Mahmood A, Kimura T, Takenaka M, Yoshida K
Department of Biological Science, Faculty of Science, Hiroshima University, Japan.
Genet Anal. 1996 Jul;13(2):25-31. doi: 10.1016/1050-3862(95)00146-8.
Trans-kingdom conjugation is an easy and efficient method for gene transfer from prokaryotes to eukaryotes since it does not require DNA extraction and purification. We constructed novel mobilizable plasmids pAY-YAC-B and pAY-YAC-E. The origin of conjugal transfer (oriT) was inserted at two different positions, pAY-YAC-B contains oriT region in between two telomeres whereas pAY-YAC-E has oriT at the cloning site of pYAC4. By conjugation, both plasmids were successfully transferred from E. coli to S. cerevisiae and S. kluyveri yeasts with the aid of helper plasmid pRH220 which harbors mob and tra genes. The plasmids were transferred more efficiently in S. cerevisiae compared to S. kluyveri. The analyses by restriction enzyme digestion and Southern hybridization indicated that both plasmids maintained their original structure and size in transconjugant yeasts, therefore, reflecting the faithful nicking and subsequent resealing of plasmids during conjugation. The comparison between conjugative transfer and transformation has also been performed and discussed.
跨王国接合是一种从原核生物向真核生物转移基因的简便高效方法，因为它无需进行DNA提取和纯化。我们构建了新型可移动质粒pAY - YAC - B和pAY - YAC - E。将接合转移起始位点（oriT）插入到两个不同位置，pAY - YAC - B在两个端粒之间包含oriT区域，而pAY - YAC - E在pYAC4的克隆位点处有oriT。通过接合作用，在携带mob和tra基因的辅助质粒pRH220的帮助下，两种质粒都成功地从大肠杆菌转移到酿酒酵母和克鲁维酵母中。与克鲁维酵母相比，这些质粒在酿酒酵母中的转移效率更高。通过限制性酶切分析和Southern杂交表明，两种质粒在接合子酵母中均保持其原始结构和大小，因此，这反映了接合过程中质粒的准确切口和随后的重新封闭。同时也进行并讨论了接合转移与转化之间的比较。
