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Spontaneous transposition in the bacteriophage lambda cro gene residing on a plasmid
R Skaliter1, Z Eichenbaum, H Shwartz
1Department of Biochemistry, Weizmann Institute of Science, Rehovot, Israel.
Mutation Research
|May 1, 1992
Summary
A new mutagenesis assay system identifies mutations in the phage lambda cro repressor gene. Insertion sequences (IS) like IS1, IS5, and IS10 are major mutagens, with RecA influencing their transposition.
Area of Science:
- Molecular Biology
- Microbial Genetics
- Bacteriophage Biology
Background:
- The phage lambda cro repressor gene plays a crucial role in regulating viral gene expression.
- Understanding mutagenesis mechanisms is vital for studying genome stability and evolution.
- Insertion sequence (IS) elements are mobile genetic elements that can cause mutations.
Purpose of the Study:
- To develop a novel mutagenesis assay system utilizing the phage lambda cro repressor gene.
- To investigate spontaneous mutation frequencies and types in Escherichia coli.
- To characterize the role of insertion sequences (IS) and the RecA protein in mutagenesis.
Main Methods:
- Development of a mutagenesis assay based on the phage lambda cro repressor gene on a plasmid.
- Detection of mutations affecting repressor-operator binding using an OR-lacZ fusion in a lambda prophage.
- Analysis of spontaneous mutants using restriction nuclease digestion and genomic Southern analysis.
- Investigation of mutation frequency changes during different growth phases (logarithmic vs. stationary).
Main Results:
- A spontaneous mutation frequency of 3-6 x 10(-6) was observed in E. coli.
- Insertion events, primarily IS1 and IS5, constituted 25-70% of mutations in wild-type strains.
- Tn10-harboring strains showed IS10 as the main insertion element, with a preference for the cro C-terminal half.
- RecA protein significantly influences IS1 and IS10 transposition, reducing their frequency.
- Mutation frequency increased in the stationary phase, with higher rates of insertion and point mutations.
- IS1 and IS5 transposition had minimal impact on chromosomal element distribution, unlike IS10, which caused multiple changes.
Conclusions:
- The developed cro repressor-based assay is effective for studying mutagenesis in E. coli.
- IS elements are significant contributors to spontaneous mutations, with RecA playing a key role in their transposition.
- IS10 transposition exhibits distinct characteristics regarding target site preference and impact on chromosomal element dynamics.