Related Experiment Videos
Bacteriophage T4 gene 17 amplification mutants: evidence for initiation by the T4 terminase subunit gp16
1Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201, USA.
Abstract:
Bacteriophage T4 genes 16 and 19 containing the 24 bp homology regions that recombine to form Hp17 mutants were cloned into plasmids. When the two homology sequences were cloned either together into one or separately into two compatible plasmids, a polymerase chain reaction assay showed that recombination occurred in vivo. The recombinant sequence was identical with that found in T4 phage Hp17 mutants, and was produced in recombination-deficient Escherichia coli. Mutational analysis revealed a requirement for functional gene 16 but not gene 17 to recombine the sequences. Moreover, gp16, the terminase small subunit, was required, since an amber gene 16 produced the recombinant sequence only when suppressed. Mutations in the gene 16 recombination sequence (3GA and 15TG) that eliminated Hp17 formation in T4 phage increased the synthesis of the large terminase subunit, gp17 in T4 infections, suggesting gp16 interaction with this site. gp16 binding to gene 16 and gene 19 pac-like sites may synapse the homologous sequences to lead to Hp17 mutant formation, and this suggests a synapsis mechanism for control of T4 DNA maturation and concatemer processing in packaging.
Insights
Bacteriophage T4 gene 16 protein (gp16) mediates recombination between homologous sequences, forming Hp17 mutants. This process occurs in recombination-deficient bacteria and requires functional gp16 for T4 DNA maturation.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Bacteriophage T4 DNA maturation involves specific recombination events.
- Hp17 mutants arise from recombination between homologous regions in genes 16 and 19.
- Understanding the mechanism of T4 DNA processing is crucial for phage biology.
Purpose of the Study:
- To investigate the in vivo recombination mechanism between T4 phage genes 16 and 19 homology regions.
- To identify the phage proteins involved in the formation of Hp17 mutants.
- To elucidate the role of gp16 and gp17 in T4 DNA packaging and maturation.
Main Methods:
- Cloning of T4 genes 16 and 19 homology regions into plasmids.
- Polymerase chain reaction (PCR) assay to detect in vivo recombination.
- Mutational analysis of T4 genes and recombination sequences.
- Complementation studies using suppressed amber mutations.
Main Results:
- Recombination between gene 16 and 19 homology regions occurred in vivo in plasmids, forming the Hp17 sequence.
- Recombination was observed in recombination-deficient Escherichia coli.
- Functional gene 16, specifically the terminase small subunit gp16, was required for recombination.
- Mutations in the gene 16 recombination site affected gp17 synthesis, suggesting gp16-site interaction.
Conclusions:
- Bacteriophage T4 gp16 plays a critical role in mediating recombination between homologous sequences, leading to Hp17 mutant formation.
- gp16 likely functions by binding to specific sites (pac-like) within genes 16 and 19, facilitating synapsis of homologous regions.
- This mechanism suggests a novel pathway for controlling T4 DNA maturation and concatemer processing during packaging.