Structure and dynamics of human interphase chromosome territories in vivo

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. Received: 10 November 1997 / Accepted: 24 November 1997     

Non-random chromosome distribution in radial metaphases from the Chinese hamster

Chromosome distribution was analyzed in uncultured radial metaphase cells (corneal epithelium, testicular mitotic cells, cells in diakinesis, and cells in metaphase II) from the Chinese hamster. The hypothesis of random distribution was rejected at the 0.001 level (x ₃ ² = 31.6). — Homologous association was observed for two pairs of chromosomes (3 and 10) in corneal epithelial cells. It was observed for all chromosomes in the testicular mitotic cells. Acrocentric association was observed in all four cell types. The chromosomes associated in four groups of similarly sized and shaped chromosomes. While group membership did not appear to vary, position within the group was highly variable. — An elevenpoint model of chromosome relationships was constructed from the data.     

Spatial organization of chromosome territories in the interphase nucleus of trisomy 21 cells

In the interphase cell nucleus, chromosomes adopt a conserved and non-random arrangement in subnuclear domains called chromosome territories (CTs). Whereas chromosome translocation can affect CT organization in tumor cell nuclei, little is known about how aneuploidies can impact CT organization. Here, we performed 3D-FISH on control and trisomic 21 nuclei to track the patterning of chromosome territories, focusing on the radial distribution of trisomic HSA21 as well as 11 disomic chromosomes. We have established an experimental design based on cultured chorionic villus cells which keep their original mesenchymal features including a characteristic ellipsoid nuclear morphology and a radial CT distribution that correlates with chromosome size. Our study suggests that in trisomy 21 nuclei, the extra HSA21 induces a shift of HSA1 and HSA3 CTs out toward a more peripheral position in nuclear space and a higher compaction of HSA1 and HSA17 CTs. We posit that the presence of a supernumerary chromosome 21 alters chromosome compaction and results in displacement of other chromosome territories from their usual nuclear position.     

Expansion of chromosome territories with chromatin decompaction in BAF53-depleted interphase cells

Chromosomes are compartmentalized into discrete chromosome territories during interphase in mammalian cells. A chromosome territory is generated by the tendency of chromatin to occupy the smallest shell volume, which is determined by the polymeric properties and interactions of the internal meshwork of the chromatin fiber. Here, we show that BAF53 knockdown by small interfering RNA interference led to the expansion of chromosome territories. This was accompanied by a reduction in chromatin compaction, an increase in the micrococcal nuclease sensitivity of the chromatin, and an alteration in H3-K9 and H3-K79 dimethylation. Interestingly, the BAF53 knockdown cells suffer a cell cycle defect. Despite the significant irregularity and decompaction of the polynucleosomes isolated from the BAF53 knockdown cells, the chromatin loading of H1 and core histones remained unaltered, as did the nucleosome spacing. The histone hyperacetylation and down-regulation of BRG-1, mBrm, and Tip49, the catalytic components of the SWI/SNF complex and the TIP60 complex, respectively, did not expand chromosome territories. These results indicate that BAF53 contributes to the polymeric properties and/or the internal meshwork interactions of the chromatin fiber probably via a novel mechanism.     

Generation of a Brassica oleracea composite RFLP map: linkage arrangements among various populations and evolutionary implications

Summary A composite linkage map of Brassica oleracea was developed from maps of four different populations, derived from 108 DNA, isozyme and morphological loci covering over 747 centimorgans in 11 linkage groups. Of these linkage groups, 8 were assigned to their respective chromosomes by alignment with gene synteny groups of B. oleracea. Distortions in segregation ratios increased with the level of divergence of the parents and were attributed to differentiation of parental chromosomes. Comparison of the individual maps demonstrates that the B. oleracea genome undergoes frequent chromosomal rearrangement, even at the subspecies level. Small inversions were the most frequent form of aberration followed by translocations. The former type of aberration could occur without a noticeable effect on meiotic behavior of chromosomes or on pollen fertility. The obvious deduction from the composite map is that a large fraction of the B. oleracea genome is duplicated, falling into three classes: randomly dispersed, linked-gene families, and blocks duplicated in non-homologous chromosomes. The genealogy of chromosomes sharing duplicated segments was formulated and indicates that B. oleracea is a secondary polyploid species derived from ancestral genome(s) of fewer chromosomes.     

Specific staining of human chromosomes in Chinese hamster x man hybrid cell lines demonstrates interphase chromosome territories

Summary In spite of Carl Rabl's (1885) and Theodor Boveri's (1909) early hypothesis that chromosomes occupy discrete territories or domains within the interphase nucleus, evidence in favor pf this hypothesis has been limited and indirect so far in higher plants and animals. The alternative possibility that the chromatin fiber of single chromosomes might be extended throughout the major part of even the whole interphase nucleus has been considered for many years. In the latter case, chromosomes would only exist as discrete chromatin bodies during mitosis but not during interphase. Both possibilities are compatible with Boveri's well established paradigm of chromosome individuality. Here we show that an active human X chromosome contained as the only human chromosome in a Chinese hamster x man hybrid cell line can be visualized both in metaphse plates and in interphase nuclei after in situ hybridization with either ³H- or biotin-labeled human genomic DNA. We demonstrate that this chromosome is organized as a distinct chromatin body throughout interphase. In addition, evidence for the territorial organization of human chromosomes is also presented for another hybrid cell line containing several autosomes and the human X chromosome. These findings are discussed in the context of our present knowledge of the organization and topography of interphase chromosomes. General applications of a strategy aimed at specific staining of individual chromosomes in experimental and clinical cytogenetics are briefly considered.     

Visualization of chromosome territories in interphase nuclei of ovarian nurse cells in Calliphora erythrocephala Mg. (Diptera: Calliphoridae)

Analysis of localization of chromosomes 2, 3, and 6 of Calliphora erythrocephala Mg. in ovarian nurse cell nuclei with different chromatin structure has shown that the regions of DNA probe hybridization reduced with increasing chromatin compaction. Hybridization of DNA probes of chromosomes 3 and 6 to secondary reticular nuclei demonstrated that chromosomes retain their territories in the nuclei when the chromatin acquires a reticular structure. These results suggest regular organization of the chromosomal apparatus at all stages of the endomitotic cycle, including the stage of highly polyploid reticular nuclei. FISH of DNA probe of the chromosome 2 telomeric region to secondary reticular nuclei revealed a peripheral distribution of the signal. Zones of more intensive DNA probe hybridization have been distinguished. These zones probably are the regions of accumulation of telomeric and (or) centromeric chromosome regions.     

An experimental evaluation of self-interference in Narcissus assoanus: functional and evolutionary implications

Floral traits that reduce self-pollination in hermaphroditic plants have usually been interpreted as mechanisms that limit the genetic consequences of self-fertilization. However, the avoidance of sexual conflict between female and male function (self-interference) may also represent an important selection pressure for the evolution of floral traits, particularly in self-incompatible species. Here, we use experimental manipulations to investigate self-interference in Narcissus assoanus, a self-incompatible species with a stigma-height dimorphism in which the degree of spatial separation between sex organs (herkogamy) differs strikingly between the long- and short-styled morphs (hereafter L- and S-morphs). We predicted that weak herkogamy in the L-morph would cause greater self-pollination and hence self-interference. Experimental self-pollination reduced seed set when it occurred prior to, or simultaneously with, cross-pollination in the L-morph, but only if it occurred prior to cross-pollination in the S-morph. In the field, autonomous self-pollination was greater in the L-morph than the S-morph, but we found no evidence that self-interference reduced maternal or paternal fitness in either morph. One-day-old flowers of the L-morph have reduced stigma receptivity and hence exhibit protandry, whereas stigma receptivity and anther dehiscence are concurrent in the S-morph. This suggests that the two style morphs have alternative strategies for reducing self-interference: dichogamy in the L-morph and herkogamy in the S-morph. These results provide insight into the mechanisms that reduce sexual conflict in hermaphrodite plants and are of significance for understanding the evolution and maintenance of sexual polymorphisms.     

Rise, fall and resurrection of chromosome territories: a historical perspective. Part I. The rise of chromosome territories

It is now generally accepted that chromosomes in the cell nucleus are organized in distinct domains, first called chromosome territories in 1909 by the great cytologist Theodor Boveri. Yet, even today chromosomes have remained enigmatic individuals, whose structures, arrangements and functions in cycling and post-mitotic cells still need to be explored in full detail. Whereas numerous recent reviews describe present evidence for a dynamic architecture of chromosome territories and discuss the potential significance within the functional compartmentalization of the nucleus, a comprehensive historical account of this important concept of nuclear organization was lacking so far. Here, we describe the early rise of chromosome territories within the context of the discovery of chromosomes and their fundamental role in heredity, covering a period from the 1870th to the early 20th century (part I, this volume). In part II (next volume) we review the abandonment of the chromosome territory concept during the 1950th to 1980th and the compelling evidence, which led to its resurrection during the 1970th to 1980th.     

Myofilament lengths of cat skeletal muscle: theoretical considerations and functional implications.

The cat is the primary model for neuromuscular research. However, sarcomere geometry, in particular thin-myofilament lengths of cat skeletal muscles, is not known, thus preventing adequate muscle modeling on the sarcomere level. The purpose of this study was to determine thin-myofilament lengths in cat skeletal muscle. It was found that average thin-myofilament lengths of cat tibialis anterior muscles (1.12 microns) were larger than the average values reported for frog (approximately 0.95 microns), rat (1.09 microns), and rabbit muscles (1.09 microns) and were smaller than the values reported for monkey (1.16 microns) and human skeletal muscles (1.27 microns). According to the cross-bridge theory of muscular contraction, this result implies that the range of sarcomere length on the ascending limb of the force-length relation for cat muscle is between those of frog, rat, and rabbit on the one side and monkey and human on the other side. It is speculated that the differences in thin-myofilament lengths of different animals are related to the functional demands of these muscles in everyday movement tasks. Isolated experimental observations appear to support this speculation.     

Three-dimensional reconstruction of painted human interphase chromosomes: active and inactive X chromosome territories have similar volumes but differ in shape and surface structure

This study provides a three-dimensional (3D) analysis of differences between the 3D morphology of active and inactive human X interphase chromosomes (Xa and Xi territories). Chromosome territories were painted in formaldehyde-fixed, three-dimensionally intact human diploid female amniotic fluid cell nuclei (46, XX) with X-specific whole chromosome compositive probes. The colocalization of a 4,6-diamidino-2- phenylindole dihydrochloride-stained Barr body with one of the two painted X territories allowed the unequivocal discrimination of the inactive X from its active counterpart. Light optical serial sections were obtained with a confocal laser scanning microscope. 3D- reconstructed Xa territories revealed a flatter shape and exhibited a larger and more irregular surface when compared to the apparently smoother surface and rounder shape of Xi territories. The relationship between territory surface and volume was quantified by the determination of a dimensionless roundness factor (RF). RF and surface area measurements showed a highly significant difference between Xa and Xi territories (P < 0.001) in contrast to volume differences (P > 0.1). For comparison with an autosome of similar DNA content, chromosome 7 territories were additionally painted. The 3D morphology of the chromosome 7 territories was similar to the Xa territory but differed strongly from the Xi territory with respect to RF and surface area (P < 0.001).     

The undiscovered country: chromosome territories and the organization of transcription

The interchromosome domain (ICD) model proposes that genes are selectively positioned at the surfaces of chromosome territories to facilitate their regulation. A paper in the May 13 issue of the Journal of Cell Biology provides evidence that supports a reinterpretation of this model.     

CpG Islands as a putative source for animal miRNAs: evolutionary and functional implications

MicroRNAs (miRs) are considered major contributors to the evolution of animal morphological complexity. Multiple bursts of novel miR families were documented throughout animal evolution, yet, their evolutionary origins are not understood. Here, we discuss two alternative genomic sources for novel miR families, namely, transposable elements, which were previously described, and a newly proposed origin: CpG islands. We show that these two origins are evolutionarily distinct and that they correspond to marked differences in several functional and genomic characteristics. Together, our results shed light on the intriguing origin of one of the major constituents of regulatory networks in animals, miRs.     

Molecular cytological differentiation of active from inactive X domains in interphase: implications for X chromosome inactivation

A fluorescence in situ hybridization method using a biotinylated DNA probe specific for the centromeric region of the human X chromosome was used to differentiate the genetically active from the inactive X in interphase cells. With this technique, we were able to interpret both the relative position and the degree of condensation of the X chromosomes within the nucleus. We first established the specificity of fluorescence labelling of the hybridized probe by comparing its location and appearance (either dense or diffuse) when associated with a sex chromatin body (SCB) in early passage normal human female fibroblasts. In these cells, where the presence of inactive X chromatin was verified by identification of a 4',6-diamidino-2-phenyl indole (DAPI)-positive SCB in 85% of the cells examined, the X chromatin fluorescence was always associated with the SCB. The signal was dense in structure in 98% and peripheral in location in 80% of the nuclei. A second type of signal, diffuse in form, was observed in 85% of the nuclei and presumably represents the location of the active X chromosome. It was located peripherally or centrally with equal frequency and was not associated with any identifiable nuclear component. This diffuse signal was the major type associated with human male fibroblasts. In rodent x human hybrid cells containing a human inactive X, the fluorescent signal was associated with an SCB-like structure in only 13% of the nuclei; it was dense in 66% of the nuclei and equally peripheral or central in location. This indicates an alteration in the interphase structure of the human inactive X chromosome in hybrid cells which may explain its known instability with respect to genetic activity in such systems.