On the Inheritance of Differentiated Traits

Cook, Peter

doi:10.1111/j.1469-185x.1974.tb01298.x

Cited by 38 publications

(16 citation statements)

References 142 publications

(109 reference statements)

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“…Their concentrations in the three viral fragments in Table II (i.e., 2:1:0) reflect the enrichments of the fragments in the pellet. Alternatively, the virus might induce an adjacent cellular sequence tc attach, perhaps by some conformational re-arrangement (Cook, 1974). Such an attachment, close to, but not within, the viral sequence is consistent with the enrichments found and the presence of viral sequences in the supernatant.…”

Section: Discussionsupporting

confidence: 83%

Active viral genes in transformed cells lie close to the nuclear cage.

Cook¹,

Lang²,

Hayday³

et al. 1982

The EMBO Journal

108

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Nuclear DNA is looped by attachment to a matrix or cage. Using nine different lines transformed by polyoma or avian sarcoma virus, we have mapped viral sequences integrated within these loops. In all lines that contain high concentrations of viral transcripts and express the transformed phenotype, the integrated viral genes lie close to the points of attachment to the cage. Integration of polyoma DNA induces outlying cellular sequences to become closely associated with the cage. The strength of this correlation between gene activity and proximity to the cage was examined using sub‐clones of one avian sarcoma virus transformant. Proviral sequences are closely associated with the cage in this transformant, much less so in two untransformed ‘flat revertants’ which contain no detectable viral transcripts but regain their close association with the cage in two retransformed derivatives.

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Section: Discussionsupporting

confidence: 83%

Active viral genes in transformed cells lie close to the nuclear cage.

Cook¹,

Lang²,

Hayday³

et al. 1982

The EMBO Journal

108

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show abstract

“…An eye-splice in a rope is maintained solely by interactions between strands. Perhaps specific interactions between identical sequences spaced along one duplex form the duplex into a series of eye-splices [28,40]. The close apposition of two identical duplexes to form a four-stranded structure in which two base pairs are themselves specifically paired (i. e. hydrogenbonded) is stereochemically possible [41].…”

Section: Discussionmentioning

confidence: 99%

“…kg-') with different doses of y-rays, ethidium was added to a final concentration of 0.2 or 8 pg/ml and the amount of bound ethidium determined from the fluorescence as described in Materials and Methods. (A) 0.2 pg/ml ethidium; (B) 8.0 pg/ml ethidium We originally predicted that the quasi-circles might be the size of chromomeres or replicons [28]. Subsequently we estimated the length of DNA (the 'target') in one quasi-circle to be much greater than this by applying target theory to curves relating the dose of y-rays to the sedimentation rate of nucleoids [4].…”

Section: The Binding Of Ethidium To Nucleoidsmentioning

confidence: 99%

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

1978

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Structures retaining many of the morphological features of nuclei may be released by lysing HeLa cells in solutions containing non-ionic detergents and high concentrations of salt. These nucleoids contain few chromatin proteins. We have shown that the DNA of nucleoids is quasicircular and supercoiled by measuring spectrofluorometrically the amount of the intercalating dye, ethidium, bound to unirradiated and y-irradiated nucleoids. Ethidium binds to nucleoids in the manner characteristic of the binding to superhelical DNA: at low concentrations more ethidium binds to unirradiated nucleoids than to their y-irradiated counterparts with broken DNA, and at higher concentrations less ethidium binds to the unirradiated nucleoids. The quasi-circles in nucleoids are 22 times less sensitive to y-irradiation than are circles of pure PM2 DNA: they must contain about 2.2 x lo5 base pairs.The constraints that maintain the quasi-circularity of nucleoid DNA are very resistant to extremes of temperature and alkali; some remain under conditions in which the duplex is denatured. The constraints are destabilised by ethidium suggesting that they are stabilised by free energy of supercoiling. Proteolytic enzymes, but not ribonucleases, remove the constraints. Possible structures for the constraining mechanism are discussed. Circular duplexes of DNA in which both strands of the duplex are covalently continuous possess one remarkable property not shared by their counterparts with broken strands: the two strands in the intact molecule are interlocked and can only be completely separated by severing one of the strands i.e. by breaking phosphodiester bonds (see [1 -31 for a fuller discussion). We have shown that the DNA of higher cells is subject to the same kind of topological constraint as that found in circular molecules of DNA. Our approach was to lyse cells in solutions containing non-ionic detergents and high concentrations of salt to release structures that retain many of the morphological features of nuclei. These nucleoids contain few of the proteins characteristic of chromatin and sediment in sucrose gradients containing ethidium in the manner characteristic of superhelical DNA. Breaking the DNA by y-irradiation abolished the characteristic behaviour [4-81 (see also [9-111). We concluded that nucleoid DNA was made quasi-circular by

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“…topoisomerases) influence transcription? Such questions provoked the suggestion that nuclear DNA might be supercoiled [61] and lie behind the continuing search for torsionally strained or 'dynamic' chromatin. Early studics showed conclusively that supercoiling stimulates transcription in uitro [62,631 ; whether it does in vivo, and whether some chromatin is torsionally strained, are now the issues.…”

Section: Loops and Supercoils In Native Chromatinmentioning

confidence: 99%

“…It could involve selective changes in chemical constitution (e. g, by methylation [157]), conformation (e.g. coiling or supercoiling in a different sense or degree [61,1581) A gcne in a loop can only be transcribed when it attaches to the polymerase (stippled rectangle) at the skeleton. For example, the globin loop is shown inactive in a fibroblast (a).…”

Section: An Attachment Hypothesis For Gene Activationmentioning

confidence: 99%

The nucleoskeleton and the topology of transcription

Cook

1989

European Journal of Biochemistry

108

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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...

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On the Inheritance of Differentiated Traits

Cited by 38 publications

References 142 publications

Active viral genes in transformed cells lie close to the nuclear cage.

Active viral genes in transformed cells lie close to the nuclear cage.

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

The nucleoskeleton and the topology of transcription

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