scholarly journals Localization of topoisomerase II in mitotic chromosomes.

1985 ◽  
Vol 100 (5) ◽  
pp. 1716-1725 ◽  
Author(s):  
W C Earnshaw ◽  
M M Heck
Keyword(s):  
Present Article ◽  
Immunoelectron Microscopy ◽  
Polyclonal Antibody ◽  
Topoisomerase Ii ◽  
Mitotic Chromosome ◽  
Experimental Protocol ◽  
Mitotic Chromosomes ◽  
Restricted Domains ◽  
Loop Domains ◽  
Chromosome Scaffold

In the preceding article we described a polyclonal antibody that recognizes cSc-1, a major polypeptide component of the chicken mitotic chromosome scaffold. This polypeptide was shown to be chicken topoisomerase II. In the experiments described in the present article we use indirect immunofluorescence and immunoelectron microscopy to examine the distribution of topoisomerase II within intact chromosomes. We also describe a simple experimental protocol that differentiates antigens that are interspersed along the chromatin fiber from those that occupy restricted domains within the chromosome. These experiments indicate that the distribution of the enzyme appears to be independent of the bulk chromatin. Our data suggest that topoisomerase II is bound to the bases of the radial loop domains of mitotic chromosomes.

scholarly journals Contribution of hCAP-D2, a Non-SMC Subunit of Condensin I, to Chromosome and Chromosomal Protein Dynamics during Mitosis

2005 ◽  
Vol 25 (2) ◽  
pp. 740-750 ◽  
Author(s):  
Erwan Watrin ◽  
Vincent Legagneux
Keyword(s):  
Rna Interference ◽  
Mitotic Spindle ◽  
Topoisomerase Ii ◽  
Mitotic Chromosome ◽  
Chromosomal Protein ◽  
Structural Components ◽  
Mitotic Chromosomes ◽  
Sister Chromatids ◽  
Chromosome Alignment ◽  
Structural Maintenance Of Chromosomes

ABSTRACT Condensins are heteropentameric complexes that were first identified as structural components of mitotic chromosomes. They are composed of two SMC (structural maintenance of chromosomes) and three non-SMC subunits. Condensins play a role in the resolution and segregation of sister chromatids during mitosis, as well as in some aspects of mitotic chromosome assembly. Two distinct condensin complexes, condensin I and condensin II, which differ only in their non-SMC subunits, exist. Here, we used an RNA interference approach to deplete hCAP-D2, a non-SMC subunit of condensin I, in HeLa cells. We found that the association of hCAP-H, another non-SMC subunit of condensin I, with mitotic chromosomes depends on the presence of hCAP-D2. Moreover, chromatid axes, as defined by topoisomerase II and hCAP-E localization, are disorganized in the absence of hCAP-D2, and the resolution and segregation of sister chromatids are impaired. In addition, hCAP-D2 depletion affects chromosome alignment in metaphase and delays entry into anaphase. This suggests that condensin I is involved in the correct attachment between chromosome kinetochores and microtubules of the mitotic spindle. These results are discussed relative to the effects of depleting both condensin complexes.

scholarly journals ScII: an abundant chromosome scaffold protein is a member of a family of putative ATPases with an unusual predicted tertiary structure.

1994 ◽  
Vol 127 (2) ◽  
pp. 303-318 ◽  
Author(s):  
N Saitoh ◽  
I G Goldberg ◽  
E R Wood ◽  
W C Earnshaw
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Topoisomerase Ii ◽  
Tertiary Structure ◽  
Coiled Coil ◽  
Scaffold Protein ◽  
The Other ◽  
Mitotic Chromosomes ◽  
Chromosome Scaffold

Here, we describe the cloning and characterization of ScII, the second most abundant protein after topoisomerase II, of the chromosome scaffold fraction to be identified. ScII is structurally related to a protein, Smc1p, previously found to be required for accurate chromosome segregation in Saccharomyces cerevisiae. ScII and the other members of the emerging family of SMC1-like proteins are likely to be novel ATPases, with NTP-binding A and B sites separated by two lengthy regions predicted to form an alpha-helical coiled-coil. Analysis of the ScII B site predicted that ScII might use ATP by a mechanism similar to the bacterial recN DNA repair and recombination enzyme. ScII is a mitosis-specific scaffold protein that colocalizes with topoisomerase II in mitotic chromosomes. However, ScII appears not to be associated with the interphase nuclear matrix. ScII might thus play a role in mitotic processes such as chromosome condensation or sister chromatid disjunction, both of which have been previously shown to involve topoisomerase II.

scholarly journals Topoisomerase II does not play a scaffolding role in the organization of mitotic chromosomes assembled in Xenopus egg extracts.

1993 ◽  
Vol 120 (3) ◽  
pp. 601-612 ◽  
Author(s):  
T Hirano ◽  
T J Mitchison
Keyword(s):  
Topoisomerase Ii ◽  
Mitotic Chromosome ◽  
Chromosome Morphology ◽  
Sperm Chromatin ◽  
Mitotic Chromosomes ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Topo Ii ◽  
Xenopus Egg Extracts

We have investigated the role of topoisomerase II (topo II) in mitotic chromosome assembly and organization in vitro using Xenopus egg extracts. When sperm chromatin was incubated with mitotic extracts, the highly compact chromatin rapidly swelled and concomitantly underwent local condensation. Further incubation induced the formation of entangled thin chromatin fibers that eventually resolved into highly condensed individual chromosomes. This in vitro system made it possible to manipulate mitotic chromosomes in their assembly condition without any isolation or stabilization steps. Two complementary approaches, immunodepletion and antibody blocking, demonstrated that topo II activity is required for chromosome assembly and condensation. Once condensation was completed, however, blocking of topo II activity had little effect on the chromosome morphology. Immunofluorescent studies showed that topo II was uniformly distributed throughout the condensed chromosomes and was not restricted to the chromosomal axis. Surprisingly, all detectable topo II molecules were easily extracted from the chromosomes under mild conditions where the shape of chromosomes was well preserved. Our results show that topo II is essential for mitotic chromosome assembly, but does not play a scaffolding role in the structural maintenance of chromosomes assembled in vitro. We also present evidence that changes of DNA topology affect the distribution of topo II in mitotic chromosomes in our system.

scholarly journals Mitotic chromosomes are constrained by topoisomerase II–sensitive DNA entanglements

2010 ◽  
Vol 188 (5) ◽  
pp. 653-663 ◽  
Author(s):  
Ryo Kawamura ◽  
Lisa H. Pope ◽  
Morten O. Christensen ◽  
Mingxuan Sun ◽  
Ksenia Terekhova ◽  
...  
Keyword(s):  
Topoisomerase Ii ◽  
Mitotic Chromosome ◽  
Specific Inhibitor ◽  
Elastic Stiffness ◽  
Type I ◽  
Mitotic Chromosomes ◽  
Relaxation Effects ◽  
Double Stranded Dna ◽  
Topo Ii ◽  
Mitotic Chromatin

We have analyzed the topological organization of chromatin inside mitotic chromosomes. We show that mitotic chromatin is heavily self-entangled through experiments in which topoisomerase (topo) II is observed to reduce mitotic chromosome elastic stiffness. Single chromosomes were relaxed by 35% by exogenously added topo II in a manner that depends on hydrolysable adenosine triphosphate (ATP), whereas an inactive topo II cleavage mutant did not change chromosome stiffness. Moreover, experiments using type I topos produced much smaller relaxation effects than topo II, indicating that chromosome relaxation by topo II is caused by decatenation and/or unknotting of double-stranded DNA. In further experiments in which chromosomes are first exposed to protease to partially release protein constraints on chromatin, ATP alone relaxes mitotic chromosomes. The topo II–specific inhibitor ICRF-187 blocks this effect, indicating that it is caused by endogenous topo II bound to the chromosome. Our experiments show that DNA entanglements act in concert with protein-mediated compaction to fold chromatin into mitotic chromosomes.

scholarly journals Smc5-Smc6-Dependent Removal of Cohesin from Mitotic Chromosomes

2009 ◽  
Vol 29 (16) ◽  
pp. 4363-4375 ◽  
Author(s):  
Emily A. Outwin ◽  
Anja Irmisch ◽  
Johanne M. Murray ◽  
Matthew J. O'Connell
Keyword(s):  
Dna Damage ◽  
Chromosome Segregation ◽  
Topoisomerase Ii ◽  
Mitotic Chromosome ◽  
Chromosome Structure ◽  
Synthetic Lethality ◽  
Mitotic Chromosomes ◽  
Interphase Chromosome ◽  
Chromosome Arm ◽  
Null Mutants

ABSTRACT The function of the essential cohesin-related Smc5-Smc6 complex has remained elusive, though hypomorphic mutants have defects late in recombination, in checkpoint maintenance, and in chromosome segregation. Recombination and checkpoints are not essential for viability, and Smc5-Smc6-null mutants die in lethal mitoses. This suggests that the chromosome segregation defects may be the source of lethality in irradiated Smc5-Smc6 hypomorphs. We show that in smc6 mutants, following DNA damage in interphase, chromosome arm segregation fails due to an aberrant persistence of cohesin, which is normally removed by the Separase-independent pathway. This postanaphase persistence of cohesin is not dependent on DNA damage, since the synthetic lethality of smc6 hypomorphs with a topoisomerase II mutant, defective in mitotic chromosome structure, is also due to the retention of cohesin on undamaged chromosome arms. In both cases, Separase overexpression bypasses the defect and restores cell viability, showing that defective cohesin removal is a major determinant of the mitotic lethality of Smc5-Smc6 mutants.

scholarly journals Replication-Coupled Topoisomerase II Templates the Mitotic Chromosome Scaffold?

Cell Cycle ◽  
2003 ◽  
Vol 2 (3) ◽  
pp. 229-231 ◽  
Author(s):  
Juan F. Gimenez-Abian ◽  
Duncan J. Clarke
Keyword(s):  
Topoisomerase Ii ◽  
Mitotic Chromosome ◽  
Chromosome Scaffold

scholarly journals Topoisomerase II is a structural component of mitotic chromosome scaffolds.

1985 ◽  
Vol 100 (5) ◽  
pp. 1706-1715 ◽  
Author(s):  
W C Earnshaw ◽  
B Halligan ◽  
C A Cooke ◽  
M M Heck ◽  
L F Liu
Keyword(s):  
Topoisomerase Ii ◽  
Mitotic Chromosome ◽  
Subcellular Fractionation ◽  
Dna Topoisomerase Ii ◽  
Structural Protein ◽  
Mitotic Chromosomes ◽  
Interphase Nuclei ◽  
Dna Topoisomerase ◽  
Sds Page ◽  
Topological States

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.

scholarly journals Centromeric Localization of Dispersed Pol III Genes in Fission Yeast

2010 ◽  
Vol 21 (2) ◽  
pp. 254-265 ◽  
Author(s):  
Osamu Iwasaki ◽  
Atsunari Tanaka ◽  
Hideki Tanizawa ◽  
Shiv I.S. Grewal ◽  
Ken-ichi Noma
Keyword(s):  
Genome Organization ◽  
Gene Locus ◽  
Mitotic Chromosome ◽  
Three Dimensional ◽  
Rrna Genes ◽  
Mitotic Chromosomes ◽  
Gene Encoding ◽  
5S Rrna Genes ◽  
Pol Iii ◽  
Mitotic Chromosome Condensation

The eukaryotic genome is a complex three-dimensional entity residing in the nucleus. We present evidence that Pol III–transcribed genes such as tRNA and 5S rRNA genes can localize to centromeres and contribute to a global genome organization. Furthermore, we find that ectopic insertion of Pol III genes into a non-Pol III gene locus results in the centromeric localization of the locus. We show that the centromeric localization of Pol III genes is mediated by condensin, which interacts with the Pol III transcription machinery, and that transcription levels of the Pol III genes are negatively correlated with the centromeric localization of Pol III genes. This centromeric localization of Pol III genes initially observed in interphase becomes prominent during mitosis, when chromosomes are condensed. Remarkably, defective mitotic chromosome condensation by a condensin mutation, cut3-477, which reduces the centromeric localization of Pol III genes, is suppressed by a mutation in the sfc3 gene encoding the Pol III transcription factor TFIIIC subunit, sfc3-1. The sfc3-1 mutation promotes the centromeric localization of Pol III genes. Our study suggests there are functional links between the process of the centromeric localization of dispersed Pol III genes, their transcription, and the assembly of condensed mitotic chromosomes.

Polytene and mitotic chromosome analysis in Ceratitis capitata (Diptera; Tephritidae)

1986 ◽  
Vol 28 (2) ◽  
pp. 180-188 ◽  
Author(s):  
D. G. Bedo
Keyword(s):  
X Chromosome ◽  
Silver Staining ◽  
Mitotic Chromosome ◽  
Ceratitis Capitata ◽  
Fat Body ◽  
Polytene Chromosomes ◽  
Chromosome Analysis ◽  
Mitotic Chromosomes ◽  
Chromosome Separation ◽  
Ectopic Pairing

Polytene chromosomes were found in several larval and pupal tissues of the Medfly, Ceratitis capitata, during a search for chromosomes suitable for detailed cytological analysis. Well-banded highly polytene chromosomes, which could be adequately separated and spread, were found in trichogen cells of the spatulate superior orbital bristles of male pupae. These chromosomes proved suitable for full polytene analysis. Thoracic trichogen cells of both male and female pupae also contain useful polytene chromosomes, although they are considerably thinner and thus more difficult to analyze. Contrasting with those in pupal trichogen cells, the chromosomes in the salivary glands, Malphighian tubules, midgut, hindgut, and fat body of larvae and pupae were difficult to prepare because of high levels of ectopic pairing and chromosome fragmentation. In hindgut preparations partial separation of up to three chromosomes was achieved, but in all other tissues no useful chromosome separation was possible. In trichogen polytene cells, five banded chromosomes and a prominent heterochromatic network associated with a nucleolus are found. The mitotic chromosomes respond to C- and Q-banding and silver staining with considerable variation. This is especially so in the X chromosome, which displays an extensive array of bands following both Q-banding and silver staining. Comparison of Q-banded metaphase and polytene chromosomes demonstrates that the five autosomes are represented by conventional polytene chromosomes, while the sex chromosomes are contained in the heterochromatic net, most of which fluoresces strongly. This suggests that the Q-bands of the mitotic X chromosome are replicated to a greater extent than the nonfluorescent material in polytene cells. This investigation shows C. capitata to have excellent cytological material for both polytene and mitotic analysis.Key words: Ceratitis capitata, Medfly, chromosomes (polytene), banding (chromosome).

scholarly journals Functional Analysis of Kinetochore Assembly in Caenorhabditis elegans

2001 ◽  
Vol 153 (6) ◽  
pp. 1209-1226 ◽  
Author(s):  
Karen Oegema ◽  
Arshad Desai ◽  
Sonja Rybina ◽  
Matthew Kirkham ◽  
Anthony A. Hyman
Keyword(s):  
Caenorhabditis Elegans ◽  
Chromosome Segregation ◽  
Mitotic Chromosome ◽  
Mitotic Chromosomes ◽  
Cell Stage ◽  
Kinetochore Assembly ◽  
Chromosomal Passenger ◽  
Complete Failure ◽  
Spindle Microtubules ◽  
The One

In all eukaryotes, segregation of mitotic chromosomes requires their interaction with spindle microtubules. To dissect this interaction, we use live and fixed assays in the one-cell stage Caenorhabditis elegans embryo. We compare the consequences of depleting homologues of the centromeric histone CENP-A, the kinetochore structural component CENP-C, and the chromosomal passenger protein INCENP. Depletion of either CeCENP-A or CeCENP-C results in an identical “kinetochore null” phenotype, characterized by complete failure of mitotic chromosome segregation as well as failure to recruit other kinetochore components and to assemble a mechanically stable spindle. The similarity of their depletion phenotypes, combined with a requirement for CeCENP-A to localize CeCENP-C but not vice versa, suggest that a key step in kinetochore assembly is the recruitment of CENP-C by CENP-A–containing chromatin. Parallel analysis of CeINCENP-depleted embryos revealed mitotic chromosome segregation defects different from those observed in the absence of CeCENP-A/C. Defects are observed before and during anaphase, but the chromatin separates into two equivalently sized masses. Mechanically stable spindles assemble that show defects later in anaphase and telophase. Furthermore, kinetochore assembly and the recruitment of CeINCENP to chromosomes are independent. These results suggest distinct roles for the kinetochore and the chromosomal passengers in mitotic chromosome segregation.

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