome Res. 2000;8(6):527-41. doi: 10.1023/a:1009223823327.
Cytosine methylation levels and susceptibility to drug-induced hypomethylation have been studied in several Nicotiana tabacum (tobacco) DNA repetitive sequences. It has been shown using HapII, MspI, BamHI and Sau3AI methylation-sensitive restriction enzymes that the degree of 5'-mCmCG-3' methylation varied significantly between different repeats. There were almost saturation levels of 5-methylcytosine at the inner (3') cytosine position and variable degrees of methylation at the outer (5') cytosine at the enzyme recognition sites. The non-transcribed high copy satellite sequences (HRS60, GRS) displayed significant heterogeneity in methylation of their basic units while middle repetitive sequences (R8.1, GRD5, 5S rDNA) were more uniformly modified at both cytosine residues. Dihydroxypropyladenine (DHPA) treatment, which is thought to reduce DNA methyltransferase activity by increasing S-adenosylhomocysteine levels, resulted in extensive demethylation of the outer cytosine in all repeats, and the partial hypomethylation of cytosines at the inner positions in less densely methylated repeats such as HRS60 and GRS. The results suggest that hypomethylation of 5'-mCmCG-3' sites with DHPA is a gradual non-random process proceeding in the direction mCmCG-->CmCG-->CCG. The 18S-5.8S-25S rDNA was remarkably hypomethylated relative to the 5S rDNA at all restriction sites studied. Fluorescence in-situ hybridization showed that DNA decondensation within and between the 18S-5.8S-25S and 5S rDNA loci was variable in different nuclei. All nuclei had condensed and decondensed sequence. The chromatin of 18S-5.8S-25S rDNA was more readily digested with micrococcal nuclease than the 5S rDNA suggesting that the overall levels of decondensation were higher for 18S-5.8S-25S rDNA. Variable decondensation patterns within and between loci were also observed for GRS and HRS60. Cytosine methylation of the tobacco repeats is discussed with respect to transcription, overall levels of condensation and overall structure.
在几种烟草（Nicotiana tabacum）DNA重复序列中，研究了胞嘧啶甲基化水平以及对药物诱导的去甲基化的敏感性。使用HapII、MspI、BamHI和Sau3AI甲基化敏感限制酶已表明，不同重复序列之间5'-mCmCG-3'的甲基化程度差异显著。在酶识别位点，内部（3'）胞嘧啶位置的5-甲基胞嘧啶几乎达到饱和水平，而外部（5'）胞嘧啶的甲基化程度各不相同。非转录的高拷贝卫星序列（HRS60、GRS）在其基本单元的甲基化方面表现出显著的异质性，而中等重复序列（R8.1、GRD5、5S rDNA）在两个胞嘧啶残基处的修饰更为均匀。二羟丙基腺嘌呤（DHPA）处理被认为通过提高S-腺苷同型半胱氨酸水平来降低DNA甲基转移酶活性，导致所有重复序列中外部胞嘧啶广泛去甲基化，以及在甲基化程度较低的重复序列（如HRS60和GRS）中内部位置的胞嘧啶部分去甲基化。结果表明，DHPA诱导的5'-mCmCG-3'位点去甲基化是一个逐渐的非随机过程，朝着mCmCG→CmCG→CCG的方向进行。在所有研究的限制位点，相对于5S rDNA，18S-5.8S-25S rDNA显著去甲基化。荧光原位杂交显示，在不同细胞核中，18S-5.8S-25S和5S rDNA基因座内部以及之间的DNA解聚情况各不相同。所有细胞核都有浓缩和解聚的序列。与5S rDNA相比，微球菌核酸酶更容易消化18S-5.8S-25S rDNA的染色质，这表明18S-5.8S-25S rDNA的总体解聚水平更高。在GRS和HRS60的基因座内部以及之间也观察到了不同的解聚模式。本文从转录、总体浓缩水平和总体结构方面对烟草重复序列的胞嘧啶甲基化进行了讨论。
