A T7 amber mutant defective in DNA-binding protein

Area of Science:

• Molecular Biology
• Virology
• Genetics

Background:

• T7 phage DNA-binding protein is crucial for viral DNA replication and stability.
• Amber mutations in essential genes can be used to study gene function.
• Bacterial suppressor mutations (sup+) can rescue amber mutations.

Purpose of the Study:

• To characterize a novel amber mutant (UP-2) in the T7 DNA-binding protein gene (gene 2.5).
• To investigate the role of T7 DNA-binding protein in DNA synthesis, replication, recombination, and repair.
• To map the mutation within the T7 genome.

Main Methods:

• Isolation of T7 amber mutants based on differential bacterial host growth.
• Analysis of mutant polypeptide size via gel electrophoresis.
• Assessment of DNA synthesis and replication in infected bacterial cells.
• Evaluation of genetic recombination and DNA repair efficiencies.
• Genetic mapping of the mutation within the T7 genome.

Main Results:

• The UP-2 mutant produces a truncated T7 DNA-binding protein (28 kDa vs. 32 kDa).
• UP-2 severely inhibits T7 DNA synthesis and represses replication in non-permissive cells.
• The mutant exhibits reduced genetic recombination and DNA repair capabilities.
• Recombination intermediates are not formed in UP-2 infected non-permissive cells.
• The mutation was mapped to gene 2.5, located between T7 genes 2 and 3.

Conclusions:

• T7 DNA-binding protein is essential for T7 DNA replication, recombination, and repair.
• Truncation of T7 DNA-binding protein significantly impairs viral genetic functions.
• Gene 2.5 plays a critical role in T7 phage viability and propagation.