Systematic mutational analysis of the yeast ACT1 gene.
Systematic mutational analysis of the yeast ACT1 gene.
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DOI：
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发表时间：
1992-10
期刊：
影响因子：
3.3
通讯作者：
K. F. Wertman;D. Drubin;D. Botstein
中科院分区：
文献类型：
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作者：
K. F. Wertman;D. Drubin;D. Botstein
我们报告了一组分布在酿酒酵母单个肌动蛋白基因的定点突变的分离和表征。通过鉴定一级序列中2个或更多个带电残基的簇，将突变系统地靶向蛋白质的表面;簇中的每个带电残基被丙氨酸取代。突变以高产率回收（36个构建的突变中的34个）为杂合二倍体。在单倍体分离体中检测到11个隐性致死突变体，16个条件致死突变体（包括温敏和盐敏突变体），7个无突变体。遗传分析表明，这两个突变构建，但没有恢复酵母可能有一个显性缺陷表型。兔肌肉肌动蛋白单体的三维结构上的突变体残基的位置证实，我们改变的大多数（81%）的带电残基位于或接近蛋白质的表面，证实了该方法的一个关键假设。许多新的act 1等位基因的特性很容易解释在光的肌动蛋白结构，并应证明在肌动蛋白功能的遗传和生物化学研究有用。
We report the isolation and characterization of a synoptic set of site-directed mutations distributed throughout the single actin gene of Saccharomyces cerevisiae. Mutations were systematically targeted to the surface of the protein by identifying clusters of 2 or more charged residues in the primary sequence; every charged residue in a cluster was replaced with alanine. Mutations were recovered in high yield (34 of 36 constructed) as heterozygous diploids. Mutant phenotypes were examined in haploid segregants: 11 were recessive lethal, 16 conditional-lethal (including temperature-sensitive and salt-sensitive) and 7 had no discernible phenotype. Genetic analysis suggested that the two mutations constructed but not recovered in yeast may have a dominant defective phenotype. Location of the mutant residues on the three-dimensional structure of the rabbit muscle actin monomer confirmed that most (81%) of the charged residues we altered lie at or near the surface of the protein, confirming a key assumption of the method. Many of the new act1 alleles have properties readily interpreted in light of the actin structure and should prove useful in both genetic and biochemical studies of actin function.