Abstract
Although a centromeric DNA fragment of tobacco (Nicotiana tabacum), Nt2-7, has been reported, the overall structure of the centromeres remains unknown. To characterize the centromeric DNA sequences, we conducted a chromatin immunoprecipitation assay using anti-NtCENH3 antibody and chromatins isolated from two ancestral diploid species (Nicotiana sylvestris and Nicotiana tomentosiformis) of N. tabacum and isolated a 178-pb fragment, Nto1 from N. tomentosiformis, as a novel centromeric DNA. Fluorescence in situ hybridization (FISH) showed that Nto1 localizes on 24 out of 48 chromosomes in some cells of a BY-2 cell line. To identify the origins of the Nt2-7 and Nto1, a tobacco bacterial artificial chromosome (BAC) library was constructed from N. tabacum, and then screened by polymerase chain reaction (PCR) with primer sets designed from the Nt2-7 and Not1 DNA sequences. Twelve BAC clones were found to localize on the centromeric regions by FISH. We selected three BAC clones for sequencing and identified two centromeric retrotransposons, NtCR and NtoCR, the DNA sequences of which are similar to that of Nt2-7 and Nto1, respectively. Quantitative PCR analysis using coprecipitated DNA with anti-NtCENH3 clearly showed coexistence of NtCENH3 with both retrotransposons. These results indicate the possibility that these two retrotransposons act as centromeric DNA sequences in tobacco. NtoCR was found to be specific to N. tomentosiformis and T genome of N. tabacum, and a NtCR-like centromeric retrotransposon (TGRIV) exists in tomato. This specificity suggests that the times of amplification of these centromeric retrotransposons were different.
| Original language | English |
|---|---|
| Pages (from-to) | 591-605 |
| Number of pages | 15 |
| Journal | Chromosome Research |
| Volume | 19 |
| Issue number | 5 |
| DOIs | |
| Publication status | Published - Jul 2011 |
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Keywords
- BAC
- centromere
- chromatin immunoprecipitation
- Nicotiana
- retrotransposon
- tobacco
ASJC Scopus subject areas
- Genetics
Cite this
Coexistence of NtCENH3 and two retrotransposons in tobacco centromeres. / Nagaki, Kiyotaka; Shibata, Fukashi; Suzuki, Go; Kanatani, Asaka; Ozaki, Souichi; Hironaka, Akiko; Kashihara, Kazunari; Murata, Minoru.
In: Chromosome Research, Vol. 19, No. 5, 07.2011, p. 591-605.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Coexistence of NtCENH3 and two retrotransposons in tobacco centromeres
AU - Nagaki, Kiyotaka
AU - Shibata, Fukashi
AU - Suzuki, Go
AU - Kanatani, Asaka
AU - Ozaki, Souichi
AU - Hironaka, Akiko
AU - Kashihara, Kazunari
AU - Murata, Minoru
PY - 2011/7
Y1 - 2011/7
N2 - Although a centromeric DNA fragment of tobacco (Nicotiana tabacum), Nt2-7, has been reported, the overall structure of the centromeres remains unknown. To characterize the centromeric DNA sequences, we conducted a chromatin immunoprecipitation assay using anti-NtCENH3 antibody and chromatins isolated from two ancestral diploid species (Nicotiana sylvestris and Nicotiana tomentosiformis) of N. tabacum and isolated a 178-pb fragment, Nto1 from N. tomentosiformis, as a novel centromeric DNA. Fluorescence in situ hybridization (FISH) showed that Nto1 localizes on 24 out of 48 chromosomes in some cells of a BY-2 cell line. To identify the origins of the Nt2-7 and Nto1, a tobacco bacterial artificial chromosome (BAC) library was constructed from N. tabacum, and then screened by polymerase chain reaction (PCR) with primer sets designed from the Nt2-7 and Not1 DNA sequences. Twelve BAC clones were found to localize on the centromeric regions by FISH. We selected three BAC clones for sequencing and identified two centromeric retrotransposons, NtCR and NtoCR, the DNA sequences of which are similar to that of Nt2-7 and Nto1, respectively. Quantitative PCR analysis using coprecipitated DNA with anti-NtCENH3 clearly showed coexistence of NtCENH3 with both retrotransposons. These results indicate the possibility that these two retrotransposons act as centromeric DNA sequences in tobacco. NtoCR was found to be specific to N. tomentosiformis and T genome of N. tabacum, and a NtCR-like centromeric retrotransposon (TGRIV) exists in tomato. This specificity suggests that the times of amplification of these centromeric retrotransposons were different.
AB - Although a centromeric DNA fragment of tobacco (Nicotiana tabacum), Nt2-7, has been reported, the overall structure of the centromeres remains unknown. To characterize the centromeric DNA sequences, we conducted a chromatin immunoprecipitation assay using anti-NtCENH3 antibody and chromatins isolated from two ancestral diploid species (Nicotiana sylvestris and Nicotiana tomentosiformis) of N. tabacum and isolated a 178-pb fragment, Nto1 from N. tomentosiformis, as a novel centromeric DNA. Fluorescence in situ hybridization (FISH) showed that Nto1 localizes on 24 out of 48 chromosomes in some cells of a BY-2 cell line. To identify the origins of the Nt2-7 and Nto1, a tobacco bacterial artificial chromosome (BAC) library was constructed from N. tabacum, and then screened by polymerase chain reaction (PCR) with primer sets designed from the Nt2-7 and Not1 DNA sequences. Twelve BAC clones were found to localize on the centromeric regions by FISH. We selected three BAC clones for sequencing and identified two centromeric retrotransposons, NtCR and NtoCR, the DNA sequences of which are similar to that of Nt2-7 and Nto1, respectively. Quantitative PCR analysis using coprecipitated DNA with anti-NtCENH3 clearly showed coexistence of NtCENH3 with both retrotransposons. These results indicate the possibility that these two retrotransposons act as centromeric DNA sequences in tobacco. NtoCR was found to be specific to N. tomentosiformis and T genome of N. tabacum, and a NtCR-like centromeric retrotransposon (TGRIV) exists in tomato. This specificity suggests that the times of amplification of these centromeric retrotransposons were different.
KW - BAC
KW - centromere
KW - chromatin immunoprecipitation
KW - Nicotiana
KW - retrotransposon
KW - tobacco
UR - http://www.scopus.com/inward/record.url?scp=80052435478&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=80052435478&partnerID=8YFLogxK
U2 - 10.1007/s10577-011-9219-2
DO - 10.1007/s10577-011-9219-2
M3 - Article
C2 - 21626175
AN - SCOPUS:80052435478
VL - 19
SP - 591
EP - 605
JO - Chromosome Research
JF - Chromosome Research
SN - 0967-3849
IS - 5
ER -