Flor-Parra Ignacio, Zhurinsky Jacob, Bernal Manuel, Gallardo Paola, Daga Rafael R
Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide-Consejo Superior de Investigaciones Científicas, Sevilla, Spain.
Yeast. 2014 Feb;31(2):61-6. doi: 10.1002/yea.2994. Epub 2013 Dec 26.
Fungal cells including yeasts are surrounded by cell wall that counteracts turgor pressure and prevents cell lysis. Many yeast experiments, including genetic manipulation of sterile strains, morphogenesis studies, nucleic acid isolation and many others, require mechanical breakage or enzymatic removal of the cell wall. Some of these experiments require the generation of live cells lacking cell walls, called protoplasts, that can be maintained in osmostabilized medium. Enzymatic digestion of cell wall proteoglycans is a commonly used method of protoplast preparation. Currently existing protocols for fission yeast cell wall digestion are time consuming and not very efficient. We developed a new rapid method for fission yeast protoplast preparation that relies on digesting cell walls with Lallzyme MMX commercial enzyme mix, which produces protoplasts from all cells in less than 10 min. We demonstrate that these protoplasts can be utilized in three commonly used fission yeast protocols. Thus, we provide the fission yeast community with a robust and efficient plasmid extraction method, a new protocol for diploid generation and an assay for protoplast recovery that should be useful for studies of morphogenesis. Our method is potentially applicable to other yeasts and fungi.
包括酵母在内的真菌细胞被细胞壁所包围，细胞壁可抵消膨压并防止细胞裂解。许多酵母实验，包括无菌菌株的基因操作、形态发生研究、核酸分离等，都需要对细胞壁进行机械破碎或酶解。其中一些实验需要产生缺乏细胞壁的活细胞，即原生质体，这些原生质体可在渗透压稳定的培养基中维持。细胞壁蛋白聚糖的酶解是制备原生质体常用的方法。目前现有的裂殖酵母细胞壁消化方案耗时且效率不高。我们开发了一种新的快速制备裂殖酵母原生质体的方法，该方法依赖于用Lallzyme MMX商业酶混合物消化细胞壁，这种酶混合物能在不到10分钟的时间内从所有细胞中产生原生质体。我们证明这些原生质体可用于三种常用的裂殖酵母实验方案。因此，我们为裂殖酵母研究群体提供了一种强大而高效的质粒提取方法、一种新的二倍体生成方案以及一种原生质体回收测定方法，这些方法对形态发生研究应该是有用的。我们的方法可能适用于其他酵母和真菌。
