Spatial genome organization

The linear sequence of genomes exists within the three-dimensional space of the cell nucleus. The spatial arrangement of genes and chromosomes within the interphase nucleus is nonrandom and gives rise to specific patterns. While recent work has begun to describe some of the positioning patterns of chromosomes and gene loci, the structural constraints that are responsible for nonrandom positioning and the relevance of spatial genome organization for genome expression are unclear. Here we discuss potential functional consequences of spatial genome organization and we speculate on the possible molecular mechanisms of how genomes are organized within the space of the mammalian cell nucleus.     

Arrangement of chromosomes in the interphase nucleus of plants

Chromosomal arrangement in the interphase nucleus has two main aspects: (1) arrangement of chromosomes with respect to nuclear polarity and to other nuclear components, and (2) arrangement of chromosomes with respect to one another. The latter aspect consists of two main types of spatial relationships; (a) relationships between different members of one chromosomal set, (b) relationships between different chromosomal sets. Data concerning various aspects of chromosomal arrangement in the interphase nucleus are presented and discussed and the genetic control as well as subcellular mechanisms which are involvled in nuclear organization, are elucidated. Evidence is presented indicating that, in common wheat, the gene system that determines the specific pattern of chromosomal arrangement in the nucleus is operating via the microtubular elements of the spindle system. The significance of ordered arrangement of chromosomes in the nucleus for the regularity of genetic activity and chromosomal behavior, is pointed out.Supported in part by a grant from the Stiftung Volkswagenwerk AZ I/34 075/76     

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.     

Dynamics of chromosome positioning during the cell cycle

The arrangement and dynamics of chromosomes inside the nucleus of mammalian cells have been studied intensively over the last two years. Although chromosomes are relatively immobile and occupy non-random positions in interphase, their dynamic movements in mitosis have traditionally been assumed to randomize this arrangement. New methods of live cell imaging now make it possible to follow chromosome movements directly and quantitatively in single cells. Such studies have generated models of chromosome positioning throughout the cell cycle and provide a new basis to address the underlying mechanisms in future experiments.     

The use of fluorochromes in the cytogenetics of the small-grained cereals (Triticeae)

Summary This paper describes some of the major advances that have been made in the cytogenetics of the small-grained cereals (Triticeae) using fluorochromes to detect nucleic acids in situ. The method, widely known as fluorescence in situ hybridization, has made a contribution in several areas including (i) chromosome mapping programmes, and (ii) cereal breeding programmes. Flow cytometry of cereal chromosomes has now been developed for the generation of chromosome enriched libraries; these libraries will ultimately be of use in both the cereal mapping and breeding programmes. Fluorescence in situ hybridization has also made a major contribution to the understanding of cereal genome structure by elucidating the distribution of different classes of DNA sequence. By using suitable nucleic acid probes whole chromosomes can now be identified in interphase nuclei. The labelling patterns have revealed a structured arrangement of chromosomes at interphase. Not only are chromosomes organized but the ribosomal RNA genes also show structured patterns of condensation and expression. Progress in each of these areas has been rapid in recent years and this progress is described.     

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.     

Alternative ends: telomeres and meiosis

Meiosis is a specialized type of cell division that halves the diploid number of chromosomes, yielding four haploid nuclei. Dramatic changes in chromosomal organization occur within the nucleus at the beginning of meiosis which are followed by the separation of homologous chromosomes at the first meiotic division. This is the case for telomeres that display a meiotic-specific behavior with gathering in a limited sector of the nuclear periphery. This leads to a characteristic polarized chromosomal configuration, called the "bouquet" arrangement. The widespread phenomenon of bouquet formation among eukaryotes has led to the hypothesis that it is functionally linked to the process of interactions between homologous chromosomes that are a unique feature of meiosis and are essential for proper chromosome segregation. Various studies in different model organisms have questioned the role of the telomere bouquet in chromosome pairing and recombination, and very recently in meiotic spindle formation, and have provided some clues about the molecular mechanisms that carry out this specific clustering of telomeres.     

Specific arrangement of chromosomes in the spermiogenesis of Gallus domesticus

On the chromosomes of the rooster the constitutive heterochromatin (C-bands) is to be found for the most part at the centromeres. The position of the centric heterochromatin in spermatids and sperm is not randomly distributed. In early, round spermatids one heterochromatic block lies at this exact position on the membrane that develops into the tip of the sperm nucleus (acrosomal chromocenter). During the elongation of the spermatid nucleus another heterochromatic block comes to lie on the basal nuclear membrane. The other centromeres arrange themselves tandem-wise between the acrosomal and the basal chromocenters. Comparisons have been made between this specific arrangement and the location of chromosomes in the sperm of amphibians and mammalians.     

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.     

Individual interphase chromosome domains revealed by in situ hybridization

Summary The position and arrangement of individual chromosomes in interphase nuclei were examined in mouse-human cell hybrids by in situ hybridization of biotinylated human DNA probes. Intense and even labeling of human chromosomes with little background was observed when polyethylene glycol and Tween-20 were included in hybridization solutions. Human interphase chromosomes were separated from each other in the nucleus, and were confined to well localized domains. Hybrid cells with a single human chromosome showed a reproducible position of this chromosome in the nucleus. Some chromosomes appeared to have a characteristic folding pattern in interphase. Optical section as well as electron microscopy of labeled regions revealed the presence of 0.2 m wide fibers in each interphase domain, as well as adjacent, locally extended 500 nm fibers. Such fibers are consistent with previously proposed structural models of interphase chromosomes.     

Heterochromatin is not an adequate explanation for close proximity of interphase chromosomes 1--Y, 9--Y, and 16--Y in human spermatozoa

Analysis of human spermatozoa and lymphocytes using C-banding techniques and in situ hybridization has shown a higher order packaging of the human genome. Chromosomes are not distributed entirely at random within the nucleus. In particular, chromosomes 1, 9, and 16, carrying large blocks of pericentromeric heterochromatin, and the Y chromosome, carrying heterochromatin in Yq12, are in close proximity to each other within the nucleus and are involved in somatic pairing with nonhomologous chromosomes. In order to determine whether the close proximity of these chromosomes in any way is attributable to the distribution of heterochromatin, double in situ hybridization was performed on chromosomes 1--Y, 9--Y, and 16--Y as well as on 1--X, 9--X, and 16--X-with chromosome X as the other gonosome carrying less heterochromatin-in human spermatozoa. Each pair was found to have a nonrandom spatial distribution. However, comparison of the arrangement of chromosomes 1--Y versus 1--X and 9--Y versus 9--X revealed that heterochromatin cannot be the only cause for the tendency of chromosome fusion, because only the results of the chromosome pair 1--Y/1--X could support this proposition. In conclusion, the heterochromatin effect cannot be, in itself, an adequate explanation for chromosome association, implicating as well other mechanisms.     

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.     

Telomere-led bouquet formation facilitates homologous chromosome pairing and restricts ectopic interaction in fission yeast meiosis

A polarized chromosomal arrangement with clustered telomeres in a meiotic prophase nucleus is often called bouquet and is thought to be important for the pairing of homologous chromosomes. Fluorescence in situ hybridization in fission yeast indicated that chromosomal loci are positioned in an ordered manner as anticipated from the bouquet arrangement. Blocking the formation of the telomere cluster with the kms1 mutation created a disorganized chromosomal arrangement, not only for the regions proximal to the telomere but also for interstitial regions. The kms1 mutation also affected the positioning of a linear minichromosome. Consistent with this cytological observation, the frequency of ectopic homologous recombination between a linear minichromosome and a normal chromosome increased in the kms1 background. Intragenic recombination between allelic loci is reduced in the kms1 mutant, but those between non-allelic loci are unaffected or slightly increased. Thus, telomere-led chromosome organization facilitates homologous pairing and also restricts irregular chromosome pairing during meiosis.     

The positioning of rye homologous chromosomes added to wheat through the cell cycle in somatic cells untreated and treated with colchicine

The arrangement of chromosome pairs 5RL and 7R added to the wild type and the ph1b mutant line of hexaploid wheat are analyzed in 2N somatic root tip cells during the cell cycle relative to the arrangement that chromosomes 5RL show in 4N tapetal cells produced after colchicine treatment. Both homologous chromosome pairs are identified at interphase and mitosis by fluorescence in situ hybridization. In nuclei at interphase, chromosomes appear as discrete domains that show the Rabl orientation. Homologous chromosomes are predominantly non-associated and their positioning seems not to be influenced by the Ph1 gene that suppresses homoeologous meiotic pairing. This pattern of arrangement contrasts with the high level of somatic pairing that sister chromosomes show in the interphase that follows chromosome duplication induced by colchicine. Disruption of pairing observed in some 4N nuclei is produced at c-anaphase which suggests no topological redistribution of homologues during conformation of the new nucleus. Homologous chromosomes show no predominant arrangement in ellipsoidal metaphase plates, which contrasts with the preferential opposite location of homologues in human prometaphase rosettes. Differences between chromosomes in the variation of the length through the cell cycle and in the chromatin morphology when the Ph1 is absent suggest different patterns of chromatin condensation in both chromosomes.     

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.     

Die Speicheldrüsenchromosomen der StechmückeCulex pipiens L. II. Ergänzungen zur Kartierung

The map of larval salivary gland chromosomes of the mosquitoCulex pipiens L. is amended as follows: The banding pattern is corrected in arm 1L, and completed in 2L. The amendments were verified on isolated entire chromosomes. Discrepancies between the three maps published previously, are ascribed to differences in method of the respective authors. In map collation, only well-spread chromosomes of a similar degree of polyteny should be used. The spatial arrangement of chromosomes in the nucleus is discussed, and indications are given for distinguishing between chromosome ends and accidental breaks.     

Identification of the genomic locations of duplicate nucleotide sequences in maize by analysis of restriction fragment length polymorphisms

While preparing a linkage map for maize based upon loci detected through the use of restriction fragment length polymorphisms (RFLPs), it was found that 62 of the 217 cloned maize sequences tested (29%) detected more than one fragment on genomic Southern blots. Thus, more than one nucleotide sequence is present within the maize genome which is in part homologous to each of these cloned sequences. The genomic locations of these ``duplicate' sequences were determined and it was found that they usually originated from different chromosomes. The process which produced them did not operate randomly as some pairs of chromosomes share many duplicate sequences while many other pairs share none. Furthermore, these shared duplicate sequences are generally arrayed in an ordered arrangement along these chromosomes. It is believed that chromosomal segments which contain several duplicate loci in a generally ordered arrangement must have had a common origin. The presence of these duplicated segments supports the idea that allopolyploidy may have been involved in the evolution of maize. Nevertheless, the duplicate loci do not primarily involve five pairs of chromosomes and thus, five pairs of homeologous chromosomes are not currently present within the maize genome. The data clearly indicate that maize is not a recent allotetraploid produced by hybridization between two individuals with similar genomic structures; however, the data are also consistent with the possibility of these shared duplicate chromosomal segments having been generated through internal duplication.     

Tissue-Specific Differences in the Spatial Interposition of X-Chromosome and 3R Chromosome Regions in the Malaria Mosquito Anopheles messeae Fall.

Spatial organization of a chromosome in a nucleus is very important in biology but many aspects of it are still generally unresolved. We focused on tissue-specific features of chromosome architecture in closely related malaria mosquitoes, which have essential inter-specific differences in polytene chromosome attachments in nurse cells. We showed that the region responsible for X-chromosome attachment interacts with nuclear lamina stronger in nurse cells, then in salivary glands cells in Anopheles messeae Fall. The inter-tissue differences were demonstrated more convincingly in an experiment of two distinct chromosomes interposition in the nucleus space of cells from four tissues. Microdissected DNA-probes from nurse cells X-chromosome (2BC) and 3R chromosomes (32D) attachment regions were hybridized with intact nuclei of nurse cells, salivary gland cells, follicle epithelium cells and imaginal disсs cells in 3D-FISH experiments. We showed that only salivary gland cells and follicle epithelium cells have no statistical differences in the interposition of 2BC and 32D. Generally, the X-chromosome and 3R chromosome are located closer to each other in cells of the somatic system in comparison with nurse cells on average. The imaginal disсs cell nuclei have an intermediate arrangement of chromosome interposition, similar to other somatic cells and nurse cells. In spite of species-specific chromosome attachments there are no differences in interposition of nurse cells chromosomes in An. messeae and An. atroparvus Thiel. Nurse cells have an unusual chromosome arrangement without a chromocenter, which could be due to the special mission of generative system cells in ontogenesis and evolution.