Mickelson-Young Leigh, Wear Emily, Mulvaney Patrick, Lee Tae-Jin, Szymanski Eric S, Allen George, Hanley-Bowdoin Linda, Thompson William
Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC 27695, USA
Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC 27695, USA.
J Exp Bot. 2016 Nov;67(21):6077-6087. doi: 10.1093/jxb/erw367. Epub 2016 Oct 3.
The duration of the DNA synthesis stage (S phase) of the cell cycle is fundamental in our understanding of cell cycle kinetics, cell proliferation, and DNA replication timing programs. Most S-phase duration estimates that exist for plants are based on indirect measurements. We present a method for directly estimating S-phase duration by pulse-labeling root tips or actively dividing suspension cells with the halogenated thymidine analog 5-ethynl-2'-deoxyuridine (EdU) and analyzing the time course of replication with bivariate flow cytometry. The transition between G and G DNA contents can be followed by measuring the mean DNA content of EdU-labeled S-phase nuclei as a function of time after the labeling pulse. We applied this technique to intact root tips of maize (Zea mays L.), rice (Oryza sativa L.), barley (Hordeum vulgare L.), and wheat (Triticum aestivum L.), and to actively dividing cell cultures of Arabidopsis (Arabidopsis thaliana (L.) Heynh.) and rice. Estimates of S-phase duration in root tips were remarkably consistent, varying only by ~3-fold, although the genome sizes of the species analyzed varied >40-fold.
细胞周期中DNA合成阶段（S期）的持续时间对于我们理解细胞周期动力学、细胞增殖和DNA复制时间程序至关重要。目前大多数针对植物S期持续时间的估计都是基于间接测量。我们提出了一种直接估计S期持续时间的方法，即通过用卤代胸腺嘧啶类似物5-乙炔基-2'-脱氧尿苷（EdU）对根尖或活跃分裂的悬浮细胞进行脉冲标记，并利用双变量流式细胞术分析复制的时间进程。通过测量标记脉冲后不同时间点EdU标记的S期细胞核的平均DNA含量，可以追踪G期和G期之间DNA含量的变化。我们将这项技术应用于玉米（Zea mays L.）、水稻（Oryza sativa L.）、大麦（Hordeum vulgare L.）和小麦（Triticum aestivum L.）的完整根尖，以及拟南芥（Arabidopsis thaliana (L.) Heynh.）和水稻的活跃分裂细胞培养物。尽管所分析物种的基因组大小差异超过40倍，但根尖中S期持续时间的估计结果非常一致，仅相差约3倍。
