title: Isolation of centromeric-tandem repetitive DNA sequences by chromatin affinity purification using a HaloTag7-fused centromere-specific histone H3 in tobacco.
abstract: The centromere is a multi-functional complex comprising centromeric DNA and a number of proteins. To isolate unidentified centromeric DNA sequences, centromere-specific histone H3 variants (CENH3) and chromatin immunoprecipitation (ChIP) have been utilized in some plant species. However, anti-CENH3 antibody for ChIP must be raised in each species because of its species specificity. Production of the antibodies is time-consuming and costly, and it is not easy to produce ChIP-grade antibodies. In this study, we applied a HaloTag7-based chromatin affinity purification system to isolate centromeric DNA sequences in tobacco. This system required no specific antibody, and made it possible to apply a highly stringent wash to remove contaminated DNA. As a result, we succeeded in isolating five tandem repetitive DNA sequences in addition to the centromeric retrotransposons that were previously identified by ChIP. Three of the tandem repeats were centromere-specific sequences located on different chromosomes. These results confirm the validity of the HaloTag7-based chromatin affinity purification system as an alternative method to ChIP for isolating unknown centromeric DNA sequences. The discovery of more than two chromosome-specific centromeric DNA sequences indicates the mosaic structure of tobacco centromeres.
authors.affiliation: Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan. nagaki@rib.okayama-u.ac.jp
authors.forename: Kiyotaka
authors.initials: K
authors.lastname: Nagaki
authors.forename: Fukashi
authors.initials: F
authors.lastname: Shibata
authors.forename: Asaka
authors.initials: A
authors.lastname: Kanatani
authors.forename: Kazunari
authors.initials: K
authors.lastname: Kashihara
authors.forename: Minoru
authors.initials: M
authors.lastname: Murata
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D002503:Centromere / Q000235:genetics* / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D002846:Chromatography, Affinity; D032461:Chromosomes, Plant / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics / Q000302:isolation & purification*; D006657:Histones / Q000378:metabolism*; D017404:In Situ Hybridization, Fluorescence; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D020080:Tandem Repeat Sequences / Q000235:genetics*; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism
chemical_list: D018744:DNA, Plant; D006657:Histones; D010940:Plant Proteins