Rubin E M, Wilson G A, Young F E
Gene. 1980 Aug;10(3):227-35. doi: 10.1016/0378-1119(80)90052-9.
The gene from Escherichia coli encoding thymidylate synthetase was cloned in the plasmid pBR322. The resulting chimeric plasmid, pER2, was effective in transforming both E. coli and Bacillus subtilis to thymine prototrophy. Uncloned linear E. coli chromosomal DNA was unable to transform thymine-requiring strains of B. subtilis to thymine independence. Linearization of the chimeric plasmid, pER2, with restriction enzymes markedly diminished its ability to transform B. subtilis auxotrophs. The Thy+ transformants derived from the transformation of B. subtilis with pER2 DNA did not contain detectable extrachromosomal DNA as demonstrated by Southern hybridization patterns and centrifugation in CsCl gradients of DNA isolated from B. subtilis colonies transformed with the chimeric plasmid. We conclude that the DNA from the chimeric plasmid was integrated into the chromosome of B. subtilis, demonstrating that extensive homology is not required for the integration of foreign DNA. This is the first reported case of a gene from a Gram-negative bacterium functioning in a Gram-positive organism.
编码胸苷酸合成酶的大肠杆菌基因被克隆到质粒pBR322中。所得的嵌合质粒pER2能有效地将大肠杆菌和枯草芽孢杆菌转化为胸腺嘧啶原养型。未克隆的线性大肠杆菌染色体DNA无法将需要胸腺嘧啶的枯草芽孢杆菌菌株转化为不依赖胸腺嘧啶的菌株。用限制酶将嵌合质粒pER2线性化显著降低了其转化枯草芽孢杆菌营养缺陷型的能力。用pER2 DNA转化枯草芽孢杆菌得到的Thy⁺转化子，通过Southern杂交图谱以及从用嵌合质粒转化的枯草芽孢杆菌菌落中分离的DNA在CsCl梯度中的离心分析表明，未检测到可检测到的染色体外DNA。我们得出结论，嵌合质粒的DNA整合到了枯草芽孢杆菌的染色体中，这表明外源DNA的整合不需要广泛的同源性。这是首次报道革兰氏阴性菌的基因在革兰氏阳性生物中发挥功能的案例。
