DISRUPTION OF THE DICTYOSTELIUM MYOSIN HEAVY-CHAIN GENE BY HOMOLOGOUS RECOMBINATION
DISRUPTION OF THE DICTYOSTELIUM MYOSIN HEAVY-CHAIN GENE BY HOMOLOGOUS RECOMBINATION
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DOI：
10.1126/science.3576222
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发表时间：
1987-05-29
期刊：
影响因子：
56.9
通讯作者：
SPUDICH, JA
中科院分区：
文献类型：
--
作者：
DELOZANNE, A;SPUDICH, JA
同源重组现象允许特定的基因转换和基因插入，可以成为研究真核细胞生物学的强大系统。所提供的数据表明，在运动真核细胞盘基网柄菌中发生通过同源重组的转染质粒的整合。使用携带G418抗性基因和肌球蛋白重链基因的氨基末端一半的质粒来转染盘基网柄菌属。所得 G418 抗性细胞的很大一部分将质粒整合到重链基因的单个基因组拷贝中。这些细胞不能表达天然肌球蛋白，但表达肌球蛋白片段，在胞质分裂中存在缺陷，并变得很大且多核。尽管不存在天然肌球蛋白，这些细胞（称为medhmm细胞）表现出多种形式的细胞运动，包括膜褶皱、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细胞恢复到野生型表型。表型的逆转是由于转化质粒的切除和丢失。回复突变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。
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 cellDictyostelium discoideum. A plasmid carrying a G418 resistance gene and the amino terminal half of the myosin heavy chain gene was used to transfectDictyostelium. 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, termedhmmcells, exhibit many forms of cell movement, including membrane ruffling, phagocytosis, and chemotaxis. Thehmmcells can aggregate but are blocked at a later stage in theDictyosteliumdevelopmental cycle. Thehmmcells revert to the wild-type phenotype. Reversion of thehmmphenotype 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.