Conjugal transfer of oncogenes from bacteria to animal celIs

癌基因从细菌到动物细胞的接合转移

基本信息

批准号：
08044292
负责人：
YOSHIDA Kazuo
金额：
$ 5.63万
依托单位：
HIROSHIMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

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.

为了实现原核大肠杆菌和真核动物培养细胞之间的跨界接合，我们构建了一个新的质粒pBASGreen，它由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中，然后在转化细胞中清楚地检测到由于GFP报告基因而产生的典型绿色荧光。然而，不幸的是，大肠杆菌供体细胞对培养细胞具有污染性和毒性。为了克服这些问题，大肠杆菌微型细胞突变体P678-54的无核和非分裂细胞被证实是有用的，因为它的毒性较低并且保持了跨界接合的活性。在夫妻癌基因转移的基础上，我们还利用Southern印迹法对肠杆菌进行了癌基因调查。另一方面，植物病原菌根癌农杆菌含有巨大的 Ti（肿瘤诱导）质粒，并通过类似接合的过程将其 T-DNA 转移到双子叶植物中。受感染的植物随后在T-DNA上产生致癌基因诱导的肿瘤。为了从分子基础上了解T-DNA转移和跨界接合之间的关系，我们分析了Ti质粒的整个基因组。 203,148bp的完整测序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。上述结果为细菌和动物细胞之间的跨界接合提供了重要线索。