Targeted transformation of Ascobolus immersus and de novo methylation of the resulting duplicated DNA sequences.

摘要: To develop a method to modify genomic sequences in Ascobolus immersus by precisely reintroducing defined DNA segments previously manipulated vitro, we investigated the effect of transforming conformation on recombination with chromosomal sequences. Circular single-stranded carrying met2 gene and double-stranded linearized cutting within both transformed protoplasts mutant strain A. prototrophy. In contrast equivalent circular DNA, which chiefly integrated at nonhomologous sites, cut DNAs recombined primarily homologous sequence. Of transformants, 65% resulted from replacement resident mutation exogenous wild-type allele. 70% double-stranded-cut one or more copies had locus, leading tandem duplications target region separated plasmid DNA. These duplicated could recombine, progeny containing only copy region. This precise if allele been retained. addition, show that newly were most often de novo methylated cytosine residues during sexual phase. Cytosine methylation was associated inactivation gene(s) segregants crosses. However, not accurately maintained each replication cycle, so Met- recovered phenotype through successive mitotic divisions. finding indicated genes silenced alone.