Topoisomerase II (TopoII) is an essential structural protein of the metaphase chromosome. It maintains the axial compaction of chromosomes during metaphase. It is localized at the axial region of chromosomes and accumulates at the centromeric region in metaphase chromosomes. However, little is known about TopoII localization and distribution in plant chromosomes, except for several publications. We used high voltage transmission electron microscopy (HVTEM) and ultra-high voltage transmission electron microscopy (UHVTEM) in conjunction with immunogold labeling and visualization techniques to detect TopoII and investigate its localization, alignment, and density on the barley chromosome at 1.4 nm scale. We found that HVTEM and UHVTEM combined with immunogold labeling is suitable for the detection of structural proteins, including a single molecule of TopoII. This is because the average size of the gold particles for TopoII visualization after silver enhancement is 8.9 ± 3.9 nm, which is well detected. We found that 31,005 TopoII molecules are distributed along the barley chromosomes in an unspecific pattern at the chromosome arms and accumulate specifically at the nucleolus organizer regions (NORs) and centromeric region. The TopoII density were 1.32-fold, 1.58-fold, and 1.36-fold at the terminal region, at the NORs, and the centromeric region, respectively. The findings of TopoII localization in this study support the multiple reported functions of TopoII in the barley metaphase chromosome.

中文翻译：

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使用 HVTEM 和 UHVTEM 对植物染色体上染色质结构蛋白拓扑异构酶 II 进行定位和定量分布

拓扑异构酶 II (TopoII) 是中期染色体的必需结构蛋白。它在中期维持染色体的轴向压缩。它位于染色体的轴向区域，并在中期染色体的着丝粒区域积累。然而，除了几篇出版物外，人们对 TopoII 在植物染色体中的定位和分布知之甚少。我们使用高压透射电子显微镜 (HVTEM) 和超高压透射电子显微镜 (UHVTEM) 结合免疫金标记和可视化技术来检测 TopoII，并在 1.4 nm 尺度上研究其在大麦染色体上的定位、排列和密度。我们发现HVTEM和UHVTEM结合免疫金标记适用于结构蛋白的检测，包括单分子TopoII。这是因为银增强后用于 TopoII 可视化的金颗粒的平均尺寸为 8.9 ± 3.9 nm，可以很好地检测到。我们发现 31,005 个 TopoII 分子沿着大麦染色体以非特异性模式分布在染色体臂上，并在核仁组织区 (NOR) 和着丝粒区特异性积累。TopoII 密度在末端区域、NOR 和着丝粒区域分别为 1.32 倍、1.58 倍和 1.36 倍。本研究中 TopoII 定位的结果支持了 TopoII 在大麦中期染色体中的多种报道功能。