Chromatin Structure and the Formation of Chromosomal Alterations (original) (raw)
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Nuclear architecture and the induction of chromosomal aberrations
1996
Progress in fluorescence in situ hybridization, three dimensional microscopy and image analysis has provided the means to study the three-dimensional structure and disla'ibution of chromosome territories within the cell nucleus. In this contribution, we summarize the present state of knowledge of the territorial organization of interphase chromosomes and their topological relationships with other macromolecular domains in the human cell nucleus, and present data from computer simulations of chromosome territory distributions. On this basis, we discuss models of chromosome territory and nuclear architecture and topological consequences for the formation of chromosome exchanges. "The idea of the nucleus as a bag of broken chromosome ends flapping around seeking new parmers...seems no longer to be tenable from our picture of the interphase nucleus" (
Microscopy Research and Technique, 1993
It is a widely held belief that the inactive X-chromosome (Xi) in female cell nuclei is strongly condensed as compared to the largely decondensed active X-chromosome (Xa). We have reconsidered this problem and painted X-chromosome domains in nuclei of subconfluent, female and male human amniotic fluid cell cultures (46,XX and 46,XY) by chromosomal in situ suppression (CISS) hybridization with biotinylated human X-chromosome specific library DNA. FITCconjugated avidin was used for probe detection and nuclei were counterstained with propidium iodide (PI). The shape of these nuclei resembling flat ellipsoids or elliptical cylinders makes them suitable for both two-dimensional (2D) and three-dimensional (3D) analyses. 2D analyses of Xi-and Xa-domains were performed in 34 female cell nuclei by outlining of the painted domains using a camera lucida. Identification of the sex chromatin body in DAPI-stained nuclei prior to CISShybridization was confirmed by its colocalization with one of the two painted X-domains. In 31 of the 34 nuclei the area Axi for the inactive X-domain was smaller than the area A, , for the active domain (mean ratio Ax,/Ax, = 1.9 * 0.8 SD, range 1.0-4.3). The signed rank test showed a highly significant (P < .0001) difference both between A, , and Axi and between the ratios r(Xa) and r(Xi), calculated by dividing the maximum length L of each X-domain by its maximum width W. In most nuclei (26134) we found r(Xa)>r(Xi) demonstrating a generally more elongated structure of Xa. For 3D analysis a confocal scanning laser fluorescence microscope (CSLFM) was used. Ten to 20 light optical sections (PI-image, FITC-image) were registered with equal spacings (approx. 0.4 pm). A thresholding procedure was applied to determine the PI-labeled nuclear and FITC-labeled X-domain areas in each section. Estimated slice volumes were used to compute total nuclear and X-domain volumes. In a series of 35 female nuclei most domains extended from the top to the bottom nuclear sections. The larger of the two X-chromosome domains comprised (3.7 t 1.7 S.D.)% of the nuclear volume. A mean ratio of 1.2 k 0.2 SD (range 1.1-2.3) was found for the volumes of the larger and the smaller X-domains in these female nuclei. In a series of 27 male amniotic fluid cell nuclei the relative X-chromosome domain volume comprised (4.0 t 2.6 S.D.)%. These findings indicate that differences in the 3D expansion of active and inactive X-chromosome domains are less pronounced than previously thought. A current model suggests that chromosome domains consist of a compact core surrounded by loosely coiled outer chromatin fiber loops. The latter fraction may be considerably larger in Xa-as compared to Xi-domains. We suggest that the interactive outlining procedure used in the 2D analyses included the loosely structured domain periphery more accurately, while the threshold algorithm applied to light optical sections delineated the more compact core of the domains, leading to smaller and more similar volume estimates of Xa and Xi. Present limitations of nuclear and chromosome domain volume measurements using confocal laser scanning microscopy are discussed. Address reprint requests to C. Cremer, Institute of Applied Physics, Univ. of posed that its constituting chromatin fiber is extended Heidelberg, Albert-Uberle-StraRe 3-5, D-6900 Heidelberg, FRG. 0 1993 WILEY-LISS. INC
Advances in Space Research, 2001
Several advances have been achieved in the knowledge of nuclear architecture and functions during the last decade, thus allowing the identification of interphase chromosome territories and sub-chromosomal domains (e.g. arm and band domains). This is an important step in the study of radiation-induced chromosome aberrations; indeed, the coupling between track-structure simulations and reliable descriptions of the geometrical properties of the target is one of the main tasks in modelling aberration induction by radiation, since it allows one to clarify the role of the initial positioning of two DNA lesions in determining their interaction probability. In the present paper, the main recent findings on nuclear and chromosomal architecture are summarised. A few examples of models based on different descriptions of interphase chromosome organisation (random-walk models, domain models and static models) are presented, focussing on how the approach adopted in modelling the target nuclei and chromosomes can influence the simulation of chromosomal aberration yields. Each model is discussed by taking into account available experimental data on chromosome aberration induction and/or interphase chromatin organisation. Preliminary results from a mechanistic model based on a coupling between radiation trackstructure features and explicitly-modelled, non-overlapping chromosome territories are presented.
Human Genetics, 1982
In 1885 Carl Rabl published his theory on the internal structure of the interphase nucleus. We have tested two predictions of this theory in fibroblasts grown in vitro from a female Chinese hamster, namely (1) the Rabl-orientation ofinterphase chromosomes and (2) the stability of the chromosome arrangement established in telophase throughout the subsequent interphase. Tests were carried out by premature chromosome condensation (PCC) and laser-UV-microirradiation of the interphase nucleus. Rabl-orientation of chromosomes was observed in G1 PCCs and G2 PCCs. The cell nucleus was microirradiated in G1 at one or two sites and pulse-labelled with SH-thymidine for 2 h. Cells were processed for autoradiography either immediately thereafter or after an additional growth period of 10 to 60h. Autoradiographs show unscheduled DNA synthesis (UDS) in the microirradiated nuclear part(s). The distribution of labelled chromatin was evaluated in autoradiographs from 1035 cells after microirradiation of a single nuclear site and from 253 cells after microirradiation of two sites. After 30 to 60 h postincubation the labelled regions still appeared coherent although the average size of the labelled nuclear area fr increased from 14.2% (0 h) to 26.5% (60 h). The relative distance dr, i.e. the distance between two microirradiated sites divided by the diameter of the whole nucleus, showed a slight decrease with increasing incubation time. Nine metaphase figures were evaluated for UDS-label after microirradiation of the nuclear edge in GI. An average of 4.3 chromosomes per cell were labelled. Several chromosomes showed joint labelling of both distal chromosome arms including the telomeres, while the centromeric region was free from label. This label pattern is interpreted as the result of a V-shaped orientation of these particular chromosomes in the interphase nucleus with their telomeric regions close to each other at the nuclear edge. Our data support the tested predictions of the RaN-model. Small time-dependent changes of the nuclear space 4 Part of this work is included in the doctoral thesis of H. Baumann to be submitted to the Faculty of Biology of the University of Heidelberg 5 Part of this work is included in the doctoraI thesis of V. Teuber to be submitted to the Faculty of Medicine of the University of Freiburg i. Br. Offprint requests to: T. Cremer occupied by single chromosomes and of their relative positions in the interphase nucleus seem possible, while the territorial organization of interphase chromosomes and their arrangement in general is maintained during interphase. The present limitations of the methods used for this study are discussed.
Structure and dynamics of human interphase chromosome territories in vivo
Human Genetics, 1998
A new approach is presented which allows the in vivo visualization of individual chromosome territories in the nuclei of living human cells. The fluorescent thymidine analog Cy3-AP3-dUTP was microinjected into the nuclei of cultured human cells, such as human diploid fibroblasts, HeLa cells and neuroblastoma cells. The fluorescent analog was incorporated during S-phase into the replicating genomic DNA. Labelled cells were further cultivated for several cell cycles in normal medium. This well-known scheme yielded sister chromatid labelling. Random segregation of labelled and unlabelled chromatids into daughter nuclei resulted in nuclei exhibiting individual in vivo detectable chromatid territories. The territories were composed of subcompartments with diameters ranging between approximately 400 and 800 nm which we refer to as subchromosomal foci. Time-resolved in vivo studies demonstrated changes of positioning and shape of territories and subchromosomal foci. The hypothesis that subchromosomal foci persist as functionally distinct entities was supported by double labelling of chromatin with CldU and IdU, respectively, at early and late S-phase and subsequent cultivation of corresponding cells for 5–10 cell cycles before fixation and immunocytochemical detection. This scheme yielded segregated chromatid territories with distinctly separated subchromosomal foci composed of either early- or late-replicating chromatin. The size range of subchromosomal foci was similar after shorter (2 h) and longer (16 h) labelling periods and was observed in nuclei of both living and fixed cells, suggesting their structural identity. A possible functional relevance of chromosome territory compartmentalization into subchromosomal foci is discussed in the context of present models of interphase chromosome and nuclear architecture.
Influence of Cell Fixation on Chromatin Topography
Analytical Biochemistry, 2000
Using in situ hybridization techniques, a fixation step must precede denaturation to prevent disintegration of the chromosomes. The analysis of nuclei fixed by paraformaldehyde, preserving the native structure (three-dimensional or 3D fixation and analysis) has become possible with the development of confocal microscopy; however, the analysis of those fixed by methanol and acetic acid, dehydrating the nuclei (two-dimensional or 2D fixation and analysis), remains a very valuable tool for practical use in diagnostics and also in many cases for research. We compared both types of fixation and analyses using different cell lines and different DNA probes. Fixation of cells by methanol and acetic acid leads to the enlargement of contact of nuclei with the slide surface, resulting in a substantial increase of nuclear diameter, flattening of the nucleus, and consequently to a distortion of gene topology. Our results indicate that chromatin structures located in the outer parts of the nuclear volume (e.g., heterochromatin of some centromeres) are relatively shifted to the membrane of these nuclei, keeping the absolute centromere-membrane distance constant. On the other hand, euchromatin located in the inner parts of the nuclear volume is not shifted outside proportionally to the increase of molecular dimensions; consequently, the relative distances for the center of nucleus to gene are smaller after methanol-acetic acid fixation. The limitations of the analysis of dehydrated preparations for practical use and in research are discussed.
Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, 1998
Various models for the nuclear architecture in interphase cell nuclei have been presented, proposing a territorial or a non-territorial organization of chromosomes. To better understand the correlation between nuclear architecture and the formation of chromosomal aberrations, we applied computer simulations to model the extent of radiation induced chromosome damage under certain geometrical constraints. For this purpose, chromosomes were described by different models, which approximate the chromatin fiber by a polymer chain, folded in different ways. Corresponding to the different condensation levels, a territorial or a non-territorial organization of chromosomes was obtained. To determine the relative frequencies of radiation induced damage, the effects of isotropic ionizing radiation and of a focused laser UV-beam were studied. For isotropic ionizing radiation the calculated translocation frequencies showed no differences between territorial and non-territorial models except for one special case. For localized irradiation, the results of both organizations were clearly different, with respect to the total number of damaged chromosomes per cell. The predictions agreed well with the experimental data available.
Chromosome spatial clustering inferred from radiogenic aberrations
International Journal of Radiation Biology, 2004
Purpose: Analysing chromosome aberrations induced by low linear energy transfer (LET) radiation in order to characterize systematic spatial clustering among the 22 human autosomes in human lymphocytes and to compare their relative participation in interchanges. Materials and methods: A multicolour fluorescence in situ hybridization (mFISH) data set, specifying colour junctions in metaphases of human peripheral blood lymphocytes 72 h after in vitro exposure to low LET radiation, was analysed separately and in combination with previously published results. Monte Carlo computer simulations and mathematical modelling guided data analysis. Results and conclusions: Statistical tests on aberration data confirmed two clusters of chromosomes, {1, 16, 17, 19, 22} and {13, 14, 15, 21, 22}, as having their members being on average closer to each other than randomness would predict. The first set has been reported previously to be near the centre of the interphase nucleus and to be formed mainly by gene-rich chromosomes, while the second set comprises the nucleolus chromosomes. The results suggest a possible interplay between chromosome positioning and transcription. A number of other clusters suggested in the literature were not confirmed and considerable randomness of chromosome-chromosome juxtapositions was present. In addition, and consistent with previous results, it was found that chromosome participation in interchanges is approximately proportional to the two-thirds power of the DNA content.