METHYLATION OF FOREIGN DNA SEQUENCES IN EUKARYOTIC CELLS
METHYLATION OF FOREIGN DNA SEQUENCES IN EUKARYOTIC CELLS
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DOI：
10.1073/pnas.77.11.6463
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发表时间：
1980-01-01
期刊：
影响因子：
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通讯作者：
CEDAR, H
中科院分区：
文献类型：
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作者：
POLLACK, Y;STEIN, R;CEDAR, H
利用疱疹病毒胸苷激酶基因，通过DNA介导的基因转移将外源DNA序列导入小鼠L细胞。然后使用限制性内切酶同工酶Hpa II和Msp I测定这些插入基因在C-C-G-G特异序列处的甲基化。尽管70%的细胞C-C-G-G位点是甲基化的，但疱疹病毒序列、质粒DNA和生长激素基因DNA在90%的含有这些基因的克隆中保持未甲基化。将已在体外用Hpa II甲基化酶甲基化的DNA也插入L细胞。然而，载体DNA中存在这种修饰并不能保证这些序列在受体克隆中保持甲基化。只有10%的转化克隆在载体DNA中含有甲基化的C-C-G-G序列，并且这些修饰在25 - 50代中是稳定的。Hha I和Mbo I用于探测这些限制性位点处的甲基，但插入的序列均未获得这些修饰。这些结果进行了讨论，提出了各种模型来解释真核细胞上的甲基化过程。
The herpesvirus thymidine kinase gene was used to introduce foreign DNA sequences into mouse L cells by DNA-mediated gene transfer. These inserted genes were then assayed for methylation at the specific sequence of C-C-G-G by using the restriction enzyme isoschizomers Hpa II and Msp I. Despite the fact that 70% of the cellular C-C-G-G sites are methylated, herpesvirus sequences, plasmid DNA, and growth hormone gene DNA remained unmethylated in 90% of the clones that contain these genes. DNA that had been methylated in vitro with Hpa II methylase was also inserted into L cells. The presence of this modification in the vector DNA did not, however, guarantee that these sequences remained methylated in the recipient clones. Only 10% of all transformed clones contained methylated C-C-G-G sequences in the vector DNA, and these modifications were stable for 25-50 generations. Hha I and Mbo I were used to probe for methyl groups at these restriction sites, but none of the inserted sequences acquired these modifications. These results are discussed in relation to various models put forth to explain the process of methylation on eukaryotic cells.