Hinrichsen R D, Pollock M, Hennessey T, Russell C
Fred Hutchinson Cancer Research Center, Division of Basic Sciences, Seattle, Washington 98104.
Genetics. 1991 Nov;129(3):717-25. doi: 10.1093/genetics/129.3.717.
We describe a suppressor of the calmodulin mutant cam1 in Paramecium tetraurelia. The cam1 mutant, which has a SER----PHE change at residue 101 of the third calcium-binding domain, inhibits the activity of the Ca(2+)-dependent K+ current and causes exaggerated behavioral responses to most stimuli. An enrichment scheme, based on an increased sensitivity to Ba2+ in cam1 cells, was used to isolate suppressors. One such suppressor, designated cam101, restores both the activity of the Ca(2+)-dependent K+ current and behavioral responses of the cells. We show that the cam101 mutant is an intragenic suppressor of cam1, based on genetic and microinjection data. The cam101 calmodulin is shown to be similar to wild-type calmodulin in terms of its ability to stimulate calmodulin-dependent phosphodiesterase at low concentrations of free calcium. However, the cam101 calmodulin has a reduced affinity for a monoclonal antibody to wild-type Paramecium calmodulin, as does the parental cam1 calmodulin, and a different mobility on acid-urea gels relative to both wild-type and cam1 calmodulin. We have been able to demonstrate that the isolation of intragenic suppressors of a calmodulin mutation is possible, which allows for the further genetic analysis of structure-function relationships in the calmodulin molecule.
我们描述了一种四膜虫中钙调蛋白突变体cam1的抑制子。cam1突变体在第三个钙结合结构域的第101位残基处发生了SER----PHE变化，它抑制了Ca(2+)依赖的K+电流的活性，并导致细胞对大多数刺激产生过度的行为反应。基于cam1细胞对Ba2+敏感性增加的富集方案被用于分离抑制子。其中一个这样的抑制子，命名为cam101，恢复了Ca(2+)依赖的K+电流的活性以及细胞的行为反应。基于遗传和显微注射数据，我们表明cam101突变体是cam1的基因内抑制子。cam101钙调蛋白在低游离钙浓度下刺激钙调蛋白依赖的磷酸二酯酶的能力方面，显示出与野生型钙调蛋白相似。然而，cam101钙调蛋白与针对野生型四膜虫钙调蛋白的单克隆抗体的亲和力降低，就像亲本cam1钙调蛋白一样，并且相对于野生型和cam1钙调蛋白，在酸性尿素凝胶上具有不同的迁移率。我们已经能够证明分离钙调蛋白突变的基因内抑制子是可能的，这使得对钙调蛋白分子结构-功能关系进行进一步的遗传分析成为可能。
