Related Experiment Videos
Aphidicolin-resistant mutants of bacteriophage phi 29: genetic evidence for altered DNA polymerase
Abstract:
Aphidicolin-resistant mutants (Aphr) of Bacillus subtilis bacteriophage phi 29 were isolated after mutagenesis with hydroxylamine. Efficiency of plating (e.o.p.) of the resistant mutants was not reduced at 500 microM aphidicolin, although e.o.p. of wild type phi 29 was less than 10(-5) at the same concentration of aphidicolin. By recombination and complementation analyses, both sites of the mutations, aph-71 and aph-101, of Aphr71 and Aphr101, respectively, were mapped in gene 2 which encodes phi 29 DNA polymerase. The activity of wild type phi 29 DNA polymerase, in a partially purified fraction, was inhibited by aphidicolin. DNA polymerases from Aphr71 and Aphr101, prepared in the same manner as that of wild type, were resistant to the drug. These results indicate that the acquisition of the aphidicolin resistance of Aphr71 and Aphr101 of bacteriophage phi 29 results from a structural alteration of phi 29 DNA polymerase which reduces sensitivity to aphidicolin.
Insights
Mutagenesis of Bacillus subtilis bacteriophage phi 29 yielded aphidicolin-resistant mutants. These mutants possess altered DNA polymerase, conferring resistance to aphidicolin, a potent inhibitor.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Bacteriophage phi 29 is a model organism for studying DNA replication.
- Aphidicolin is a specific inhibitor of certain DNA polymerases.
Purpose of the Study:
- To isolate and characterize aphidicolin-resistant mutants of bacteriophage phi 29.
- To determine the genetic basis of aphidicolin resistance in bacteriophage phi 29.
Main Methods:
- Isolation of resistant mutants using hydroxylamine mutagenesis.
- Recombination and complementation analyses to map mutation sites.
- Enzyme assays to assess DNA polymerase activity and drug sensitivity.
Main Results:
- Aphidicolin-resistant mutants (Aphr) of phi 29 were obtained.
- Mutations conferring resistance were mapped to gene 2, encoding phi 29 DNA polymerase.
- Purified DNA polymerases from resistant mutants showed reduced sensitivity to aphidicolin.
Conclusions:
- Aphidicolin resistance in phi 29 mutants is due to structural alterations in the DNA polymerase.
- These alterations reduce the enzyme's sensitivity to aphidicolin inhibition.