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Physical and biological properties of phage phi 29 deoxyribonucleic acid
Abstract:
Deoxyribonucleic acid (DNA) molecules having a mean length of 5.8 mum were released from purified Bacillus subtilis bacteriophage phi29 with 2 m sodium perchlorate. Small 0.1 to 0.2-mum molecules were also detected in these DNA preparations. Since intact single chains annealed to form linear duplex molecules, phage phi29 DNA was found to be nonpermuted. The molecular weights of single chains of phi29 DNA were approximately half that of native DNA, as determined by analytical band sedimentation in CsCl, indicating that phi29 DNA is composed of two continuous polynucleotide chains. The molecular weight values of native and annealed phi29 DNA from sedimentation agreed with the molecular weight values obtained from electron microscopy. The infectivity of phi29 DNA was reduced to a low level by alkaline denaturation and was partially restored by annealing.
Insights
Bacillus subtilis bacteriophage phi29 DNA consists of two continuous polynucleotide chains. Annealing intact single chains reforms linear duplex molecules, confirming nonpermuted DNA structure and partial infectivity restoration.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Bacillus subtilis bacteriophage phi29 is a model organism for studying DNA replication and packaging.
- Understanding the structure and properties of bacteriophage DNA is crucial for genetic engineering and therapeutic applications.
Purpose of the Study:
- To characterize the physical and chemical properties of purified Bacillus subtilis bacteriophage phi29 DNA.
- To determine the structure (linear vs. circular, permuted vs. nonpermuted) and molecular weight of phi29 DNA.
- To investigate the infectivity of phi29 DNA and the effect of denaturation and annealing.
Main Methods:
- Release of DNA from purified bacteriophage phi29 using 2 M sodium perchlorate.
- Electron microscopy for determining DNA length and molecular weight.
- Analytical band sedimentation in Cesium Chloride (CsCl) for molecular weight determination.
- Alkaline denaturation and annealing experiments to assess DNA integrity and infectivity.
Main Results:
- Deoxyribonucleic acid (DNA) molecules with a mean length of 5.8 µm were isolated, alongside smaller 0.1-0.2 µm fragments.
- Intact single DNA chains annealed to form linear duplex molecules, indicating nonpermuted DNA.
- Sedimentation analysis revealed that single chains of phi29 DNA have approximately half the molecular weight of native DNA, confirming a two-continuous-chain structure.
- Molecular weights obtained from sedimentation and electron microscopy were consistent.
- Alkaline denaturation significantly reduced phi29 DNA infectivity, which was partially restored upon annealing.
Conclusions:
- Bacillus subtilis bacteriophage phi29 DNA is a linear, nonpermuted molecule composed of two continuous polynucleotide chains.
- The DNA structure is stable under specific conditions, with infectivity dependent on its duplex form.
- Annealing of denatured DNA can restore partial infectivity, highlighting the importance of DNA integrity.