De Buck S, Depicker A
Departement Plantengenetica, Vlaams Interuniversitair Instituut voor Biotechnologie, Universiteit Gent, Belgium.
Mol Genet Genomics. 2001 Aug;265(6):1060-8. doi: 10.1007/s004380100503.
The transgene locus KH15, which is highly susceptible to silencing in Arabidopsis thaliana, contains two inversely repeated beta-glucuronidase (gus) genes separated by a palindromic sequence and has a low GUS activity, was found to be heavily methylated in the gus coding sequence and in the center of the inverted repeat. The locus KHsb67, which is less prone to silencing, was found to be less densely methylated in the non-repetitive region that separates the inversely repeated gus genes. After the removal of one of the gus genes by Cre-mediated recombination, methylation in both loci decreased or was totally lost. Despite the presence of a 732-bp palindromic sequence in the deletion line derived from KH15, this sequence was not methylated. Whereas the KH15 locus triggers methylation of homologous gus genes when placed in trans to them, the deletion derivative did not, suggesting that the capacity for cross-talk was severely affected by disruption of the palindromic arrangement. This result suggests that the transcribed palindromic sequences are required to maintain the methylation of both symmetrically and non-symmetrically arranged cytosines.
转基因位点KH15在拟南芥中极易发生沉默，它包含两个反向重复的β-葡萄糖醛酸酶（gus）基因，中间由一个回文序列隔开，且GUS活性较低。研究发现，该位点在gus编码序列以及反向重复序列的中心区域存在高度甲基化。而沉默倾向较低的位点KHsb67，在分隔反向重复gus基因的非重复区域甲基化程度较低。通过Cre介导的重组去除其中一个gus基因后，两个位点的甲基化程度均降低或完全消失。尽管源自KH15的缺失系中存在一个732 bp的回文序列，但该序列并未发生甲基化。当KH15位点与同源gus基因反式排列时会引发后者的甲基化，而缺失衍生物则不会，这表明回文排列的破坏严重影响了相互作用的能力。该结果表明，转录的回文序列对于维持对称和非对称排列的胞嘧啶的甲基化是必需的。
