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An engineered nonsense URA3 allele provides a versatile system to detect the presence absence and appearance of the PSI+ prion in Saccharomyces cerevisiae

机译:工程无意义的URA3等位基因提供了一种多功能系统可检测啤酒酵母中PSI + ion病毒的存在不存在和外观

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摘要

Common methods to identify yeast cells containing the prion form of the Sup35 translation termination factor, [PSI+], involve a nonsense suppressor phenotype. Decreased function of Sup35p in [PSI+] cells leads to readthrough of certain nonsense mutations in a few auxotrophic markers, for example, ade1-14. This readthrough results in growth on adenine deficient media. While this powerful tool has dramatically facilitated the study of [PSI+], it is limited to a narrow range of laboratory strains and cannot easily be used to screen for cells that have lost the [PSI+] prion. Therefore we have engineered a nonsense mutation in the widely used URA3 gene, termed the ura3-14 allele. Introduction of the ura3-14 allele into an array of genetic backgrounds, carrying a loss-of-function URA3 mutation and [PSI+], allows for growth on media lacking uracil, indicative of decreased translational termination efficiency. This ura3-14 allele is able to distinguish various forms of the [PSI+] prion, called variants and is able to detect the de novo appearance of [PSI+] in strains carrying the prion form of Rnq1p, [PIN+]. Furthermore, 5-fluoorotic acid, which kills cells making functional Ura3p, provides a means to select for [psi] derivatives in a population of [PSI+] cells marked with the ura3-14 allele, making this system much more versatile than previous methods.
机译:鉴定包含the病毒形式的Sup35翻译终止因子[PSI + ]的酵母细胞的常见方法涉及无意义的抑制子表型。 Sup35p在[PSI + ]细胞中的功能降低导致通读一些营养缺陷型标记(例如ade1-14)中的某些无意义突变。这种通读导致腺嘌呤缺乏培养基上的生长。尽管此功能强大的工具极大地促进了[PSI + ]的研究,但它仅限于实验室菌株的狭窄范围,并且不能轻易用于筛选丢失了[PSI + ] ion病毒。因此,我们在广泛使用的URA3基因中设计了一个无意义的突变,称为ura3-14等位基因。将ura3-14等位基因引入一系列遗传背景,携带功能丧失的URA3突变和[PSI + ],可在缺乏尿嘧啶的培养基上生长,表明翻译终止效率降低。 ura3-14等位基因能够区分[PSI + ] ion病毒的各种形式,称为变异体,并且能够检测到[PSI + ]的从头出现。携带Rnq1p的1病毒形式的菌株,[PIN + ]。此外,5-氟乳清酸可杀死产生功能性Ura3p的细胞,它提供了一种在[ PSI + 群体中选择[psi -]衍生物的手段。 / sup>]细胞标记有 ura3-14 等位基因,使得该系统比以前的方法更具通用性。

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