Nishikawa M, Yoshida K
Research Institute for Biological Sciences, Okayama, Japan.
Genet Anal. 1998 Jan;14(3):65-73. doi: 10.1016/s1050-3862(97)10003-1.
Gene targeting is one of the powerful techniques used to investigate eukaryotic genes. In a typical eukaryotic microbe, Saccharomyces cerevisiae yeast, we examined trans-kingdom conjugation between Escherichia coli bacterium and yeast as a gene targeting tool. Here, it is shown that trans-kingdom conjugation effectively induced gene replacement even on yeast's target loci (e.g. ura3-52 allele) which is never targeted by conventional transformation. This clearly indicates that trans-kingdom conjugation offers a very powerful gene targeting tool in yeasts. In fact, Southern hybridization analysis of transconjugants distinctly verified the accuracy in the conjugative gene replacement. The efficiency of gene replacement was about 0.4 x 10(-7) per recipient yeast. This is enough to sustain gene targeting with gene replacement by trans-kingdom conjugation. We also discuss the mechanism of conjugative gene replacement.
基因打靶是用于研究真核基因的强大技术之一。在典型的真核微生物酿酒酵母中，我们研究了大肠杆菌与酵母之间的跨界接合作为一种基因打靶工具。在此表明，跨界接合即使在酵母的靶位点（如ura3-52等位基因）上也能有效诱导基因替换，而传统转化从未针对过该位点。这清楚地表明，跨界接合在酵母中提供了一种非常强大的基因打靶工具。事实上，对接合子的Southern杂交分析明确证实了接合性基因替换的准确性。基因替换效率约为每受体酵母0.4×10⁻⁷。这足以通过跨界接合维持基因打靶和基因替换。我们还讨论了接合性基因替换的机制。
