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RNA structure and heterologous recombination in the double-stranded RNA bacteriophage phi 6
S Onodera1, X Qiao, P Gottlieb
1Department of Microbiology, Public Health Research Institute, New York, New York 10016.
Abstract:
Bacteriophage phi 6 has a genome of three segments of double-stranded RNA, designated L, M, and S. A 1.2-kbp kanamycin resistance gene was inserted into segment M but was shown to be genetically unstable because of a high recombination rate between segment M and the 3' ends of segments S and L. The high rate of recombination is due to complementary homopolymer tracts bounding the kan gene. Removal of one arm of this potential hairpin stabilizes the insertion. The insertion of a 241- or 427-bp lacZ' gene into segment M leads to a stable Lac+ phage. The insertion of the same genes bounded by complementary homopolymer arms leads to recombinational instability. A stable derivative of this phage was shown to have lost one of the homopolymer arms. Several other conditions foster recombination. The truncation of a genomic segment at the 3' end prevents replication, but such a damaged molecule can be rescued by recombination. Similarly, insertion of the entire 3-kb lacZ gene prevents normal formation of virus, but the viral genes can be rescued by recombination. It appears that conditions leading to the retardation or absence of replication of a particular genomic segment facilitate recombinational rescue.
Insights
Genetic instability in bacteriophage phi 6 is caused by complementary homopolymer tracts. Removing these tracts or truncating genomic segments stabilizes insertions and facilitates recombinational rescue.
Area of Science:
- Molecular biology
- Virology
- Genetics
Background:
- Bacteriophage phi 6 possesses a three-segmented double-stranded RNA genome (L, M, S).
- Genetic insertions into the M segment can exhibit instability due to high recombination rates.
- Complementary homopolymer tracts flanking inserted genes are implicated in this instability.
Purpose of the Study:
- To investigate the mechanisms underlying genetic instability in bacteriophage phi 6.
- To determine the role of homopolymer tracts in recombination rates.
- To identify conditions that promote or stabilize genetic insertions.
Main Methods:
- Insertion of kanamycin resistance (kan) and lacZ' genes into the M segment of bacteriophage phi 6.
- Manipulation of flanking homopolymer tracts to assess their effect on genetic stability.
- Analysis of phage replication and recombination rates under various conditions.
Main Results:
- Insertion of the kan gene was unstable due to flanking homopolymer tracts, but stabilization occurred upon removal of one tract.
- Insertion of lacZ' genes resulted in stable Lac+ phages, with instability arising from complementary homopolymer arms.
- Truncation of genomic segments or insertion of large genes leading to replication defects facilitated recombinational rescue.
Conclusions:
- Complementary homopolymer tracts are key drivers of recombinational instability in bacteriophage phi 6.
- Genetic engineering strategies can stabilize insertions by modifying these tracts.
- Recombination serves as a rescue mechanism for replication-deficient or damaged viral genomic segments.