Mechanisms of chromosome banding : VI. Whole mount electron microscopy of banded metaphase chromosomes and a comparison with pachytene chromosomes

Chinese hamster chromosomes, banded by exposure to actinomycin D during the G 2 period, were examined by whole mount electron microscopy. Bands of condensed chromatin were present in unstained preparations that were not fixed with methanol-acetic acid indicating that the differential condensation of chromatin plays a role in banding by this technique. There was a tendency for interdigitation of the chromatin of the homologous bands on sister chromatids. Since previous studies had shown that the bands of mitotic chromosomes matched the chromomeres of meiotic chromosomes, whole mount electron microscope preparations of pachytene chromosomes were also examined. These suggest that in addition to condensation the chromatin of the chromomeres may also have a higher density of attachment sites to the lateral element of the synaptonemal complex, and probably to the nuclear membrane in interphase cells.     

Electron microscopy and biochemical analysis of mouse metaphase chromosomes after digestion with restriction endonucleases

Electron microscopy (EM) of whole mounted mouse chromosomes, light microscopy (LM), and agarose gel electrophoresis of DNA were used to investigate the cytological effect on chromosomes of digestion with the restriction endonucleases (REs) AluI, HinfI, HaeIII and HpaII. Treatment with AluI produces C-banding as seen by LM, cuts DNA into small fragments, and reduces the density of centromeres and disperses the chromatin of the arms as determined by EM. Treatment with HinfI produces C-banding, cuts DNA into slightly larger fragments than does AluI and increases the density of centromeres and disperses the fibres in the chromosomal arms. Exposure to HaeIII produces G- + C-banding, cuts the DNA into large fragments, and results in greater density of centromeres and reduced density of arms. Finally HpaII digestion produces G-like bands, cuts the DNA into the largest fragments found and results in greater density of centromeres and the best preservation of chromosomal arms detected by EM. These results provide evidence for: (1) REs producing identical effects in the LM (AluI and HinfI) produce different effects in the EM. (2) All enzymes appear to affect C-bands but while REs such as AluI reduce the density of these regions, other enzymes such as HpaII, HaeIII or HinfI increase their density. Conformational changes in the chromatin could explain this phenomenon. (3) The appearance of chromosomes in the EM is related to the action of REs on isolated DNA. The more the DNA is cut by the enzyme, the greater the alteration of the chromosomal ultrastructure.     

Synaptosomal cytoskeleton visualized by whole mount electron microscopy

Organization of synaptosomal cytoskeleton was reproducibly visualized by the technique of whole mount electron microscopy. Synaptosomes from rat cerebrums were immobilized on the formvar membrane of the electron microscopic grid, partly solubilized by detergents of various kinds, and treated with chemicals to reveal cytoskeletons and their characteristics. Synaptosomal cytoskeletons consisted of three types: (1) pre-synaptic fiber network structure whose composite fiber was 15–20 nm in diameter and formed 60–100 nm circular rings. The rings had small particles inside and were organized into three-dimensional networks. The pre-synaptic network was different from the Triton-unextractable structure of mitochondria. (2) Post-synaptic fiber aggregate was constructed of 10-nm filaments that were typically visualized as deoxycholate- or N-lauroyl sarcosinate-unextractable cytoskeletons. The aggregate was a major structure in the Triton-unextractable cytoskeleton of synaptic plasma membrane (more than 95%). (3) Fiber connecting individual clusters of synaptosomal cytoskeletons which was probably an artifactual product formed during and after synaptosomal isolation. Existence of actin was indicated both in pre- and post-synaptic cytoplasm.     

Electron microscopy of G-banded human mitotic chromosomes

Trypsin-treated human metaphase chromosomes stained with Giemsa and uranyl acetate showed clear, reproducible band structures under the transmission electron microscope (TEM). The banding pattern observed with TEM corresponded very closely to the G-band pattern visualized by light microscopy. The TEM images were used for karyotype analyses. Trypsin-treated chromosomes stained with uranyl acetate alone also showed clear G-bands under TEM. Shadow casting in addition to uranyl acetate staining revealed more structural detail of the chromosomes. Chromosome fibers, 200 Å–300 Å in diameter, were observed in the interband regions. Most chromosomes showed the major G-bands under the higher TEM magnification wit0out any trypsin treatment.     

Whole mount electron microscopy of meiotic chromosomes and the synaptonemal complex

The mechanism by which homologous chromosomes pair and crossover has been a major unsolved problem in genetics. Thin section electron microscopy of the synaptonemal complex has not provided enough details to allow any significant insight into this problem. Whole mount preparations of the testis of mice, quail, crayfish, and frogs provided a striking improvement in visualization of the morphological features of meiotic chromosomes. These studies, when combined with the use of deoxyribonuclease and trypsin allowed the following conclusions. 1. The synaptonemal complex (lateral and central elements with connecting L-C fibers) is composed of protein. 2. Contrary to common speculation the central element is not the pairing surface of homologous chromosomes. 3. The L-C fibers, averaging 75–100 Å in width, extend from the lateral elements and meet to form the central element which is usually composed of four fibers. 4. During leptotene, homologous axial elements, although unpaired for most of their length, attach next to each other at the nuclear membrane. 5. Short segments of the chromatin fibers attach to the lateral elements. These points of attachment are clustered, producing the chromomeres seen by light microscopy. 6. The chromatin fibers extend out from the lateral element as loops. Lampbrush chromosomes are thus not restricted to oogenesis but are common to all meiotic chromosomes.Since the morphological features of the central element of the synaptonemal complex persist despite extensive deoxyribonuclease digestion, pairing is perhaps best visualized as a two-step process consisting of a) chromosomal pairing during which the proteinaceous synaptonemal complex pulls homologous chromosomes into approximate association with each other, and b) molecular pairing, which probably takes place in the area around the synaptonemal complex.Supported by NIH Grants GM-15886 and C-2568, and The Charles and Henrietta Detoy Research Fellowship.     

Imaging of human metaphase chromosomes by atomic force microscopy in liquid

Human metaphase chromosomes were observed using an intermittent contact mode of atomic force microscopy (AFM) in a phosphate-buffered saline solution to clarify their conformation close to that in the physiological state. In the AFM images in liquid, symmetric alternating ridges and grooves were evident on their surface of the paired sister chromatids. The number of the ridges and grooves were rather specific to the type of the chromosome. The structural changes of chromosomes caused by trypsin treatment were also directly observable using AFM in liquid. These results suggest that the intermittent contact mode AFM is useful not only for analyzing the structure of chromosomes in a liquid condition but also for studying the effect of chemical treatments on chromosomes in relation to their structural changes.     

Electron microscopic analysis of the structure of metaphase chromosomes after liposome treatment

The fine structure of the methaphase chromosomes from the Chinese hamster cell culture was studied after the incubation with lyposomes isolated from hen egg yolk phosphatidilcholine, or from the total rat liver phospholipid. It has been found that during incubation lyposomes surround chromosomes, form a multifold covering considerably decondensing the chromosome matrix. The data obtained indicate that the analogues of cell membrane - lyposomes - may be involved in the regulation of the structural organization of metaphase chromosomes.     

Electron microscopy of gold-labeled human and equine chromosomes

We present an immunochemical technique for the detection of 5-bromo-2'-deoxyuridine (BrdU) incorporated discontinuously into the chromosomal DNA. A monoclonal anti-BrdU antibody and a protein A-gold complex were used to produce chromosome banding of human and equine chromosomes, specific for electron microscopy (EM). Well-defined bands, symmetry of sister chromatids, concordance between homologues, and band patterns similar to those observed by light microscopy facilitate chromosome identification and karyotyping. From prophase to late metaphase, chromosomes condense and bands appear to fuse. The fusion appears to be owing to chromatin reorganization. Our results underline the value of using immunogold reagents, which are ideal probes for antigen localization on chromosomes.     

Immunoelectronmicroscopy of metaphase chromosomes

Summary A method for the preparation of ultrathin sections of metaphase chromosomes is described. This method was applied to human metaphase chromosomes, which were immunocytochemically stained with anti-DNA and anti-ribonucleoprotein antibodies, derived from patients with auto-immune disease. Conventionally prepared metaphase spreads as well as cytocentrifuge preparations of chromosome suspensions were studied. The results indicate that the ultrastructure of chromosomes and the immunoreactivity of chromosomal constituents are influenced by the applied preparation methods. In comparison with whole mount preparations, ultrathin sections of immunostained chromosomes allow higher resolution and more precise localization of immunoreactive sites within the chromosomal structure.     

Immunoelectronmicroscopy of metaphase chromosomes

A method for the preparation of ultrathin sections of metaphase chromosomes is described. This method was applied to human metaphase chromosomes, which were immunocytochemically stained with anti-DNA and anti-ribonucleoprotein antibodies, derived from patients with auto-immune disease. Conventionally prepared metaphase spreads as well as cytocentrifuge preparations of chromosome suspensions were studied. The results indicate that the ultrastructure of chromosomes and the immunoreactivity of chromosomal constituents are influenced by the applied preparation methods. In comparison with whole mount preparations, ultrathin sections of immunostained chromosomes allow higher resolution and more precise localization of immunoreactive sites within the chromosomal structure.     

Atomic force microscopy and scanning near-field optical microscopy studies on the characterization of human metaphase chromosomes

A better knowledge of biochemical and structural properties of human chromosomes is important for cytogenetic investigations and diagnostics. Fluorescence in situ hybridization (FISH) is a commonly used technique for the visualization of chromosomal details. Localizing specific gene probes by FISH combined with conventional fluorescence microscopy has reached its limit. Also, microdissecting DNA from G-banded human metaphase chromosomes by either a glass tip or by laser capture needs further improvement. By both atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM), local information from G-bands and chromosomal probes can be obtained. The final resolution allows a more precise localization compared to standard techniques, and the extraction of very small amounts of chromosomal DNA by the scanning probe is possible. Besides new strategies towards a better G-band and fluorescent probe detection, this study is focused on the combination of biochemical and nanomanipulation techniques which enable both nanodissection and nanoextraction of chromosomal DNA.     

Electron microscopy of membrane-associated folded chromosomes of Escherichia coli

Membrane-associated folded chromosomes were purified from log-phase cultures of Escherichia coli 15 TAU-bar and prepared for electron microscopy by aqueous spreading techniques. A spectrum of structures was observed, ranging from condensed structures with no DNA fibers visible, to extended structures with DNA fibers. In the extended structures, loops of DNA radiated from residual envelope, the loops sometimes appeared super-coiled, and both their number and apparent contour length approximated previous estimates from physical and biochemical data. It is proposed that the structures with free DNA arose from the condensed structures.     

Electron microscopy of membrane-free folded chromosomes from Escherichia coli

Membrane-free folded chromosomes were purified from log-phase cultures of Escherichia coli and prepared for electron microscopy by aqueous (Kleinschmidt and Zahn) spreading. The appearance of the chromosomes depended on the salt concentrations in spreading. At certain salt concentration, the chromosomes resembled rosettes, with supercoiled loops of DNA radiating from a central core containing RNA. The rosettes support previous models deduced from physical studies of folded chromosomes. Apparently, cores contain must of the visible RNA, and the organization of the core is linked to the organization of the DNA loops.Submitted in celebration of Julius Marmur's birthday—his teachings made this study possible     

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.