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Double-length, circular, single-stranded DNA from filamentous phage
Abstract:
Wild-type and gene 3 mutant filamentous phage stocks, containing different relative amounts of multiple-length particles, were treated exhaustively with DNase and then were highly purified. The phage DNA was extracted and examined by electron microscopy. In all cases, about 0.03% of the molecules were circular dimers. (3)H-labeled phage DNA was separated as to size by sedimentation in a preformed CsCl density gradient. Individual fractions were then examined in the electron microscope, and the percentage of linear and circular monomer and dimer DNAs was determined. A peak of double-length, circular molecules (with the expected sedimentation constant of 38S) was found ahead of the 24S monomer peak. The double-length molecules had been purified 65-fold. As previously found for single-stranded DNA, the contour length of these molecules was strongly dependent upon ionic strength. Possible artifacts were ruled out, and it was shown that the double-length molecules arose from phage particles.
Insights
Researchers found double-length circular DNA molecules in filamentous phage particles. These molecules, representing about 0.03% of the total DNA, were purified and characterized, confirming their origin from the phage.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Filamentous phage are single-stranded DNA viruses.
- Previous studies have investigated the structure and DNA content of these phages.
Purpose of the Study:
- To investigate the presence and characteristics of double-length DNA molecules in filamentous phage.
- To determine the origin and properties of these double-length DNA molecules.
Main Methods:
- Purification of wild-type and mutant filamentous phage stocks.
- DNase treatment and exhaustive purification of phage particles.
- DNA extraction, followed by electron microscopy and size separation using CsCl density gradients.
- Characterization of DNA molecules by sedimentation analysis and contour length measurements.
Main Results:
- Approximately 0.03% of purified phage DNA molecules were identified as circular dimers.
- A distinct peak of double-length, circular DNA molecules (38S) was observed, sedimenting ahead of the monomer peak (24S).
- These double-length molecules were purified 65-fold and their contour length was found to be dependent on ionic strength.
- Artifacts were ruled out, confirming that these double-length molecules originate from phage particles.
Conclusions:
- Filamentous phage particles contain a small but significant population of double-length circular DNA molecules.
- These molecules are a genuine component of the phage, not an artifact of the purification process.
- The findings contribute to understanding the structural diversity and DNA packaging mechanisms within filamentous phage.