Figure 5 - uploaded by Igor Kireev
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Chromosome 13 after DHT ( a ) and stained according to the standard GTG procedure ( b ). Size-distribution of chromosome 13 in control preparations ( c ) or after DHT ( d ).
Source publication
In this study we used a novel technique to reveal both longitudinal and transverse differentiation within mammalian mitotic chromosomes. Structural changes in chromosomes that we term 'differential decondensation' were produced in cells that were first incubated in hypotonic medium (15% Hanks' solution), then adapted to normotonic conditions and th...
Contexts in source publication
Context 1
... analysis of chromosome 13 revealed signi¢cant topological correspondence of axial glo- bules to G-bands (Figure 5a, b). At the same time, measurement of the length of this marker chromo- some demonstrated that repeated hypotonic treat- ment caused about 30% elongation compared with control (Figure 5c, d). ...
Context 2
... analysis of chromosome 13 revealed signi¢cant topological correspondence of axial glo- bules to G-bands (Figure 5a, b). At the same time, measurement of the length of this marker chromo- some demonstrated that repeated hypotonic treat- ment caused about 30% elongation compared with control (Figure 5c, d). We conclude that these treatments caused not only transverse but also longitudinal decondensation of chromosomes. ...
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Citations
... It is supposed that a certain amount of constitutive heterochromatin is essential in multicellular organisms at two levels of organization: chromosomal and nuclear. At the chromosomal level, TRs can be of profound structural as well as evolutionary importance, as genomic regions with a high density of TRs (e.g., telomeric, centromeric, and heterochromatic regions) often have specific properties such as alternative DNA structure and packaging (Kobliakova, Zatsepina, Stefanova, Polyakov, & Kireev, 2005;Podgornaya, Voronin, Enukashvily, Matveev, & Lobov, 2003;Ushiki & Hoshi, 2008;Vogt, 1990). ...
Much of tandem repeats' functional nature in any genome remains enigmatic because there are only few tools available for dissecting and elucidating the functions of repeated DNA. The large tandem repeat arrays (satellite DNA) found in two mouse whole-genome shotgun assemblies were classified into 4 superfamilies, 8 families, and 62 subfamilies. With the simplified variant of chromosome positioning of different tandem repeats, we noticed the nonuniform distribution instead of the positions reported for mouse major and minor satellites. It is visible that each chromosome possesses a kind of unique code made up of different large tandem repeats. The reference genomes allow marking only internal tandem repeats, and even with such a limited data, the colored "bar code" made up of tandem repeats is visible. We suppose that tandem repeats bare the mechanism for chromosomes to recognize the regions to be associated. The associations, initially established via RNA, become fixed by histone modifications (the histone or chromatin code) and specific proteins. In such a way, associations, being at the beginning flexible and regulated, that is, adjustable, appear as irreversible and inheritable in cell generations. Tandem repeat multiformity tunes the developed nuclei 3D pattern by sequential steps of associations. Tandem repeats-based chromosome bar code could be the carrier of the genome structural information; that is, the order of precise tandem repeat association is the DNA morphogenetic program. Tandem repeats are the cores of the distinct 3D structures postulated in "gene gating" hypothesis.
... The computation approaches to the genome-wide TR analysis gradually appear with the genome sequencing advanced [5,6,[44][45][46]. At the chromosomal level TR can be of profound structural as well as evolutionary importance, since genomic regions with a high density of TR, e.g., telomeric, centromeric, and heterochromatic regions, often have specific properties such as alternative DNA structure and packaging [47][48][49]. At the nuclear level of organization, constitutive heterochromatin may help maintain the proper spatial relationships necessary for the efficient operation of the cell through the stages of mitosis and meiosis. ...
Functional and morphological studies of tandem DNA repeats, that combine high portion of most genomes, are mostly limited due to the incomplete characterization of these genome elements. We report here a genome wide analysis of the large tandem repeats (TR) found in the mouse genome assemblies.
Using a bioinformatics approach, we identified large TR with array size more than 3 kb in two mouse whole genome shotgun (WGS) assemblies. Large TR were classified based on sequence similarity, chromosome position, monomer length, array variability, and GC content; we identified four superfamilies, eight families, and 62 subfamilies - including 60 not previously described. 1) The superfamily of centromeric minor satellite is only found in the unassembled part of the reference genome. 2) The pericentromeric major satellite is the most abundant superfamily and reveals high order repeat structure. 3) Transposable elements related superfamily contains two families. 4) The superfamily of heterogeneous tandem repeats includes four families. One family is found only in the WGS, while two families represent tandem repeats with either single or multi locus location. Despite multi locus location, TRPC-21A-MM is placed into a separated family due to its abundance, strictly pericentromeric location, and resemblance to big human satellites. To confirm our data, we next performed in situ hybridization with three repeats from distinct families. TRPC-21A-MM probe hybridized to chromosomes 3 and 17, multi locus TR-22A-MM probe hybridized to ten chromosomes, and single locus TR-54B-MM probe hybridized with the long loops that emerge from chromosome ends. In addition to in silico predicted several extra-chromosomes were positive for TR by in situ analysis, potentially indicating inaccurate genome assembly of the heterochromatic genome regions.
Chromosome-specific TR had been predicted for mouse but no reliable cytogenetic probes were available before. We report new analysis that identified in silico and confirmed in situ 3/17 chromosome-specific probe TRPC-21-MM. Thus, the new classification had proven to be useful tool for continuation of genome study, while annotated TR can be the valuable source of cytogenetic probes for chromosome recognition.
... Standard fixation uses methanolacetic acid or formaldhyde. The former operates by dehydration and is particularly damaging to 3D architecture, causing a flattened nucleus and damaged chromosome morphology [2,3]. Whilst formaldehyde is thought to be more satisfactory, the tendency of this fixative to trigger retraction of cells' cytoskeletal protrusions urges for caution [4]. ...
Despite the distinctive structure of mitotic chromosomes, it has not been possible to visualise individual chromosomes in living interphase cells, where chromosomes spend over 90% of their time. Studies of interphase chromosome structure and dynamics use fluorescence in-situ hybridisation (FISH) on fixed cells, which potentially damages structure and loses dynamic information. We have developed a new methodology, involving photoactivation of labelled histone H3 at mitosis, to visualise individual and specific human chromosomes in living interphase cells. Our data revealed bulk chromosome volume and morphology are established rapidly after mitosis, changing only incrementally after the first hour of G1. This contrasted with the behaviour of specific loci on labelled chromosomes, which showed more progressive reorganisation, and revealed that "looping out" of chromatin from chromosome territories is a dynamic state. We measured considerable heterogeneity in chromosome decondensation, even between sister chromatids, which may reflect local structural impediments to decondensation and could potentially amplify transcriptional noise. Chromosome structure showed tremendous resistance to inhibitors of transcription, histone deacetylation and chromatin remodelling. Together, these data indicate steric constraints determine structure, rather than innate chromosome architecture or function-driven anchoring, with interphase chromatin organisation governed primarily by opposition between needs for decondensation and the space available for this to happen.
... Additionally, a different distribution of Scaffold Associated Regions (SARs) (Heng et al. (2004)) in alpha satellite DNA than in gene domains might be assumed. Kobliakova et al. (2005) introduced a model of mitotic chromosome organisation based on the morphology of differentially decondensed chromosomes and the distribution of early and late replication sites. There, in case of standard G-and R-band staining typical longitudinal differentiation was revealed. ...
... In undamaged chromosomes the scaffold proteins provide the compaction of higher order chromatin fibre. Early replication is a feature of nearly all transcriptionally active genes (Kobliakova et al. (2005)). ...
The hypothesis that distinct chromatin domains expand and are remodelled differently when they undergo transcription, replication or cell cycle processes is well accepted. The condensation changes by which chromosomes are transformed at the metaphase–interphase transition are especially interesting and therefore extensively studied by light microscopy; however, quantitative information of the size on specific small chromatin domains during the cell cycle is scarce. In this respect, a serious problem is the determination of structural features close to the resolution limit. In this report we use a novel approach to quantify the lateral extent of the 8q24/c-myc gene domain and the centromeric region of chromosome 8 in doubly labelled normal human foreskin fibroblasts using confocal laser scanning microscopy (CLSM). The domains were analysed in both metaphase spreads and interphase nuclei. These high precision measurements revealed a somewhat smaller (few 10s of nm) lateral extension of the centromere region in metaphase compared to interphase. Surprisingly, within the same cells the lateral extension of the 8q24/c-myc region was significantly smaller in interphase than in metaphase. For comparison the centromere size was more condensed in metaphase than in interphase. This implies a different folding behaviour for specific chromatin domains with opposite condensation behaviour.
... Interphase nuclei in such preparations are flattened and have an increased diameter ( Kozubek et al. 2000 and present study). Hypotonic treatment ( Kobliakova et al. 2005) and other changes in ion concentration ( Belmont et al. 1989) have been shown to disturb chromosome morphology. Distributions of core histones and of the splicing factor SC-35 were observed to change substantially under these conditions ( Hendzel and Bazett-Jones 1997), suggesting a redistribution of chromatin components. ...
The nuclear organization of specific endogenous chromatin regions can be investigated only by fluorescence in situ hybridization (FISH). One of the two fixation procedures is typically applied: (1) buffered formaldehyde or (2) hypotonic shock with methanol acetic acid fixation followed by dropping of nuclei on glass slides and air drying. In this study, we compared the effects of these two procedures and some variations on nuclear morphology and on FISH signals. We analyzed mouse erythroleukemia and mouse embryonic stem cells because their clusters of subcentromeric heterochromatin provide an easy means to assess preservation of chromatin. Qualitative and quantitative analyses revealed that formaldehyde fixation provided good preservation of large-scale chromatin structures, while classical methanol acetic acid fixation after hypotonic treatment severely impaired nuclear shape and led to disruption of chromosome territories, heterochromatin structures, and large transgene arrays. Our data show that such preparations do not faithfully reflect in vivo nuclear architecture.
Electronic supplementary material
Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00412-006-0084-2 and is accessible for authorized users.
To visualize characteristic chromatin distortions we have distinguished first among regularly occurring intermediates of chromatin structures in mammalian (Indian muntjac, CHO, murine preB, rat liver, rat myeloid leukemia, K562 human erythroid leukemia) and Drosophila nuclei. Fluorescence microscopy of chromatin structures isolated from nuclei of reversibly permeable cells revealed a common pathway of chromatin condensation in mammalian cells. Different intermediates in mammalian and Drosophila cells indicate alternative mechanisms of chromosome condensation. Genotoxic agents such as irradiation (alpha, gamma, UV-B) and heavy metals (Cd, Pb, Ni, Hg, Ag) caused alterations in chromatin structures leading to apoptosis. Injury-specific chromatin changes manifested at significantly lower concentrations than non-specific signs of cellular toxicity, suggesting that preapoptotic events are useful indicators of genotoxicity.
