Kanabrocki J A, Saimi Y, Preston R R, Haynes W J, Kung C
Laboratory of Molecular Biology, University of Wisconsin-Madison 53706.
Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10845-9. doi: 10.1073/pnas.88.23.10845.
An Ile-136----Thr substitution in calmodulin reduces the Ca(2+)-dependent K+ currents of cam2, a behavioral mutant of Paramecium tetraurelia, and renders it overly susceptible to BaCl2. DNA fragments carrying the wild-type CAM gene injected into cam2 macronuclei reverted these phenotypes in the clonal descendants of the recipients. Tetrahymena telomeric sequences, added in vitro to the fragment termini before injection, enhanced the efficiency and quality of transformation. Five times 10(4) copies of such fragments consistently restored the phenotypes to near normal; even 10(3) or fewer copies could still effect weak transformation. The restored phenotypes were stable for greater than 20 fissions in many clones and were lost after autogamy. We examined the fate of the injected fragments in the transformed clones and discuss the possible application of this efficient transformation in the cloning of other genes of P. tetraurelia.
钙调蛋白中异亮氨酸 -136 到苏氨酸的替换降低了草履虫行为突变体 cam2 的钙依赖性钾电流，并使其对氯化钡过度敏感。将携带野生型 CAM 基因的 DNA 片段注射到 cam2 大核中，可使受体的克隆后代恢复这些表型。在注射前体外添加到片段末端的四膜虫端粒序列提高了转化效率和质量。5×10⁴ 个这样的片段拷贝始终能将表型恢复到接近正常水平；即使 10³ 个或更少的拷贝仍能产生微弱的转化。恢复的表型在许多克隆中超过 20 次分裂都是稳定的，并在自体受精后消失。我们研究了转化克隆中注射片段的命运，并讨论了这种高效转化在克隆四膜虫其他基因中的可能应用。
