USE OF RESTRICTION ENZYMES TO STUDY EUKARYOTIC DNA METHYLATION .1. METHYLATION PATTERN IN RIBOSOMAL DNA FROM XENOPUS-LAEVIS
USE OF RESTRICTION ENZYMES TO STUDY EUKARYOTIC DNA METHYLATION .1. METHYLATION PATTERN IN RIBOSOMAL DNA FROM XENOPUS-LAEVIS
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DOI：
10.1016/0022-2836(78)90242-5
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发表时间：
1978-01-01
影响因子：
5.6
通讯作者：
SOUTHERN, EM
中科院分区：
文献类型：
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作者：
BIRD, AP;SOUTHERN, EM
限制性内切酶HpaII、AvaI、HhaI和HaeII能区分X.从扩增的rDNA中扩增出的红细胞rDNA。扩增的rDNA在许多位点被每种酶切割，而体细胞rDNA相对耐消化。这些差异归因于5-甲基胞嘧啶的存在，使得体细胞rDNA中的许多限制性位点对核酸酶攻击具有抗性。真核DNA中的主要甲基化序列是CpG，并且所有4种酶的识别位点都含有该序列。由此鉴定的CpG酶用于研究红细胞rDNA中甲基化的模式。通过定位脆弱的限制性位点定位未甲基化的CpG，并确定CpG甲基化的分布和水平。对于大多数检测到的CpG，甲基化水平很高（约99%）。在30-60%的rDNA重复单元中有1个位点未甲基化。在非转录间隔区中也有一个区域，其中HpaII和Aviral的未甲基化位点集中，尽管还不确定这是否是由于甲基化不足。
Restriction enzymes HpaII, AvaI, HhaI and HaeII can distinguish X. laevis somatic (erythrocyte) rDNA from amplified rDNA. Amplified rDNA is cleaved at many sites by each enzyme while somatic rDNA is relatively resistant to digestion. The differences is attributed to the presence of 5-methylcytosine rendering many restriction sites in somatic rDNA resistant to nuclease attack. The major methylated sequence in eukaryotic DNA is CpG, and recognition sites for all 4 enzymes contain this sequence. The CpG enzymes thus identified were used to study the pattern of methylation in erythrocyte rDNA. Unmethylated CpG were located by mapping vulnerable restriction sites and the distribution and level of CpG methylation was determined. For most detected CpG the level of methylation is high (about 99%). There is 1 site which is unmethylated in 30-60% of rDNA repeat units. There is also a region in the non-transcribed spacer within which unmethylated sites for HpaII and AvaI are concentrated, though it is not yet certain whether this is due to undermethylation.