Size fractionation of double-stranded DNA by precipitation with polyethylene glycol

Lis, John T.; Schleif, Robert

doi:10.1093/nar/2.3.383

Cited by 213 publications

(128 citation statements)

References 8 publications

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“…1C). To test whether changes in replication timing coincide with nuclease sensitivity changes, chromatin from ESCs before and after differentiation to NPCs was digested with MNase and separated into more sensitive (low-molecular-mass DNA) and less sensitive (high-molecular-mass DNA) fractions by polyethylene glycol (PEG) precipitation (20). These two fractions were differentially labeled and hybridized to a mouse comparative genomic hybridization (CGH) array, and the signal ratio for each probe was plotted as a function of chromosomal position (Fig.…”

Section: Resultsmentioning

confidence: 99%

Chromatin-interaction compartment switch at developmentally regulated chromosomal domains reveals an unusual principle of chromatin folding

Takebayashi

Dileep

Ryba

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Several 400- to 800-kb murine chromosome domains switch from early to late replication during loss of pluripotency, accompanied by a stable form of gene silencing that is resistant to reprogramming. We found that, whereas enhanced nuclease accessibility correlated with early replication genome-wide, domains that switch replication timing during differentiation were exceptionally inaccessible even when early-replicating. Nonetheless, two domains studied in detail exhibited substantial changes in transcriptional activity and higher-order chromatin unfolding confined to the region of replication timing change. Chromosome conformation capture (4C) data revealed that in the unfolded state in embryonic stem cells, these domains interacted preferentially with the early-replicating chromatin compartment, rarely interacting even with flanking late-replicating domains, whereas after differentiation, these same domains preferentially associated with late-replicating chromatin, including flanking domains. In both configurations they retained local boundaries of self-interaction, supporting the replication domain model of replication-timing regulation. Our results reveal a principle of developmentally regulated, large-scale chromosome folding involving a subnuclear compartment switch of inaccessible chromatin. This unusual level of regulation may underlie resistance to reprogramming in replication-timing switch regions.

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Section: Resultsmentioning

confidence: 99%

Chromatin-interaction compartment switch at developmentally regulated chromosomal domains reveals an unusual principle of chromatin folding

Takebayashi

Dileep

Ryba

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…This could be predicted especially for formulations containing Tween and TX whereby both surfactants have long hydrophilic polyoxyethylene side chains. This favourable interaction could be similar to the interaction between polyethylene glycol (PEG) and DNA, which results in precipitating of DNA molecules [21]. Microspheres prepared with Span alone (F2) did not show similar preservation efficiency.…”

Section: Discussionmentioning

confidence: 97%

Effect of Surfactants on Plasmid DNA Stability and Release from Poly (D,L-lactide-co-glycolide) Microspheres

Doolaanea¹,

Ismail²,

Mansor³

et al. 2015

Trop. J. Pharm Res

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“…quence determination methodology of Maxam and Gilbert (14) with the DNase I protection method of Galas and Schmitz (15) to determine the (8) by EcoRI endonuclease digestion and polyethylene glycol precipitation (16). CRP was purified as described (12), and araC protein was purified as described (11), with the substitution of a hydroxyapatite column for the DEAE-Sephadex column.…”

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confidence: 99%

The Escherichia coli L-arabinose operon: binding sites of the regulatory proteins and a mechanism of positive and negative regulation.

Ogden¹,

Haggerty²,

Stoner³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

168

123

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The locations of DNA binding by the proteins involved with positive and negative regulation of transcription initiation of the L-arabinose operon in Escherichia coli have been determined by the DNase I protection method. Two cyclic AMP receptor protein sites were found, at positions -78 to -107 and -121 to -146, an araCprotein-arabinose binding site was found at position -40 to -78, and an araC protein-fucose binding site was found at position -106 to -144. These locations, combined with in vivo data on induction of the two divergently oriented arabinose promoters, suggest the following regulatory mechanism: induction of the araBAD operon occurs when cyclic AMP receptor protein, araC protein, and RNA polymerase are all present and able to bind to DNA. Negative regulation is accomplished by the repressing form of araC protein binding to a site in the regulatory region such that it simultaneously blocks access of cyclic AMP receptor protein to two sites on the DNA, one site of which serves each of the two promoters. Thus, from a single operator site, the negative regulator represses the two outwardly oriented ara promoters. This regulatory mechanism explains the known positive and negative regulatory properties of the ara promoters.Studies on the L-arabinose operon of Escherichia coli have established the following important facts on regulation of the divergently oriented ara promoters PC and PBAD (see Fig. 1).The activity of both promoters is stimulated by the cyclic AMP (cAMP) receptor protein (CRP) in the presence of cyclic AMP (1-4). The promoter PBAD is positively regulated by araC protein in the presence of arabinose-i.e., the protein is required for activity of the promoter (1, 2, 5). Under noninducing conditions, the araC protein instead acts negatively to repress both the PC and PBAD promoters (1-3, 6). At least part of the DNA site necessary for repression of PBAD lies upstream from all of the sites necessary for induction of PBAD, as shown by the existence of deletions entering the ara regulatory region from the Pc side that abolish repression of PBAD without affecting induction of PBAD (6, 7).The requisite components are now available for in vitro studies of the mechanism of regulation of the ara operon. The regulatory region DNA has been isolated, and its nucleotide sequence has been determined (8,9) and found to contain elements similar to the RNA polymerase-binding sites seen in other E. coli promoters at about 10 and 35 bases before the start sites of transcription (8). The sequence also contains several stretches that resemble the CRP-binding site in the gal operon (10). Also available are the proteins involved in the regulation of the ara operon: araC protein (11), CRP (12), and RNA polymerase (13).In the work reported here, we have utilized the DNA se- MATERIALS AND METHODS DNA fragments for sequence determination and protection were obtained from plasmid pMB9ara440 (8) by EcoRI endonuclease digestion and polyethylene glycol precipitation (16). CRP was purified as described (12), and ara...

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Size fractionation of double-stranded DNA by precipitation with polyethylene glycol

Cited by 213 publications

References 8 publications

Chromatin-interaction compartment switch at developmentally regulated chromosomal domains reveals an unusual principle of chromatin folding

Chromatin-interaction compartment switch at developmentally regulated chromosomal domains reveals an unusual principle of chromatin folding

Effect of Surfactants on Plasmid DNA Stability and Release from Poly (D,L-lactide-co-glycolide) Microspheres

The Escherichia coli L-arabinose operon: binding sites of the regulatory proteins and a mechanism of positive and negative regulation.

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