Clustering of multiple transgene integrations in highly-unstable Ascobolus immersus transformants

Abstract

A large proportion of Ascobolus immersus transformants are highly unstable in crosses: the phenotype conferred by the transgene is not transmitted to the progeny, irrespective of the endogenous or foreign origin of the transgene. They all have integrated multiple transgene copies, clustered at a single chromosomal site or at tightly-linked sites. Clustered non-homologous integrations are always rearranged. Yet they never escape the "methylation induced premeiotically" (MIP) process. This always results in gene silencing, even when the transgene is partially repeated, accounting for the high instability of these transformants.

Pezizomycetes genomes reveal the molecular basis of ectomycorrhizal truffle lifestyle

“…boudieri. Strain RN42 (CBS 140666) of A. immersus was grown as described in Rhounim et al 22 . Strain S1 (ATCC MYA-4762) of Te.…”

Masc2, a gene from Ascobolus encoding a protein with a DNA‐methyltransferase activity in vitro, is dispensable for in vivo methylation

“…For this, we used strains harbouring an hph transgene previously methylated and silenced by MIP (Maloisel and Rossignol, 1998). When active, the hph transgene confers hygromycin resistance to Ascobolus (Rhounim et al ., 1994). In contrast, strains harbouring a silenced hph transgene exhibit the same sensitivity to hygromycin as untransformed strains.…”

“…The demethylating agent 5‐azacytidine (5‐AC) (Jones and Taylor, 1980) facilitates the re‐expression of genes that had been methylated and silenced by MIP (Goyon and Faugeron, 1989; Rhounim et al ., 1992; Rhounim et al ., 1994). However, the demethylation induced by 5‐AC is neither total nor permanent.…”

“…Standard Ascobolus genetic techniques, transformation procedures and media were used (Rhounim et al ., 1994; and references therein).…”

DNA Methyltransferases and Structural-Functional Specificity of Eukaryotic DNA Modification

“…Unlike the case of tandem duplications, the methylation efficiency of unconnected duplications smoothly correlates with their size [119]. Methylation of cytosine residues inside the repeating genes results in their reversible silencing which can be, in particular, removed with the demethylating agent 5 azacytidine [117,122]. Methylation of fungal genes during MIP and RIP decreases the content of corresponding mRNAs or fully eliminates them [123].…”