title: Differential methylation of the circular DNA in geminiviral minichromosomes.
abstract: Geminiviral minichromosomes were purified to explore epigenetic modifications. The levels of methylation in their covalently closed circular DNA were examined with the help of methylation-dependent restriction (MdR). DNA with 12 superhelical turns was preferentially modified, indicating minichromosomes with 12 nucleosomes leaving an open gap. MdR digestion yielded a specific product of genomic length, which was cloned and Sanger-sequenced, or amplified following ligation-mediated rolling circle amplification and deep-sequenced (circomics). The conventional approach revealed a single cleavage product indicating specific methylations at the borders of the common region. The circomics approach identified considerably more MdR sites in a preferential distance to each other of ~200 nts, which is the DNA length in a nucleosome. They accumulated in regions of nucleosome-free gaps, but scattered also along the genomic components. These results may hint at a function in specific gene regulation, as well as in virus resistance.
authors.affiliation: Institut für Biomaterialien und biomolekulare Systeme, Abteilung für Molekularbiologie und Virologie der Pflanzen, Universität Stuttgart, Pfaffenwaldring 57, D-70550 Stuttgart, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Deuschle
authors.affiliation: Institut für Biomaterialien und biomolekulare Systeme, Abteilung für Molekularbiologie und Virologie der Pflanzen, Universität Stuttgart, Pfaffenwaldring 57, D-70550 Stuttgart, Germany.
authors.forename: Gabi
authors.initials: G
authors.lastname: Kepp
authors.affiliation: Institut für Biomaterialien und biomolekulare Systeme, Abteilung für Molekularbiologie und Virologie der Pflanzen, Universität Stuttgart, Pfaffenwaldring 57, D-70550 Stuttgart, Germany. Electronic address: holger.jeske@bio.uni-stuttgart.de.
authors.forename: Holger
authors.initials: H
authors.lastname: Jeske
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D053839:Begomovirus / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002875:Chromosomes / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation; D004270:DNA, Circular / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D016679:Genome, Viral; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D004270:DNA, Circular; D004279:DNA, Viral; D014764:Viral Proteins
keywords: Abutilon mosaic virus; Begomovirus; DNA methylation; Geminiviridae; Ligation-mediated RCA; Minichromosome purification; Nicotiana benthamiana; tomato yellow leaf curl Sardinia virus