Spectrofluorometric Measurement of the Binding of Ethidium to Superhelical DNA from Cell Nuclei

Cook, Peter R.; Brazell, I.A.

doi:10.1111/j.1432-1033.1978.tb12188.x

Cited by 108 publications

(47 citation statements)

References 47 publications

(46 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…In fact, gamma rays reverse the DNAse I sensitivity of p-globin gene in chicken erythrocytes (35). In HeLa nucleoids, gamma rays increased the ethidium binding capacity of irradiated versus unirradiated DNA, through the release of supercoils (6). In HeLa nuclei stained with ethidium bromide, an increase in fluorescence intensity could be observed only after gamma irradiation with a very high dose (271, suggesting that a great number of nicks, about one every 200 base pairs, were necessary for the stainability changes to occur.…”

Section: Discussionmentioning

confidence: 99%

“…Although a direct comparison cannot be made between eukaryotic chromatin and viral DNA, their similarity in supercoiling was already shown by the thermodynamic properties of nicked versus intact DNA. Accordingly, differences in sedimentation velocity, as well as in ethidium bromide binding, were reported between the supercoiled and the relaxed form of DNA in HeLa nucleoids (5,6) and in the Drosophila genome prepared in 0.9 M NaCl (3). Our results have been obtained in nuclei retaining the whole complement of histones (3,131, suggesting that DNA relaxation, as assessed by stainability properties, occurs in nucleosome-free regions, consistently with the evidence that DNAse-hypersensitive sites are devoid of histones (14).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Nuclease‐induced DNA structural changes assessed by flow cytometry with the intercalating dye propidium iodide

1991

View full text Add to dashboard Cite

A flow cytometric analysis of DNA structural changes induced by cleavage with nucleases was performed on isolated HeLa nuclei by assessing changes in stainability with the DNA-specific fluorochrome propidium iodide (PI). After mild digestion with DNAse I, micrococcal nuclease, or with the single-strandspecific S1 and Neurospora crassa nucleases, fluorescence intensity of nuclei stained with PI increased by about 15-30% above the value of undigested control samples. No significant modifications were observed with the restriction enymzes Eco RI, Alu I, and Not I. The DNAse I-induced increase in fluorescence intensity was also observed with the non-intercalating dye Hoechst 33258, but not with mithramycin. Nucleaseinduced fluorescence intensity changes as determined with PI were found to be dependent on the dye concentration. A constant increase (about 20%) was measured at dye/DNA-P ratios > 0.11. Below this value (2 pg/ml PI), the fluorescence intensity of digested samples was 1530% lower than that of undigested controls. This behaviour towards intercalating dyes is similar to that of the relaxed (nicked) vs. the supercoiled (intact) form of circular DNA. These results suggest that conformation-but not sequence-specific nucleases induce a relaxation of DNA supercoils.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Nuclease‐induced DNA structural changes assessed by flow cytometry with the intercalating dye propidium iodide

1991

View full text Add to dashboard Cite

show abstract

“…2b, c) [46,47], suggesting that this linear DNA is looped and attached. Supercoiling in the loop stabilises any righthanded interwound superhelix at its base [43]. Supercoils arise because dissociation of nucleosome cores in 2 M NaCl leaves their imprints in DNA if it is looped [48,491.…”

Section: Eurly Evidencementioning

confidence: 99%

“…Supercoiled DNA has distinctive properties [36]; these are shared by 'nucleoids' isolated by stripping off histones with 2 M NaCl [37]. Both sediment biphasically in gradients containing intercalators [37 -421 and they bind ethidium [43], denature [43] and change shape [44, 451 similarly. Electron microscopy of spread nucleoids reveals supercoiled fibres attached to a collapsed nuclear 'cage' (Fig.…”

Section: Eurly Evidencementioning

confidence: 99%

The nucleoskeleton and the topology of transcription

Cook

1989

European Journal of Biochemistry

Self Cite

108

View full text Add to dashboard Cite

Transcription is conventionally believed to occur by passage of a mobile polymerase along a fixed template. Evidence for this model is derived almost entirely from material prepared using hypotonic salt concentrations. Studies on subnuclear structures isolated using hypertonic conditions, and more recently using conditions closer to the physiological, suggest an alternative. Transcription occurs as the template moves past a polymerase attached to a nucleoskeleton; this skeleton is the active site of transcription. Evidence for the two models is summarised. Much of it is consistent with the polymerase being attached and not freely diffusible. Some consequences of such a model are discussed.The accepted model for transcription contains three essential participants -the template, polymerase and nascent RNA [I, 21. Transcription is initiated by a diffusible polymerase binding to a promoter; the polymerase then processes along the DNA (Fig. 1 A). As knowledge has increased, other occasional participants have been added (e. g. transcription factors, topoisomerases) but not any other central players. Evidence for this model comes almost entirely from studies using hypotonic salt concentrations, largely because chromatin aggregates in physiological concentrations and so becomes difficult to use. In contrast, studies on preparations isolated using hyper-or iso-tonic salt concentrations suggest that active RNA polymerase is associated with a nucleoskeleton and is not freely diffusible. This nucleoskeleton is seen as the active site of RNA synthesis and transcription occurs by movement of DNA past the attachcd polymerase (Fig. 1 B).Ultimately models will be distinguished by reconstructing efficient transcription in vitro from purified constituents. Proof of the model involving an attached polymerase will require measurement ofits association constant for a skeleton. In the meantime, the two models can be distinguished operationally by determining whether the polymerase is freely diffusible or attached to a larger structure. Much evidence for the 'text-book' model is consistent with the alternative; this alternative has a number of important consequences and these are examined. Discussion centrcs on RNA polymerase 11, the enzyme that transcribes most eukaryotic genes, but applies equally to other polymerases. ARTEFACTSStructure within isolated nuclei cannot be discussed sensibly without some consideration of problems caused by artefacts. They arise because nuclei and chromatin aggregate in physiological salt concentrations 131 ; therefore unphysiological conditions are almost invariably used. ControversyCorrespondence to P. R. Cook, Sir William Dunn School of Pathology, Universlty of Oxford, South Parks Road, Oxford OX1 3RE. UK centres on whether structures seen In vitro are any more than isolation artefacts. RNA, DNA and protein, each concentrated in the nucleus at about 0.1 g/ml, might be expected to aggregate as soon as ion concentrations are altered. (For reviews of the controversy, see [4-61.) Nuclei are usually isol...

show abstract

“…Results from a number of laboratories suggestthat the chromatin fiber of interphase chromosomes is also topologically constrained in loops (16)(17)(18)(19)(20)(21)(22). ILcorre~t, this model implies that DNA topoisomerases might be required for the termination of replication.…”

mentioning

confidence: 99%

Topoisomerase II is a structural component of mitotic chromosome scaffolds.

Earnshaw¹,

Halligan²,

Cooke³

et al. 1985

The Journal of Cell Biology

709

286

View full text Add to dashboard Cite

We have obtained a polyclonal antibody that recognizes a major polypeptide component of chicken mitotic chromosome scaffolds. This polypeptide migrates in SDS PAGE with Mr 170,000. Indirect immunofluorescence and subcellular fractionation experiments confirm that it is present in both mitotic chromosomes and interphase nuclei. Two lines of evidence suggest that this protein is DNA topoisomerase II, an abundant nuclear enzyme that controls DNA topological states: anti-scaffold antibody inhibits the strand-passing activity of DNA topoisomerase II; and both anti-scaffold antibody and an independent antibody raised against purified bovine topoisomerase II recognize identical partial proteolysis fragments of the 170,000-mol-wt scaffold protein in immunoblots. Our results suggest that topoisomerase II may be an enzyme that is also a structural protein of interphase nuclei and mitotic chromosomes.It has been known for many years that the chromatin fiber of both meiotic (1-4) and mitotic (5) chromosomes is folded into loops. 1978, Laemmli and co-workers (6-8) proposed that in mitotic chromosomes these loops are formed as a result of interactions between the nucleohistone fiber and a subgroup of chromosomal nonhistone proteins. A residual structure thought to be enriched in these nonhistone proteins was isolated by extraction of nuclease-digested chromosomes under conditions that solubilized >90% of the chromosomal proteins, including all of the histones (9). Because it retained the size and approximate shape of the mitotic chromosome, this residual structure, was termed the "chromosome scaffold." More recent studies have shown that the scaffold fraction is greatly enriched in two high molecular weight polypeptides, Sc-I (170,000 mol wt) and Sc-2 (135,000 mol wt; see reference 10).The existence of a discrete scaffold substructure in intact mitotic chromosomes remains a subject of some debate. The isolated residual scaffold comprises only 3-4% of the chromosome mass (10). Therefore, it is not surprising that direct inspection of whole mounts (11, 12) or thin sections (13) of intact chromosomes does not permit ready visualization of a scaffold substructure, although under certain conditions axial structures can be seen (14). Localization of scaffold components in intact chromosomes has been forced to wait for the availability of anti-scaffold antibodies (15). 1706Results from a number of laboratories suggestthat the chromatin fiber of interphase chromosomes is also topologically constrained in loops (16)(17)(18)(19)(20)(21)(22). ILcorre~t, this model implies that DNA topoisomerases might be required for the termination of replication. For example, the final stages of SV40 replication involve production of catenated dimers (23,24). In the absence of nicks, such strucures can only be separated by a type II DNA topoisomerase. This class of enzyme changes the linking number of circular DNA molecules in steps of two, in an ATP-dependent reaction that proceeds via production of protein-linked double strand DNA breaks ...

show abstract

Spectrofluorometric Measurement of the Binding of Ethidium to Superhelical DNA from Cell Nuclei

Cited by 108 publications

References 47 publications

Nuclease‐induced DNA structural changes assessed by flow cytometry with the intercalating dye propidium iodide

Nuclease‐induced DNA structural changes assessed by flow cytometry with the intercalating dye propidium iodide

The nucleoskeleton and the topology of transcription

Topoisomerase II is a structural component of mitotic chromosome scaffolds.

Contact Info

Product

Resources

About