TAGGING DEVELOPMENTAL GENES IN DICTYOSTELIUM BY RESTRICTION ENZYME-MEDIATED INTEGRATION OF PLASMID DNA
TAGGING DEVELOPMENTAL GENES IN DICTYOSTELIUM BY RESTRICTION ENZYME-MEDIATED INTEGRATION OF PLASMID DNA
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DOI：
10.1073/pnas.89.18.8803
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发表时间：
1992-09-15
影响因子：
11.1
通讯作者：
LOOMIS, WF
中科院分区：
文献类型：
--
作者：
KUSPA, A;LOOMIS, WF
限制性内切酶与线性化质粒一起引入，导致所得转化子中高比例的质粒 DNA 整合在基因组限制性位点上。我们发现，将 BamHI 或 EcoRI 与用相同酶切割的 Pyr5-6 质粒一起电穿孔，可刺激盘基网柄菌中的转化效率，比单独引入质粒 DNA 时的转化效率高 20 倍以上。限制性内切酶介导的整合以明显随机的方式产生基因组限制性位点的插入，其中一些导致突变。大约四百分之一的盘基网柄菌转化体表现出发育停滞或异常。从其中一些突变体中切下整合的质粒以及侧翼基因组DNA，克隆到大肠杆菌中，并用于转化其他盘基网柄菌属细胞。侧翼序列内的同源重组导致与原始突变体表现出相同的表型，直接证明受影响的基因负责特定的形态表型。这种插入诱变方法应该可用于标记和随后克隆许多发育上重要的基因，这些基因可以通过其突变表型来识别。
Introduction of restriction enzyme along with linearized plasmid results in integration of plasmid DNA at genomic restriction sites in a high proportion of the resulting transformants. We have found that electroporating BamHI or EcoRI together with pyr5-6 plasmids cut with the same enzyme stimulates the efficiency of transformation in Dictyostelium discoideum more than 20-fold over the rate seen when plasmid DNA alone is introduced. Restriction enzyme-mediated integration generates insertions into genomic restriction sites in an apparently random manner, some of which cause mutations. About 1 in 400 of the Dictyostelium transformants displayed arrested or aberrant development. The integrated plasmid, along with flanking genomic DNA, was excised from some of these mutants, cloned in Escherichia coli, and used to transform other Dictyostelium cells. Homologous recombination within the flanking sequences resulted in the same phenotypes displayed by the original mutants, directly demonstrating that the affected genes were responsible for the specific morphological phenotypes. This method of insertional mutagenesis should be useful for tagging, and subsequent cloning, of many developmentally important genes that can be identified by their mutant phenotypes.