Plasterk R H, Kanaar R, van de Putte P
Proc Natl Acad Sci U S A. 1984 May;81(9):2689-92. doi: 10.1073/pnas.81.9.2689.
Inversion of the G segment in the DNA of Escherichia coli phage Mu depends on the Mu Gin protein and alters the host range of the phage. The frequency of the inversion reaction is low both in the lysogenic state and during lytic growth. A sensitive assay was developed to detect low levels of G inversion: the E. coli lac operon was inserted within the invertible G segment in such a way that the lac operon was expressed only by G(-) clones. As a result Gin-catalyzed inversion from G(+) to G(-) can be monitored as a lactose-negative to lactose-utilizing switch. Using a crude extract from a Gin-overproducing strain and this assay plasmid, we could detect a low level of G inversion in vitro (1% in 30 min). The reaction depends on Mg2+ and a supercoiled substrate. Under optimized reaction conditions over 15% of the plasmids had the G segment inverted after incubation with Gin in vitro. The inversion was then visualized by agarose gel analysis of plasmid DNA digested by restriction endonucleases. The Gin protein retains its catalytic properties upon partial purification. The mechanism of this genetic switch can now be studied in vitro.
大肠杆菌噬菌体Mu DNA中G片段的倒位依赖于Mu Gin蛋白，并改变噬菌体的宿主范围。在溶原状态和裂解生长期间，倒位反应的频率都很低。我们开发了一种灵敏的检测方法来检测低水平的G倒位：将大肠杆菌乳糖操纵子插入可倒位的G片段中，使得乳糖操纵子仅由G(-)克隆表达。结果，Gin催化的从G(+)到G(-)的倒位可以作为乳糖阴性到利用乳糖的转变来监测。使用来自Gin高产菌株的粗提物和该检测质粒，我们可以在体外检测到低水平的G倒位（30分钟内为1%）。该反应依赖于Mg2+和超螺旋底物。在优化的反应条件下，超过15%的质粒在体外与Gin孵育后G片段发生了倒位。然后通过对限制性内切酶消化的质粒DNA进行琼脂糖凝胶分析来观察倒位情况。Gin蛋白在部分纯化后仍保留其催化特性。现在可以在体外研究这种遗传开关的机制。
