Kuspa A, Loomis W F
Department of Biology, University of California, San Diego, La Jolla 92093.
Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8803-7. doi: 10.1073/pnas.89.18.8803.
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.
将限制性内切酶与线性化质粒一同导入，会使质粒DNA在很大比例的转化子中整合到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质粒一起进行电穿孔处理，相较于单独导入质粒DNA时，能将盘基网柄菌的转化效率提高20倍以上。限制性内切酶介导的整合以一种明显随机的方式在基因组限制性位点产生插入，其中一些会导致突变。约每400个盘基网柄菌转化子中就有1个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，用于转化其他盘基网柄菌细胞。侧翼序列内的同源重组导致了与原始突变体相同的表型，直接证明了受影响的基因导致了特定的形态表型。这种插入诱变方法对于标记以及随后克隆许多可通过其突变表型鉴定的发育重要基因应该是有用的。
