Haynes W J, Vaillant B, Preston R R, Saimi Y, Kung C
Laboratory of Molecular Biology, University of Wisconsin, Madison, Wisconsin 53706, USA.
Genetics. 1998 Jun;149(2):947-57. doi: 10.1093/genetics/149.2.947.
The genetic dissection of a simple avoidance reaction behavior in Paramecium tetraurelia has shown that ion channels are a critical molecular element in signal transduction. Pawn mutants, for example, were originally selected for their inability to swim backward, a trait that has since been shown to result from the loss of a voltage-dependent calcium current. The several genes defined by this phenotype were anticipated to be difficult to clone since the 800-ploid somatic macronucleus of P. tetraurelia is a formidable obstacle to cloning by complementation. Nonetheless, when the macronucleus of a pawn mutant (pwA/pwA) was injected with total wild-type DNA or a fractional library of DNA, its clonal descendants all responded to stimuli like the wild type. By sorting a fractional library, we cloned and sequenced a 2.3-kb fragment that restores the Ca2+ current and excitability missing in pawn-A. Data from RNase protection assays, followed by the sequencing of mutant alleles and cDNA clones, established an open reading frame. The conceptually translated product suggests a novel protein that may be glycophosphatidylinositol anchored. We also discuss the general usefulness of this method in cloning other unknown DNA sequences from Paramecium that are functionally responsible for various mutant phenotypes.
对四膜虫简单回避反应行为的遗传学剖析表明，离子通道是信号转导中的关键分子元件。例如，“兵卒”突变体最初是因其无法向后游动而被筛选出来的，后来发现该性状是由于电压依赖性钙电流缺失所致。由于四膜虫800倍体的体细胞大核是通过互补进行克隆的巨大障碍，预计由这种表型定义的几个基因很难克隆。尽管如此，当向“兵卒”突变体（pwA/pwA）的大核中注射总野生型DNA或DNA片段文库时，其克隆后代都像野生型一样对刺激作出反应。通过对片段文库进行筛选，我们克隆并测序了一个2.3 kb的片段，该片段可恢复“兵卒-A”中缺失的Ca2+电流和兴奋性。核糖核酸酶保护分析的数据，随后对突变等位基因和cDNA克隆进行测序，确定了一个开放阅读框。从概念上翻译的产物表明这是一种可能通过糖基磷脂酰肌醇锚定的新型蛋白质。我们还讨论了该方法在克隆四膜虫中其他未知DNA序列方面的普遍实用性，这些序列在功能上导致了各种突变表型。
