Conjugal transfer of oncogenes from bacteria to animal celIs

为了实现原核大肠杆菌与真核动物培养细胞的跨界接合，我们构建了一个新的质粒pBAS绿色，该质粒由oriT、SV 40-ori/promoter、pBR 322-oriV、Ap、Neo和GFP（绿色荧光蛋白）基因组成。将该质粒成功地导入动物培养细胞COS 1和NIH 3 T3中，然后在这些细胞中清楚地检测到由于GFP报告基因引起的典型的绿色荧光。然而，大肠杆菌供体细胞不幸地是污染物并且对培养的细胞有毒。为了克服这些问题，大肠杆菌小细胞突变体P678-54的无核和非分裂细胞被证实是有用的，因为它毒性较小并且保持跨界接合的活性。在癌基因接合转移的基础上，我们还用Southern印迹法对肠杆菌进行了癌基因的检测。另一方面，植物病原细菌根癌农杆菌（Agrobacterium tumefaciens）携带一个巨大的Ti（tumor inducing）质粒，并通过类接合的方式将其T-DNA导入双子叶植物。为了从分子水平上了解T-DNA转移与跨界接合的关系，我们对Ti质粒的全基因组进行了分析。203，148 bp的全序列测定和分析清楚地表明接合和T-DNA转移系统的独立性。我们还发现根癌农杆菌对动物培养细胞和植物细胞的毒性较小。上述结果为细菌与动物细胞间的跨界接合提供了重要线索。

For trans-kingdom conjugation between prokaryotic Escherichia coli and eukaryotic animal cultured cells, we have constructed a novel plasmid, pBASGreen which consists of oriT,SV40-ori/promoter, pBR322-oriV,Ap, Neo, and GFP (green fluorescent protein) genes. The plasmid was successfully introduced into animal cultured cells, COS1 and NIH3T3 then typical green fluorescence due to the GFP reporter gene was clearly detected in the transformant celIs. However, E.coli donor celIs were unfortunately contaminant and toxic against cultured celIs. To overcome the problems, anucleated and non-dividing celIs of E.coli mini-cell mutant P678-54 was verified to be useful because it is less toxic and keeps the activity of trans-kingdom conjugation. On the basis of conjugal oncogene transfer, we also carried out oncogenes survey in enterobacteria by using Southern blotting. On the other hand, phytopathogenic bacterium Agrobacterium tumefaciens harbors a giant Ti (tumor inducing) plasmid and transfers it's T-DNA into dicots by conjugation-like process. The infected plants subsequently have tumors induced by oncogenes on T-DNA.In order to know the relationship between T-DNA transfer and trans-kingdom conjugation on the molecular basis, we analyzed the entire genome of Ti plasmid. The complete sequencing and analyzing of 203,148bp clearly indicate the independence of conjugation and T-DNA transfer system. We also found that A.tumefaciens is less toxic against animal cultured cells as well as plant celIs. The above results offer important clues on trans-kingdom conjugation between bacterial and animal celIs.