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DNA of Myxococcus bacteriophage MX-1: macromolecular properties and restriction fragments
Abstract:
1. Bacteriophage MX-1 is a virulent DNA phage whose hosts include strains of Myxococcus xanthus, M. fulvus and M. virescens. DNA was extracted from purified phage preparations. The molecular weight of phage DNA was measured by sedimentation-velocity and by rate-zonal ultracentrifugation. The apparent molecular weight was found to vary for reasons discussed in the text. From rate-zonal ultracentrifugation, using calibrated sucrose gradients, the molecular weight was calculated to be 149 (+/-22) X 10(6) daltons. The base composition of the DNA was estimated by different methods and was found to be 50-52% (G + C). The DNA demonstrated an anomalous thermal denaturation profile in dilute buffer. Denatured DNA was fractionated by ion-exchange chromatography and by buoyant-density centrifugation. No significant strand separation was obained and it was concluded that overall base compositions of the two strands are very similar. 2. DNA from bacteriophage MX-1 was hydrolysed with restriction endonucleases R. EcoRI, R.EcoRII and R. HindIII. The restriction fragments were catelogued and their apparent molecular weights calculated from electrophoresis gels calibrated with fragments from the DNA of coliphage lambda. From the total fragments obtained with nuclease R.EcoRI, the minimum apparent molecular weight of MX-1 DNA was found to be 130 X 10(6) daltons.
Insights
Bacteriophage MX-1 DNA has a molecular weight of approximately 149 million daltons and a GC content of 50-52%. Restriction endonuclease analysis confirmed a minimum molecular weight of 130 million daltons.
Area of Science:
- Microbiology
- Molecular Biology
- Virology
Background:
- Bacteriophage MX-1 is a virulent DNA phage infecting Myxococcus species.
- Understanding phage DNA is crucial for microbial genetics and biotechnology.
Purpose of the Study:
- To characterize the physical and chemical properties of bacteriophage MX-1 DNA.
- To determine the molecular weight, base composition, and strand similarity of MX-1 DNA.
Main Methods:
- DNA extraction from purified phage particles.
- Molecular weight determination using sedimentation-velocity and rate-zonal ultracentrifugation.
- Base composition analysis via thermal denaturation and chromatography.
- Restriction endonuclease digestion (EcoRI, EcoRII, HindIII) and fragment analysis via gel electrophoresis.
Main Results:
- Rate-zonal ultracentrifugation yielded a molecular weight of 149 (±22) x 10^6 daltons.
- DNA base composition was estimated at 50-52% Guanine-Cytosine (G+C).
- Restriction endonuclease analysis, particularly with EcoRI, indicated a minimum molecular weight of 130 x 10^6 daltons.
- Anomalous thermal denaturation and chromatographic/centrifugation fractionation suggested similar base compositions between DNA strands.
Conclusions:
- Bacteriophage MX-1 DNA possesses a molecular weight around 149 million daltons.
- The DNA has a balanced base composition with strands of similar overall composition.
- Restriction enzyme mapping provides further validation of the DNA's molecular weight.