Perpetuation of cytosine methylation in Ascobolus immersus implies a novel type of maintenance methylase.

In the ascomycete Ascobolus immersus, duplicated DNA segments are subject to the methylation induced premeiotically (MIP) process. Affected sequences are heavily methylated at their cytosine residues. We used the bisulphite genomic sequencing method to determine the methylation status of every cytosine residue in a gene which had undergone MIP. Several individual DNA molecules, all issued from the replication of a single molecule initially subject to MIP, were sequenced. In each molecule, methylation extended over almost the whole length of the previously duplicated segment. The methylation extent was precisely delimited and constant in each of the molecules, leaving unmethylated a nearly 100-nucleotide region next to each end. In none of the molecules did methylation resulting from MIP extend beyond the ends. Although the DNA molecules were not all methylated with the same intensity, all cytosine residues in the methylated portion could be methylated, most of them belonging to non-symmetrical sequences. This finding contrasts with the situation in higher eukaryotes in which most, if not all, methylation is at short symmetrical sequences such as CpG or CpNpG, ensuring perpetuation of methylation. Methylation at non-symmetrical sequences implies that in A. immersus maintenance involves a novel sequence-non-specific methyltransferase.