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Heterologous recombination in the double-stranded RNA bacteriophage phi 6
1Department of Microbiology, Public Health Research Institute, New York, New York 10016.
Abstract:
Bacteriophage phi 6 contains three double-stranded RNA genomic segments. We have constructed a virus with an insertion of a kanamycin resistance gene in genomic RNA segment M. The virus forms small, turbid plaques, and its genome is unstable. Virus from a single plaque contained from about 0.1 to 10% large clear-plaque forms of the virus; these were usually missing the kanamycin resistance gene, and in many cases, the resulting segment M was larger or smaller than its normal size. Sequence analysis of the genomic RNA of the apparent deletions showed that they were formed by recombination events between segment M and either segment S or L. These heterologous recombination events resulted in the loss of the kanamycin resistance gene from segment M and the replacement of the 3' end of segment M with the 3' end of segment S or L. Although the 3' ends of the single-stranded RNA transcripts of the genomic segments appear to have extensive secondary structure, the sequences at the 3' ends are not involved in the specificity of genomic packaging.
Insights
Researchers modified bacteriophage phi 6 by inserting a kanamycin resistance gene into its M segment. This genetic modification led to genome instability and gene loss through recombination with other segments.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Bacteriophage phi 6 possesses a genome composed of three double-stranded RNA segments.
- Genetic manipulation of viral genomes is crucial for understanding gene function and viral evolution.
Purpose of the Study:
- To investigate the stability of the bacteriophage phi 6 genome after insertion of a foreign gene.
- To analyze the mechanisms underlying genomic instability and gene loss in bacteriophages.
Main Methods:
- Construction of a recombinant bacteriophage phi 6 with a kanamycin resistance gene inserted into genomic segment M.
- Analysis of plaque morphology and genome composition of progeny viruses.
- Sequence analysis of genomic RNA to identify recombination events.
Main Results:
- The engineered virus exhibited genome instability, producing clear-plaque variants lacking the kanamycin resistance gene.
- Sequence analysis revealed that these variants arose from heterologous recombination between segment M and segments S or L.
- Recombination resulted in the loss of the inserted gene and altered segment M size, with replacement of its 3' end by sequences from other segments.
Conclusions:
- Heterologous recombination is a significant factor in the instability of the bacteriophage phi 6 genome.
- The 3' end sequences of genomic segments, despite their secondary structures, are not solely responsible for genomic packaging specificity.
- Viral genome engineering can reveal fundamental mechanisms of viral genetic dynamics.