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偶联，我们构建了一种新的质粒pBASGreen，该质粒由oriT、SV40-ori/启动子、pBR322-oriV、Ap、Neo和GFP（绿色荧光蛋白）基因组成。将质粒成功导入动物培养细胞，COS1和NIH3T3在转化细胞中明显检测到典型的绿色荧光，这是由于GFP报告基因。然而，不幸的是，大肠杆菌供体细胞对培养的细胞具有污染和毒性。为了克服这些问题，我们利用大肠杆菌迷你细胞突变体P678-54的无核非分裂细胞，证明其毒性较小，并且保持了跨界偶联的活性。在偶联癌基因转移的基础上，采用Southern印迹法对肠杆菌进行了癌基因调查。另一方面，植物致病菌农杆菌含有一个巨大的Ti（肿瘤诱导）质粒，并通过偶联过程将其T-DNA转移到肿瘤中。受感染的植株随后由T-DNA上的致癌基因诱导产生肿瘤。为了从分子基础上了解T-DNA转移与跨界偶联之间的关系，我们分析了Ti质粒的全基因组。203,148bp的完整测序和分析清楚地表明偶联和T-DNA转移系统的独立性。我们还发现，对动物培养细胞和植物细胞的毒性较低。以上结果为研究细菌与动物细胞的跨界结合提供了重要线索。

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.