Irelan J T, Selker E U
Institute of Molecular Biology, University of Oregon, Eugene 97403-1229, USA.
Genetics. 1997 Jun;146(2):509-23. doi: 10.1093/genetics/146.2.509.
Repeated DNA sequences are frequently mutated during the sexual cycle in Neurospora crassa by a process named repeat-induced point mutation (RIP). RIP is often associated with methylation of cytosine residues in and around the mutated sequences. Here we demonstrate that this methylation can silence a gene located in nearby, unique sequences. A large proportion of strains that had undergone RIP of a linked duplication flanking a single-copy transgene, hph (hygromycin B phosphotransferase), showed partial silencing of hph. These strains were all heavily methylated throughout the single-copy hph sequences and the flanking sequences. Silencing was alleviated by preventing methylation, either by 5-azacytidine (5AC) treatment or by introduction of a mutation (eth-I) known to reduce intracellular levels of S-adenosylmethionine. Silenced strains exhibited spontaneous reactivation of hph at frequencies of 10(4) to 0.5. Reactivated strains, as well as cells that were treated with 5AC, gave rise to cultures that were hypomethylated and partially hygromycin resistant, indicating that some of the original methylation was propagated by a maintenance mechanism. Gene expression levels were found to be variable within a population of clonally related cells, and this variation was correlated with epigenetically propagated differences in methylation patterns.
在粗糙脉孢菌的有性生殖周期中，重复DNA序列经常通过一种名为重复序列诱导点突变（RIP）的过程发生突变。RIP通常与突变序列及其周围的胞嘧啶残基甲基化有关。在此我们证明，这种甲基化能够使位于附近的独特序列中的基因沉默。很大一部分经历了单拷贝转基因hph（潮霉素B磷酸转移酶）侧翼连锁重复序列RIP的菌株，表现出hph的部分沉默。这些菌株在整个单拷贝hph序列及其侧翼序列中都高度甲基化。通过5-氮杂胞苷（5AC）处理或引入已知可降低细胞内S-腺苷甲硫氨酸水平的突变（eth-I）来防止甲基化，可减轻沉默现象。沉默的菌株以10⁻⁴至0.5的频率表现出hph的自发重新激活。重新激活的菌株以及用5AC处理的细胞，产生了甲基化程度降低且对潮霉素部分抗性的培养物，这表明一些原始甲基化是通过维持机制传播的。在克隆相关细胞群体中发现基因表达水平存在差异，并且这种差异与甲基化模式的表观遗传传播差异相关。
