Using cell cycle analysis to estimate in situ growth rate of the dinoflagellate Dinophysis acuminata:: drawbacks of the DNA quantification method
Using cell cycle analysis to estimate in situ growth rate of the dinoflagellate Dinophysis acuminata:: drawbacks of the DNA quantification method
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DOI：
10.3354/meps184055
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发表时间：
1999-01-01
影响因子：
2.5
通讯作者：
Carlsson, P
中科院分区：
文献类型：
--
作者：
Gisselson, LÅ;Granéli, E;Carlsson, P
为了尝试利用细胞周期分析来估计甲藻尖甲藻（Dinophysis acuminata）的原位生长速率，采用荧光显微镜结合图像分析系统来测量DAPI染色的尖甲藻（Dinophysis acuminata）的DNA相对含量。尖核为了能够用这种方法估计生长速率，细胞合成第二个DNA拷贝和完成有丝分裂（细胞周期中终末事件的持续时间）所需的时间必须是已知的或可测量的。终端事件的持续时间通常是从相分数曲线的日变化图形估计。在这项研究中，没有检测到相位分数的日变化，因此无法获得对终末事件持续时间的可靠估计。其主要缺点是很难在仅仅基于几百个细胞的DNA直方图中描绘S期。在我们的48小时研究中没有明确的S期最大值。双核细胞和具有双基因组的细胞（G2 + M期细胞）的持续高百分比（23至43%）的存在表明，在没有细胞可以停滞在G2或M期的假设下，群体正在活跃地分裂，但与昼夜周期不明显同步。Chang & Carpenter（1991）先前估计该物种的终末事件（S + G2 + M期的持续时间）为11至13小时。在本研究中，终末事件持续12 h将产生0.69至0.75 d（-1）的比生长率。我们的结论是，可以使用落射荧光显微镜（几百个样品）测量的细胞数量太低，允许检测的同步程度低，特别是关于S期。使用细胞周期技术估计同步性差的浮游植物种群的原位生长速率将需要对每个样品数千个细胞进行DNA测量，例如使用流式细胞术或自动图像细胞术。
In an attempt to use cell cycle analysis to estimate in situ gross growth rate of the dinoflagellate Dinophysis acuminata, epifluorescence microscopy in combination with an image analysis system was used to measure the relative DNA content of DAPI stained D. acuminata nuclei. To be able to estimate growth rate with this method, the time it takes for a cell to synthesise a second DNA copy and complete mitosis (the duration of the terminal event in the cell cycle) must be known or measurable. The duration of the terminal event is normally estimated graphically from diurnal variations in the phase fraction curves. No diurnal variation in the phase fractions was detected in this study, and consequently no reliable estimate of the duration of the terminal event could be obtained. The main drawback was the difficulty in delineating the S phase in DNA histograms based on only a few hundred cells. No dear S phase maximum could be obtained during our 48 h study. The presence of double-nucleated cells and a constantly high percentage (23 to 43%) of cells with double genomes (G2 + M phase cells) suggests, under the assumption that no cells can arrest in the G2 or M phase, that the population was actively dividing, but not clearly in phase with a diurnal cycle. Chang & Carpenter (1991) previously estimated the duration of the terminal event (the duration of the S + G2 + M phases) in this species to be 11 to 13 h. A 12 h duration of the terminal event in this study would yield specific growth rates of 0.69 to 0.75 d(-1). We conclude that the number of cells that can be measured using epifluorescence microscopy (a few hundred per sample) is too low to allow detection of a low degree of synchronisation, especially with regard to the S phase. Estimations of in situ growth rate of poorly synchronised populations of phytoplankton using the cell cycle technique will require DNA measurements on several thousand cells per sample, e.g. using flow cytometry or automated image cytometry.