Howlett N G, Avery S V
Department of Biology, Georgia State University, Atlanta 30303, USA.
FEMS Microbiol Lett. 1999 Jul 15;176(2):379-86. doi: 10.1111/j.1574-6968.1999.tb13687.x.
The variable stress-sensitivity of individual cells within pure cultures is widely noted but generally unexplained. Here, factors determining the heterogeneous susceptibility to copper toxicity in Saccharomyces cerevisiae were examined with a rapid non-perturbing approach based on flow cytometry. By determination of the DNA content (with propidium iodide) in cell fractions gated by forward angle light scatter (an indicator of the cell volume), it was shown that forward angle light scatter measurements gave an approximation of the cell cycle stage. Thus, our observation that cells in different forward angle light scatter fractions displayed differing Cu-sensitivities indicated that heterogeneous Cu-sensitivity is a function of the cell cycle stage. Furthermore, cells sorted by their Cu-sensitivity and-resistance and subsequently analyzed for DNA content were found predominantly to occupy G1/S and G2/M cell cycle stages, respectively. The oxidant-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate was used to show that the Cu-sensitivity of G2/M phase S. cerevisiae was correlated with greater levels of pre-existing reactive oxygen species in these cells. The results indicate that differential Cu-sensitivity in a S. cerevisiae culture is linked to the cell cycle stage and this link may be determined partly by cell cycle-dependent fluctuations in basal reactive oxygen species generation.
纯培养物中单个细胞的可变应激敏感性已被广泛注意到，但通常无法解释。在此，我们基于流式细胞术采用一种快速非侵入性方法，研究了酿酒酵母中决定对铜毒性异质性敏感性的因素。通过测定前向角光散射（细胞体积的指标）门控的细胞组分中的DNA含量（用碘化丙啶），结果表明前向角光散射测量给出了细胞周期阶段的近似值。因此，我们观察到不同前向角光散射组分中的细胞表现出不同的铜敏感性，这表明异质性铜敏感性是细胞周期阶段的函数。此外，按铜敏感性和抗性分选的细胞随后进行DNA含量分析，发现主要分别处于G1/S和G2/M细胞周期阶段。使用氧化敏感探针2',7'-二氯二氢荧光素二乙酸酯表明，G2/M期酿酒酵母的铜敏感性与这些细胞中预先存在的更高水平的活性氧有关。结果表明，酿酒酵母培养物中的差异铜敏感性与细胞周期阶段有关，这种联系可能部分由基础活性氧生成的细胞周期依赖性波动决定。
