Related Experiment Videos
Isolation and preliminary characterization of amber mutants of bacteriophage phi W-14 defective in DNA synthesis
Abstract:
Of 42 amber mutants of bacteriophage phi W-14, 6 were defective in DNA synthesis. Three of the mutants synthesized DNA in the nonpermissive host, but were defective in post-replicational modification of the DNA. The DNA synthesized by two of these mutants, am36 and am42, contained more thymine and less alpha-putrescinylthymine than did wild-type DNA; that synthesized by the third mutant, am37, contained the normal amount of thymine, no alpha-putrescinylthymine, and hydroxymethyluracil. The properties of these mutants suggested that the presence of the normal amount of alpha-putrescinylthymine in phi W-14 DNA was essential for the production of viable progeny. Three of the mutants, am6, am35, and am45, failed to synthesize DNA in the nonpermissive host. These mutants were analogous to the DNA off mutants of T4. Nonpermissive cells infected with DNA off mutants accumulated dATP, dGTP, dCTP, and hydroxymethyl dUTP, but not dTTP or alpha-putrescinyldeoxythymidine triphosphate, confirming that both thymine and alpha-putrescinylthymidine in phi W-14 DNA are formed from hydroxymethyluracil at the polynucleotide level. The synthesis of phi W-14 DNA is unusual because (i) thymine is formed from hydroxymethyluracil at the polynucleotide level, (ii) the hypermodification forming alpha-putrescinylthymine is essential, and (iii) thymine and alpha-putrescinylthymine must be made in the correct proportions. Complementation tests showed that the mutants defined three genes involved in DNA polymerization and two genes involved in post-replicational modification.
Insights
Bacteriophage phi W-14 DNA synthesis requires specific modifications, including alpha-putrescinylthymine, essential for viable progeny. Mutants revealed unusual DNA synthesis pathways and gene functions.
Area of Science:
- Microbiology
- Molecular Biology
- Virology
Background:
- Bacteriophage phi W-14 possesses a unique DNA structure with modified bases.
- Understanding bacteriophage DNA replication and modification is crucial for virology and molecular biology.
Purpose of the Study:
- To investigate the genetic basis of DNA synthesis and modification in bacteriophage phi W-14.
- To identify genes involved in DNA polymerization and post-replicational modification.
Main Methods:
- Isolation and characterization of amber mutants in bacteriophage phi W-14.
- Analysis of DNA synthesis and base composition in mutant strains.
- Complementation tests to identify genes involved in DNA replication and modification.
Main Results:
- Six mutants were defective in DNA synthesis, with three showing defects in post-replicational DNA modification.
- Mutants am36 and am42 produced DNA with altered thymine and alpha-putrescinylthymine ratios.
- Mutant am37 produced DNA lacking alpha-putrescinylthymine and containing hydroxymethyluracil.
- Mutants am6, am35, and am45 failed to synthesize DNA, accumulating specific nucleotide precursors.
- Complementation tests identified three genes for DNA polymerization and two for post-replicational modification.
Conclusions:
- The presence of alpha-putrescinylthymine in phi W-14 DNA is essential for producing viable progeny.
- Thymine and alpha-putrescinylthymine are formed from hydroxymethyluracil at the polynucleotide level.
- Phi W-14 DNA synthesis involves unusual steps: thymine formation from hydroxymethyluracil, essential alpha-putrescinylthymine hypermodification, and precise base proportions.