Rhounim L, Rossignol J L, Faugeron G
Institut de Génétique et Microbiologie, CNRS URA 1354, Université, Paris-Sud, Orsay, France.
EMBO J. 1992 Dec;11(12):4451-7. doi: 10.1002/j.1460-2075.1992.tb05546.x.
Ascobolus immersus artificial gene repeats were shown previously to be subject premeiotically to both cytosine methylation and inactivation. We studied sexual progenies of strains harbouring two wild type copies of the endogenous met2 gene lying either in tandem array or at ectopic unlinked positions, by (i) investigating the methylation status, (ii) searching for mutations and (iii) analysing the inheritance of inactivation both in mitotic and sexual offspring. 100% of the 'tandem' progeny and 64% of the 'ectopic' progeny had methylated repeats and displayed gene inactivation. Similar methylation patterns involving all or most of the cytosine residues within the repeats were observed in both arrangements. The inactivated met2 copies were totally devoid of mutation, as deduced from: (i) extensive restriction site analysis and DNA sequencing; (ii) the finding that all the Met- derivatives tested reverted to prototrophy in selective conditions; and (iii) the finding that an inactivated copy of met2 stripped of its methylation through amplification in Escherichia coli regained activity when reintroduced in A.immersus. In the absence of selection, gene silencing and methylation were faithfully maintained through mitotic divisions and through five successive sexual cycles. Altogether, these data show the epimutational nature of this methylation induced premeiotically (MIP) process.
先前已表明，浸没状粪壳菌的人工基因重复序列在减数分裂前会发生胞嘧啶甲基化和失活。我们研究了携带两个内源性met2基因野生型拷贝的菌株的有性子代，这两个拷贝要么呈串联排列，要么位于异位非连锁位置，通过（i）研究甲基化状态，（ii）寻找突变，以及（iii）分析有丝分裂和有性后代中失活的遗传情况。100%的“串联”子代和64%的“异位”子代具有甲基化重复序列并表现出基因失活。在两种排列中都观察到了涉及重复序列内所有或大部分胞嘧啶残基的相似甲基化模式。从以下方面推断，失活的met2拷贝完全没有突变：（i）广泛的限制性位点分析和DNA测序；（ii）发现所有测试的Met-衍生物在选择性条件下都恢复为原养型；以及（iii）发现通过在大肠杆菌中扩增而去除甲基化的met2失活拷贝，当重新引入浸没状粪壳菌时恢复了活性。在没有选择的情况下，基因沉默和甲基化通过有丝分裂以及五个连续的有性周期得以忠实地维持。总之，这些数据显示了这种减数分裂前诱导的甲基化（MIP）过程的表观突变性质。
