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. 2008 Oct 1;3(10):e3306. doi: 10.1371/journal.pone.0003306

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Berdasco et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Flowchart for identification of hypermethylated growth-associated genes. We used Arabidopsis cell suspensions after 5 µM ADC treatments followed by cRNA hybridization to a 22,500-oligonucleotide microarray. We obtained over 1,794 unique sequences overexpressed after treatments. Of these, 1,719 corresponded to known genes and 75 to repeat elements. We selected fourteen genes to test for promoter hypermethylation by direct bisulfite sequencing; five of them were found to be methylated in Arabidopsis cell suspensions but not in differentiated tissues such as roots and shoots. (B) Quantification of DNA methylation as: overall 5-methyl-cytosine (5 mC) using high-performance capillary electrophoresis (Left panel), and percentage of methylated CpGs using the methyl acceptor assay (Right panel). (C) Reactivation of genes in Arabidopsis cell suspensions after treatment with the demethylating drug ADC. Upper panel, scatterplots showing expression profiles of control cells and cells treated with ADC obtained by Affymetrix GeneChip technology; Lower panel, relative percentage of overexpressed and repressed genes after treatment with ADC.