Genetic analysis of staphylococcal nuclease: identification of three intragenic "global" suppressors of nuclease-minus mutations.
Genetic analysis of staphylococcal nuclease: identification of three intragenic "global" suppressors of nuclease-minus mutations.
复制标题
葡萄球菌核酸酶的遗传分析：鉴定核酸酶缺失突变的三个基因内“全局”抑制因子。
DOI：
10.1093/genetics/110.4.539
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发表时间：
1985
期刊：
影响因子：
3.3
通讯作者：
Lin,B
中科院分区：
文献类型：
--
作者：
Shortle,D;Lin,B
收集了77个独特的错义突变分布在整个基因编码葡萄球菌核酸酶（nuc）已组装。这些突变是由克隆基因的随机缺口错修复突变引起的，并在E.大肠杆菌转化体表达降低水平的核酸酶活性。将改变酶的活性位点区域之外的位置处的氨基酸残基的Fournuc-mutations提交给第二轮诱变，并且对几个独立的NUC+分离株的表征导致在nucgene的蛋白质编码序列内鉴定出三个第二位点抑制突变。在与最初被抑制的突变分离并与许多其他突变重组时，所有三个抑制子都显示出“全局”抑制的性质，即，多个不同等位基因的核酸酶阴性特征的表型抑制。一个简单的和普遍适用的策略是用来获得有效的同源重组质粒之间的目的定位nuc-mutations，定位第二个位点的抑制和构建双突变体组合从对单突变。
A collection of 77 unique missense mutations distributed across the gene encoding staphylococcal nuclease (nuc) has been assembled. These mutations were induced by random gap misrepair mutagenesis of the cloned gene and were identified inE. colitransformants expressing reduced levels of nuclease activity. Fournuc-mutations which alter amino acid residues at positions outside of the active site region of the enzyme were submitted to a second round of mutagenesis, and characterization of several independent NUC+isolates lead to the identification of three second-site suppressor mutations within the protein-coding sequence of thenucgene. On separation from the mutation originally suppressed and recombination with a number of othernuc-mutations, all three suppressors displayed the property of "global" suppression,i.e., phenotypic suppression of the nuclease-minus character of multiple different alleles. A simple and generally applicable strategy was used to obtain efficient homologous recombination between plasmids for purposes of mappingnuc-mutations, mapping second-site suppressors and constructing double mutant combinations from pairs of single mutations.