Metaphase Chromosome Structure: The Role of Nonhistone Proteins

Laemmli, Ulrich Karl; Cheng, S. M.; Adolph, Kenneth W.; Paulson, James R.; Brown, Janice A.; Baumbach, W R

doi:10.1101/sqb.1978.042.01.036

Cited by 187 publications

(95 citation statements)

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“…This scaffold has been visualized in the electron microscope by histone-depleting chromosomes; the resulting micrographs show Ϸ40-kb DNA loops dangling from a protein-rich aggregate, which is the shape of the original chromosome (3). Isolated scaffolds were shown to contain nonhistone proteins (5) including topoisomerase II (6) and structural maintenance of chromosomes (7). Further studies indicated that the metaphase scaffold is helically folded (8) and contains AT-rich DNA sequences (9,10).…”

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confidence: 99%

“…It has been proposed that metaphase chromatin is organized into loops attached to an underlying protein-rich structure called the metaphase scaffold (3)(4)(5). This scaffold has been visualized in the electron microscope by histone-depleting chromosomes; the resulting micrographs show Ϸ40-kb DNA loops dangling from a protein-rich aggregate, which is the shape of the original chromosome (3).…”

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confidence: 99%

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Mitotic chromosomes are chromatin networks without a mechanically contiguous protein scaffold

Poirier

Marko

2002

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

159

167

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Isolated newt (Notophthalmus viridescens) chromosomes were studied by using micromechanical force measurement during nuclease digestion. Micrococcal nuclease and short-recognition-sequence blunt-cutting restriction enzymes first remove the native elastic response of, and then to go on to completely disintegrate, single metaphase newt chromosomes. These experiments rule out the possibility that the mitotic chromosome is based on a mechanically contiguous internal non-DNA (e.g., protein) ''scaffold''; instead, the mechanical integrity of the metaphase chromosome is due to chromatin itself. Blunt-cutting restriction enzymes with longer recognition sequences only partially disassemble mitotic chromosomes and indicate that chromatin in metaphase chromosomes is constrained by isolated chromatin-crosslinking elements spaced by Ϸ15 kb.

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confidence: 99%

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confidence: 99%

Mitotic chromosomes are chromatin networks without a mechanically contiguous protein scaffold

Poirier

Marko

2002

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

159

167

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“…172 kb, comparable to the approx. 80 kb loops on cellular chromosomes; Laemmli et al, 1977) makes it an attractive system to study the relationship between chromatin structure and function. METHODS B95-8 cells were grown in RPMI 1640 medium containing 10~ foetal calf serum.…”

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confidence: 99%

Chromatin Structure of Epstein-Barr Virus

Dyson¹,

Farrell²

1985

Journal of General Virology

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SUMMARYThe episomal copies of Epstein-Barr virus (EBV) DNA are in a chromatin structure in the lymphoblastoid cell line B95-8. Nucleosomes on EBV DNA have the same spacing as those in cellular chromatin. Some of the EBV DNA is not in nucleosomes; this probably corresponds to DNA which is being packaged. Several DNase hypersensitive sites have been mapped on the EBV genome and patterns of CpG methylation are examined.

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“…Starting with the hypothesis that nonhistone proteins-may play a role in determining the superstructure of interphase and metaphase chromosomes, we have recently developed an approach that permits dissection of the structural contribution made by the histones and certain nonhistone proteins. Our general approach toward uncovering a structural role for the nonhistone proteins has been to gently remove the histones from chromosomes by competition with dextran sulfate and heparin, without destroying the continuity of the DNA fiber (2). In this way, all the histones and many nonhistone proteins are removed, leaving behind some tightly bound nonhistone proteins that keep the chromosomal DNA highly organized and compact.…”

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confidence: 99%

Isolation of a protein scaffold from mitotic HeLa cell chromosomes

Adolph

Cheng

Paulson

et al. 1977

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

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168

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We have recently shown that, after the histones and most of the nonhistone proteins are gently removed from HeLa metaphase chromosomes, the chromosomal DNA is still highly organized and relatively compact. The structure of these histone-depleted chromosomes is due to the presence of a number of nonhistone proteins that form a central scaffold that retains the approximate size and shape of intact chromosomes and to which the DNA is attached, predominantly forming loops. We now demonstrate that the protein scaffold may be iso ated independently of the DNA by treating HeLa chromosomes with micrococcal nuclease before removing the histones.The chromosomal scaffolds may be isolated by sucrose density gradient centrifugation as a well-defined peak that is stable in 2 M sodium chloride, but is dissociated by treatment with proteases, 4 M urea, or 0.1% sodium dodecyl sulfate. Polyacrylamide gel electrophoresis reveals that the protein content of scaffold preparations is identical to that of histone-depleted chromosomes. Fluorescence microscopy of purified scaffolds in isolation buffer shows that the particles still possess the familiar chromosome morphology. When the scaffolds are examined in the electron microscope, a fibrous structure with the approximate size and shape of intact, paired chromatids is seen. Less than 0.1I% of the chromosomal DNA and virtually no histones are associated with the purified scaffold structures. Mitotic chromosomes are composed of the five histones and a large number of nonhistone proteins that keep the DNA in a highly compact arrangement. Electron microscopy has revealed that the nucleoprotein fiber in mitotic chromosomes is a "knobby" fiber with a diameter of about 200-300 A and appears in a complex, net-like arrangement (e.g., ref. 1). The complexity of this network and the tendency of the chromatin fibers to adhere to each other make it difficult to study the higher-order folding of the nucleoprotein fiber in chromosomes.A great deal has been learned in the last few years about the structural role played by the histones in chromatin, but little is known about a possible involvement of nonhistone proteins in chromosome structure. Starting with the hypothesis that nonhistone proteins-may play a role in determining the superstructure of interphase and metaphase chromosomes, we have recently developed an approach that permits dissection of the structural contribution made by the histones and certain nonhistone proteins. Our general approach toward uncovering a structural role for the nonhistone proteins has been to gently remove the histones from chromosomes by competition with dextran sulfate and heparin, without destroying the continuity of the DNA fiber (2). In this way, all the histones and many nonhistone proteins are removed, leaving behind some tightly bound nonhistone proteins that keep the chromosomal DNA highly organized and compact. This dissecting procedure hasThe costs of publication of this article were defrayed in part by the payment of page charges. This article mu...

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Metaphase Chromosome Structure: The Role of Nonhistone Proteins

Cited by 187 publications

References 0 publications

Mitotic chromosomes are chromatin networks without a mechanically contiguous protein scaffold

Mitotic chromosomes are chromatin networks without a mechanically contiguous protein scaffold

Chromatin Structure of Epstein-Barr Virus

Isolation of a protein scaffold from mitotic HeLa cell chromosomes

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