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Isolation of mutants in a DNA methyltransferase through mcrB-mediated restriction
R M Blumenthal1, M M Cotterman
1Department of Microbiology, Medical College of Ohio, Toledo 43699.
Abstract:
A procedure has been developed that permits the positive selection of mutants in a DNA methyltransferase (MTase) gene. The stringency of this selection can be varied so as to yield null mutants only, or a mixture of null and partially defective mutants. The procedure was developed with the PvuII MTase gene (pvuIIM), which was subcloned into a bacteriophage lambda vector. Growth of this lambda pvuIIM construct on an mcrB+ host selected for non-methylating mutants, and the stringency of selection was proportional to the number of consecutive lytic cycles. Many cytosine MTases have been found to generate substrates for mcrB-mediated restriction, and this procedure should be applicable to a number of cytosine MTase genes.
Insights
A new positive selection method identifies mutants in DNA methyltransferase (MTase) genes. This technique allows for variable stringency, enabling the isolation of null or partially defective MTase mutants for various cytosine MTase genes.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA methyltransferases (MTases) play crucial roles in gene regulation and DNA repair.
- Identifying and characterizing MTase mutants is essential for understanding their functions.
- Existing methods for mutant selection can be inefficient or lack tunable stringency.
Purpose of the Study:
- To develop a novel positive selection procedure for DNA methyltransferase (MTase) mutants.
- To enable variable stringency for selecting either null or partially defective MTase mutants.
- To demonstrate the applicability of this method to cytosine MTase genes.
Main Methods:
- Subcloning of the PvuII MTase gene (pvuIIM) into a bacteriophage lambda vector.
- Utilizing an mcrB+ host for positive selection of non-methylating mutants.
- Varying the stringency of selection by adjusting the number of consecutive lytic cycles.
Main Results:
- A functional positive selection procedure for DNA MTase mutants was successfully developed.
- The selection stringency was found to be directly proportional to the number of lytic cycles.
- The method effectively selected for non-methylating mutants of the PvuII MTase gene.
Conclusions:
- The developed procedure provides an efficient means for positive selection of DNA MTase mutants.
- This method offers tunable stringency, allowing for the isolation of different mutant classes.
- The technique is potentially applicable to a wide range of cytosine MTase genes.