Structure of human chromosomes studied by atomic force microscopy. Part II. Relationship between structure and cytogenetic bands

In the first part of this work, human chromosomes were characterized by atomic force microscopy (AFM) in air and in aqueous solution. The analysis of the images suggests that the last level of organization consists of a radial arrangement of chromatin loops which are anchored to a fiber which is folded giving a pattern of bands which differs in volume. Here the pattern of bands observed by AFM is compared to the cytogenetic map at the 850-band level. Thus thicker and thinner bands are identified as G and R bands, respectively. Finally a model is proposed which links genome sequence, cytogenetics, and chromosome structure.     

Helical Structure Revealed upon the Condensation and Decondensation of Chromosomes of Vicia faba

By using conventional and stereo electron microscopy, helical structures were revealed in prophase and telophase chromosomes of root tip cells of Vicia faba. Longitudinal and transverse sections of the chromosomes showed that both prophase and telophase chromosomes were composed of chromatin fibres about 0.5μm in diameter and among the 0.5μm chromatin fibre thinner chromatin fibres with a diameter of about 0.2μm were found. In transverse sections, prophase chromosomes appeared to be a circular structure which contained a low electron density centre encircled by the 0.5 μm fibre. In longitudinal section of the chromosomes, the 0.5 μm fibres were seen to be orientated parallel to each other while constituted roughly a right angle to the long axis of the chromosome. Helical coils consisting of the 0.5μm fibre were identified easily by stereo electron microscopy. In transverse sections of telophase chromosomes, both the circular structure similar to that of the prophase chromosomes and the hoof-shaped structure composed of the 0.5μm fibre were observed, demonstrating4 the de-spiralization of the helical coils in the decondensation of the chromosomes. Based on these observations, the radial loop. model and the multiple coiling model are discussed.     

Scanning electron microscopy of the centromeric region of L-cell chromosomes after treatment with Hoechst 33258 combined with 5-bromodeoxyuridine

When mouse L-cells were treated with a combination of 5-bromodeoxyuridine (BrdUrd) and Hoechst 33258, the metaphase chromosomes revealed undercondensation of the chromatin fibers in the sister centromeres. The application of the osmium-thiocarbohydrazide technique to the air-dried chromosome preparations made it possible to elucidate the ultrastructure of the undercondensed centromeric region at the level of the 30 nm chromatin fiber. Scanning electron microscopy revealed that the undercondensed region consisted of a coiled fiber with a diameter of about 400 nm, and a gyre diameter of approximately 600 nm. The coiled fiber was composed of the 30 nm chromatin fiber loops. These findings indicate that a continuous coiled structure, which is the final higher order structure of the condensed chromatin fiber, exists throughout the entire length of the mouse L-cell metaphase chromosome.     

Packaging the genome: the structure of mitotic chromosomes

Mitotic chromosomes are essential structures for the faithful transmission of duplicated genomic DNA into two daughter cells during cell division. Although more than 100 years have passed since chromosomes were first observed, it remains unclear how a long string of genomic DNA is packaged into compact mitotic chromosomes. Although the classical view is that human chromosomes consist of radial 30 nm chromatin loops that are somehow tethered centrally by scaffold proteins, called condensins, cryo-electron microscopy observation of frozen hydrated native chromosomes reveals a homogeneous, grainy texture and neither higher-order nor periodic structures including 30 nm chromatin fibres were observed. As a compromise to fill this huge gap, we propose a model in which the radial chromatin loop structures in the classic view are folded irregularly toward the chromosome centre with the increase in intracellular cations during mitosis. Consequently, compact native chromosomes are made up primarily of irregular chromatin networks cross-linked by self-assembled condensins forming the chromosome scaffold.     

Complementary visualization of mitotic barley chromatin by field-emission scanning electron microscopy and scanning force microscopy

The surface structure of mitotic barley chromatin was studied by field-emission scanning electron microscopy (FESEM) and scanning force microscopy (SFM). Different stages of the cell cycle were accessible after a cell suspension was dropped onto a glass surface, chemical fixed, and critically point dried. Imaging was carried out with metal-coated specimen or uncoated specimen (only for SFM). The spatial contour of the chromatin could be resolved by SFM correlating to FESEM data. The experimentally determined volume of the residue chromatin during mitosis was within the range of 65-85 microm(3). A comparison with the theoretically calculated volume indicated a contribution of about 40% of internal cavities. Decondensation of chromosomes by proteinase K led to a drastic decrease in the chromosome volume, and a 3-D netlike architecture of the residue nucleoprotein material, similar to that in the intact chromosome, was obvious. Incubation of metaphase chromosomes in citrate buffer permitted access to different levels of chromatin packing. We imaged intact chromosomes in liquid by SFM without any intermediate drying step. A granular surface was obvious but with an appreciably lower resolution. Under similar imaging conditions proteinase K-treated chromosomes exhibited low topographic contrast but were susceptible to plastic deformations.     

Scaffold-like structures in mouse chromosomes revealed by restriction endonuclease digestion and electron microscopy

A scaffold-like structure is observed under the electron microscope when mouse chromosomes are digested with the restriction endonuclease Hae III. This structure, located in the inner part of chromatids, may correspond to those fragments of chromatin loops anchored to the chromosome scaffold and is obtained when chromosomes are treated either in suspension or attached to grids. The width of the structure is correlated with the extent of digestion in chromosomes treated in suspension. Those treated on grids show this structure whenever chromatids do not collapse. These results agree with the model of chromosome organization based on a non-histone protein scaffold.     

Assembly of chromatin fibers into metaphase chromosomes analyzed by transmission electron microscopy and scanning electron microscopy.

The higher-order assembly of the approximately 30 nm chromatin fibers into the characteristic morphology of HeLa mitotic chromosomes was investigated by electron microscopy. Transmission electron microscopy (TEM) of serial sections was applied to view the distribution of the DNA-histone-nonhistone fibers through the chromatid arms. Scanning electron microscopy (SEM) provided a complementary technique allowing the surface arrangement of the fibers to be observed. The approach with both procedures was to swell the chromosomes slightly, without extracting proteins, so that the densely-packed chromatin fibers were separated. The degree of expansion of the chromosomes was controlled by adjusting the concentration of divalent cations (Mg2+). With TEM, individual fibers could be resolved by decreasing the Mg2+ concentration to 1.0-1.5 mM. The predominant mode of fiber organization was seen to be radial for both longitudinal and transverse sections. Using SEM, surface protuberances with an average diameter of 69 nm became visible after the Mg2+ concentration was reduced to 1.5 mM. The knobby surface appearance was a variable feature, because the average diameter decreased when the divalent cation concentration was further reduced. The surface projections appear to represent the peripheral tips of radial chromatin loops. These TEM and SEM observations support a "radial loop" model for the organization of the chromatin fibers in metaphase chromosomes.     

Architecture of metaphase chromosomes and chromosome scaffolds

We have developed procedures for depositing intact mitotic chromosomes and isolated residual scaffolds on electron microscope grids at controlled and reproducible levels of compaction. The chromosomes were isolated using a recently developed aqueous method. Our study has addressed two different aspects of chromosome structure. First, we present a method for improved visualization of radial chromatin loops in undisrupted mitotic chromosomes. Second, we have visualized a nonhistone protein residual scaffold isolated from nuclease-digested chromosomes under conditions of low salt protein extraction. These scaffolds, which have an extremely simple protein composition, are the size of chromosomes, are fibrous in nature, and are found to retain differentiated regions that appear to derive from the kinetochores and the chromatid axis. When our standard preparation conditions were used, the scaffold appearance was found to be very reproducible. If the ionic conditions were varied, however, the scaffold appearance underwent dramatic changes. In the presence of millimolar concentrations of Mg++ or high concentrations of NaCl, the fibrous scaffold protein network was observed to undergo a lateral aggregation or assembly into a coarse meshlike structure. The alteration of scaffold structure was apparently reversible. This observation is consistent with a model in which the scaffolding network plays a dynamic role in chromosome condensation at mitosis.     

Cohesin Smc1beta determines meiotic chromatin axis loop organization

Meiotic chromosomes consist of proteinaceous axial structures from which chromatin loops emerge. Although we know that loop density along the meiotic chromosome axis is conserved in organisms with different genome sizes, the basis for the regular spacing of chromatin loops and their organization is largely unknown. We use two mouse model systems in which the postreplicative meiotic chromosome axes in the mutant oocytes are either longer or shorter than in wild-type oocytes. We observe a strict correlation between chromosome axis extension and a general and reciprocal shortening of chromatin loop size. However, in oocytes with a shorter chromosome axis, only a subset of the chromatin loops is extended. We find that the changes in chromatin loop size observed in oocytes with shorter or longer chromosome axes depend on the structural maintenance of chromosomes 1β (Smc1β), a mammalian chromosome–associated meiosis-specific cohesin. Our results suggest that in addition to its role in sister chromatid cohesion, Smc1β determines meiotic chromatin loop organization.     

Differential silver staining of chromatin in metaphase chromosomes

The silver techniques used to demonstrate nucleolar organizer regions and cores in chromosomes can also differentially stain chromatin within chromosomes. Direct silver staining of mouse and human chromosomes resulted in preferential staining of centromeric regions and non-nucleolar secondary constrictions, both of which are composed of constitutive heterochromatin. After C-banding, these regions were no longer silver-stainable, suggesting that the biochemical constituents (presumably non-histone proteins) which contain the reaction sites for silver are extracted during the banding treatment. Light and electron microscopy of chromosomes G-banded with trypsin and then silver-stained revealed heavier deposits of silver over the condensed aggregates of chromatin within the band regions than over the more dispersed interband chromatin. At the ultrastructural level, chromatin fibres were covered with silver grains, indicating that there are many reaction sites for this metal along the fibres. These results suggest that the degree of silver staining in any region of the chromosome may be contingent upon the concentration of chromatin in that region. This finding may have important implications concerning the nature of the silver-stained core-like structure in chromosomes. If a preferential dispersion of chromatin fibres occurs at the periphery of the chromosome during slide preparation, leaving the central region of each chromatid relatively undispersed, this difference in the concentration of chromatin may account for the differential silver staining of these regions and the consequent appearance of a core-like structure.     

Differential distribution and association of repeat DNA sequences in the lateral element of the synaptonemal complex in rat spermatocytes

The synaptonemal complex (SC) is an evolutionarily conserved structure that mediates synapsis of homologous chromosomes during meiotic prophase I. Previous studies have established that the chromatin of homologous chromosomes is organized in loops that are attached to the lateral elements (LEs) of the SC. The characterization of the genomic sequences associated with LEs of the SC represents an important step toward understanding meiotic chromosome organization and function. To isolate these genomic sequences, we performed chromatin immunoprecipitation assays in rat spermatocytes using an antibody against SYCP3, a major structural component of the LEs of the SC. Our results demonstrated the reproducible and exclusive isolation of repeat deoxyribonucleic acid (DNA) sequences, in particular long interspersed elements, short interspersed elements, long terminal direct repeats, satellite, and simple repeats. The association of these repeat sequences to the LEs of the SC was confirmed by in situ hybridization of meiotic nuclei shown by both light and electron microscopy. Signals were also detected over the chromatin surrounding SCs and in small loops protruding from the lateral elements into the SC central region. We propose that genomic repeat DNA sequences play a key role in anchoring the chromosome to the protein scaffold of the SC. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Selective cleaning of the cell debris in human chromosome preparations studied by scanning force microscopy

The chromosome structure is one of most challenging biological structures to be discovered. Most evidence about the structure comes from optical microscopy. Scanning force microscopy (SFM) can achieve molecular resolution and allows imaging in liquids. However, little information about the chromosome structure has been revealed by SFM. In this work, a mild enzymatic treatment is applied to the chromosomes to remove selectively the RNA and proteins coming from the cell. The resulting SFM images indicate that a protein film with embedded RNA molecules covers chromosomes in standard cytogenetic preparations. The thickness of the protein layer is 15-35 nm and the RNA adheres preferentially to the chromosome surface. The cell material film results in a quite smooth chromosome surface without evidence of any structural detail. After treatment, the chromosome was cleaned from cell residues and individual chromatin fibers at the surface were resolved. Furthermore, insights about the higher order structure of the chromosome can be inferred.     

Anti-topoisomerase II recognizes meiotic chromosome cores

At meiotic prophase the chromatin becomes arranged in loops on newly formed chromosome cores. The cores of homologous chromosomes become aligned in parallel and thus form the synaptonemal complex (SC), a structure found in the meiocytes of nearly all recombinationally competent, sexually reproducing organisms. We report that two polyclonal antibodies against topoisomerase II (topo II), which recognize the mitotic metaphase chromosome scaffold give, at pachytene, a positive immunocytological reaction with the chromatin and, predominantly, with the cores and centromeric regions of the paired chromosomes. It therefore appears that during meiotic prophase, topo II — a DNA-binding enzyme implicated in transient double-strand breaks, chromosome condensation, and anaphase separation — is associated with the chromatin and SCs of the pachytene and diplotene chromosomes.     

A three-dimensional approach to mitotic chromosome structure: evidence for a complex hierarchical organization

We describe findings on the architecture of Drosophila melanogaster mitotic chromosomes, made using a three-dimensional-oriented structural approach. Using high-voltage and conventional transmission electron microscopy combined with axial tomography and digital contrast-enhancement techniques, we have for the first time visualized significant structural detail within minimally perturbed mitotic chromosomes. Chromosomes prepared by several different preparative procedures showed a consistent size hierarchy of discrete chromatin structural domains with cross-sectional diameters of 120, 240, 400-500, and 800-1,000 A. In fully condensed, metaphase-arrested chromosomes, there is evidence for even larger-scale structural organization in the range of 1,300-3,000-A size. The observed intrachromosomal arrangements of these higher-order structural domains show that both the radial loop and sequential helical coiling models of chromosome structure are over-simplifications of the true situation. Finally, our results suggest that the pathway of chromatin condensation through mitosis consists of concurrent changes occurring at several levels of chromatin organization, rather than a strictly sequential folding process.     

Localization of topoisomerase II in mitotic chromosomes

In the preceding article we described a polyclonal antibody that recognizes cSc-1, a major polypeptide component of the chicken mitotic chromosome scaffold. This polypeptide was shown to be chicken topoisomerase II. In the experiments described in the present article we use indirect immunofluorescence and immunoelectron microscopy to examine the distribution of topoisomerase II within intact chromosomes. We also describe a simple experimental protocol that differentiates antigens that are interspersed along the chromatin fiber from those that occupy restricted domains within the chromosome. These experiments indicate that the distribution of the enzyme appears to be independent of the bulk chromatin. Our data suggest that topoisomerase II is bound to the bases of the radial loop domains of mitotic chromosomes.     

Electron Tomography of Metaphase Nucleolar Organizer Regions: Evidence for a Twisted-Loop Organization

Metaphase nucleolar organizer regions (NORs), one of four types of chromosome bands, are located on human acrocentric chromosomes. They contain r-chromatin, i.e., ribosomal genes complexed with proteins such as upstream binding factor and RNA polymerase I, which are argyrophilic NOR proteins. Immunocytochemical and cytochemical labelings of these proteins were used to reveal r-chromatin in situ and to investigate its spatial organization within NORs by confocal microscopy and by electron tomography. For each labeling, confocal microscopy revealed small and large double-spotted NORs and crescent-shaped NORs. Their internal three-dimensional (3D) organization was studied by using electron tomography on specifically silver-stained NORs. The 3D reconstructions allow us to conclude that the argyrophilic NOR proteins are grouped as a fiber of 60–80 nm in diameter that constitutes either one part of a turn or two or three turns of a helix within small and large double-spotted NORs, respectively. Within crescent-shaped NORs, virtual slices reveal that the fiber constitutes several longitudinally twisted loops, grouped as two helical 250- to 300-nm coils, each centered on a nonargyrophilic axis of condensed chromatin. We propose a model of the 3D organization of r-chromatin within elongated NORs, in which loops are twisted and bent to constitute one basic chromatid coil.     

Cytogenetic stability of chicken T-cell line transformed with Marek's disease virus: atomic force microscope, a new tool for investigation

The Marek's disease virus (MDV) integration may induce a novel organization of chromatin architecture with a modified genetic expression. In our opinion it is worthwhile trying to relate cytogenetic stability to functional modifications. Recently, atomic force microscopy technique was applied to study the structure of chromosomes at a nanoscale level. This high resolution allows to investigate the different structure of chromatin in order to study cytogenetic stability and chromosome aberrations due to MDV insertion. In this paper data are presented indicating a duplication [78,WZ,dup(1p)(p22-p23)] and a deletion [78,WZ cht del(3)(q2.10)] of Chromosomes 1 and 3 relatively. Relationships between GTG (G-bands by Trypsin using Giemsa) bands and the topography of chromosomes are also discussed, naming them Topographic Banding. The architecture of chromosomes observed by AFM can be related to the data obtained with classic banding techniques thus overcoming the optical resolution limits. The presence of chromatin bridges between sister chromatids at most of the heterochromatic regions is also evidenced. Besides, we present different studies of the longitudinal and transversal symmetry of the hetero and euchromatic regions to clearly demonstrate a different underlying architecture of these regions. It is indeed evident that the heterochromatic bands are more symmetrical than euchromatic bands.     

Engineered chromosome regions with altered sequence composition demonstrate hierarchical large-scale folding within metaphase chromosomes

Mitotic chromosome structure and DNA sequence requirements for normal chromosomal condensation remain unknown. We engineered labeled chromosome regions with altered scaffold-associated region (SAR) sequence composition as a formal test of the radial loop and other chromosome models. Chinese hamster ovary cells were isolated containing high density insertions of a transgene containing lac operator repeats and a dihydrofolate reductase gene, with or without flanking SAR sequences. Lac repressor staining provided high resolution labeling with good preservation of chromosome ultrastructure. No evidence emerged for differential targeting of SAR sequences to a chromosome axis within native chromosomes. SAR sequences distributed uniformly throughout the native chromosome cross section and chromosome regions containing a high density of SAR transgene insertions showed normal diameter and folding. Ultrastructural analysis of two different transgene insertion sites, both spanning less than the full chromatin width, clearly contradicted predictions of simple radial loop models while providing strong support for hierarchical models of chromosome architecture. Specifically, an approximately 250-nm-diam folding subunit was visualized directly within fully condensed metaphase chromosomes. Our results contradict predictions of simple radial loop models and provide the first unambiguous demonstration of a hierarchical folding subunit above the level of the 30-nm fiber within normally condensed metaphase chromosomes.