The kinetochore is part of the metaphase chromosome scaffold.

Earnshaw, William C.; Halligan, Nadine; Cooke, Carol; Rothfield, Naomi F.

doi:10.1083/jcb.98.1.352

Cited by 119 publications

(68 citation statements)

References 20 publications

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“…One unrelated scaffold protein has been characterized in some detail, however, This is CENP-B, an 80,000-mol-wt human centromere protein that is detected by autoimmune sera from certain patients with rheumatic diseases (29,31). Both electron microscopy (14) and a combination of immunoblotting and immunofluorescence using the autoimmune sera (3 l) indicate that centromere components are retained in chromosome scaffolds prepared by our methods.…”

Section: Comparison With Other Scaffold Proteinsmentioning

confidence: 92%

“…as previously described (31), except that hepatoma and chick embryo fibroblast cultures were grown on polylysine-coated coverslips. Before processing for immunofluorescence, the coverslips were centrifuged at 1,000 g for 2 rain to increase adherence of mitotic cells.…”

Section: Production Of Antibody To Bovine Topoisomerase IImentioning

confidence: 99%

“…Before processing for immunofluorescence, the coverslips were centrifuged at 1,000 g for 2 rain to increase adherence of mitotic cells. Subsequent processing was as described previously (31). Rhodamine-conjugated anti-guinea pig IgG was obtained from Capp¢l Laboratories (Cochranville, PA).…”

Section: Production Of Antibody To Bovine Topoisomerase IImentioning

confidence: 99%

“…Procedure a involved spotting solutions containing antigen onto nitrocellulose strips and processing these with iodinated protein A as for normal immunoblots (31). After drying, the strips were sliced up and counted in a gamma counter.…”

Section: Production Of Antibody To Bovine Topoisomerase IImentioning

confidence: 99%

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Topoisomerase II is a structural component of mitotic chromosome scaffolds.

Earnshaw¹,

Halligan²,

Cooke³

et al. 1985

The Journal of Cell Biology

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709

286

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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 ...

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Section: Comparison With Other Scaffold Proteinsmentioning

confidence: 92%

Section: Production Of Antibody To Bovine Topoisomerase IImentioning

confidence: 99%

Section: Production Of Antibody To Bovine Topoisomerase IImentioning

confidence: 99%

Section: Production Of Antibody To Bovine Topoisomerase IImentioning

confidence: 99%

See 2 more Smart Citations

Topoisomerase II is a structural component of mitotic chromosome scaffolds.

Earnshaw¹,

Halligan²,

Cooke³

et al. 1985

The Journal of Cell Biology

Self Cite

709

286

View full text Add to dashboard Cite

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“…Microtubule organizing centers, which include kinetochores and centrosomes with their centrioles (36), are one such target for these autoantibodies. Anticentromere antibodies, which occur in the CREST (calcinosis, Raynaud's phenomenon, esophageal motility disorder, sclerodactyly, telangiectases) syndrome variant of systemic sclerosis (35), react specifically with kinetochore proteins (37,38). Autoantibodies to centrioles and to centrosomes have also been described (39)(40)(41).…”

Section: Discussionmentioning

confidence: 99%

Anticytoskeletal autoantibody to microfilament anchorage sites recognizes novel focal contact proteins.

Senécal¹,

Fortin²,

Roussin³

et al. 1987

J. Clin. Invest.

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Actin microfilaments are anchored to the plasma membrane at focal contacts. Using an indirect immunofluorescence method, we detected an autoantibody reactive with focal contacts in PtK2, HEp-2, and BHK-21 cells in serum from two patients with early systemic sclerosis. With double immunofluorescence, using the actin-binding drug phalloidin, we localized the plaques decorated by these sera specifically at the termini of microfilament bundles. The reactive antigens were identified by immunoblotting as proteins of 80,000-and 75,300-mol wt in PtK2, and of 53,500-mol wt in HEp-2 and BHK-2i cells. The 53,500-mol wt protein was also identified in rat skeletal, myocardial, and smooth muscle tissues. The detergent solubility of these proteins suggested that they may be linked to the plasma membrane. The autoantigens were immunologically distinct from vinculin and a-actinin, two major proteins also known to be concentrated at the ends of microfilament bundles. Our observations suggest that this novel anticytoskeletal autoantibody may identify a novel family of vertebrate cell proteins involved in the linkage of microfilamnents to the plasma membrane at focal contacts.

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Mitotic chromosome structure

Earnshaw

1988

BioEssays

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SummaryThe various models of chromatin fiber folding that have been proposed over the years are considered and evaluated. It is concluded that the radial loop/scafold model is strongly supported by the available evidence, although the term 'scafold' may be an unfortunate one. The scafold is not a solid rod running the length of the chromatid but rather appears to be an aggregation of discrete anchoring complexes for the loops of the fiber. Despite support for this model, there is a need for more evidence to resolve the questions that remain.

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The kinetochore is part of the metaphase chromosome scaffold.

Cited by 119 publications

References 20 publications

Topoisomerase II is a structural component of mitotic chromosome scaffolds.

Topoisomerase II is a structural component of mitotic chromosome scaffolds.

Anticytoskeletal autoantibody to microfilament anchorage sites recognizes novel focal contact proteins.

Mitotic chromosome structure

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