We have obtained a polyclonal antibody that recognizes major polypeptide component of chicken mitotic chromosome scaffolds. This migrates in SDS PAGE with Mr 170,000. Indirect immunofluorescence and subcellular fractionation experiments confirm it is present both chromosomes interphase nuclei. Two lines evidence suggest this protein DNA topoisomerase II, an abundant nuclear enzyme controls topological states: anti-scaffold inhibits the strand-passing activity II; independent raised against...

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

We have used an antibody probe to measure the levels of topoisomerase II in several transformed and developmentally regulated normal cell types. Transformed cells contain roughly 1 X 10(6) copies enzyme. During erythropoiesis chicken embryos enzyme level drops from 7.8 10(4) per erythroblast less than 300 erythrocyte concomitant with cessation mitosis blood. Cultured myoblasts also lose upon fusion into nonproliferating myotubes. When peripheral blood lymphocytes (which lack detectable II)...

We have utilized antibody probes to examine the expression of DNA topoisomerases I and II chromosome scaffold protein Sc-2 in normal transformed cells. Neither topoisomerase nor shows significant fluctuations content or stability across cell cycle. In contrast, undergoes cycle-dependent alterations both amount stability. As cells progress from mitosis into G1, much is degraded. During first 2 hr half life decreased that measured asynchronous populations by a factor 7. This suggests...

We describe a novel set of polypeptide antigens that shows dramatic change in structural localization during mitosis. Through metaphase these define new chromosomal substructure is located between the sister chromatids. Because are concentrated pericentromeric region, we have provisionally termed them INCENPs (inner centromere proteins). The (two polypeptides 155 and 135 kD) were identified with monoclonal antibody was raised against bulk proteins mitotic chromosome scaffold fraction. These...

We report here that disruption of a recently discovered kinesin-like protein in Drosophila melanogaster, KLP61F, results mitotic mutation lethal to the organism. show absence KLP61F function, spindle poles fail separate, resulting formation monopolar spindles. The phenotype metaphase arrest with polyploid cells is reminiscent seen fungal bimC and cut7 mutations, where it has also been shown pole bodies are not segregated. specifically expressed proliferating tissues during embryonic larval...

DNA from Drosophila egg chambers undergoing chorion gene amplification was analyzed using the two-dimensional gel technique of Brewer and Fangman. At stage 10, 34% molecules maximally amplified region third chromosome cluster contained replication forks or bubbles. These nonlinear forms were intermediates in process amplification; they confined to follicle cells, found only within replicating during time amplification. Multiple origins gave rise these intermediates, since three separate...

The SMC proteins are found in nearly all living organisms examined, where they play crucial roles mitotic chromosome dynamics, regulation of gene expression, and DNA repair. We have explored the phylogenetic relationships from prokaryotes eukaryotes, as well their relationship to similar ABC ATPases, using maximum-likelihood analyses. also investigated coevolution different domains eukaryotic attempted account for evolutionary patterns we observed terms available structural data. Based on...

We have examined the level of incorporation 32P into DNA topoisomerase II in vivo chicken lymphoblastoid cells that were fractionated various cell cycle phases by centrifugal elutriation. find is phosphorylated vivo, with being approximately 3.5-fold higher G2 + M fraction than earlier cycle. Our antibody studies revealed antigen exists as a number discrete polypeptide species these cells. Of these, 170-kDa intact 4.5-fold more several antigenic fragments actually comprise bulk at mitosis....

Background Glucocorticoid-mediated inhibition of angiogenesis is important in physiology, pathophysiology and therapy. However, the mechanisms through which glucocorticoids inhibit growth new blood vessels have not been established. This study addresses hypothesis that physiological levels by directly preventing tube formation endothelial cells. Methodology/Principal Findings Cultured human umbilical vein (HUVEC) aortic (HAoEC) cells were used to determine influence on tube-like structure...

Background A complex relationship exists between diet and sleep but despite its impact on human health, this remains uncharacterized poorly understood. Drosophila melanogaster is an important model for the study of metabolism behaviour, however effect upon largely unaddressed. Methodology/Principal Findings Using automated behavioural monitoring, a capillary feeding assay pharmacological treatments, we examined dietary yeast sucrose sleep-wake behaviour three consecutive days. We found that...

The precise mechanism of chromosome condensation and decondensation remains a mystery, despite progress over the last 20 years aimed at identifying components essential to mitotic compaction genome. In this study, we analyse localization role CAP-D2 non-SMC condensin subunit its effect on stability complex. We demonstrate that complex exists in Drosophila embryos, containing CAP-D2, anticipated SMC2 SMC4 proteins, CAP-H/Barren CAP-G (non-SMC) subunits. show is nuclear protein throughout...

Previously, we discovered a conserved interaction between RB proteins and the Condensin II protein CAP-D3 that is important for ensuring uniform chromatin condensation during mitotic prophase. The Drosophila melanogaster homologs RBF1 dCAP-D3 co-localize on non-dividing polytene chromatin, suggesting existence of shared, non-mitotic role these two proteins. Here, show absence alters expression many same genes in larvae adult flies. Strikingly, most affected by loss are not classic cell cycle...

The cell cycle is widely known to be regulated by networks of phosphorylation and ubiquitin-directed proteolysis. Here, we describe IX-14/invadolysin, a novel metalloprotease present only in metazoa, whose activity appears essential for mitotic progression. Mitotic neuroblasts Drosophila melanogaster IX-14 mutant larvae exhibit increased levels nuclear envelope proteins, monopolar asymmetric spindles, chromosomes that appear hypercondensed length with surrounding halo loosely condensed...

Abstract The condensin complex has been implicated in the higher-order organization of mitotic chromosomes a host model eukaryotes from yeasts to flies and vertebrates. Although paradoxically appear condense mutants, chromatids are not properly resolved, resulting chromosome segregation defects during anaphase. We have examined role different components interphase chromatin function by examining effects various mutations on position-effect variegation Drosophila melanogaster. Surprisingly,...

BackgroundThe MCM2-7 proteins are crucial components of the pre replication complex (preRC) in eukaryotes. Since they significantly more abundant than other preRC components, we were interested determining whether entire cellular content was necessary for DNA vivo.Methodology/Principle FindingsWe performed a systematic depletion MCM Drosophila S2 cells using dsRNA-interference. Reducing MCM2-6 levels by >95–99% had no significant effect on cell cycle distribution or viability. Depletion MCM7...

Two alleles of the Drosophila melanogaster Rfc4(DmRfc4) gene, which encodes subunit 4 replication factor C (RFC) complex, cause striking defects in mitotic chromosome cohesion and condensation. These mutations produce larval phenotypes consistent with a role DNA but also result chromosomal appearing either as premature condensation-like or precocious sister chromatid separation figures. Though DmRFC4 protein localizes to all replicating nuclei, it is dispersed from chromatin mitosis. Thus...

Invadolysin is a metalloprotease conserved in many different organisms, previously shown to be essential Drosophila with roles cell division and migration. The gene seems ubiquitously expressed four distinct splice variants have been identified human cells but not most other species examined. Immunofluorescent detection of invadolysin cultured reveals the protein associated surface lipid droplets. By means subcellular fractionation, we independently confirmed association We thus identify as...

The rheumatic diseases are characterized by the production of autoantibodies that usually directed againts components cell nucleus. In this communication, we describe recognize DNA topoisomerase II (anti-topoII) present in serum a patient with systemic lupus erythematosus. Several lines evidence indicate antibody recognizes II. First, it binds to native enzyme soluble extracts prepared from isolated chromosomes and effectively depletes such active enzyme. Second, immunoblots mitotic...