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A genetic switch in vitro: DNA inversion by Gin protein of phage Mu
Abstract:
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.
Insights
The Escherichia coli phage Mu Gin protein catalyzes G segment inversion, altering phage host range. A new assay allows in vitro study of this low-frequency genetic switch.
Area of Science:
- Molecular Biology
- Genetics
- Virology
Background:
- The G segment of Escherichia coli phage Mu DNA undergoes inversion, mediated by the Mu Gin protein.
- This inversion event influences the phage's host range.
- The inversion frequency is typically low during both lysogenic and lytic growth phases.
Purpose of the Study:
- To develop a sensitive assay for detecting low levels of G segment inversion.
- To investigate the in vitro activity of the Mu Gin protein in catalyzing G segment inversion.
- To enable mechanistic studies of this genetic switch.
Main Methods:
- Development of a novel assay utilizing the E. coli lac operon inserted within the invertible G segment.
- Monitoring inversion via a lactose-negative to lactose-utilizing switch.
- In vitro reaction using crude extracts from a Gin-overproducing strain and supercoiled plasmid DNA.
- Analysis of inverted DNA by restriction endonuclease digestion and agarose gel electrophoresis.
Main Results:
- A sensitive assay was established to detect low levels of G inversion.
- In vitro, Gin-catalyzed inversion was detected at 1% in 30 minutes using crude extracts.
- Optimized conditions yielded over 15% inversion in vitro.
- The reaction requires Mg2+ and supercoiled substrate.
- Gin protein retained catalytic activity after partial purification.
Conclusions:
- A functional in vitro system for studying Mu G segment inversion has been established.
- The developed assay facilitates the detection and analysis of low-frequency inversion events.
- Further mechanistic studies of the Gin-catalyzed genetic switch are now feasible in vitro.