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首页> 外文期刊>Molecular and Cellular Biology >Targeted transformation of Ascobolus immersus and de novo methylation of the resulting duplicated DNA sequences.
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Targeted transformation of Ascobolus immersus and de novo methylation of the resulting duplicated DNA sequences.

机译：有针对性的转化Ascobolus浸泡和从头甲基化的重复的DNA序列。

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To develop a method to modify genomic sequences in Ascobolus immersus by precisely reintroducing defined DNA segments previously manipulated in vitro, we investigated the effect of transforming DNA conformation on recombination with chromosomal sequences. Circular single-stranded DNA carrying the met2 gene and double-stranded DNA linearized by cutting within the met2 gene both transformed protoplasts of a met2 mutant strain of A. immersus to prototrophy. In contrast to the equivalent circular double-stranded DNA, which chiefly integrated at nonhomologous chromosomal sites, single-stranded and double-stranded cut DNAs recombined primarily with the homologous chromosomal met2 sequence. Of the single-stranded DNA transformants, 65% resulted from replacement of the resident met2 mutation by the exogenous wild-type allele. In 70% of the double-stranded-cut DNA transformants, one or more copies of the transforming DNA had integrated at the met2 locus, leading to tandem duplications of the met2 target region separated by plasmid DNA. These duplicated sequences could recombine, leading to progeny containing only one copy of the met2 region. This resulted in a precise gene replacement if the wild-type allele had been retained. In addition, we show that newly duplicated sequences were most often de novo methylated at the cytosine residues during the sexual phase. Cytosine methylation was associated with inactivation of the integrated met2 gene(s) in segregants of crosses. However, methylation was not accurately maintained at each DNA replication cycle, so that Met- segregants recovered a wild-type phenotype through successive mitotic divisions. This finding indicated that met2 genes were silenced by methylation alone.
机译：为了开发一种方法，通过精确地重新引入先前在体外操作过的确定的DNA片段来修饰拟南芥中的基因组序列，我们研究了转化DNA构象对与染色体序列重组的影响。携带met2基因的环状单链DNA和通过在met2基因内切割而线性化的双链DNA均将浸入曲霉的met2突变株的原生质体转化为原生质体。与主要在非同源染色体位点整合的同等环状双链DNA相反，单链和双链切割DNA主要与同源染色体met2序列重组。在单链DNA转化子中，有65％是由外源野生型等位基因替代了常驻的met2突变。在70％的双链DNA转化子中，转化DNA的一个或多个副本已整合到met2基因座，导致由质粒DNA分离的met2目标区域串联重复。这些重复的序列可以重组，导致后代仅包含一个拷贝的met2区。如果保留了野生型等位基因，这将导致精确的基因替换。此外，我们表明，新复制的序列最常在性阶段在胞嘧啶残基处从头甲基化。胞嘧啶甲基化与杂交分离子中整合的met2基因失活有关。但是，甲基化不能在每个DNA复制周期中精确维持，因此Metsegregants通过连续的有丝分裂分裂恢复了野生型表型。这一发现表明，仅通过甲基化使met2基因沉默。

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