Estimation of domain length of chicken erythrocyte chromatin
TL;DR: Chromatin of chicken erythrocyte nuclei was extracted by digestion with micrococcal nuclease and the length distribution of the soluble chromatin was determined by gel electrophoresis and electron microscopy to fitted with a theoretical distribution.
Abstract: Chromatin of chicken erythrocyte nuclei was extracted by digestion with micrococcal nuclease. The length distribution of the soluble chromatin was determined by gel electrophoresis and electron microscopy. These results were fitted with a theoretical distribution which was an outcome of the domain model proposed by Igo-Kemenes and Zachau (Igo-Kemenes, T. and H.G. Zachau (1977) Cold Spring Harbour Symp. Quant. Biol. 42, 109-118). A domain length of 45 kbp was obtained.
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TL;DR: A novel approach for mapping the DNA loop anchorage sites that seems to constitute a unique tool for the analysis of topological organization of chromosomal DNA in living cells is presented and rationales for the supposition that DNA loops constitute basic units of eukaryotic genome organization and evolution are considered.
Abstract: In this chapter the specificity of chromosomal DNA partitioning into topological loops is discussed. Different experimental approaches used for the analysis of the above problem are critically reviewed. This discussion is followed by presentation of a novel approach for mapping the DNA loop anchorage sites that we have developed. This approach, based on the excision of the whole DNA loops by topoisomerase II-mediated DNA cleavage at matrix attachment sites, seems to constitute a unique tool for the analysis of topological organization of chromosomal DNA in living cells. We also discuss experimental results indicating that the DNA-loop anchorage sites form „weak points” in chromosomes that are preferentially sensitive to cleavage with both endogenous and exogenous nucleases. In connection with this discussion, rationales for the supposition that DNA loops constitute basic units of eukaryotic genome organization and evolution are considered. The chapter concludes by suggesting a new model of spatial organization of eukaryotic genome within the cell nucleus that resolves apparent contradictions between different data on the specificity of DNA interaction with the nuclear matrix.
96 citations
TL;DR: The large thermal activation energy and the limited DNA size reduction suggest the possible involvement of thermal denaturation of a nuclear polypeptide in the production of these nuclear lesions.
Abstract: Induction of DNA lesions in the nucleus of Chinese hamster ovary (CHO) cells was observed at hyperthermic temperatures using the alkaline filter elution and the alkaline sucrose gradient sedimentation methods. These lesions were observed principally at temperatures greater than 45 degrees C with an activation energy of 140 kcal/mole. On alkaline sucrose gradients the cell genome was reduced to a 140 S or 2 X 10(8) dalton subunit of DNA independent of increasing exposure time at temperatures above 45 degrees C. The large thermal activation energy and the limited DNA size reduction suggest the possible involvement of thermal denaturation of a nuclear polypeptide in the production of these nuclear lesions.
31 citations
TL;DR: Although there is no significant sequence conservation among various chromatin domain boundaries, these elements show functional conservation across the species and are discussed mechanistic aspects of how chromatindomain boundaries may function in organizing and regulating eukaryotic genome.
Abstract: Understanding of how the eukaryotic genome is packaged into chromatin and what the functional consequences of this organization are has begun to emerge recently. The concept of ‘chromatin domains’ — the topologically independent structural unit — is the basis of higher order chromatin organization. The idea that this structural unit may also coincide with the functional unit, offers a useful framework in dissecting the structure-function relationship. Boundaries that define these domains have been identified and several assays have been developed to test themin vivo. We have used genetic means to identify and analyse such boundary elements in the bithorax complex ofDrosophila melanogaster. In this review we discuss chromatin domain boundaries identified in several systems using different means. Although there is no significant sequence conservation among various chromatin domain boundaries, these elements show functional conservation across the species. Finally, we discuss mechanistic aspects of how chromatin domain boundaries may function in organizing and regulating eukaryotic genome.
17 citations
TL;DR: The higher-order organisation of chromatin in chicken erythrocyte nuclei as a function of the ionic strength of the nuclear suspension buffer and also of the time of incubation in this buffer prior to nuclease digestion has been investigated.
Abstract: The higher-order organisation of chromatin in chicken erythrocyte nuclei as a function of the ionic strength of the nuclear suspension buffer and also of the time of incubation in this buffer prior to nuclease digestion has been investigated. This organisation is described in terms of a physical parameter called the domain length. The 45-kbp-long domains of control nuclei were unravelled to give rise to domains of length 150 kbp on overnight equilibration at 0°C of the nuclei in standard isolation buffer containing 0.135 M NaCl prior to nuclease digestion. However, transition to the equilibrium state was preceded by a metastable and irregular domain architecture when the nuclei were incubated for only 1 h. In contrast, the domain length remained unchanged when nuclei were incubated in the isolation buffer alone for identical periods of time. The proteins dissociated at the higher ionic strength were characterised and their role in stabilising the domain structure is discussed.
2 citations
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TL;DR: The structure of nucleus and linker is described and the path followed by the DNA is described, revealing the arrangement of Histones and their role in theogenesis.
Abstract: STRUCTURE OF NUCLEOSOMES 940 Core and Linker 940 Arrangement of Histones 942 Path Followed by the DNA 944 Conformation of Histones 948
1,156 citations
TL;DR: Chromatin prepared by a method involving limited nuclease digestion contains the same repeating structure as chromatin in the nucleus, whereas chromatin preparation by conventional methods involving shear does not.
Abstract: Chromatin prepared by a method involving limited nuclease digestion contains the same repeating structure as chromatin in the nucleus, whereas chromatin prepared by conventional methods involving shear does not.
349 citations
TL;DR: If chromatin is treated in the presence of the resin with NaCl at concentrations of 650 mM or more, the structural organization of the Chromatin is decreased, yielding fibers of irregular appearance.
Abstract: Removal of histone H1 produces a transition in the structure of chromatin fibers as observed by electron microscopy. Chromatin containing all histone proteins appears as fibers with a diameter of about 250 A. The nucleosomes within these fibers are closely packed. If histone H1 is selectively removed with 50–100 mM NaCl in 50 mM sodium phosphate buffer (pH 7.0) in the presence of the ion-exchange resin AG 50 W − X2, chromatin appears as "beads-on-a-string" with the nucleosomes separated from each other by distances of about 150–200 A. If chromatin is treated in the presence of the resin with NaCl at concentrations of 650 mM or more, the structural organization of the chromatin is decreased, yielding fibers of irregular appearance.
271 citations
TL;DR: Nucleoids derived from adult hen erythrocytes differ from the other nucleoids studied in that their sedimentation rate does not vary in the manner characteristic of supercoiled DNA.
Abstract: We have investigated DNA superstructure in a wide range of nuclei of higher cells by gently lysing cells to release structures that resemble nuclei but are depleted of nuclear proteins. The sedimentation properties of these structures, which we call nucleoids, have been examined in sucrose gradients containing the intercalating agent, ethidium. The sedimentation rate of nucleoids derived from the growing cells of mammals, birds, amphibians and insects varies in the manner characteristic of circular and superhelical molecules of DNA. These characteristic changes in sedimentation rate are abolished by irradiating the nucleoids with low doses of gamma-rays, a procedure known to introduce single-strand scissions into DNA. We have also investigated by similar means DNA superstructure in nucleoids derived from a variety of different chick cells. Nucleoids derived from adult hen erythrocytes differ from the other nucleoids studied in that their sedimentation rate does not vary in the manner characteristic of supercoiled DNA.
243 citations
TL;DR: A detailed space-filling model for the higher order DNA coiling in chromatin is constructed, starting with the symmetrical nucleosome core previously described and proposing that histone H1 binds to the 100 A diameter superhelix and coils it into tightly packed, 110 A pitch super-superhelices of variable diameter.
Abstract: Electron microscopic examination of the histone H1-depleted, folded genomes of Drosophila melanogaster reveals that they are composed of long cylindrical cables of about 100 A diameter. Limited single-strand nicking with DNAase I relaxes the 100 A fibers to a "beads-on-a-string" structure, showing the nucleosomes and internucleosome DNA. Based on these results and other available data, we have constructed a detailed space-filling model for the higher order DNA coiling in chromatin, starting with the symmetrical nucleosome core previously described (Weintraub, Worcel and Alberts, 1976). The model defines the path of the DNA helix and the nucleosome arrangement along the DNA coil for both the 100 A and the 200–300 A fibers. Following Sobell et al. (1976), we believe that the DNA is coiled in the 100 A nucleofilament in a uniform left-handed supercoil of about 90 base pairs (bp) per turn and 47 A pitch; the 140 bp symmetrical nucleosome cores align themselves along this uniform DNA superhelix so that the isologous outer surfaces of adjacent nucleosomes touch and the internucleosome spacer DNA coils between them. A few single-strand discontinuities [about one nick per 85 kilobases (kb); Benyajati and Worcel, 1976] in the H1-depleted 100 A fiber can thus relax the negatively supercoiled internucleosome DNA generating the "beads-on -a-string" appearance. We propose that histone H1 binds to the 100 A diameter superhelix and coils it into tightly packed, 110 A pitch super-superhelices ("solenoids;" Finch and Klug, 1976) of variable diameter (between 200–300 A). In our model, the "thick" 200–300 A fiber is stabilized at metaphase by histone H1-H1 heterologous interactions between adjacent helical turns of the nucleofilament, and the internucleosome spacer DNA is located on the outside. Symmetry considerations demand that changes in the length of the repeat should lead to variations in the number of nucleosomes per helical turn and in the handedness of these turns in the 200–300 A metaphase fiber.
176 citations