A Technique for the Analysis of Non-Replicating and Replicating Forms of R6K Plasmid DNA in Escherichia Coli: Relationship to the Folded Chromosome

Sheehy, Ronald J.; Clark, C. W.; Archibold, Errol R.

doi:10.1007/978-3-642-81064-0_32

Cited by 2 publications

(9 citation statements)

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“…1B are the results obtained when similar samples of 30-s pulsed-labeled cells were sedimented in CLOS gradients in the absence of an intermediate layer of RNase. As can be seen, there was a significant reduction in the recovery of 58S monomer molecules, when compared with the RNase layered gradients, to ap- We have previously presented data showing the results of CsCl-EtBr analysis of folded chromosomal material found at the bottom of CLOS gradients (16,17) as depicted in Fig. 1A and B.…”

Section: Resultsmentioning

confidence: 71%

“…1A, where cells were sedimented through a layer of RNase, the 3H pulse-label released on the gradient sedimented as a broad peak with a mean sedimentation coefficient of 79S. We have previously shown that these rapidly labeled molecules are replicative intermediates (17). A comparison of the 3H and 14C counts recovered in plaid DNA (Fig.…”

Section: Resultsmentioning

confidence: 88%

“…In both parts A and B of Fig. 1, the 14C label in plasmid DNA was present predominantly as 58S circular duplex monomers (16,17) and served as an internal sedimentation marker. To compare recoveries of the pulse and long-term counts in plasmid DNA, both radioactivities were nornalized by reference to their incorporation into chromosomal DNA.…”

Section: Resultsmentioning

confidence: 99%

“…The synthetic medium used was a minimal salts solution (6) supplemented with 0.5% Casamino Acids, 0.5% glucose, and 2 /g of thiamine hydrochloride per ml. Thymidine at 20 jsg/ml was included in the synthetic medium for nonradioactive experiments and at 4 ug/ml where radioactive thymidine crose" (CLOS) gradients was essentially as described by Sheehy et al (16,17). For pulse-labeling experiments, the cells from overnight growth were diluted 1:100 into 10 to 25 ml of the supplemented synthetic medium.…”

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confidence: 99%

“…Immediately thereafter, the gradients were centrifuged at 50C in Spinco SW50.1 (5-ml capacity) swinging bucket rotors in Beckman L5-50 or L5-65 ultracentrifuges. The gradients were collected on strips or in trays and handled as indicated by Sheehy et al (16,17).…”

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confidence: 99%

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Relationship of R6K replicating forms to the folded chromosome of Escherichia coli

Archibold¹,

Clark²,

Sheehy³

1978

J Bacteriol

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An examination of the relationship of both nonreplicating and replicating forms of R6K plasmid DNA to the Escherichia coli folded chromosome showed that both forms cosediment with the chromosome in neutral sucrose gradients. Approximately 20% of the nonreplicating molecules was found as freely sedimenting forms when the folded configuration of the chromosomes was preserved. However, under the same conditions negligible amounts of the replicating forms were found as freely sedimenting molecules. Thus, it is concluded that the replicating forms, when compared with nonreplicating molecules, are preferentially associated with the folded chromosomal structure. Exposure of the folded chromosomal structure to RNase resulted in an unfolding of the chromosome and a concomitant increase in the amount of freely sedimenting replicating and nonreplicating forms of R6K DNA. Analyses of the single-stranded length of RNase-released nascent molecules suggest that they replicate in continuous association with the folded chromosome complex. Nonenzymatic unfolding of the chromosomes by progressively lowering the sodium ion concentration during lysis resulted in a progressive increase in the release of nonreplicating molecules. Replicating molecules were not released by unfolding the chromosome in this fashion. was used. Streptomycin (200,ug/ml) and ampicillin (40 ytg/ml) were used to select for cells harboring R6K.

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Section: Resultsmentioning

confidence: 71%

Section: Resultsmentioning

confidence: 88%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Relationship of R6K replicating forms to the folded chromosome of Escherichia coli

Archibold¹,

Clark²,

Sheehy³

1978

J Bacteriol

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Membrane-bound fractions of R6K plasmid DNA in Escherichia coli

Archibold¹,

Wilson²,

Allison³

et al. 1983

J Bacteriol

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The intracellular location of plasmid DNA has been of interest in an effort to understand the maintenance of these molecules. We have employed a simple procedure which enables us to isolate from exponentially grown cells on sucrose gradients membrane-complexed forms of R6K plasmid DNA. Electron micrographs identified the complexing of membrane fractions to circular forms of R6K DNA. Biochemical studies of the complexed R6K molecules showed the presence of membrane-specific proteins and suggested that complexing of R6K DNA was primarily with inner membrane fractions of Escherichia coli.

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A Technique for the Analysis of Non-Replicating and Replicating Forms of R6K Plasmid DNA in Escherichia Coli: Relationship to the Folded Chromosome

Cited by 2 publications

References 14 publications

Relationship of R6K replicating forms to the folded chromosome of Escherichia coli

Relationship of R6K replicating forms to the folded chromosome of Escherichia coli

Membrane-bound fractions of R6K plasmid DNA in Escherichia coli

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