DIFFERENTIAL LOCALIZATION OF THE CENTROMERIC HISTONE H3 VARIANT IN THE MAJOR CENTROMERIC SATELLITE OF Arabidopsis thaliana

¹ Core Research for Evolutionary Science and Technology, Japan Science and Technology Corporation, Kawaguchi, 332-0012, Japan

² Research Institute for Bioresources, Okayama University, 2-20-1 Chuo, Kurashiki 710-0046, Japan

The 180-bp family of tandem repeating sequences is the major centromere satellite in Arabidopsis thaliana thought to confer centromere function; however, no direct evidence for this has yet been obtained. In the Arabidopsis cell culture, drastic changes in chromosome number and structure occurred; nevertheless, no chromosomes without 180-bp repeats were found. To assess the centromere activities of these repeats, we performed indirect fluorescence immunolabeling with antibodies against centromeric histone H3 variant (HTR12) and others. The immunosignals from anti-HTR12 appeared on all sites of the 180-bp repeats detected by fluorescence in situ hybridization (FISH). In addition to this colocalization of the antibodies with the 180-bp repeats, anaphase-bridge formation by the putative dicentric chromosomes carrying two 180-bp sites indicated a close relationship between the 180-bp repeats and centromere activity. However, some of the 180-bp repeat clusters were not entirely covered by the signals from anti-HTR12, when the clusters were long or stretched at interphase. Chromatin-fiber immunolabeling revealed clearly that these centromere proteins localize only at knobs on the extended chromatin fibers, which make up a limited part of the continuous 180-bp clusters. Furthermore, the localization of outer HTR12 and inner phosphohistone H3 at kinetochores of metaphase chromosomes suggests that two kinds of histone H3 (centromere variant and phosphorylated H3) play differential roles in the centromeres.