Rosebrock Adam P
Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
Cold Spring Harb Protoc. 2017 Jan 3;2017(1):2017/1/pdb.prot088724. doi: 10.1101/pdb.prot088724.
The cell cycle of budding yeast can be arrested at specific positions by different genetic and chemical methods. These arrests enable study of cell cycle phase-specific phenotypes that would be missed during examination of asynchronous cultures. Some methods for arrest are reversible, with kinetics that enable release of cells back into a synchronous cycling state. Benefits of chemical and genetic methods include scalability across a large range of culture sizes from a few milliliters to many liters, ease of execution, the absence of specific equipment requirements, and synchronization and release of the entire culture. Of note, cell growth and division are decoupled during arrest and block-release experiments. Cells will continue transcription, translation, and accumulation of protein while arrested. If allowed to reenter the cell cycle, cells will do so as a population of mixed, larger-than-normal cells. Despite this important caveat, many aspects of budding yeast physiology are accessible using these simple chemical and genetic tools. Described here are methods for the block and release of cells in G phase and at the M/G transition using α-factor mating pheromone and the temperature-sensitive cdc15-2 allele, respectively, in addition to methods for arresting the cell cycle in early S phase and at G/M by using hydroxyurea and nocodazole, respectively.
通过不同的遗传和化学方法，芽殖酵母的细胞周期可以在特定位置被阻断。这些阻断使得能够研究细胞周期阶段特异性表型，而这些表型在异步培养物的检测过程中会被遗漏。一些阻断方法是可逆的，其动力学特性能够使细胞重新回到同步循环状态。化学和遗传方法的优点包括可扩展性，能适用于从几毫升到许多升的大范围培养规模，易于实施，无需特定设备要求，以及能使整个培养物同步化和释放。值得注意的是，在阻断和阻断释放实验过程中，细胞生长和分裂是解偶联的。细胞在被阻断时会继续进行转录、翻译和蛋白质积累。如果允许重新进入细胞周期，细胞会以一群混合的、比正常细胞大的细胞群体形式进行。尽管有这个重要的注意事项，但使用这些简单的化学和遗传工具仍可了解芽殖酵母生理学的许多方面。本文除了分别介绍使用α因子交配信息素和温度敏感型cdc15 - 2等位基因在G期和M/G转换期阻断和释放细胞的方法外，还介绍了分别使用羟基脲和诺考达唑在早S期和G/M期阻断细胞周期的方法。
