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

Abstract

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 met 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 me2 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 me2 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 me2 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.A procedure has recently been developed in our laboratory for integrative transformation of the filamentous ascomycete Ascobolus immersus (2); this procedure is based on the complementation of an auxotrophic met2 mutation by the wild-type cloned gene (3). Transformation of a met2 mutant strain of A. immersus by circular double-stranded (ds) DNA carrying the met2 gene (2) results either in the integration of the transforming DNA at nonhomologous chromosomal sites or in the substitution of the endogenous met2 mutation by the transforming wild-type allele. The relative frequency of both events varies according to the vector sequences carrying the transforming met2 gene, although nonhomologous integrations are the most often obtained. Homologous integration of the transforming plasmid by a reciprocal exchange at the chromosomal met2 locus has never been observed.We determined whether changing the conformation of the transforming DNA might have an effect on the transformation modalities of A. immersus and might eventually force homologous recombination with chromosomal sequences. We report on the ability of circular single-stranded (ss) DNA and linear ds DNA carrying the met2 gene to transform protoplasts of a met2 mutant strain of A. immersus. In contrast to circular ds DNA, they both most often participate in homolog...

Histone H1 Is Dispensable for Methylation-Associated Gene Silencing in Ascobolus immersusand Essential for Long Life Span

“…Most experimental procedures were as described previously (13). Other standard techniques were as in reference 45.…”

“…This organism displays DNA methylation, and MIP provides a convenient tool to methylate and silence at will endogenous genes (10,13,43). The cloning and characterization of the H1-like gene from Ascobolus, henceforth named H1, allowed us to inactivate the expression of this gene and to construct strains lacking Ascobolus histone H1.…”

Genomic Evolution of the Long Terminal Repeat Retrotransposons in Hemiascomycetous Yeasts

“…known to occur in fungi, in Ascobolus (Goyon and Faugeron 1989) and Neurospora (Cambareri et al 1989), in which they silence or inactivate retrotransposons by generating an accumulation of point mutations as a result of G-C to A-T transitions.…”

The RING finger/B-Box factor TAM-1 and a retinoblastoma-like protein LIN-35 modulate context-dependent gene silencing in Caenorhabditis elegans

“…Alternatively, the organism could possess a mechanism allowing repeated structures to be recognized by virtue of repeated character and not of any specific sequence. The latter seems to be the situation in Neurospora (Selker et al 1987;Cambareri et al 1989;Selker 1997) and Ascobolus (Goyon and Faugeron 1989;Rossignol and Faugeron 1994), whereas the limited number of transgenic constructs tested for repeat-dependent silencing in the other systems leave both models open.…”