Multiple Restraints to the Unfolding of Spermidine Nucleoids from Escherichia coli

Murphy, Lizabeth D.; Zimmerman, Steven B.

doi:10.1006/jsbi.2000.4306

Cited by 20 publications

(11 citation statements)

References 66 publications

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“…40,41 There appear to be several factors that influence the nucleoid compaction, including supercoiling of the DNA, macromolecular crowding, RNAs, cellular polyamines, and histone-like proteins. 42 DNA looping could be the lowest level in bacterial nucleoid organizational hierarchy, playing a structural role similar to that of nucleosome in eukaryotes.…”

Section: The "Flip-over" Problemmentioning

confidence: 99%

“Antiparallel” DNA Loop in Gal Repressosome Visualized by Atomic Force Microscopy

Virnik

Lyubchenko

Karymov

et al. 2003

Journal of Molecular Biology

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Section: The "Flip-over" Problemmentioning

confidence: 99%

“Antiparallel” DNA Loop in Gal Repressosome Visualized by Atomic Force Microscopy

Virnik

Lyubchenko

Karymov

et al. 2003

Journal of Molecular Biology

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“…Since the introduction of the first protocol by Stonington and Pettijohn (1971), detergents (non-ionic Brij-58 and anionic deoxycholate) at a high salt concentration (1 M NaCl) or spermidine have been used in most studies Burgi, 1972, 1974;Kornberg et al, 1974;Drlica and Worcel, 1975;Meijer et al, 1976;Materman and van Gool, 1978;Murphy and Zimmerman, 2000;Foley et al, 2010). Depending on the lysis temperature, the detergent-salt method produced membrane-attached (at 10°C) or membrane-free (at 25°C) ''particles'' as analyzed by sedimentation through sucrose gradients (Worcel and Burgi, 1974) or by ''visualization'' with the electron microscope (Delius and Worcel, 1974).…”

Section: Introductionmentioning

confidence: 99%

Characterization of Escherichia coli nucleoids released by osmotic shock

Wegner

Alexeeva

Odijk

et al. 2012

Journal of Structural Biology

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“…The stable, round shape of nucleoids in chloramphenicol-treated cells (11,17,27,32,35) provides structures that can be readily identified by light microscopy. In addition, nucleoids from chloramphenicoltreated cells are unusually stable (20,21), further adding to their utility as test objects.Nalidixate treatment can cause the nucleoids within cells to form elongated, crystalline bodies (14). Nalidixate is an inhibitor of DNA gyrase (8, 29); presumably for this reason, nalidixate-treated cells often contain nucleoids which have failed to segregate properly (28).…”

mentioning

confidence: 99%

“…First, the nucleoid DNA underwent the partial decompaction that is associated with cell lysis (21). Second, large amounts of residual envelope remained with the nucleoid DNA, so that many such preparations are more accurately described as lysed or broken cell preparations than as isolated nucleoids (see footnote 6 of reference 20).…”

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Release of Compact Nucleoids with Characteristic Shapes from Escherichia coli

Zimmerman

Murphy

2001

J Bacteriol

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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.The genomic DNA of Escherichia coli is localized in one or two compact bodies, known as nucleoids, per cell (3,15,31,36,38,40). There have been many attempts to isolate representative nucleoids outside of cells (24,25). However, the various DNA-containing structures that have been isolated generally have failed to meet this goal in two important ways. First, the nucleoid DNA underwent the partial decompaction that is associated with cell lysis (21). Second, large amounts of residual envelope remained with the nucleoid DNA, so that many such preparations are more accurately described as lysed or broken cell preparations than as isolated nucleoids (see footnote 6 of reference 20).Evaluation of the released nucleoids is made difficult by a lack of information on the structure of the original nucleoids within cells. Here we use retention of the general shape of nucleoids in initial cells as a criterion for evaluation of a new procedure for nucleoid isolation. There have been few reports on the shapes of isolated nucleoids; the doublet-shaped highsalt nucleoids (nucleoids released in the presence of high salt concentrations) described by Hecht et al. (10) and Van Ness and Pettijohn (33) and the large DNA double structure found in high-salt nucleoids of a DNA gyrase mutant by Steck and Drlica (28) are the only instances of which we are aware.We have used the distinctively shaped nucleoids formed in cells that have been exposed to antibiotics (4, 5, 11, 12) as test objects. The three antibiotics used here, chloramphenicol, nalidixate, and rifampin, all promote the coalescence of nucleoids in cells and can cause changes in nucleoid morphology and stability:The nucleoid coalescence caused by chloramphenicol (17, 32) has been attributed to a loss of cotranslational insertion linkages between the DNA and the cell envelope when protein synthesis is inhibited; the los...

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Multiple Restraints to the Unfolding of Spermidine Nucleoids from Escherichia coli

Cited by 20 publications

References 66 publications

“Antiparallel” DNA Loop in Gal Repressosome Visualized by Atomic Force Microscopy

“Antiparallel” DNA Loop in Gal Repressosome Visualized by Atomic Force Microscopy

Characterization of Escherichia coli nucleoids released by osmotic shock

Release of Compact Nucleoids with Characteristic Shapes from Escherichia coli

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