Cambareri E B, Foss H M, Rountree M R, Selker E U, Kinsey J A
Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City 66160-7420, USA.
Genetics. 1996 May;143(1):137-46. doi: 10.1093/genetics/143.1.137.
An unstable allele of the Neurospora am (GDH) gene resulting from integration of the retrotransposon Tad3-2 into 5' noncoding sequences was found in previous work. We report that reversion to Am+ depends on DNA methylation within and upstream of Tad. Levels of methylation were correlated with the proportion of Am+ conidia, whether the cultures were derived from Am- or Am+ isolates. Reversion to Am+ did not occur when conidia were plated on 5-azacytidine, which reduces DNA methylation. The mutation dim-2, which appears to abolish DNA methylation, also prevented reversion to Am+. The native am allele, in a strain that lacked Tad elements, was replaced with am::Tad3-2 or with a deletion derivative that prevents transposition of Tad. Transformants of both classes showed instability comparable with that of the original isolates, which contain multiple Tad elements. Deletion of the upstream enhancer-like sequences, URSam alpha and beta, did not prevent the instability of am::Tad3-2. The results suggest that am expression is dependent on DNA methylation but not on proliferation or transposition of the Tad element and that the instability does not require the upstream sequences of am.
在之前的研究中发现，由于反转录转座子Tad3-2整合到5'非编码序列中，导致了粗糙脉孢菌am（GDH）基因的一个不稳定等位基因。我们报告称，回复到Am+依赖于Tad内部和上游的DNA甲基化。甲基化水平与Am+分生孢子的比例相关，无论培养物是来自Am-还是Am+分离株。当分生孢子接种在可降低DNA甲基化的5-氮杂胞苷上时，不会发生回复到Am+的情况。似乎能消除DNA甲基化的突变体dim-2也会阻止回复到Am+。在一个缺乏Tad元件的菌株中，将天然的am等位基因替换为am::Tad3-2或一个可阻止Tad转座的缺失衍生物。这两类转化体都表现出与原始分离株相当的不稳定性，原始分离株含有多个Tad元件。删除上游增强子样序列URSamα和β并不会阻止am::Tad3-2的不稳定性。结果表明，am的表达依赖于DNA甲基化，而不依赖于Tad元件的增殖或转座，并且这种不稳定性不需要am的上游序列。
