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Results for “chromosome assembly”

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At least 19 recordsLinked to original sources
PubMed1993

Topoisomerase II does not play a scaffolding role in the organization of mitotic chromosomes assembled in 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.

Amino Acid Sequence↗
PubMed2004

Distinct functions of condensin I and II in mitotic chromosome assembly.

Condensin is a protein complex associated with mitotic chromosomes that has been implicated in chromosome condensation. In vertebrates, two types of condensin complexes have recently been identified, called condensin I and II. Here, we show that in mammalian cells condensin II associates with chromatin in prophase, in contrast to condensin I which is cytoplasmic and can thus interact with chromosomes only after nuclear envelope breakdown. RNA interference experiments in conjunction with imaging of live and fixed cells revealed that condensin II is required for chromosome condensation in early prophase, whereas condensin I appears to be dispensable at this stage. By contrast, condensin I is required for the complete dissociation of cohesin from chromosome arms, for chromosome shortening and for normal timing of progression through prometaphase and metaphase, whereas normal condensin II levels are dispensable for these processes. After depletion of both condensin complexes, the onset of chromosome condensation is delayed until the end of prophase, but is then initiated rapidly before nuclear envelope breakdown. These results reveal that condensin II and I associate with chromosomes sequentially and have distinct functions in mitotic chromosome assembly.

Cell Cycle Proteins↗
PubMed2004

Spatial and temporal regulation of Condensins I and II in mitotic chromosome assembly in human cells.

Two different condensin complexes make distinct contributions to metaphase chromosome architecture in vertebrate cells. We show here that the spatial and temporal distributions of condensins I and II are differentially regulated during the cell cycle in HeLa cells. Condensin II is predominantly nuclear during interphase and contributes to early stages of chromosome assembly in prophase. In contrast, condensin I is sequestered in the cytoplasm from interphase through prophase and gains access to chromosomes only after the nuclear envelope breaks down in prometaphase. The two complexes alternate along the axis of metaphase chromatids, but they are arranged into a unique geometry at the centromere/kinetochore region, with condensin II enriched near the inner kinetochore plate. This region-specific distribution of condensins I and II is severely disrupted upon depletion of Aurora B, although their association with the chromosome arm is not. Depletion of condensin subunits causes defects in kinetochore structure and function, leading to aberrant chromosome alignment and segregation. Our results suggest that the two condensin complexes act sequentially to initiate the assembly of mitotic chromosomes and that their specialized distribution at the centromere/kinetochore region may play a crucial role in placing sister kinetochores into the back-to-back orientation.

Adenosine Triphosphatases↗
PubMed2003

A two-step scaffolding model for mitotic chromosome assembly.

Topoisomerase IIalpha (topoIIalpha) and 13S condensin are both required for mitotic chromosome assembly. Here we show that they constitute the two main components of the chromosomal scaffold on histone-depleted chromosomes. The structural stability and chromosomal shape of the scaffolding toward harsh extraction procedures are shown to be mediated by ATP or its nonhydrolyzable analogs, but not ADP. TopoIIalpha and 13S condensin components immunolocalize to a radially restricted, longitudinal scaffolding in native-like chromosomes. Double staining for topoIIalpha and condensin generates a barber pole appearance of the scaffolding, where topoIIalpha- and condensin-enriched "beads" alternate; this structure appears to be generated by two juxtaposed, or coiled, chains. Cell cycle studies establish that 13S condensin appears not to be involved in the assembly of prophase chromatids; they lack this complex but contain a topoIIalpha-defined (-mediated?) scaffolding. Condensin associates only during the pro- to metaphase transition. This two-step assembly process is proposed to generate the barber pole appearance of the native-like scaffolding.

Adenosine Diphosphate↗
PubMed2001

pEg2 aurora-A kinase, histone H3 phosphorylation, and chromosome assembly in Xenopus egg extract.

In eukaryotes cell division is accompanied by phosphorylation of histone H3 at serine 10. In this work we have studied the kinase activity responsible for this histone H3 modification by using cell-free extracts prepared from Xenopus eggs. We have found that the Xenopus aurora-A kinase pEg2, immunoprecipitated from the extract, is able to phosphorylate specifically histone H3 at serine 10. The enzyme is incorporated into chromatin during in vitro chromosome assembly, and the kinetics of this incorporation parallels that of histone H3 phosphorylation. Recombinant pEg2 phosphorylates efficiently histone H3 at serine 10 in reconstituted nucleosomes and in sperm nuclei decondensed in heated extracts. These data identify pEg2 as a good candidate for mitotic histone H3 kinase. However, immunodepletion of pEg2 does not interfere with the chromosome assembly properties of the extract nor with the pattern of H3 phosphorylation, suggesting the existence of multiple kinases involved in this H3 modification in Xenopus eggs. This hypothesis is supported by in gel activity assay experiments using extracts from Saccharomyces cerevisiae.

Animals↗
PubMed2003

The highly conserved Ndc80 complex is required for kinetochore assembly, chromosome congression, and spindle checkpoint activity.

We show that the Xenopus homologs of Ndc80/Tid3/HEC1 (xNdc80) and Nuf2/MPP1/Him-10 (xNuf2) proteins physically interact in a 190-kD complex that associates with the outer kinetochore from prometaphase through anaphase. Injecting function-blocking antibodies to either xNdc80 or xNuf2 into XTC cells caused premature exit from mitosis without detectable chromosome congression or anaphase movements. Injected cells did not arrest in response to microtubule drugs, showing that the complex is required for the spindle checkpoint. Kinetochores assembled in Xenopus extracts after immunodepletion of the complex did not contain xRod, xZw10, xP150 glued (Dynactin), xMad1, xMad2, xBub1, and xBub3, demonstrating that the xNdc80 complex is required for functional kinetochore assembly. In contrast, function-blocking antibodies did not affect the localization of other kinetochore proteins when added to extracts containing previously assembled kinetochores. These extracts with intact kinetochores were deficient in checkpoint signaling, suggesting that the Ndc80 complex participates in the spindle checkpoint. We also demonstrate that the spindle checkpoint can arrest budding yeast cells lacking Ndc80 or Nuf2, whereas yeast lacking both proteins fail to arrest in mitosis. Systematic deletion of yeast kinetochore genes suggests that the Ndc80 complex has a unique role in spindle checkpoint signaling. We propose that the Ndc80 complex has conserved roles in kinetochore assembly, chromosome congression, and spindle checkpoint signaling.

Anaphase↗
PubMed2004

Chromosome-wide regulation of meiotic crossover formation in Caenorhabditis elegans requires properly assembled chromosome axes.

Most sexually reproducing organisms depend on the regulated formation of crossovers, and the consequent chiasmata, to accomplish successful segregation of homologous chromosomes at the meiosis I division. A robust, chromosome-wide crossover control system limits chromosome pairs to one crossover in most meioses in the nematode Caenorhabditis elegans; this system has been proposed to rely on structural integrity of meiotic chromosome axes. Here, we test this hypothesis using a mutant, him-3(me80), that assembles reduced levels of meiosis-specific axis component HIM-3 along cohesin-containing chromosome axes. Whereas pairing, synapsis, and crossing over are eliminated when HIM-3 is absent, the him-3(me80) mutant supports assembly of synaptonemal complex protein SYP-1 along some paired chromosomes, resulting in partial competence for chiasma formation. We present both genetic and cytological evidence indicating that the him-3(me80) mutation leads to an increased incidence of meiotic products with two crossovers. These results indicate that limiting the amount of a major axis component results in a reduced capacity to communicate the presence of a (nascent) crossover and/or to discourage others in response.

Animals↗
PubMed1988

Visualization of chromosome assembly during the S and G2 stages of the cycle and chromosome disassembly during the G1 stage in semithin sections of mouse duodenal crypt cells and other cells.

Previous examination of dividing cells in the isthmus of the mouse pyloric antrum by using semithin (0.5-micron-thick) Epon sections revealed that the prophasic condensation of chromosomes began early in the DNA-synthesizing (S) stage. In order to examine whether the same observation could be made in other proliferating cell types, the crypt base columnar cells in mouse duodenum and the hepatocytes of the rat 48 hr after partial hepatectomy were investigated by morphologic and radioautographic techniques. When crypt base columnar cells were studied in semithin Epon sections, the four phases of mitosis showed the characteristic features described by classical cytologists. Moreover, the proportion of cells in prophase and telophase was high. To relate the mitotic phases to the stages of the cell cycle, the "frequency of labeled mitoses method" provided the duration of the cell cycle, 12.3 hr, and of the S stage, 7.3 hr. From the frequency of the occurrence of mitotic phases, it was estimated that metaphase lasted 0.3 hr and anaphase 0.11 hr, in line with previous estimates. However, the durations of prophase and telophase were long, 5.9 and 1.9 hr, respectively. The whole mitotic process took over 8 hr. From the duration of prophase and cycle stages, it was calculated that 67% of the S stage was occupied by prophasic cells. In fair agreement with this estimate, 68% of the labeled cells 10 min after a 3H-thymidine injection were found to be in prophase. In regenerating hepatocytes, the morphological features and frequency of prophase and telophase cells were similar to those observed in duodenal crypt cells. While the cycle time was not measured and, therefore, the duration of cycle stages and mitotic phases could not be estimated, it is likely that their duration would be of the same order of magnitude. In conclusion, the mitotic process in duodenal crypt cells takes over 8 hr. Moreover, the crypt cells, like antral isthmal cells, show features of early prophase soon after they enter the S stage of the cycle.

Animals↗
PubMed2006

The X and Y chromosomes assemble into H2A.Z-containing [corrected] facultative heterochromatin [corrected] following meiosis.

Spermatogenesis is a complex sequential process that converts mitotically dividing spermatogonia stem cells into differentiated haploid spermatozoa. Not surprisingly, this process involves dramatic nuclear and chromatin restructuring events, but the nature of these changes are poorly understood. Here, we linked the appearance and nuclear localization of the essential histone variant H2A.Z with key steps during mouse spermatogenesis. H2A.Z cannot be detected during the early stages of spermatogenesis, when the bulk of X-linked genes are transcribed, but its expression begins to increase at pachytene, when meiotic sex chromosome inactivation (MSCI) occurs, peaking at the round spermatid stage. Strikingly, when H2A.Z is present, there is a dynamic nuclear relocalization of heterochromatic marks (HP1beta and H3 di- and tri-methyl K9), which become concentrated at chromocenters and the inactive XY body, implying that H2A.Z may substitute for the function of these marks in euchromatin. We also show that the X and the Y chromosome are assembled into facultative heterochromatic structures postmeiotically that are enriched with H2A.Z, thereby replacing macroH2A. This indicates that XY silencing continues following MSCI. These results provide new insights into the large-scale changes in the composition and organization of chromatin associated with spermatogenesis and argue that H2A.Z has a unique role in maintaining sex chromosomes in a repressed state.

Animals↗
PubMed1998

Depletion of a Drosophila homolog of yeast Sup35p disrupts spindle assembly, chromosome segregation, and cytokinesis during male meiosis.

In the course of a genetic screen for male-sterile mutations in Drosophila affecting chromosome segregation during the meiotic divisions in spermatocytes, we identified the mutation dsup35(63D). Examination of mutant testes showed that chromosome misbehavior was a consequence of major disruptions in meiotic spindle assembly. These perturbations included problems in aster formation, separation, and migration around the nuclear envelope; aberrations in spindle organization and integrity; and disappearance of the ana/telophase central spindle, which in turn disrupts cytokinesis. The dsup35(63D) mutation is caused by a P element insertion that affects, specifically in the testis, the expression of a gene (dsup35) encoding the Drosophila homolog of the yeast Sup35p and Xenopus eRF3 proteins. These proteins are involved in the termination of polypeptide synthesis on ribosomes, but previous studies have suggested that Sup35p and closely related proteins of the same family also interact directly with microtubules. An affinity-purified antibody directed against the product of the dsup35 gene was prepared; interestingly, this antibody specifically labels primary spermatocytes in one or two discrete foci of unknown structure within the nucleoplasm. We discuss how depletion of the dsup35 gene product in spermatocytes might lead to the global disruptions in meiotic spindle assembly seen in mutant spermatocytes.

Amino Acid Sequence↗
PubMed1991

Chromosome assembly in vitro: topoisomerase II is required for condensation.

The role of topoisomerase II (topo II) in chromosome condensation was studied in a mitotic extract derived from Xenopus eggs by specific immunodepletion. HeLa nuclei, which have a high complement of endogenous topo II, are converted to mitotic chromosomes in the topo II-depleted extract equally well as in the control. Chicken erythrocyte nuclei, however, which have a very low content of topo II, do not convert to condensed chromosomes in the depleted extract, although their condensation is normal upon addition of purified topo II. Dosage experiments support the possible notion of a structural involvement of topo II in chromosome condensation. In the topo II-depleted extract the erythrocyte nuclei progress to precondensation chromosomes, which lack the nuclear membrane-lamina complex and consist of a cluster of swollen chromatids.

Animals↗
PubMed2005

The canine sarcoglycan delta gene: BAC clone contig assembly, chromosome assignment and interrogation as a candidate gene for dilated cardiomyopathy in Dobermann dogs.

Dilated cardiomyopathy (DCM) is a common disease of the myocardium recognized in human, dog and experimental animals. Genetic factors are responsible for a large proportion of cases in humans, and 17 genes with DCM causing mutations have been identified. The genetic origin of DCM in the Dobermann dogs has been suggested, but no disease genes have been identified to date. In this paper, we describe the characterization and evaluation of the canine sarcoglycan delta (SGCD), a gene implicated in DCM in human and hamster. Bacterial artificial chromosomes (BACs) containing the canine SGCD gene were isolated with probes for exon 3 and exons 4-8 and were characterized by Southern blot analysis. BAC end sequences were obtained for four BACs. Three of the BACs overlapped and could be ordered relative to each other and the end sequences of all four BACs could be anchored on the preliminary assembly of the dog genome sequence (www. ensembl.org). One of the BACs of the partial contig was localized by fluorescent in situ hybridization to canine chromosome 4q22, in agreement with the dog genome sequence. Two highly informative polymorphic microsatellite markers in intron 7 of the SGCD gene were identified. In 25 DCM-affected and 13 non DCM-affected dogs seven different haplotypes could be distinguished. However, no association between any of the SGCD variants and the disease locus was apparent.

Animals↗
PubMed2000

The murine SCP3 gene is required for synaptonemal complex assembly, chromosome synapsis, and male fertility.

During meiosis, the homologous chromosomes pair and recombine. An evolutionarily conserved protein structure, the synaptonemal complex (SC), is located along the paired meiotic chromosomes. We have studied the function of a structural component in the axial/lateral element of the SC, the synaptonemal complex protein 3 (SCP3). A null mutation in the SCP3 gene was generated, and we noted that homozygous mutant males were sterile due to massive apoptotic cell death during meiotic prophase. The SCP3-deficient male mice failed to form axial/lateral elements and SCs, and the chromosomes in the mutant spermatocytes did not synapse. While the absence of SCP3 affected the nuclear distribution of DNA repair and recombination proteins (Rad51 and RPA), as well as synaptonemal complex protein 1 (SCP1), a residual chromatin organization remained in the mutant meiotic cells.

Animals↗
PubMed2006

Methods for studying spindle assembly and chromosome condensation in Xenopus egg extracts.

Methods are presented for preparing cytoplasmic extracts from Xenopus laevis eggs and their utilization to reconstitute and monitor events of the cell cycle in vitro. Addition of sperm nuclei to crude extracts and "cycling" of the reaction through interphase and back into metaphase promotes formation of bipolar spindles capable of segregating their duplicated chromosomes. Reactions can be "spun down" onto cover slips for immunofluorescence analysis. High-speed extracts support mitotic chromosome condensation, which can be observed live by fluorescence time-lapse video microscopy. Because of the biochemically accessible nature of the egg extract system, a wide array of biochemical techniques can be combined with spindle and chromosome assembly reactions to evaluate the roles of specific proteins in these processes.

Animals↗
PubMed1994

A heterodimeric coiled-coil protein required for mitotic chromosome condensation in vitro.

We report here a chromosomal protein that plays an essential role in mitotic chromosome condensation in Xenopus egg extracts. Two polypeptides, designated XCAP-C and XCAP-E, were found to associate with each other in the extracts, presumably forming a heterodimer. During chromosome assembly in mitotic extracts, XCAP-C/E was recruited to the chromatin and formed a discrete internal structure within assembled chromosomes. Antibody blocking experiments showed that XCAP-C function is required for both assembly and structural maintenance of mitotic chromosomes in vitro. Deduced amino acid sequences revealed that the two polypeptides share common structural motifs, consisting of an N-terminal NTP-binding domain, two central coiled-coil regions, and a C-terminal conserved domain. These motifs are highly conserved in a protein family, members of which have been identified recently in both prokaryotes and eukaryotes.

Amino Acid Sequence↗
PubMed2006

ORC is necessary at the interphase-to-mitosis transition to recruit cdc2 kinase and disassemble RPA foci.

The origin-recognition complex (ORC) has an essential role in defining DNA replication origins and in chromosome segregation. Recent studies in Drosophila orc2 mutants, and in human cells depleted of ORC2, have suggested that this factor is also implicated in mitotic chromosome assembly. We asked whether ORC was required for M phase chromosome assembly independently of its function in DNA replication. We performed depletion assays and reconstitution experiments in Xenopus egg extracts, in conditions of M phase chromosome assembly coupled or uncoupled from DNA replication. We show that, although ORC is dispensable for mitotic chromosome condensation, it is necessary at the interphase-mitosis transition for proper mitotic chromosome assembly to occur in a reaction not strictly dependent on DNA replication. This function involves the recruitment to chromatin of cdc2 kinase and the chromatin disassembly of interphasic replication protein A (RPA) foci. Furthermore, we show that mutations of RPA at the cdc2 kinase site prevents RPA dissociation from chromatin and impairs mitotic chromosome assembly without affecting DNA replication. Our results support the conclusion that in addition to its role in the assembly of prereplication complexes (pre-RCs), at the G1-S transition, ORC is also required for their disassembly at mitotic entry.

Animals↗
PubMed2026

Chromosome-level assembly and annotation of the Jaguar (Panthera onca) genome.

OBJECTIVES: The Jaguar (Panthera onca) is a large cat species native to the Americas. Despite being successful predators, jaguar populations have declined due to habitat loss. Genome resources can help in conservation efforts as well as in understanding the interesting biology of these Felids. Beside contiguity, a well annotated reference genome provides contextual information for variants that will benefit the design of appropriate conservation programs. DATA DESCRIPTION: We sequenced material from two individuals using a combination of ONT reads and Illumina PE. The resulting nuclear genome assembly has a larger contig N50 (48.04 Mb) compared with the existing annotated chromosome-level assembly published by the DNA Zoo project. Using public Hi-C data, we obtained an improved chromosome-level assembly of the Jaguar genome (mPanOnc3.5) with larger contigs, 99.85% of the sequence assigned to chromosomes and 25,267 protein coding genes annotated. Overall, this improved assembly provides a better reference to study this threatened species.

Animals↗