Stein R, Razin A, Cedar H
Proc Natl Acad Sci U S A. 1982 Jun;79(11):3418-22. doi: 10.1073/pnas.79.11.3418.
The effect of DNA methylation on the expression of the hamster adenine phosphoribosyltransferase (aprt) gene in mouse cells has been examined. This gene was methylated in vitro at all of its C-C-G-G sites by using Hpa II methylase and was inserted into mouse Ltk- aprt- L cells by cotransformation, with the herpes virus thymidine kinase gene as a selectable vector. Whereas clones carrying unmethylated aprt sequences were found to have an aprt+ phenotype as shown by their ability to grow in azaserine-containing medium, almost all clones carrying methylated aprt sequences were shown to be phenotypically aprt-. Blot hybridization analysis demonstrated that both the methylated and unmethylated aprt sequences were integrated into the cellular genome to the same extent and that the in vitro modification was stably maintained in these cells for many generations. When clones containing methylated aprt genes were exposed to conditions that select for the expression of the aprt gene, a low frequency of reversion to the aprt+ phenotype was observed. In all of these clones, this reversion was accompanied by reorganization and undermethylation of the aprt sequences. These results show that the expression of certain genes may be inhibited by site-specific methylation of these sequences and suggest that methylation may play a direct role in the regulation of gene expression.
已研究了DNA甲基化对仓鼠腺嘌呤磷酸核糖转移酶（aprt）基因在小鼠细胞中表达的影响。通过使用Hpa II甲基化酶，该基因在体外其所有C-C-G-G位点均被甲基化，并通过共转化与疱疹病毒胸苷激酶基因作为选择载体一起插入小鼠Ltk- aprt- L细胞中。发现携带未甲基化aprt序列的克隆具有aprt+表型，这可通过它们在含氮杂丝氨酸的培养基中生长的能力来证明，而几乎所有携带甲基化aprt序列的克隆在表型上均为aprt-。印迹杂交分析表明，甲基化和未甲基化的aprt序列均以相同程度整合到细胞基因组中，并且体外修饰在这些细胞中稳定维持了许多代。当含有甲基化aprt基因的克隆暴露于选择aprt基因表达的条件下时，观察到向aprt+表型的低频回复。在所有这些克隆中，这种回复伴随着aprt序列的重组和去甲基化。这些结果表明，某些基因的表达可能会被这些序列的位点特异性甲基化所抑制，并表明甲基化可能在基因表达的调控中起直接作用。
