Construction and genetic characterization of temperature-sensitive mutant alleles of the yeast actin gene.
Construction and genetic characterization of temperature-sensitive mutant alleles of the yeast actin gene.
复制标题
酵母肌动蛋白基因温度敏感突变等位基因的构建和遗传特征。
DOI：
10.1073/pnas.81.15.4889
复制
发表时间：
1984
影响因子：
11.1
通讯作者：
Botstein,D
中科院分区：
文献类型：
--
作者：
Shortle,D;Novick,P;Botstein,D
通过体外诱变，将酵母的肌动蛋白单基因（ACT1）整合转化到酵母细胞中，构建了两个温度敏感突变。使用等位基因替代策略，允许隐性突变在初始转化子中表型表达，从而简化了大量独立转化细胞的筛选。通过遗传方法确认了几个ts突变位于肌动蛋白结构基因内，克隆了这些突变等位基因，并通过DNA序列分析确定了改变的氨基酸残基。这两个独特的突变导致脯氨酸-32被亮氨酸取代，丙氨酸-58被苏氨酸取代。在分离这些突变的过程中，观察到通过球质体法转化外源DNA的酵母细胞中有很大一部分由于与转化DNA无关的遗传变化而对生长温度敏感。
Two temperature-sensitive mutations have been constructed in the single actin gene (ACT1) of the yeast Saccharomyces cerevisiae by in vitro mutagenesis of the cloned gene followed by integrative transformation of mutagenized DNA into yeast cells. A strategy of allele replacement was used that allowed recessive mutations to be phenotypically expressed in the initial transformants, thus simplifying the screening of large numbers of independently transformed cells. After confirming that several ts mutations were located within the actin structural gene by genetic methods, these mutant alleles were cloned, and the altered amino acid residues were defined by DNA sequence analysis. The two unique mutations resulted in substitution of proline-32 with leucine and alanine-58 with threonine. In the course of isolating these mutations, the observation was made that a high proportion of yeast cells transformed with exogenous DNA by the spheroplast method are temperature sensitive for growth because of genetic changes unrelated to the transforming DNA.