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A centromeric DNA sequence colocalized with a centromere-specific histone H3 in tobacco

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Abstract

Centromeres play an important role in segregating chromosomes into daughter cells, and centromeric DNA assembles specific proteins to form a complex referred to as the kinetochore. Among these proteins, centromere-specific histone H3 (CENH3) is one of the most characterized and found to be located only on active centromeres. We isolated four different CENH3-coding complementary DNAs (cDNAs), two from Nicotiana tabaccum and one each from the ancestral diploid species, Nicotiana sylvestris and Nicotiana tomentosiformis and raised an antibody against N-terminal amino acid sequences deduced from the cDNAs. Immunostaining with the antibody revealed the preferential centromere localization, indicating that the cDNAs cloned in this study encode authentic tobacco CENH3. A tobacco centromeric DNA sequence (Nt2-7) was also identified by chromatin immunoprecipitation cloning using the antibody.

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Acknowledgments

This work was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology in Japan (#18770007). Seeds of N. sylvestris and N. tomentosiformis were kindly provided by Japan Tobacco Inc.

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Authors and Affiliations

  1. Research Institute for Bioresources, Okayama University, Kurashiki, 710-0046, Japan

    Kiyotaka Nagaki, Kazunari Kashihara & Minoru Murata

Authors
  1. Kiyotaka Nagaki
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  2. Kazunari Kashihara
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  3. Minoru Murata
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Corresponding author

Correspondence to Kiyotaka Nagaki.

Additional information

Communicated by I. Schubert

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Figure S1

Alignment of CENH3 cDNAs from Nicotiana species. CENH3 cDNAs isolated in this study (NtCENH3-1 and -2, NsCENH3 and NtoCENH3) and found in the gene index project database (BP526496) were aligned. Start and stop codons are marked by red and blue boxes, respectively. Primer positions and SalI and AfeI sites used for CAPS analyses are indicated by arrows and green boxes, respectively. (GIF 363 KB)

Figure S2

Alignment of CENH3s from plants. CENH3s identified in this study (NtCENH3-1 and -2, NsCENH3 and NtoCENH3) were aligned with HTR12 (Arabidopsis), LnCENH3 (Luzula), OsCENH3 (rice), MaizeCENH3, SoCENH3-1 (sugarcane CA127217), SoCENH3-2 (sugarcane TC19006), and OsHistoneH3 (canonical histone H3 from rice). The red box indicates the peptide sequence used to raise the anti-NtCENH3 antibody. (GIF 287 KB)

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Nagaki, K., Kashihara, K. & Murata, M. A centromeric DNA sequence colocalized with a centromere-specific histone H3 in tobacco. Chromosoma 118, 249–257 (2009). https://doi.org/10.1007/s00412-008-0193-1

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  • DOI: https://doi.org/10.1007/s00412-008-0193-1

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