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DNA methylation has been implicated in the control of a number of cellular processes in eukaryotes, including transcription (1), imprinting (2), transposition (3), DNA repair (4), chromatin organization (5, 6), and a variety of epigenetic phenomena (7), although the function of DNA methylation in eukaryotes remains the subject of continued debate. Using DNA methylation mutants to study the functions of DNA modification in eukaryotes is an attractive approach that avoids some of the limitations associated with correlative and methylation inhibitor studies. Li et al. (8) recently reported that mouse DNA methyltransferase mutants, generated by gene disruption, are unable to complete embryogenesis, although homozygous mutant cell lines are viable and apparently normal. We report here the isolation and characterization of DNA methylation mutants in the flowering plant Arabidopsis thaliana.
We identified A. thaliana DNA methylation mutants by screening directly for plants that contained hypomethylated genomic DNA using Southern (DNA) blot analysis. Filters containing Hpa II-digested genomic DNA prepared from leaves of mutagenized A. thaliana plants were hybridized with a cloned 180-bp repeat found in long tandem arrays at all five A. thaliana centromeres (9). Centromeric repeat arrays isolated from wild-type plants are resistant to Hpa II (CCGG) digestion (10, 11) because of extensive methylation of cytosines at the CpG and CpNpG sites, which is characteristic of plants (12) [ sup 5m CCGG and C sup 5m CGG are not digested by Hpa II (13)]. DNA methylation mutants, on the other hand, were predicted to contain hypomethylated centromeric arrays susceptible to Hpa II digestion. We examined Southern filters containing DNA from 79 pools representing approximately 2000 plants from ethyl methanesulfonate-mutagenized M sub 2 populations (ecotype Columbia) to look for centromere repeat multimers of low molecular weight that were clipped from the long arrays by Hpa II. Individual plants from candidate pools were then screened to identify DNA hypomethylation mutants. Three independent mutant lines were isolated, and the mutations carried by these lines were designated ddm to refer to their decreased DNA methylation phenotype.
A variety of genomic DNA sequences are hypomethylated in the ddm mutants, including highly reiterated and low copy number sequences. Most of the cytosine methylation in the A. thaliana genome is found in the highly repeated DNA fraction (14), a large portion of which...