Schläppi M, Smith D, Fedoroff N
Carnegie Institution of Washington, Department of Embryology, Baltimore, Maryland 21210.
Genetics. 1993 Apr;133(4):1009-21. doi: 10.1093/genetics/133.4.1009.
The maize Suppressor-mutator (Spm) transposable element is subject to epigenetic inactivation in transgenic tobacco, as it is in maize. Spm inactivation in tobacco is correlated with increased methylation of sequences near the element's transcription start site. To determine whether element-encoded gene products can promote the reactivation of an inactive element, we investigated the effects of introducing individual CaMV 35S promoter-driven cDNAs for tnpA, tnpB, tnpC and tnpD, the element's four known protein-coding sequences. Introduction of the tnpA cDNA promoted the reactivation of the inactive resident Spm element, as judged by the appearance of regenerants with very early excision events and transposed elements. By contrast, the tnpB, tnpC and tnpD cDNAs had no affect on the activity of the resident Spm element. Similar results were obtained when the element-encoded cDNAs were introduced either by Agrobacterium-mediated retransformation or by a genetic cross. Reactivation of an inactive Spm by the tnpA cDNA is accompanied by reduced methylation of several methylation-sensitive restriction sites near the element's transcription start site. Maintenance of the reactivated Spm element in an active state requires the continued presence of the tnpA cDNA. Elimination of the tnpA cDNA locus by genetic segregation generally results in decreased element activity, as judged by a low frequency of excision events, and is accompanied by increased methylation of the element's 5'-end. Exceptions resembling the phenomenon of "presetting" are also observed in which progeny plants that did not receive the tnpA cDNA locus after meiotic segregation maintain high excision activity and exhibit low methylation levels.
玉米抑制子突变体（Spm）转座元件在转基因烟草中会发生表观遗传失活，就像在玉米中一样。烟草中Spm的失活与元件转录起始位点附近序列甲基化增加有关。为了确定元件编码的基因产物是否能促进失活元件的重新激活，我们研究了分别导入由CaMV 35S启动子驱动的tnpA、tnpB、tnpC和tnpD（该元件四个已知的蛋白质编码序列）的cDNA的效果。导入tnpA cDNA促进了失活的驻留Spm元件的重新激活，从具有非常早期切除事件和转座元件的再生植株的出现可以判断出来。相比之下，tnpB、tnpC和tnpD的cDNA对驻留Spm元件的活性没有影响。当通过农杆菌介导的重新转化或遗传杂交导入元件编码的cDNA时，也得到了类似的结果。tnpA cDNA使失活的Spm重新激活，同时元件转录起始位点附近几个对甲基化敏感的限制性位点的甲基化减少。使重新激活的Spm元件保持活跃状态需要tnpA cDNA的持续存在。通过遗传分离消除tnpA cDNA位点通常会导致元件活性降低，从切除事件的低频率可以判断出来，并且伴随着元件5'端甲基化增加。也观察到了类似“预设”现象的例外情况，即减数分裂分离后没有获得tnpA cDNA位点的后代植株保持高切除活性并表现出低甲基化水平。
