Mapping sequences in loops of nuclear DNA by their progressive detachment from the nuclear cage

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

doi:10.1093/nar/8.13.2895

Cited by 102 publications

(44 citation statements)

References 28 publications

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“…If attachments are generated artefactually, there should be no specificity in the aggregate and any given sequence will neither be enriched nor depleted. In fact, CI globin sequences in HeLa nuclcoids can be enriched cightfold whereas B and y genes are depleted; c( globin must lie closer to the attachment site than / 3 or y [53]. This 'detachment mapping' has been extended to many different preparations (e. g. matrices and scaffolds), but with variable results [5].…”

Section: Specific Attachmentsmentioning

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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Section: Specific Attachmentsmentioning

confidence: 99%

The nucleoskeleton and the topology of transcription

Cook

1989

European Journal of Biochemistry

108

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“…The 300-A (30-nm) chromatin fiber is thought to be organized into 30-to 100-kilobase-pair (kbp) loops that are anchored to a protein structure variously termed the nuclear matrix (7,8,37,(41)(42)(43)48), scaffold (9, 15-17, 21, 22, 25, 33, 34). or cage (10,11,29 (7) and ovalbumin, a gene that is tissue specific and highly transcribed (8, 37). On the other hand, when histones are extracted by lithium 3',5-diiodosalicylate (LIS) (33), attachment to the nuclear scaffold has been shown to occur at specific regions, which implies that loop structure is constant from one cell to the next.…”

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Chromatin loop structure of the human X chromosome: relevance to X inactivation and CpG clusters.

Beggs¹,

Migeon²

1989

Mol. Cell. Biol.

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Part of the higher-order structure of chromatin is achieved by constraining DNA in loops ranging in size from 30 to 100 kilobase pairs; these loops have been implicated in defining functional domains and replicons and possibly in facilitating transcription. Because the human active and inactive X chromosomes differ in transcriptional activity and replication, we looked for differences in their chromatin loop structures. Since the islands of CpG-rich DNA at the 5' ends of X-linked housekeeping genes are the regions where functional differences in DNA methylation and nuclease sensitivity are found, we looked for scaffold association of these sequences after extraction of histones with lithium diiodosalicylate. Specifically, we examined the 5' CpG islands within the hypoxanthine phosphoribosyltransferase, glucose 6-phosphate dehydrogenase, P3, GdX, phosphoglycerate kinase type 1, and a-galactosidase loci in human lymphoblasts obtained from individuals with 1 to 4 X chromosomes. Although we detected no scaffold-associated regions near these genes, we found several such regions at the ornithine transcarbamylase and blood clotting factor IX loci. Our results suggest that the CpG islands are excluded from the nuclear scaffold and that even though transcriptionally active, housekeeping genes are less likely than X-linked tissue-specific genes to be scaffold associated. In all cases, the pattern of scaffold association was the same for loci on active and inactive X chromosomes.Sex dosage compensation in mammalian females is achieved by random inactivation of one X chromosome (reviewed in reference 26). Active and inactive X chromosomes differ in a number of characteristics, including degree of condensation (3), timing of replication (18), and transcriptional activity (19). In addition, genes that are constitutively expressed are methylated differently on the two chromosomes (24,46,49,50). The functional differences in DNA methylation reside in the clusters of CpG dinucleotides, the so-called CpG islands, that are commonly found at the 5' ends of many housekeeping genes (4). These clusters are methylated only in genes on the inactive X chromosome, and methylation helps maintain the inactive state (23). Furthermore, since these clusters are nuclease hypersensitive in active but not in inactive genes (51), the chromatin structures of the two chromosomes must differ. Finally, methylation and gene activity are coordinately regulated at three CpG clusters near the glucose 6-phosphate dehydrogenase (G6PD) locus (46), which suggests that CpG islands may help define functional domains along the chromosome.Although the differences in structure of the homologous X chromosomes during interphase are striking, little is known about the organization of X chromatin at the molecular level. The 300-A (30-nm) chromatin fiber is thought to be organized into 30-to 100-kilobase-pair (kbp) loops that are anchored to a protein structure variously termed the nuclear matrix (7,8,37,(41)(42)(43)48), scaffold (9, 15-17, 21, 22, 25, 33, 34). or cage...

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“…6 could most likely be explained by binding of the E1Aa protein to a nuclear matrix-like structure, particularly since DNase treatment of nuclear extracts before loading onto the glycerol gradient did not alter sedimentation (data not shown). It has been shown previously that adenovirus DNA is associated with the nuclear matrix (48), and specific transcription units for induced ovalbumin genes and other mammalian genes have been detected on the matrix (8,30,39). Therefore, we performed a fractionation procedure to obtain a nuclear matrix fraction free of both DNA and soluble proteins.…”

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confidence: 99%

Localization of the adenovirus E1Aa protein, a positive-acting transcriptional factor, in infected cells infected cells.

Feldman¹,

Nevins²

1983

Mol. Cell. Biol.

102

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The function of the adenovirus E1Aa protein (the product of the 13S ElA mRNA) during a productive viral infection is to activate transcription of the six early viral transcription units. To study the mechanism of action of this protein, a peptide which was 13 amino acids long and had a sequence unique to the protein product of the adenovirus 13S ElA mRNA (pElAa) was coupled to keyhole limpet hemocyanin and used to raise an antibody in rabbits. The resulting antiserum was specific to this protein and did not react with the protein product of the 12S ElA mRNA, which shares considerable sequence with the E1Aa protein.This antiserum was used to probe for the E1Aa protein in situ by indirect immunofluorescence and in extracts of infected HeLa cells. We found that the protein was associated with large cellular structures both in the nucleus and in the cytoplasm. The nuclear form of the protein was analyzed further and was found to purify with the nuclear matrix.

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Mapping sequences in loops of nuclear DNA by their progressive detachment from the nuclear cage

Cited by 102 publications

References 28 publications

The nucleoskeleton and the topology of transcription

The nucleoskeleton and the topology of transcription

Chromatin loop structure of the human X chromosome: relevance to X inactivation and CpG clusters.

Localization of the adenovirus E1Aa protein, a positive-acting transcriptional factor, in infected cells infected cells.

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