McGeady M L, Jhappan C, Ascione R, Vande Woude G F
Mol Cell Biol. 1983 Mar;3(3):305-14. doi: 10.1128/mcb.3.3.305-314.1983.
The transforming activity of cloned Moloney sarcoma virus (MSV) proviral DNA was inhibited by in vitro methylation of the DNA at cytosine residues, using HpaII and HhaI methylases before transfection into NIH 3T3 cells. The inhibition of transforming activity due to HpaII methylation was reversed by treatment of the transfected cells with 5-azacytidine, a specific inhibitor of methylation. Analysis of the genomic DNA from the transformed cells which resulted from the transfection of methylated MSV DNA revealed that the integrated MSV proviral DNA was sensitive to HpaII digestion in all cell lines examined, suggesting that loss of methyl groups was necessary for transformation. When cells were infected with Moloney murine leukemia virus at various times after transfection with methylated MSV DNA, the amount of transforming virus produced indicated that the loss of methyl groups occurred within 24 h. Methylation of MSV DNA at HhaI sites was as inhibitory to transforming activity as methylation at HpaII sites. In addition, methylation at both HpaII and HhaI sites did not further reduce the transforming activity of the DNA. These results suggested that; whereas methylation of specific sites on the provirus may not be essential for inhibiting the transforming activity of MSV DNA, methylation of specific regions may be necessary. Thus, by cotransfection of plasmids containing only specific regions of the MSV provirus, it was determined that methylation of the v-mos gene was more inhibitory to transformation than methylation of the viral long terminal repeat.
在将克隆的莫洛尼氏肉瘤病毒（MSV）前病毒DNA转染到NIH 3T3细胞之前，使用HpaII和HhaI甲基化酶对DNA中的胞嘧啶残基进行体外甲基化，可抑制其转化活性。由HpaII甲基化导致的转化活性抑制可通过用5-氮杂胞苷（一种甲基化特异性抑制剂）处理转染细胞来逆转。对由甲基化MSV DNA转染产生的转化细胞的基因组DNA分析表明，在所检测的所有细胞系中，整合的MSV前病毒DNA对HpaII消化敏感，这表明甲基基团的丢失对于转化是必要的。在用甲基化MSV DNA转染后的不同时间用莫洛尼氏鼠白血病病毒感染细胞时，产生的转化病毒量表明甲基基团的丢失发生在24小时内。MSV DNA在HhaI位点的甲基化对转化活性的抑制作用与在HpaII位点的甲基化相同。此外，在HpaII和HhaI位点的甲基化都不会进一步降低DNA的转化活性。这些结果表明，虽然前病毒上特定位点的甲基化可能对于抑制MSV DNA的转化活性不是必需的，但特定区域的甲基化可能是必要的。因此，通过共转染仅含有MSV前病毒特定区域的质粒，确定v-mos基因的甲基化对转化的抑制作用比病毒长末端重复序列的甲基化更强。
