Species-specific reorganization of the interphase chromosome architecture in generative tissue as a special type of chromosomal mutations associated with speciation

Epigenetic mechanisms of speciation are considered, including heterochromatic modifications and changes in spatial chromosome organization in the generative cell systems. The value of lamina, topoisomerase II, and a DNA polypurine tract in the attachment of chromosomes to the nuclear envelope is discussed. It is postulated that the main event leading to species-specific fixation of gene mutations, chromosomal mutations, and heterochromatin modifications in speciation is the rearrangement of spatial chromosome organization in the nucleus. The change in interchromosomal relationships associated with the reorganization of the system of chromosomal contacts with the nuclear envelope and the rearrangement of the chromocenter apparatus of the interphase nucleus is estimated as a systemic mutation directly related to speciation.     

Size-dependent positioning of human chromosomes in interphase nuclei

By using a fluorescence in situ hybridization technique we revealed that for nine different q-arm telomere markers the positioning of chromosomes in human G(1) interphase nuclei was chromosome size-dependent. The q-arm telomeres of large chromosomes are more peripherally located than telomeres on small chromosomes. This highly organized arrangement of chromatin within the human nucleus was discovered by determining the x and y coordinates of the hybridization sites and calculating the root-mean-square radial distance to the nuclear centers in human fibroblasts. We demonstrate here that global organization within the G(1) interphase nucleus is affected by one of the most fundamental physical quantities-chromosome size or mass-and propose two biophysical models, a volume exclusion model and a mitotic preset model, to explain our finding.     

An interphase model for mitotic chromosome organization in eukaryota

A model for the spatial relationship of the arrangement of the chromosomes in the nucleus in eukaryota is presented. Evidence is derived from light and electron microscopic studies, application of autoradiographic and banding techniques; on the organization, structure and behaviour of chromosomes during interphase and other stages of cell cycle. This model visualizes the entire chromosomal DNA as a single uninemic multirepliconic continuum where the chromosomes with their centromeres and telomeres have a predetermined arrangement among themselves as well as in relation to the nucleolus and nuclear membrane. This orderly arrangement is presumably maintained through interchromosomal connections. The impact of this model on the interpretation of various cytogenetic phenomena is discussed.     

Rabl's model of the interphase chromosome arrangement tested in Chinise hamster cells by premature chromosome condensation and laser-UV-microbeam experiments

Summary 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 of interphase 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 ³H-thymidine for 2h. 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 60h postincubation the labelled regions still appeared coherent although the average size of the labelled nuclear area fr increased from 14.2% (0h) to 26.5% (60h). 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 G1. 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 Rabl-model. Small time-dependent changes of the nuclear space 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.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 HeidelbergPart of this work is included in the doctoral thesis of V. Teuber to be submitted to the Faculty of Medicine of the University of Freiburg i. Br.     

An ordered arrangement of chromosomes in the somatic nucleus of common wheat Triticum aestivum L.

Spatial relationships between chromosomes of the same genome, both homologous and non-homologous, were studied in root-tip cells of common wheat, Triticum aestivum (2n = 6x = 42). Mean distance between members of all the 21 homologous pairs (seven in each of the three genomes) and of 45 out of the 63 possible non-homologous combinations of two (21 in each genome) were determined. To minimize disruption of nuclear chromosomal arrangement, the cells were pretreated with cold temperature either in tap water or in a physiological medium (White solution) and distances between cytologically marked chromosomes were measured at metaphase. Comparison of distances for homologues with those for non-homologues indicated clearly that, within each genome, the homologous chromosomes were significantly closer to one another than were the non-homologues. Distances between homologues were similar in all three genomes, as were distances between non-homologues. The data are consistent with the hypothesis that the chromosomes of each genome of common wheat are arranged in the somatic nucleus in a highly specific ordered pattern. In this hypothetical arrangement, homologous chromosomes are closely associated, while the nonhomologues occupy definite positions with respect to one another. The universality of the phenomenon and its cellular mechanism and biological significance are discussed.     

Conservation of relative chromosome positioning in normal and cancer cells

Chromosomes exist in the interphase nucleus as individual chromosome territories. It is unclear to what extent chromosome territories occupy particular positions with respect to each other and how structural rearrangements, such as translocations, affect chromosome organization within the cell nucleus. Here we analyze the relative interphase positioning of chromosomes in mouse lymphoma cells compared to normal splenocytes. We show that in a lymphoma cell line derived from an ATM(-/-) mouse, two translocated chromosomes are preferentially positioned in close proximity to each other. The relative position of the chromosomes involved in these translocations is conserved in normal splenocytes. Relative positioning of chromosomes in normal splenocytes is not due to their random distribution in the interphase nucleus and persists during mitosis. These observations demonstrate that the relative arrangement of chromosomes in the interphase nucleus can be conserved between normal and cancer cells and our data support the notion that physical proximity facilitates rearrangements between chromosomes.     

Mitotic spatial model: Arrangement of homologous chromosomes

Two different mitotic spatial models (Lavania & Sharma, Biosystems 14: 171–178, 1981: Ashley & Pocock, Genetica 55: 161–169, 1981) for the arrangement of the chromosomes in the nucleus are analysed for the sake of comparison and assessment. In general, the two models propose nearly the same orientation regarding chromosomal organization. However, they differ with respect to the position of homologous chromosomes, suggesting respectively their adjacent or opposite location in somatic arrangement. The two proposals have been analysed in the light of experimental evidence as well as organizational interpretations. The arguments presented here support the adjacent location of somatic homologous chromosomes.     

The state of the chromosomes in the interphase nucleus

In the living interphase nucleus no chromosomal structures are visible. Yet in the injured cell and after treatment with most histological fixatives chromatin structures become apparent. Under certain conditions this appearance of structure in the living interphase nucleus is reversible. We have found that this change in the interphase nucleus is the result of a change in the state of the chromosomes. In the living nucleus the chromosomes are in a greatly extended state, filling the entire nucleus. Upon injury the chromosomes condense and therefore become visible. At the same time the nuclear volume decreases. This behavior of the chromosomes is connected with their content of desoxyribonucleic acid (DNA). This view is based on the following observations: (a) Distribution of DNA in the Nucleus.-(1) The living interphase nucleus of uninjured cells absorbs diffusely at 2537 A. No chromosomal structures are visible in ultraviolet photographs unless they are also distinct in ordinary light. If the chromosomes are made to condense they become visible and the absorption at 2537 A is now localized in these structures. (2) After fixation with formalin and osmic acid interphase nuclei stain diffusely with Feulgen. These fixatives preserve the extended state of the chromosomes. (3) If nuclei are teased out in non-electrolytes (sucrose, glycerin) the chromosomes are extended. Such nuclei stain homogeneously with methyl green. On adding salts the chromosomes condense and the methyl green is now restricted to the visible structures. (b) Extension and Condensation of Isolated Chromosomes.-When chromosomes isolated from interphase nuclei of calf thymus are suspended in sucrose, their volume is four to five times larger than in saline, but they retain their characteristic shapes. Chromosomes from which DNA and histone have been removed do not show this reversible extension and condensation, neither do lampbrush chromosomes of frog oocytes which contain very little DNA. During mitosis a partial condensation of the DNA occurs in prophase, so that the mitotic chromosomes now occupy a much smaller volume of the nucleus. At telophase the chromosomes swell again to fill the entire nucleus.     

Chromosome pairing does not contribute to nuclear architecture in vegetative yeast cells

There are several reports of a closer-than-random colocalization of homologous chromosomes in the vegetative nuclei of diploid budding yeast. Here, we studied by fluorescence in situ hybridization (FISH) the nuclear distribution of chromosomes and found a slight tendency toward closer proximity between homologous (allelic) loci than between any nonhomologous chromosomal regions. We show that most of this preferential association is not due to vegetative (also known as somatic) pairing but is caused by the polar orientation of interphase chromosomes (Rabl orientation). We quantified the occasional loss of detectable fluorescence signals that is inherent to the FISH method. Signal loss leads to the occurrence of a single signal that may be misinterpreted as the close association of two homologous chromosomal sites. The nuclear distribution of homologous loci, when corrected for the influence of nuclear architecture and methodological faults, was not different or was only marginally different from a random relative positioning as predicted by computer simulation. We discuss here several possibilities for the residual homologous proximity that do not invoke homology-dependent vegetative pairing, and we conclude that, in diploid budding yeast, constitutive vegetative pairing is a negligible factor for the organization of the interphase nucleus.     

Localization of the gene-richest and the gene-poorest isochores in the interphase nuclei of mammals and birds

At a resolution of 850 bands, human chromosomes comprise two subsets of bands, the GC-richest H3⁺ and the GC-poorest L1⁺ bands, accounting for about 17 and 26%, respectively, of all bands. The former are a subset of the R bands and the latter are a subset of the G bands. These bands showed the highest and the lowest gene densities, respectively, as well as a number of other distinct features. Here we report that human and chicken interphase nuclei are characterized by the following features. (1) The gene-richest/GC-richest chromosomal regions are predominantly distributed in internal locations, whereas the gene-poorest/GC-poorest DNA regions are close to the nuclear envelope. (2) The interphase chromosomes seem to be characterized by a polar arrangement, because the gene-richest/GC-richest bands and the gene-poorest/GC-poorest bands are predominantly located in the distal and proximal regions, respectively, of chromosomes, and because interphase chromosomes are extremely long. While this polar arrangement is evident in the larger chromosomes, it is not displayed by the chicken microchromosomes and by some small human chromosomes, namely by chromosomes that are almost only composed by GC-rich or by GC-poor DNA. (3) The gene-richest chromosomal regions display a much more spread-out conformation compared to the gene-poorest regions in human nuclei. This finding has interesting implications for the formation of GC-rich isochores of warm-blooded vertebrates.     

Methods of molecular cytogenetics for studying interphase chromosomes in human brain cells

One of the main genetic factors determining the functional activity of the genome in somatic cells, including brain nerve cells, is the spatial organization of chromosomes in the interphase nucleus. For a long time, no studies of human brain cells were carried out until high-resolution methods of molecular cytogenetics were developed to analyze interphase chromosomes in nondividing somatic cells. The purpose of the present work was to assess the potential of high-resolution methods of interphase molecular cytogenetics for studying chromosomes and the nuclear organization in postmitotic brain cells. A high efficiency was shown by such methods as multiprobe and quantitative fluorescence in situ hybridization (Multiprobe FISH and QFISH), ImmunoMFISH (analysis of the chromosome organization in different types of brain cells), and interphase chromosome-specific multicolor banding (ICS-MCB). These approaches allowed studying the nuclear organization depending on the gene composition and types of repetitive DNA of specific chromosome regions in certain types of brain cells (in neurons and glial cells, in particular). The present work demonstrates a high potential of interphase molecular cytogenetics for studying the structural and functional organizations of the cell nucleus in highly differentiated nerve cells. Analysis of interphase chromosomes of brain cells in the normal and pathological states can be considered as a promising line of research in modern molecular cytogenetics and cell neurobiology, i. e., molecular neurocytogenetics.     

Order and disorder in the nucleus

Fluorescence in situ hybridization combined with three-dimensional microscopy has shown that chromosomes are not randomly strewn throughout the nucleus but are in fact fairly well organized, with different loci reproducibly found in different regions of the nucleus. At the same time, increasingly sophisticated methods to track and analyze the movements of specific chromosomal loci in vivo using four-dimensional microscopy have revealed that chromatin undergoes extensive Brownian motion. However, the diffusion of interphase chromatin is constrained, implying that chromosomes are physically anchored within the nucleus. This constraint on diffusion is the result of interactions between chromatin and structural elements within the nucleus, such as nuclear pores or the nuclear lamina. The combination of defined positioning with constrained diffusion has a strong impact on interactions between chromosomal loci, and appears to explain the tendency of certain chromosome rearrangements to occur during the development of cancer.     

Identification of chromosome markers in interphase nuclei

The chromosome complement of the mosquito Cuilseta longiareolata (2n=6) reveals distinguishable centromeric regions and one telomere of the Y chromosome by using light-induced differentiation and autoradiographic techniques in mitotic and premeiotic interphase nuclei. The localization of these cytological markers and their spatial relationships appear to be very similar in the two types of nuclei and suggest an interphase arrangement where centromeric regions are clustered together in a chromocenter like structure, close to the nuclear membrane, with the telomeres lying on the opposite pole of the nucleus.     

Mechanistic modelling allows to assess pathways of DNA lesion interactions underlying chromosome aberration formation

The knowledge of radiation-induced chromosomal aberration (CA) mechanisms is required in many fields of radiation genetics, radiation biology, biodosimetry, etc. However, these mechanisms are yet to be quantitatively characterised. One of the reasons is that the relationships between primary lesions of DNA/chromatin/chromosomes and dose-response curves for CA are unknown because the pathways of lesion interactions in an interphase nucleus are currently inaccessible for direct experimental observation. This article aims for the comparative analysis of two principally different scenarios of formation of simple and complex interchromosomal exchange aberrations: by lesion interactions at chromosome territories?? surface vs. in the whole space of the nucleus. The analysis was based on quantitative mechanistic modelling of different levels of structures and processes involved in CA formation: chromosome structure in an interphase nucleus, induction, repair and interactions of DNA lesions. It was shown that the restricted diffusion of chromosomal loci, predicted by computational modelling of chromosome organization, results in lesion interactions in the whole space of the nucleus being impossible. At the same time, predicted features of subchromosomal dynamics agrees well with in vivo observations and does not contradict the mechanism of CA formation at the surface of chromosome territories. On the other hand, the ??surface mechanism?? of CA formation, despite having certain qualities, proved to be insufficient to explain high frequency of complex exchange aberrations observed by mFISH technique. The alternative mechanism, CA formation on nuclear centres is expected to be sufficient to explain frequent complex exchanges.     

Nuclear matrix proteins (with molecular masses of 38 and 50 kDa) are transported by chromosomes in mitosis

Using the immunofluorescence method, sera M-68 and K-43 from patients with autoimmune diseases were shown to stain interphase nuclei and the periphery of mitotic chromosomes of pig embryo kidney cells. Western blotting revealed a polypeptide with a molecular mass of 50 kDa in M-68 serum and polypeptide with a molecular mass 38 kDa in K-43 serum. In the nuclear protein matrix, the antibodies to protein with a molecular mass of 38 kDa stained only the nucleolar periphery, while the antibodies to protein with a molecular mass of 50 kDa stained not only the nucleolar periphery, but also all interphase nuclei. It was shown that, among all components of the nuclear protein matrix (lamina, internuclear network, residual nucleoli), only the nucleolar periphery contained the 38-kDa protein, while the 50-kDa protein was part of the residual nucleolar periphery and participated in the formation of a nuclear-protein network. Both proteins in interphase cell in situ were located in nuclei, but one of them with a molecular mass of 50 kDa was in the form of small, clearly outlined granules, while the other protein (38 kDa) was in the form of small, bright granules on a background of a diffusely stained nucleus. Both proteins also were revealed as a continuous rim around the nucleolar periphery. During all mitotic stages, the 50-kDa protein was seen over the whole chromosomal periphery as a sheath, while the 38-kDa protein formed individual fragments and granules around them. After the decondensation of the nucleus and chromosomes induced by hypotonic treatment, both antibodies stained interphase nuclei diffusely, whereas, in mitotic cells, they stained the surfaces of swollen chromosomes. Polypeptide with a molecular mass of 50 kDa maintained a strong connection with the periphery of the chromosome in the norm during decondensation induced by hypotonic treatment and during subsequent recondensation in isotonic medium, while, during recondensation, protein with a molecular mass of 38 kDa partially lost contact with the chromosome and, at the same time, appeared in the form of granules in the cytoplasm. The obtained data allow one to conclude that nuclear matrix proteins can be transferred with peripheral chromosomal material; similar to the main nucleolar proteins (fibrillarin, B-23, nucleolin, et al.) and some non-nucleolar components of the nuclear protein matrix, they can also have connections of different stabilities with chromosomal periphery.     

Analysis of chromosome positions in the interphase nucleus of Chinese hamster cells by laser-UV-microirradiation experiments

Summary Unsynchronized cells of an essentially diploid strain of female Chinese hamster cells derived from lung tissue (CHL) were laser-UV-microirradiated (=257 nm) in the nucleus either at its central part or at its periphery. After 7–9 h postincubation with 0.5 mM caffeine, chromosome preparations were made in situ. Twenty-one and 29 metaphase spreads, respectively, with partial chromosome shattering (PCS) obtained after micro-irradiation at these two nuclear sites, were Q-banded and analyzed in detail. A positive correlation was observed between the frequency of damage of chromosomes and both their DNA content and length at metaphase. No significant difference was observed between the frequencies of damage obtained for individual chromosomes at either site of microirradiation. The frequency of joint damage of homologous chromosomes was low as compared to nonhomologous ones. Considerable variation was noted in different cells in the combinations of jointly shattered chromosomes. Evidence which justifies an interpretation of these data in terms of an interphase arrangement of chromosome territories is discussed. Our data strongly argue against somatic pairing as a regular event, and suggest a considerable variability of chromosome positions in different nuclei. However, present data do not exclude the possibility of certain non-random chromosomal arrangements in CHL-nuclei. The interphase chromosome distribution revealed by these experiments is compared with centromere-centromere, centromere-center and angle analyses of metaphase spreads and the relationship between interphase and metaphase arrangements of chromosomes is discussed.     

Molecular cytogenetic methods for studying interphase chromosomes in human brain cells

One of the main genetic factors determining the functional activity of the genome in somatic cells, including brain nerve cells, is the spatial organization of chromosomes in the interphase nucleus. For a long time, no studies of human brain cells were carried out until high-resolution methods of molecular cytogenetics were developed to analyze interphase chromosomes in nondividing somatic cells. The purpose of the present work was to assess the potential of high-resolution methods of interphase molecular cytogenetics for studying chromosomes and the nuclear organization in postmitotic brain cells. A high efficiency was shown by such methods as multiprobe and quantitative fluorescence in situ hybridization (Multiprobe FISH and QFISH), ImmunoMFISH (analysis of the chromosome organization in different types of brain cells), and interphase chromosome-specific multicolor banding (ICS-MCB). These approaches allowed studying the nuclear organization depending on the gene composition and types of repetitive DNA of specific chromosome regions in certain types of brain cells (in neurons and glial cells, in particular). The present work demonstrates a high potential of interphase molecular cytogenetics for studying the structural and functional organizations of the cell nucleus in highly differentiated nerve cells. Analysis of interphase chromosomes of brain cells in the normal and pathological states can be considered as a promising line of research in modern molecular cytogenetics and cell neurobiology, i. e., molecular neurocytogenetics.