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1.
Individual chromosomes are not directly visible within the interphase nuclei of most somatic cells; they can only be seen during mitosis. We have developed a method that allows DNA strands to be observed directly in living cells, and we use it to analyze how mitotic chromosomes form. A fluorescent analogue (e.g., Cy5-dUTP) of the natural precursor, thymidine triphosphate, is introduced into cells, which are then grown on the heated stage of a confocal microscope. The analogue is incorporated by the endogenous enzymes into DNA. As the mechanisms for recognizing and removing the unusual residues do not prevent subsequent progress around the cell cycle, the now fluorescent DNA strands can be followed as they assemble into chromosomes, and segregate to daughters and granddaughters. Movies of such strands in living cells suggest that chromosome axes follow simple recognizable paths through their territories during G2 phase, and that late replicating regions maintain their relative positions as prophase chromosomes form. Quantitative analysis confirms that individual regions move little during this stage of chromosome condensation. As a result, the gross structure of an interphase chromosome territory is directly related to that of the prophase chromosome. 相似文献
2.
Visualization of early chromosome condensation: a hierarchical folding, axial glue model of chromosome structure 总被引:10,自引:0,他引:10
Kireeva N Lakonishok M Kireev I Hirano T Belmont AS 《The Journal of cell biology》2004,166(6):775-785
Current models of mitotic chromosome structure are based largely on the examination of maximally condensed metaphase chromosomes. Here, we test these models by correlating the distribution of two scaffold components with the appearance of prophase chromosome folding intermediates. We confirm an axial distribution of topoisomerase IIalpha and the condensin subunit, structural maintenance of chromosomes 2 (SMC2), in unextracted metaphase chromosomes, with SMC2 localizing to a 150-200-nm-diameter central core. In contrast to predictions of radial loop/scaffold models, this axial distribution does not appear until late prophase, after formation of uniformly condensed middle prophase chromosomes. Instead, SMC2 associates throughout early and middle prophase chromatids, frequently forming foci over the chromosome exterior. Early prophase condensation occurs through folding of large-scale chromatin fibers into condensed masses. These resolve into linear, 200-300-nm-diameter middle prophase chromatids that double in diameter by late prophase. We propose a unified model of chromosome structure in which hierarchical levels of chromatin folding are stabilized late in mitosis by an axial "glue." 相似文献
3.
Eukaryotic chromosomes are organized into two large and distinct domains, euchromatin and heterochromatin, which are cytologically
characterized by different degrees of chromatin compaction during interphase/prophase and by post-synthesis modifications
of histones and DNA methylation. Typically, heterochromatin remains condensed during the entire cell cycle whereas euchromatin
is decondensed at interphase. However, a fraction of the euchromatin can also remain condensed during interphase and appears
as early condensing prophase chromatin. 5S and 45S rDNA sites and telomere DNA were used to characterize these regions in
metaphase and interphase nuclei. We investigated the chromosomal distribution of modified histones and methylated DNA in the
early and late condensing prophase chromatin of two species with clear differentiation between these domains. Both species,
Costus spiralis and Eleutherine bulbosa, additionally have a small amount of classical heterochromatin detected by CMA/DAPI staining. The distribution of H4 acetylated
at lysine 5 (H4K5ac), H3 phosphorylated at serine 10 (H3S10ph), H3 dimethylated at lysine 4 or 9 (H3K4me2, H3K9me2), and 5-methylcytosine
was compared in metaphase, prophase, and interphase cells by immunostaining with specific antibodies. In both species, the
late condensing prophase chromatin was highly enriched in H4K5ac and H3K4me2 whereas the early condensing chromatin was very
poor in these marks. H3K9me2 was apparently uniformly distributed along the chromosomes whereas the early condensing chromatin
was slightly enriched in 5-methylcytosine. Signals of H3S10ph were restricted to the pericentromeric region of all chromosomes.
Notably, none of these marks distinguished classical heterochromatin from the early condensing euchromatin. It is suggested
that the early condensing chromatin is an intermediate type between classical heterochromatin and euchromatin. 相似文献
4.
Whether chromosomes maintain their nuclear positions during interphase and from one cell cycle to the next has been controversially discussed. To address this question, we performed long-term live-cell studies using a HeLa cell line with GFP-tagged chromatin. Positional changes of the intensity gravity centers of fluorescently labeled chromosome territories (CTs) on the order of several microm were observed in early G1, suggesting a role of CT mobility in establishing interphase nuclear architecture. Thereafter, the positions were highly constrained within a range of approximately 1 microm until the end of G2. To analyze possible changes of chromosome arrangements from one cell cycle to the next, nuclei were photobleached in G2 maintaining a contiguous zone of unbleached chromatin at one nuclear pole. This zone was stably preserved until the onset of prophase, whereas the contiguity of unbleached chromosome segments was lost to a variable extent, when the metaphase plate was formed. Accordingly, chromatin patterns observed in daughter nuclei differed significantly from the mother cell nucleus. We conclude that CT arrangements were stably maintained from mid G1 to late G2/early prophase, whereas major changes of CT neighborhoods occurred from one cell cycle to the next. The variability of CT neighborhoods during clonal growth was further confirmed by chromosome painting experiments. 相似文献
5.
Giorgio Bernardi 《PloS one》2015,10(11)
How the same DNA sequences can function in the three-dimensional architecture of interphase nucleus, fold in the very compact structure of metaphase chromosomes and go precisely back to the original interphase architecture in the following cell cycle remains an unresolved question to this day. The strategy used to address this issue was to analyze the correlations between chromosome architecture and the compositional patterns of DNA sequences spanning a size range from a few hundreds to a few thousands Kilobases. This is a critical range that encompasses isochores, interphase chromatin domains and boundaries, and chromosomal bands. The solution rests on the following key points: 1) the transition from the looped domains and sub-domains of interphase chromatin to the 30-nm fiber loops of early prophase chromosomes goes through the unfolding into an extended chromatin structure (probably a 10-nm “beads-on-a-string” structure); 2) the architectural proteins of interphase chromatin, such as CTCF and cohesin sub-units, are retained in mitosis and are part of the discontinuous protein scaffold of mitotic chromosomes; 3) the conservation of the link between architectural proteins and their binding sites on DNA through the cell cycle explains the “mitotic memory” of interphase architecture and the reversibility of the interphase to mitosis process. The results presented here also lead to a general conclusion which concerns the existence of correlations between the isochore organization of the genome and the architecture of chromosomes from interphase to metaphase. 相似文献
6.
The phases of mitosis were examined in the columnar cells at the base of duodenal crypts in adult male mice given an intravenous injection of 3H-thymidine and sacrificed 20 min later. The duodenum was fixed by immersion into glutaraldehyde-formaldehyde, and the cells were examined in the electron microscope, with or without processing for radioautography. Interphase nuclei are characterized by the distribution of chromatin; aside from the cortical chromatin spread along nuclear envelope and nucleolus, there are chromatin accumulations that belong mainly in two different classes: 1) numerous chromatin "specks" ranging in size from about 5 to 70 nm and averaging 47 nm; 2) a few roughly circular or elongated chromatin "packets" measuring from 70 to 230 nm. Early prophase nuclei differ mainly by a large increase in the number of chromatin packets to 20-30 or more per nuclear profile; their average diameter is 128 nm. During mid-prophase, the chromatin packets enlarge gradually to an average 221 nm diameter. Between mid- and late prophase, there is a further increase in diameter to 679 nm. At metaphase, the packets take on the appearance of mature chromosomes, and their diameter increases to 767 nm. At anaphase, daughter chromosomes migrate to each pole, where they fuse into a compact chromatin mass. At telophase, nucleoplasmic areas progressively enlarge within the chromatin mass and separate strands of chromatin, which gradually become segmented into chromatin clumps. Counts of mitotic cells show a high proportion of prophase and telophase nuclei. Calculation from the counts yields the duration of the phases, that is, 5.6, 0.2, 0.1, and 1.6 hr, respectively, for pro-, meta-, ana-, and telophase. Finally, radioautography 20 min after 3H-thymidine injection shows labeling in 54% of the interphase nuclei, 85% of early prophase nuclei, and 73% of mid-prophase nuclei, while there is no label in late prophase, metaphase, anaphase and telophase nuclei. In confirmation of previous light microscopic work, the S stage of the cycle begins when a cell is in interphase and continues through the early prophase and part of mid-prophase. Moreover, the main sites of DNA synthesis are the chromatin specks during interphase and the cortical chromatin during early and mid-prophase. The chromosome condensation taking place in the meantime may be separated into two main steps: 1) a slow, moderate condensation of the chromatin packets during early and mid-prophase and 2) a rapid, pronounced one during late prophase and prometaphase when the packets become chromosomes. 相似文献
7.
Steffensen S Coelho PA Cobbe N Vass S Costa M Hassan B Prokopenko SN Bellen H Heck MM Sunkel CE 《Current biology : CB》2001,11(5):295-307
BACKGROUND: Faithful segregation of the genome during mitosis requires interphase chromatin to be condensed into well-defined chromosomes. Chromosome condensation involves a multiprotein complex known as condensin that associates with chromatin early in prophase. Until now, genetic analysis of SMC subunits of the condensin complex in higher eukaryotic cells has not been performed, and consequently the detailed contribution of different subunits to the formation of mitotic chromosome morphology is poorly understood. RESULTS: We show that the SMC4 subunit of condensin is encoded by the essential gluon locus in Drosophila. DmSMC4 contains all the conserved domains present in other members of the structural-maintenance-of-chromosomes protein family. DmSMC4 is both nuclear and cytoplasmic during interphase, concentrates on chromatin during prophase, and localizes to the axial chromosome core at metaphase and anaphase. During decondensation in telophase, most of the DmSMC4 leaves the chromosomes. An examination of gluon mutations indicates that SMC4 is required for chromosome condensation and segregation during different developmental stages. A detailed analysis of mitotic chromosome structure in mutant cells indicates that although the longitudinal axis can be shortened normally, sister chromatid resolution is strikingly disrupted. This phenotype then leads to severe chromosome segregation defects, chromosome breakage, and apoptosis. CONCLUSIONS: Our results demonstrate that SMC4 is critically important for the resolution of sister chromatids during mitosis prior to anaphase onset. 相似文献
8.
9.
Kurchashova S. Yu. Prusov A. N. Gulak P. V. Kireev I. I. Polyakov V. Yu. 《Russian Journal of Developmental Biology》2000,31(6):362-371
In the interphase nucleus, the chromatin associated with the nuclear envelope is represented by a layer of anchorosomes, granules with a diameter of 20–25 nm. Biochemically, the fraction of chromatin directly associated with the nuclear envelope is characterized by resistance against decondensing influences, a low level of DNA methylation, and presence of specific acid-soluble proteins. However, the mechanisms lying at the base of chromatin-nuclear envelope interaction have been insufficiently studied. Specifically, it is unknown whether anchorosomes are constant structures or subject to reversible disassembly, when the contacts between chromatin and nuclear envelope are destroyed. We obtained immune serum recognizing a 68 kDa protein from the nuclear envelopes fraction and studied the localization of this protein in interphase and mitotic cells. We show that this protein, present in the NE/anchorosomal fraction, does not remain bound with chromosomes during mitosis. It dissociates from chromosomes at the beginning of the prophase and then can be identified again at the periphery of the newly forming nuclei in the telophase. 相似文献
10.
Chromatin associated with the nuclear envelope appears in the interphase nuclei as a layer of anchorosomes, granules 20-25 nm in diameter. The fraction of chromatin directly associated with the nuclear envelope is resistant to decondensation, shows a low level of DNA methylation, and contains specific acid-soluble proteins. However, mechanisms underlying the interaction of chromatin with the nuclear envelope are not fully understood. Specifically, it is not known whether anchorosomes are permanent structures or if they undergo reversible disassembly during mitosis, when contacts between chromatin and the nuclear envelope are destroyed. We obtained immune serum recognizing a 68 kDa protein from the nuclear envelopes fraction and studied the localization of this protein in interphase and mitotic cells. We show that this protein present in the NE/anchorosomal fraction does not remain bound with chromosomes during mitosis. It dissociates from chromosomes at the beginning of the prophase and then can be identified again at the periphery of the newly forming nuclei in the telophase. 相似文献
11.
The organization and the mechanisms of condensation of mitotic chromosomes remain unsolved despite many decades of efforts. The lack of resolution, tight compaction, and the absence of function-specific chromatin labels have been the key technical obstacles. The correlation between DNA sequence composition and its contribution to the chromosome-scale structure has been suggested before; it is unclear though if all DNA sequences equally participate in intra- or inter-chromatin or DNA-protein interactions that lead to formation of mitotic chromosomes and if their mitotic positions are reproduced radially. Using high-resolution fluorescence microscopy of live or minimally perturbed, fixed chromosomes in Drosophila embryonic cultures or tissues expressing MSL3-GFP fusion protein, we studied positioning of specific MSL3-binding sites. Actively transcribed, dosage compensated Drosophila genes are distributed along the euchromatic arm of the male X chromosome. Several novel features of mitotic chromosomes have been observed. MSL3-GFP is always found at the periphery of mitotic chromosomes, suggesting that active, dosage compensated genes are also found at the periphery of mitotic chromosomes. Furthermore, radial distribution of chromatin loci on mitotic chromosomes was found to be correlated with their functional activity as judged by core histone modifications. Histone modifications specific to active chromatin were found peripheral with respect to silent chromatin. MSL3-GFP-labeled chromatin loci become peripheral starting in late prophase. In early prophase, dosage compensated chromatin regions traverse the entire width of chromosomes. These findings suggest large-scale internal rearrangements within chromosomes during the prophase condensation step, arguing against consecutive coiling models. Our results suggest that the organization of mitotic chromosomes is reproducible not only longitudinally, as demonstrated by chromosome-specific banding patterns, but also radially. Specific MSL3-binding sites, the majority of which have been demonstrated earlier to be dosage compensated DNA sequences, located on the X chromosomes, and actively transcribed in interphase, are positioned at the periphery of mitotic chromosomes. This potentially describes a connection between the DNA/protein content of chromatin loci and their contribution to mitotic chromosome structure. Live high-resolution observations of consecutive condensation states in MSL3-GFP expressing cells could provide additional details regarding the condensation mechanisms. 相似文献
12.
Reversible chromosome condensation induced in Drosophila embryos by anoxia: visualization of interphase nuclear organization 总被引:19,自引:9,他引:10
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We have studied the morphology of nuclei in Drosophila embryos during the syncytial blastoderm stages. Nuclei in living embryos were viewed with differential interference-contrast optics; in addition, both isolated nuclei and fixed preparations of whole embryos were examined after staining with a DNA-specific fluorescent dye. We find that: (a) The nuclear volumes increase dramatically during interphase and then decrease during prophase of each nuclear cycle, with the magnitude of the nuclear volume increase being greatest for those cycles with the shortest interphase. (b) Oxygen deprivation of embryos produces a rapid developmental arrest that is reversible upon reaeration. During this arrest, interphase chromosomes condense against the nuclear envelope and the nuclear volumes increase dramatically. In these nuclei, individual chromosomes are clearly visible, and each condensed chromosome can be seen to adhere along its entire length to the inner surface of the swollen nuclear envelope, leaving the lumen of the nucleus devoid of DNA. (c) In each interphase nucleus the chromosomes are oriented in the "telophase configuration," with all centromeres and all telomeres at opposite poles of the nucleus; all nuclei at the embryo periphery (with the exception of the pole cell nuclei) are oriented with their centromeric poles pointing to the embryo exterior. 相似文献
13.
Masatoki Taga Minoru Murata Hans D. VanEtten 《Fungal genetics and biology : FG & B》1999,26(3):169-177
Supernumerary chromosomes, termed "conditionally dispensable" (CD) chromosomes, are known in Nectria haematococca. Because these CD chromosomes had been revealed solely by pulsed-field gel electrophoresis, their morphological properties were unknown. In this study, we visualized a 1.6-Mb CD chromosome of this fungus by three different types of fluorescence in situ hybridization. The CD chromosome at mitotic metaphase was similar in its appearance to the other chromosomes in the genome. Heterochromatic condensation was not distinct in the CD chromosome, suggesting that it is primarily euchromatic. It was also evident that the CD chromosome is unique and not a duplicate of other chromosomes in the genome. At interphase and prophase, the CD chromosome was not dispersed throughout the nucleus, but occupied a limited domain. Occasionally, occurrence of two distinct unattached copies of the CD chromosome were observed during interphase and metaphase. 相似文献
14.
Proteome analysis of human metaphase chromosomes 总被引:7,自引:0,他引:7
Uchiyama S Kobayashi S Takata H Ishihara T Hori N Higashi T Hayashihara K Sone T Higo D Nirasawa T Takao T Matsunaga S Fukui K 《The Journal of biological chemistry》2005,280(17):16994-17004
DNA is packaged as chromatin in the interphase nucleus. During mitosis, chromatin fibers are highly condensed to form metaphase chromosomes, which ensure equal segregation of replicated chromosomal DNA into the daughter cells. Despite >1 century of research on metaphase chromosomes, information regarding the higher order structure of metaphase chromosomes is limited, and it is still not clear which proteins are involved in further folding of the chromatin fiber into metaphase chromosomes. To obtain a global view of the chromosomal proteins, we performed proteome analyses on three types of isolated human metaphase chromosomes. We first show the results from comparative proteome analyses of two types of isolated human metaphase chromosomes that have been frequently used in biochemical and morphological analyses. 209 proteins were quantitatively identified and classified into six groups on the basis of their known interphase localization. Furthermore, a list of 107 proteins was obtained from the proteome analyses of highly purified metaphase chromosomes, the majority of which are essential for chromosome structure and function. Based on the information obtained on these proteins and on their localizations during mitosis as assessed by immunostaining, we present a four-layer model of metaphase chromosomes. According to this model, the chromosomal proteins have been newly classified into each of four groups: chromosome coating proteins, chromosome peripheral proteins, chromosome structural proteins, and chromosome fibrous proteins. This analysis represents the first compositional view of human metaphase chromosomes and provides a protein framework for future research on this topic. 相似文献
15.
In addition to genetic information, mitotic chromosomes transmit essential components for nuclear assembly and function in
a new cell cycle. A specialized chromosome domain, called the perichromosomal layer, perichromosomal sheath, chromosomal coat,
or chromosome surface domain, contains proteins required for a variety of cellular processes, including the synthesis of messenger
RNA, assembly of ribosomes, repair of DNA double-strand breaks, telomere maintenance, and apoptosis regulation. The layer
also contains many proteins of unknown function and is a major target in autoimmune disease. Perichromosomal proteins are
found along the entire length of chromosomes, excluding centromeres, where sister chromatids are paired and spindle microtubules
attach. Targeting of proteins to the perichromosomal layer occurs primarily during prophase, and they generally remain associated
until telophase. During interphase, perichromosomal proteins localize to nucleoli, the nuclear envelope, nucleoplasm, heterochromatin,
centromeres, telomeres, and/or the cytoplasm. It has been suggested that the perichromosomal layer may contribute to chromosome
structure, as several of the associated proteins have functions in chromatin remodeling during interphase. We review the identified
proteins associated with this chromosome domain and briefly discuss their known functions during interphase and mitosis. 相似文献
16.
电镜原位观察结合图象分析研究了多头绒泡菌Physarum polycephalum Schw间期细胞核和中期染色体中银染蛋白的形状、大小和分布。结果看到,银染蛋白主要呈颗粒状存在于间期细胞核和中期染色体中。银粒的大小不一,分布不均匀。间期细胞核中存在众多直径在5~15nm的银粒,其中10nm以上的较大银粒主要分布于核仁,集缩染色质和核基质部分10nm以上银粒不多。中期细胞核内10nm以上的较大银粒主要分布于染色体中。染色体中除含有一些较大银粒外,多数银粒的直径为5~10nm。本文结果提示,构成染色体骨架的嗜银蛋白可能来自间期细胞核的染色质、核基质和核仁。 相似文献
17.
Strokov AA 《Genetika》2007,43(11):1468-1477
The qualitative and quantitative changes in molecular chromatin structures during the meiotic prophase I were studied. The following patterns were discovered: (1) unlike somatic cells, the syntheses of total histone and DNA and its integration into the chromatin occur independently and asynchronously: DNA replication is completed by the interphase, whereas the synthesis of histone and its integration into the chromatin continue to late meiotic prophase I, and (2) individual histone fractions are synthesized and integrated into the chromatin during meiotic prophase independently and asynchronously. Chromatin hydrolysis with nucleases DNI, STN, and SI demonstrated considerable differences in the hydrolysis products obtained at different stages of the meiotic prophase I; presumably, this reflects the differences between the structures of initial chromatin at different stages of the meiotic prophase I. 相似文献
18.
C. P. Pussell 《Genetica》1984,62(3):193-201
A model for the arrangement of chromosomes in interphase nuclei is proposed. The model assumes that interphase chromosomes have a Rabl orientation (a relic telophase arrangement). During interphase and prophase telomeres are attached to the nuclear envelope often in pairs. The association of telomeres, homologous or nonhomologous, is based on similarity of arm lengths and occurs at the time the nuclear envelope reforms. At this stage arm lengths will vary to some extent due to the amount of uncoiling etc. The sequence of chromosomes resulting from telomere-telomere pairing may vary among cells, but the number of arrangements will be restricted by arm length similarities.The ramifications of this model on melotic pairing, the constant attachment of chromosomes to some structure throughout the cell cycle, the distribution of genes within nuclei, and chromosome evolution are raised. 相似文献
19.
A. A. Strokov 《Russian Journal of Genetics》2007,43(11):1227-1235
The qualitative and quantitative changes in molecular chromatin structures during the meiotic prophase I were studied. The following patterns were discovered: (1) unlike somatic cells, the syntheses of total histone and DNA and its integration into the chromatin occur independently and asynchronously: DNA replication is completed by the interphase, whereas the synthesis of histone and its integration into the chromatin continue to late meiotic prophase I, and (2) individual histone fractions are synthesized and integrated into the chromatin during meiotic prophase independently and asynchronously. Chromatin hydrolysis with nucleases DNI, STN, and SI demonstrated considerable differences in the hydrolysis products obtained at different stages of the meiotic prophase I; presumably, this reflects the differences between the structures of initial chromatin at different stages of the meiotic prophase I. 相似文献
20.
Ashwathi Rajeevan Riya Keshri Sukriti Kapoor Sachin Kotak 《Molecular biology of the cell》2020,31(22):2437
NuMA is an abundant long coiled-coil protein that plays a prominent role in spindle organization during mitosis. In interphase, NuMA is localized to the nucleus and hypothesized to control gene expression and chromatin organization. However, because of the prominent mitotic phenotype upon NuMA loss, its precise function in the interphase nucleus remains elusive. Here, we report that NuMA is associated with chromatin in interphase and prophase but released upon nuclear envelope breakdown (NEBD) by the action of Cdk1. We uncover that NuMA directly interacts with DNA via evolutionarily conserved sequences in its C-terminus. Notably, the expression of the DNA-binding–deficient mutant of NuMA affects chromatin decondensation at the mitotic exit, and nuclear shape in interphase. We show that the nuclear shape defects observed upon mutant NuMA expression are due to its potential to polymerize into higher-order fibrillar structures. Overall, this work establishes the spindle-independent function of NuMA in choreographing proper chromatin decompaction and nuclear shape by directly associating with the DNA. 相似文献