Dorweiler Jane E, Manogaran Anita L
Department of Biological Sciences, Marquette University, Milwaukee, WI, USA.
Bio Protoc. 2021 Sep 5;11(17):e4146. doi: 10.21769/BioProtoc.4146.
Cytoduction, and a related technique referred to as plasmiduction, have facilitated substantial advancements in the field of yeast prion biology by providing a streamlined method of transferring prions from one yeast strain to another. Prions are cytoplasmic elements consisting of aggregated misfolded proteins, and as such, they exhibit non-Mendelian patterns of inheritance. While prion transfer through mating and sporulation, or through protein transformation, is possible, these approaches yield non-isogenic strains or are technically complex, respectively. Cytoduction is a mating-based technique that takes advantage of a mutation with impaired nuclear fusion (karyogamy). It is a straightforward method for introducing a prion to any yeast strain (referred to as the recipient) by mating it with a donor strain containing the prion of interest. The only absolute requirement is that one of these two strains (donor or recipient) must carry the mutation to limit nuclear fusion. The resulting cytoductant contains the original nucleus of the recipient strain, but a cytoplasm reflecting a mix of all elements from the donor and the recipient. Modifications to the basic cytoduction strategy provide several options for successful cytoduction, including when working with slow growing or respiratory deficient strains. A significant advantage of the plasmiduction protocol presented is the ability to transfer a plasmid encoding a fluorescently tagged version of the prion protein, which allows for the direct verification of the prion state through visual protein aggregates. Graphic abstract: Transfer of Yeast Cytoplasmic Elements such as Prions using Cytoduction.
细胞导入以及一种相关技术——质粒导入,通过提供一种将朊病毒从一种酵母菌株转移到另一种酵母菌株的简化方法,推动了酵母朊病毒生物学领域的重大进展。朊病毒是由聚集的错误折叠蛋白质组成的细胞质元件,因此,它们表现出非孟德尔遗传模式。虽然通过交配和孢子形成或通过蛋白质转化进行朊病毒转移是可能的,但这些方法分别会产生非同基因菌株或技术复杂。细胞导入是一种基于交配的技术,利用了核融合(核配)受损的突变。这是一种通过将含有感兴趣朊病毒的供体菌株与受体菌株交配,将朊病毒引入任何酵母菌株(称为受体)的直接方法。唯一的绝对要求是这两种菌株(供体或受体)中的一种必须携带限制核融合的突变。产生的细胞融合体包含受体菌株的原始细胞核,但细胞质反映了供体和受体所有元素的混合。对基本细胞导入策略的修改为成功进行细胞导入提供了几种选择,包括在处理生长缓慢或呼吸缺陷菌株时。所介绍的质粒导入方案的一个显著优点是能够转移编码朊病毒蛋白荧光标记版本的质粒,这允许通过可视化蛋白质聚集体直接验证朊病毒状态。图形摘要:使用细胞导入转移酵母细胞质元件如朊病毒。