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 language | English |
|---|---|
| Pages (from-to) | 771-779 |
| Number of pages | 9 |
| Journal | Plant Cell Reports |
| Volume | 31 |
| Issue number | 4 |
| DOIs | |
| Publication status | Published - Apr 2012 |
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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 journal › Article
}
TY - JOUR
T1 - Isolation of centromeric-tandem repetitive DNA sequences by chromatin affinity purification using a HaloTag7-fused centromere-specific histone H3 in tobacco
AU - Nagaki, Kiyotaka
AU - Shibata, Fukashi
AU - Kanatani, Asaka
AU - Kashihara, Kazunari
AU - Murata, Minoru
PY - 2012/4
Y1 - 2012/4
N2 - 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.
AB - 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.
KW - CENH3
KW - Centromere-specific histone H3
KW - Centromeric DNA sequence
KW - Chromatin immunoprecipitation and ChIP
KW - HaloTag
UR - http://www.scopus.com/inward/record.url?scp=84858286841&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84858286841&partnerID=8YFLogxK
U2 - 10.1007/s00299-011-1198-4
DO - 10.1007/s00299-011-1198-4
M3 - Article
C2 - 22147136
AN - SCOPUS:84858286841
VL - 31
SP - 771
EP - 779
JO - Plant Cell Reports
JF - Plant Cell Reports
SN - 0721-7714
IS - 4
ER -