De Lozanne A, Spudich J A
Science. 1987 May 29;236(4805):1086-91. doi: 10.1126/science.3576222.
The phenomenon of homologous recombination, which allows specific gene conversion and gene insertion, can be a powerful system for the study of eukaryotic cell biology. Data are presented demonstrating that integration of a transfected plasmid by homologous recombination occurs in the motile eukaryotic cell Dictyostelium discoideum. A plasmid carrying a G418 resistance gene and the amino terminal half of the myosin heavy chain gene was used to transfect Dictyostelium. A large fraction of the resultant G418-resistant cells had the plasmid integrated into the single genomic copy of the heavy chain gene. These cells, which fail to express the native myosin but express the myosin fragment, are defective in cytokinesis and become large and multinucleate. In spite of the absence of native myosin, these cells, termed hmm cells, exhibit many forms of cell movement, including membrane ruffling, phagocytosis, and chemotaxis. The hmm cells can aggregate but are blocked at a later stage in the Dictyostelium developmental cycle. The hmm cells revert to the wild-type phenotype. Reversion of the hmm phenotype is due to excision and loss of the transforming plasmid. The revertant cells express native myosin, are G418 sensitive, and have a normal developmental cycle. These results constitute genetic proof that the intact myosin molecule is required for cytokinesis and not for karyokinesis.
同源重组现象可实现特定的基因转换和基因插入，它可能成为研究真核细胞生物学的强大系统。本文给出的数据表明，转染质粒通过同源重组整合到运动性真核细胞盘基网柄菌中。携带G418抗性基因和肌球蛋白重链基因氨基端一半的质粒被用于转染盘基网柄菌。大部分产生的G418抗性细胞中，质粒整合到了重链基因的单基因组拷贝中。这些细胞无法表达天然肌球蛋白，但能表达肌球蛋白片段，在胞质分裂方面存在缺陷，会变大并形成多核。尽管缺乏天然肌球蛋白，这些被称为hmm细胞的细胞仍表现出多种细胞运动形式，包括膜波动、吞噬作用和趋化性。hmm细胞可以聚集，但在盘基网柄菌发育周期的后期被阻断。hmm细胞可恢复为野生型表型。hmm表型的恢复是由于转化质粒的切除和丢失。回复细胞表达天然肌球蛋白，对G418敏感，并且具有正常的发育周期。这些结果构成了遗传学证据，证明完整的肌球蛋白分子是胞质分裂所必需的，而不是核分裂所必需的。
