Bacterial chromosome is assembled and folded into one or several nucleoids, depending on the metabolic status of the cell. Development of reliable nucleoid isolation protocols has always been an objective for researchers. A rapid and reproducible procedure for isolation of E. coli nucleoids is described here, while the cell envelope is maintained. Membrane dispersions and vesicles were prepared by lysozyme-EDTA treatment with subsequent rupture of the spheroplasts by electric field. Under these conditions the yield of electroreleased nucleoids was around 90%. The extent of DNA-envelope contacts was determined by light microscopy employing phase contrast and fluorescence modes.

• MeSH
• buněčné jádro ultrastruktura   MeSH
• EDTA MeSH
• elektřina MeSH
• Escherichia coli ultrastruktura   MeSH
• frakcionace buněk metody   MeSH
• muramidasa MeSH
• organely ultrastruktura   MeSH
• sféroplasty ultrastruktura   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• EDTA MeSH
• muramidasa MeSH

Minicells of Escherichia coli P678-54 containing plasmid RIdrd19 were submitted to careful controlled lysis. By sedimentation of the resulting lyzate in a sucrose gradient, the material absorbing at 260 nm was separated into three distinct bands. Among the most rapidly sedimenting particles, double-stranded topological circles of DNA attached to patches of membrane were visualized by electron microscopy, while single-stranded molecules (probably RNA) with associated proteins were detected in the medium band. Covalently closed and open circles of the RIdrd19 DNA were found at the top of the gradient. Their contour lengths correspond to the size of the DNA sedimenting together with the membrane in the first peak. This finding implies a direct intracellular interaction between RIdrd19 DNA and membrane in E. coli minicelle.

• MeSH
• bakteriolýza MeSH
• buněčná membrána analýza   ultrastruktura   MeSH
• centrifugace - gradient hustoty MeSH
• DNA bakterií analýza   MeSH
• elektronová mikroskopie MeSH
• Escherichia coli analýza   ultrastruktura   MeSH
• kruhová DNA analýza   MeSH
• R-plasmidy * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA bakterií MeSH
• kruhová DNA MeSH