Isolation of centromeric-tandem repetitive DNA sequences by chromatin affinity purification using a HaloTag7-fused centromere-specific histone H3 in tobacco

Kiyotaka Nagaki, Fukashi Shibata, Asaka Kanatani, Kazunari Kashihara, Minoru Murata

Research output: Contribution to journalArticle

9 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)771-779
Number of pages9
JournalPlant Cell Reports
Volume31
Issue number4
DOIs
Publication statusPublished - Apr 2012

Fingerprint

centromeres
histones
chromatin
tobacco
nucleotide sequences
antibodies
chromosomes
tandem repeat sequences
retrotransposons
DNA
antibody formation
proteins

Keywords

  • CENH3
  • Centromere-specific histone H3
  • Centromeric DNA sequence
  • Chromatin immunoprecipitation and ChIP
  • HaloTag

ASJC Scopus subject areas

  • Plant Science
  • Agronomy and Crop Science

Cite this

Isolation of centromeric-tandem repetitive DNA sequences by chromatin affinity purification using a HaloTag7-fused centromere-specific histone H3 in tobacco. / Nagaki, Kiyotaka; Shibata, Fukashi; Kanatani, Asaka; Kashihara, Kazunari; Murata, Minoru.

In: Plant Cell Reports, Vol. 31, No. 4, 04.2012, p. 771-779.

Research output: Contribution to journalArticle

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