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Dissecting the first and the second meiotic divisions using a marker-less drug-hypersensitive fission yeast

机译：使用无标记药物过度敏感的裂变酵母解剖第一个和第二个减数分裂分裂

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Faithful chromosome segregation during meiosis is indispensable to prevent birth defects and infertility. Canonical genetic manipulations have not been very useful for studying meiosis II, since mutations of genes involved in cell cycle regulation or chromosome segregation may affect meiosis I, making interpretations of any defects observed in meiosis II complicated. Here we present a powerful strategy to dissect meiosis I and meiosis II, using chemical inhibitors in genetically tractable model organism fission yeast (Schizosaccharomyces pombe). As various chemical probes are not active in fission yeast, mainly due to an effective multidrug resistance (MDR) response, we have recently developed a drug-hypersensitive MDR-sup strain by suppression of the key genes responsible for MDR response. We further developed the MDR-supML (marker-less) strain by deleting 7 MDR genes without commonly used antibiotic markers. The new strain makes fluorescent tagging and gene deletion much simpler, which enables effective protein visualization in varied genetic backgrounds. Using the MDR-supML strain with chemical inhibitors and live cell fluorescence microscopy, we established cell cycle arrest at meiosis I and meiosis II and examined Aurora-dependent spindle assembly checkpoint (SAC) regulation during meiosis. We found that Aurora B/Ark1 kinase activity is required for recruitment of Bub1, an essential SAC kinase, to unattached kinetochore in prometaphase I and prometaphase II as in mitosis. Thus, Aurora's role in SAC activation is likely conserved in mitosis, meiosis I, and meiosis II. Together, our MDR-supML strain will be useful to dissect complex molecular mechanisms in mitosis and 2 successive meiotic divisions.
机译：减数分裂过程中忠实的染色体分离对于防止先天缺陷和不育是必不可少的。规范的基因操作对于研究减数分裂II并不是很有用，因为参与细胞周期调控或染色体分离的基因突变可能影响减数分裂I，使对减数分裂II中观察到的任何缺陷的解释变得复杂。在这里，我们提出了一种在遗传易处理的模型生物裂变酵母（Schizosaccharomyces pombe）中使用化学抑制剂来分解减数分裂I和减数分裂II的有效策略。由于各种化学探针在裂变酵母中不起作用，主要是由于有效的多药耐药性（MDR）反应，我们最近通过抑制引起MDR反应的关键基因，开发出了对药物过敏的MDR-sup菌株。我们通过删除7个没有常用抗生素标记的MDR基因，进一步开发了MDR-supML（无标记）菌株。新菌株使荧光标记和基因删除变得更加简单，从而可以在各种遗传背景下有效地进行蛋白质可视化。使用具有化学抑制剂的MDR-supML菌株和活细胞荧光显微镜，我们在减数分裂I和减数分裂II处建立了细胞周期停滞期，并研究了减数分裂过程中Aurora依赖性纺锤体装配检查点（SAC）的调控。我们发现Aurora B / Ark1激酶活性是募集必需的SAC激酶Bub1到前中期I和前中期II上独立的动粒，就像在有丝分裂中一样。因此，极光在SAC激活中的作用可能在有丝分裂，减数分裂I和减数分裂II中得以保留。总之，我们的MDR-supML菌株将有助于剖析有丝分裂和2个连续减数分裂的复杂分子机制。

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