Rapid method for isolation and screening of cytochrome c oxidase-deficient mutants of Saccharomyces cerevisiae.
Rapid method for isolation and screening of cytochrome c oxidase-deficient mutants of Saccharomyces cerevisiae.
复制标题
快速分离和筛选酿酒酵母细胞色素c氧化酶缺陷突变体的方法。
DOI：
10.1128/jb.161.3.831-835.1985
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发表时间：
1985
影响因子：
3.2
通讯作者：
Poyton,RO
中科院分区：
文献类型：
--
作者：
McEwen,JE;Cameron,VL;Poyton,RO
我们在此描述了一种特异性分离酿酒酵母细胞色素 c 氧化酶缺陷突变体的新方法。该方法的一个独特之处是使用四甲基对苯二胺作为酵母菌落的细胞色素 C 氧化酶活性染色剂。四甲基对苯二胺对酵母菌落的染色依赖于功能性细胞色素c氧化酶，并且不受呼吸中其他损伤的影响。由于四甲基对苯二胺菌落染色反应快速且简单，因此极大地促进了细胞色素 c 氧化酶缺陷突变体的鉴定和表征。该方法的另一个特点是使用 op1 亲本菌株来分离特定蛋白质编码基因中的核 pet 或线粒体 mit 突变体，这是通过四甲基对苯二胺菌落染色实现的。携带该标记的亲本菌株会针对多效性呼吸缺陷突变体的 rho0 或 rho 类进行选择，因为这些突变体在 op1 菌株中是致命的。我们使用这种方法分离了 123 个独立衍生的细胞色素 c 氧化酶缺陷 pet 突变体和 300 个独立衍生的 mit 突变体。
We describe here a new method for the specific isolation of cytochrome c oxidase-deficient mutants of Saccharomyces cerevisiae. One unique feature of the method is the use of tetramethyl-p-phenylenediamine as a cytochrome c oxidase activity stain for yeast colonies. The staining of yeast colonies by tetramethyl-p-phenylenediamine is dependent upon a functional cytochrome c oxidase and is unaffected by other lesions in respiration. Since the tetramethyl-p-phenylenediamine colony staining reaction is rapid and simple, it greatly facilitates both the identification and characterization of cytochrome c oxidase-deficient mutants. Another feature of the method, which is made possible by the tetramethyl-p-phenylenediamine colony stain, is the use of an op1 parent strain for the isolation of nuclear pet or mitochondrial mit mutants in specific protein-coding genes. A parent strain that carries this marker selects against rho0 or rho- classes of pleiotropic respiratory-deficient mutants, since these are lethal in op1 strains. We have used this method to isolate 123 independently derived cytochrome c oxidase-deficient pet mutants and 300 independently derived mit mutants.