ade1和ade2突变用于开发一种多功能的红色/白色测定法，用于酿酒酵母中淀粉样蛋白诱导的氧化应激。,Yeast

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ade1和ade2突变用于开发一种多功能的红色/白色测定法，用于酿酒酵母中淀粉样蛋白诱导的氧化应激。
Yeast ( IF 2.2 ) Pub Date : 2016-09-23 , DOI: 10.1002/yea.3209
Vidhya Bharathi ₁ , Amandeep Girdhar ₁ , Archana Prasad ₁ , Meenkshi Verma ₂ , Vibha Taneja ₃ , Basant K Patel ₁

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腺嘌呤生物合成途径基因ADE1和ADE2的突变通常已用于对酵母中的ion病毒[PSI +]进行评分。如果存在包含提前终止密码子的ade1-14突变等位基因，则由于红色中间化合物在液泡中积累，[psi-]酵母在YPD培养基上显示为红色。在[PSI +]酵母中，由于淀粉样蛋白的聚集，翻译终止因子Sup35蛋白的部分失活可以使ade1-14终止密码子通读，并且由于红色中间色素没有积累，酵母显示为白色。ade1和ade2突变型酵母中的红色显影需要还原型谷胱甘肽，这有助于将中间代谢物P-核糖基氨基咪唑羧酸盐转运到液泡中，从而产生红色。这里，我们假设淀粉样蛋白诱导的氧化应激会耗尽减少的谷胱甘肽水平，从而阻碍ade1或ade2酵母中红色的发展。确实，当我们过表达形成淀粉样蛋白的人类蛋白TDP-43，Aβ-42和Poly-Gln-103以及酵母病毒蛋白Rnq1时，否则红色的ade1酵母会产生一些白色菌落。此外，关闭淀粉样蛋白的表达后，白色最终恢复为红色。同样，带有聚集体的酵母在带有还原剂的培养基上生长时，氧化应激增加，白色表型酵母恢复为红色。此外，还可以在ade2-1，ade2Δ和ade1Δ突变酵母中以及在具有增加细胞壁通透性的erg6基因缺失的ade1-14突变体中模拟红色/白色测定。该模型将是针对一般淀粉样蛋白诱导的氧化应激和毒性进行药物筛选的有用工具。版权所有©2016 John Wiley＆Sons，Ltd.

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Use of ade1 and ade2 mutations for development of a versatile red/white colour assay of amyloid-induced oxidative stress in saccharomyces cerevisiae.

Mutations in adenine biosynthesis pathway genes ADE1 and ADE2 have been conventionally used to score for prion [PSI+ ] in yeast. If ade1-14 mutant allele is present, which contains a premature stop codon, [psi- ] yeast appear red on YPD medium owing to accumulation of a red intermediate compound in vacuoles. In [PSI+ ] yeast, partial inactivation of the translation termination factor, Sup35 protein, owing to its amyloid aggregation allows for read-through of the ade1-14 stop codon and the yeast appears white as the red intermediate pigment is not accumulated. The red colour development in ade1 and ade2 mutant yeast requires reduced-glutathione, which helps in transport of the intermediate metabolite P-ribosylaminoimidazole carboxylate into vacuoles, which develops the red colour. Here, we hypothesize that amyloid-induced oxidative stress would deplete reduced-glutathione levels and thus thwart the development of red colour in ade1 or ade2 yeast. Indeed, when we overexpressed amyloid-forming human proteins TDP-43, Aβ-42 and Poly-Gln-103 and the yeast prion protein Rnq1, the otherwise red ade1 yeast yielded some white colonies. Further, the white colour eventually reverted back to red upon turning off the amyloid protein's expression. Also, the aggregate-bearing yeast have increased oxidative stress and white phenotype yeast revert to red when grown on media with reducing agent. Furthermore, the red/white assay could also be emulated in ade2-1, ade2Δ, and ade1Δ mutant yeast and also in an ade1-14 mutant with erg6 gene deletion that increases cell-wall permeability. This model would be useful tool for drug-screening against general amyloid-induced oxidative stress and toxicity. Copyright © 2016 John Wiley & Sons, Ltd.

更新日期：2019-11-01

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