Release of compact nucleoids with characteristic shapes from Escherichia coli.
The genomic DNA of bacteria is contained in one or a few compact bodies known as nucleoids. We describe a simple procedure that retains the general shape and compaction of nucleoids from Escherichia coli upon cell lysis and nucleoid release from the cell envelope. The procedure is a modification of that used for the preparation of spermidine nucleoids (nucleoids released in the presence of spermidine) (T. Kornberg, A. Lockwood, and A. Worcel, Proc. Natl. Acad. Sci. USA 71:3189--3193, 1974). Polylysine is added to prevent the normal decompaction of nucleoids which occurs upon cell lysis. Nucleoids retained their characteristic shapes in lysates of exponential-phase cells or in lysates of cells treated with chloramphenicol or nalidixate to alter nucleoid morphology. The notably unstable nucleoids of rifampin-treated cells were obtained in compact, stable form in such lysates. Nucleoids released in the presence of polylysine were easily processed and provided well-defined DNA fluorescence and phase-contrast images. Uniform populations of nucleoids retaining characteristic shapes could be isolated after formaldehyde fixation and heating with sodium dodecyl sulfate.
Zimmerman
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SB
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0560, USA. stevenz@bdg5.niddk.nih.gov
Murphy
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LD
D004269:DNA, Bacterial / Q000187:drug effects / Q000648:ultrastructure*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000648:ultrastructure; D005557:Formaldehyde; D007202:Indicators and Reagents; D008854:Microscopy, Electron; D009113:Muramidase / Q000378:metabolism; D009268:Nalidixic Acid / Q000494:pharmacology; D009690:Nucleic Acid Conformation; D011107:Polylysine; D012293:Rifampin / Q000494:pharmacology; D012967:Sodium Dodecyl Sulfate; D013095:Spermidine
D004269:DNA, Bacterial; D007202:Indicators and Reagents; D005557:Formaldehyde; D011107:Polylysine; D012967:Sodium Dodecyl Sulfate; D009268:Nalidixic Acid; D009113:Muramidase; D013095:Spermidine; D012293:Rifampin