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Construction of a microphage variant of filamentous bacteriophage
L Specthrie1, E Bullitt, K Horiuchi
1Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons, Columbia University, New York, NY 10032.
Abstract:
The intergenic region in the genome of the Ff class of filamentous phage (comprising strains fl, fd and M13) genome constitutes 8% of the viral genome, and has essential functions in DNA replication and phage morphogenesis. The functional domains of this region may be inserted into separate sites of a plasmid to function independently. Here, we demonstrate the construction of a plasmid containing, sequentially, the origin of (+)-strand synthesis, the packaging signal and a terminator of (+)-strand synthesis. When host cells harboring this plasmid (pLS7) are infected with helper phage they produce a microphage particle containing all the structural elements of the mature, native phage. The microphage is 65 A in diameter and about 500 A long. It contains a 221-base single-stranded circle of DNA coated by about 95 copies of the major coat protein (gene 8 protein).
Insights
Researchers engineered a plasmid to create microphage particles. These particles mimic native filamentous phage (Ff, fl, fd, M13) structure, containing essential DNA replication and morphogenesis elements.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- The intergenic region of Ff-class filamentous phage (fl, fd, M13) is crucial for DNA replication and phage morphogenesis.
- This 8% viral genome segment contains functional domains that can operate independently when placed on a plasmid.
Purpose of the Study:
- To construct a plasmid (pLS7) containing key functional domains of the Ff phage intergenic region.
- To demonstrate the production of functional microphage particles from host cells harboring the engineered plasmid.
Main Methods:
- Sequential insertion of the origin of (+)-strand synthesis, packaging signal, and terminator into a plasmid.
- Infection of host cells containing the engineered plasmid (pLS7) with a helper phage.
Main Results:
- Successful construction of plasmid pLS7 with sequential functional domains.
- Production of microphage particles upon helper phage infection of pLS7-harboring cells.
- Characterization of the microphage: 65 Å diameter, 500 Å length, containing a 221-base single-stranded DNA circle coated by major coat protein (gene 8).
Conclusions:
- The engineered plasmid successfully recapitulates essential Ff phage intergenic region functions.
- This demonstrates the feasibility of creating synthetic microphages with native structural components.
- The study provides insights into phage assembly and DNA replication mechanisms.