USE OF RESTRICTION ENZYMES TO STUDY EUKARYOTIC DNA METHYLATION .2. SYMMETRY OF METHYLATED SITES SUPPORTS SEMI-CONSERVATIVE COPYING OF METHYLATION PATTERN
USE OF RESTRICTION ENZYMES TO STUDY EUKARYOTIC DNA METHYLATION .2. SYMMETRY OF METHYLATED SITES SUPPORTS SEMI-CONSERVATIVE COPYING OF METHYLATION PATTERN
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DOI：
10.1016/0022-2836(78)90243-7
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发表时间：
1978-01-01
影响因子：
5.6
通讯作者：
BIRD, AP
中科院分区：
文献类型：
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作者：
BIRD, AP
用X. laevis rDNA中的碱基配对序列证明了这一点。**图形 **。或者两个胞嘧啶都被甲基化，或者都不被甲基化。未发现半甲基化位点。第一个实验涉及将标记的甲基化rDNA限制性片段（4.65-RI）与过量的未甲基化片段重新结合，以使任何半甲基化位点暴露于CpG酶消化。随后的治疗与HpaII和HhaI表明，甲基化/未甲基化的rDNA杂交是没有更敏感的消化比天然rDNA。不到2%的甲基化位点是半甲基化的。在第二个实验中，红细胞rDNA被变性并允许自行重新缔合。该方法使4.65内切酶EcoRI片段在除HhaI热点以外的所有位点上对HpaII、Avalidine和HhaI的消化免疫。由于天然片段在退火前在许多位点被HpaII、Avalidine和HhaI部分消化，其随后的免疫性表明大多数配对的CpG是对称甲基化的。该结果还验证了HhaI的特异性低甲基化识别序列的存在。最后，它被证明为X。在DNA复制后，新的甲基基团被添加到子代链中。亲本链未被[甲基-3H]甲硫氨酸可检测地标记。细胞基因组中甲基化和非甲基化配对CpG的任何模式都可能被该细胞的后代遗传。
Two experiments with X. laevis rDNA have demonstrated that in the base-paired sequence .**GRAPHIC**. either both cytosines are methylated or neither is methylated. Half-methylated sites are not found. The 1st experiment involved reassociation of a labeled methylated rDNA restriction fragment (4.65-RI) with an excess of the unmethylated fragment to expose any half-methylated sites to CpG-enzyme digestion. Subsequent treatment with HpaII and HhaI showed that methylated/unmethylated rDNA hybrids are no more sensitive to digestion than the native rDNA. Fewer than 2% of methylated sites are half-methylated. In the 2nd experiment erythrocyte rDNA was denatured and allowed to self-reassociate. This procedure rendered the 4.65 kilobase EcoRI fragment immune to digestion with HpaII, AvaI and HhaI at all sites except the HhaI hot-spot. Since the native fragment was partially digested at many sites by HpaII, AvaI and HhaI prior to annealing, its subsequent immunity indicates that most paired CpG are symmetrically methylated. The result also verifies the presence of a specific undermethylated recognition sequence for HhaI. Finally it has been shown for X. laevis cultured cells that following DNA replication new methyl groups are added to the progeny strand. The parental strand was not detectably labeled with [methyl- 3H]methionine. Any pattern of methylated and unmethylated paired CpG in the genome of a cell will probably be inherited by descendants of that cell.