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Site-specific recombination by Gin of bacteriophage Mu: inversions and deletions
Abstract:
A 3000-bp invertible segment in the DNA of bacteriophage Mu determines the host range of the phage. The inversion is catalyzed by the phage-coded protein Gin; the recombination sites are short inverted repeats. Gin protein is only made in low amounts by Mu. To further investigate the Gin-mediated recombination reaction a Gin overproducing strain was constructed. The gin gene was cloned on a plasmid behind the PL-promotor of phage lambda. This results in a 100-fold higher inversion frequency of a Mu gin phage as compared to the situation when Gin is expressed from its own promoter. A test system was developed suitable for the detection of Gin action in vivo and in vitro: the lacZ gene of E. coli was cloned within the invertible region in such a way that it is only expressed when the region is in one specific orientation. Thus inversions can be detected or selected as a switch from Lac- to Lac+. This system was used to determine the inversion frequency under different experimental conditions. The ability of Gin to catalyze deletions was investigated by inverting in vitro one of the two recombination sites using restriction enzymes and genetically marking the DNA between those sites. Deletions do occur, although at a lower frequency than inversions.
Insights
Bacteriophage Mu
Area of Science:
- Molecular Biology
- Genetics
- Virology
Background:
- Bacteriophage Mu's host range is determined by a 3000-bp invertible DNA segment.
- This inversion is catalyzed by the phage-coded Gin protein, acting at short inverted repeats.
- Gin protein is typically produced in low quantities by the phage.
Purpose of the Study:
- To investigate the Gin-mediated recombination reaction further.
- To develop a system for detecting Gin action in vivo and in vitro.
- To determine inversion frequency under various experimental conditions and explore Gin's deletion-catalyzing ability.
Main Methods:
- Constructed a Gin overproducing strain by cloning the gin gene behind the lambda phage PL-promoter on a plasmid.
- Developed a test system using the E. coli lacZ gene within the invertible region for Lac- to Lac+ switching detection.
- Investigated deletions by inverting recombination sites in vitro and genetically marking intervening DNA.
Main Results:
- The Gin overproducing strain resulted in a 100-fold higher inversion frequency compared to native Gin expression.
- The developed test system effectively detected and selected Gin-mediated inversions.
- Gin was found to catalyze deletions, albeit at a lower frequency than inversions.
Conclusions:
- Overproduction of Gin significantly enhances bacteriophage Mu DNA inversion frequency.
- A novel reporter system allows for efficient detection and selection of Gin-mediated inversions.
- Gin exhibits a dual capability in catalyzing both DNA inversions and deletions.