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Mutagenesis of plasmid DNA with hydroxylamine: isolation of mutants of multi-copy plasmids
Abstract:
An investigation of in vitro mutagenesis of plasmid DNA with hydroxylamine is described. The treated plasmid DNA was used to transform Escherichia coli K12. Mutants of the plasmid NTP3, which codes for resistance to ampicillin and sulphonamides, were isolated and characterised. They were classified according to the reduction in level of their beta-lactamase activity. Hydroxylamine-induced mutants of NTP14 were also isolated. This plasmid codes for ampicillin resistance, synthesis of colicin E1, and the EcoRI restriction and modification enzymes. One class of mutants is lethal to the host strain at temperatures above 33 degrees C, but carrier strains grow well at 28 degrees C. There is evidence that these mutants code for a temperature-sensitive EcoRI modification activity: the lethal effect probably results from the cleavage of the host-cell DNA by the restriction enzyme at non-permissive temperatures. The possible genetic uses of the mutant plasmids for the production of hybrid plasmids in the bacterial cell are discussed.
Insights
Hydroxylamine mutagenesis created plasmid DNA mutants in Escherichia coli. These mutants, affecting antibiotic resistance and enzyme activity, show potential for genetic engineering and hybrid plasmid production.
Area of Science:
- Molecular Biology
- Microbial Genetics
Background:
- Plasmid DNA is crucial for bacterial genetic exchange and adaptation.
- Hydroxylamine is a chemical mutagen used to induce mutations in DNA.
Purpose of the Study:
- To investigate in vitro mutagenesis of plasmid DNA using hydroxylamine.
- To characterize hydroxylamine-induced mutants of specific plasmids in Escherichia coli K12.
Main Methods:
- In vitro treatment of plasmid DNA with hydroxylamine.
- Transformation of Escherichia coli K12 with mutagenized plasmid DNA.
- Isolation and characterization of plasmid mutants based on altered phenotypes.
Main Results:
- Mutants of plasmid NTP3 exhibited reduced beta-lactamase activity, affecting ampicillin and sulphonamide resistance.
- Hydroxylamine-induced mutants of plasmid NTP14 displayed temperature-sensitive EcoRI modification activity, leading to host lethality above 33°C.
- Lethality in NTP14 mutants is likely due to EcoRI restriction enzyme activity cleaving host DNA at elevated temperatures.
Conclusions:
- Hydroxylamine is effective for generating diverse plasmid mutants.
- Mutant plasmids, particularly those with temperature-sensitive restriction enzyme activity, offer valuable tools for genetic manipulation.
- These findings support the use of mutant plasmids for producing hybrid plasmids within bacterial cells.