Isolation and characterization of DNA from the entomopathogenBeauveria bassiana |
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| Institution: | 1. Department of Plant Pathology, Nanjing Agricultural University, Nanjing, China;2. Department of Entomology, Nanjing Agricultural University, Nanjing, China;1. Institute of Sciences of Food Production (ISPA), National Research Council of Italy (CNR), Bari, Italy;2. Department of Agricultural and Environmental Science, University of Bari Aldo Moro, Bari, Italy;1. Australian Centre for Research on Separation Science, School of Chemistry, Monash University, Wellington Road, 3800 Victoria, Australia;2. School of Chemistry, Monash University, Wellington Road, 3800 Victoria, Australia;1. Department of Neuroscience, Facultad de Medicina, Universidad de Chile, Chile;2. Biomedical Neuroscience Institute (BNI), Facultad de Medicina, Universidad de Chile, Chile;1. Faculty of Biology, University of Athens, Panepistimioupolis, Athens 15784, Greece;2. Department of Microbiology, Imperial College London, London SW7 2AZ, United Kingdom;3. Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Bâtiment 400, 91405 Orsay Cedex, France |
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| Abstract: | Beauveria bassiana chromosomal (chr) and mitochondrial (mt) DNA were isolated, purified, and characterized. Modification of a recent rapid method of total DNA extraction was followed by DNA purification on CsCl-bisbenzimide gradients. Cytosine methylation at 5′-CpG-3′ doublets was undetectable in the total DNA as demonstrated byMspI andHpaII restriction enzyme digests. The chrDNA had a bouyant density of 1.7130 g/ml (G + C ratio of 53.4%) and a Tm of 92.6°C (G + C ratio of 56.9%). Mitochondrial DNA was isolated from total DNA or purified mitochondria and had a buoyant density of 1.7120 g/ml (G + C ratio of 52.3%) and a biphasic DNA melting curve. A minor melt at 76.2°C (16.8% G + C ratio) yielded a 10% increase in absorbance while the major melt at 93.1°C (58% G + C ratio) yielded a 30% increase in absorbance. The mtDNA was estimated by gel electrophoresis to be 17.6 ± 0.6 MDa and determined to be 28.5 kb by restriction enzyme analysis. Electron microscopy of the mtDNA revealed circular molecules having an average contour length of 8.84 ± 0.51 μm. A physical map of the mtDNA was generated by double restriction enzyme digestion using the restriction enzymesClaI,EcoRI,SalI,BstEII, andXhoI. |
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