Scanning electron microscopy of amphibian lampbrush chromosomes

A protein having a molecular weight of 73,000 daltons has been purified from the nuclear membranes of preleptotene, leptotene, and zygotene cells. It has been named the leptotene protein (L-protein) because of its role in suppressing the replication of zygotene DNA sequences through leptotene until the initiation of zygotene DNA synthesis. The protein has been found to be highly specific in its inhibitory activity. S-phase replication in somatic nuclei and in microspore nuclei are unaffected by the protein. Only zygotene DNA sequences appear to be affected. L-protein binds specifically to zygotene DNA. The binding is limited to a relatively short DNA segment, probably no longer than 90 base pairs (bp). Chloroplast and mitochondrial DNA do not bind to the protein, but a low level of binding is displayed by DNA from several other eukaryotic species. The L-protein also has the capacity to nick the bound DNA in the presence of ATP. Nicking does not occur in the absence of binding. Using supercoiled plasmids with zygotene DNA inserts as substrates, the nicking has been found to be confined to a small region of the plasmid and to occur in only one of the strands. The L-protein is considered to be one of the principal factors responsible for the irreversible commitment of cells to meiosis at the end of the preleptotene S-phase. It is also proposed that its endonucleolytic activity plays a role in the initiation of synapsis.     

Heterochromatin regions of the chromosomes of the hen and Japanese quail in mitosis and at the lampbrush stage

The mitotic and lampbrush chromosomes of the domestic fowl and Japanese quail were analysed by fluorochrome staining technique. The lampbrush chromosomes of both the subjects displayed a typical "loop-chromomere" structure. Three distinct kinds of loops were distinguished in Gallus g. domesticus--normal, telomeric bows, and lumps. The former are distributed along the whole chromosome length. The latter and the bows were observed in subtelomeric and telomeric regions. By DNA/RNA specific acridine orange staining it was shown that each loop (especially, "lumpy" loops) contained a rich RNP matrix. A comparative analysis of the chromomycin A3/distamycin A banding pattern of mitotic and lampbrush chromosomes shows that the telomeric "bows" and "lumps" are special loops developed in telomeric heterochromatic bands. In Coturnix c. japonica, the CMA/DA-positive bands were not observed in telomeres of mitotic macrochromosomes, except a smallest band in the 2p-arm telomere. The absence of telomeric heterochromatic bands which can be visualized in the quail mitotic chromosomes coincides with the absence of "bow"-like loops. Only small lump-like structures were seen in some telomeres of macroautosomes. The biological significance of loop formation and RNA synthesis in heterochromatic band loops in growing oocytes is briefly discussed.     

Electron microscopy of chromatin subunit particles

Electron microscope studies of PS-particles obtained by partial nuclease digestion of calf thymus chromatin shows them to be very similar in size and shape to particles which have been detected in native chromatin. Furthermore, measurement of the spacing between pairs of such particles, together with the known length of the DNA coiled in the particles gives an estimate of about 200 base pairs for the chromatin “subunit”.     

Higher-order structure of chromatin and chromosomes

The linear array of nucleosomes that comprises the primary structure of chromatin is folded and condensed to varying degrees in nuclei and chromosomes forming 'higher order structures'. We discuss the recent findings from novel experimental approaches that have yielded significant new information on the different hierarchical levels of chromatin folding and their functional significance.     

Loop size in newt lampbrush chromosomes

Lampbrush chromosome 11 from the newtTaricha granulosa was studied by scanning electron microscopy (SEM) to determine the size of all loops in this bivalent. Measurements with an XY digitizer revealed a mean loop length of 14.9 µm, with a large standard deviation and a skewed distribution toward higher values. The size of the loops at this stage of diplotene extension is similar to that reported in other eukaryotes studies with different approaches. We estimated, from the DNA content of chromosome 11, that between 0.4% and 2.2% of the DNA is found in the loops while the rest of the DNA must remain in the compact chromomeres.     

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.     

Electron microscopy of whole mount metaphase chromosomes

Whole mount metaphase chromosomes, from cultured L cells, have been centrifuged onto grids and examined by electron microscopy. Compact and dispersed chromosome forms provide extensive ultrastructural information. Condensed chromosome arms appear as packed fibers with centromeric heterochromatin identifiable because it stains more intensely than the rest of the chromosome. Kinetochores are readily visible in these preparations. Under appropriate isolation conditions, it is possible to obtain mitotic spindles in which bundles of microtubules remain attached to kinetochores, suggesting that the kinetochores retain basic structural integrity throughout the isolation procedure. Dispersal of metaphase chromosomes by treatment with formalin and distilled water shows that these chromosomes are composed of a basic fiber that is normally highly condensed. This fiber is made up of regularly repeating 70-90 A diameter nucleoprotein granules separated from neighboring granules by a 20-40 A diameter fiber whose continuity is maintained by DNA. This structural arrangement is totally analagous to that reported for interphase chromatin from a variety of sources.     

Electron microscopy in cell-matrix research

Tissue development in multicellular animals relies on the ability of cells to synthesise an extracellular matrix (ECM) containing spatially-organised fibrous assemblies, the most widespread of which is based on collagen fibrils whose length greatly exceeds that of individual cells. The importance of the correct regulation of fibril deposition is exemplified in diseases such as osteogenesis imperfecta (caused by mutations in collagen genes), fibrosis (caused by ectopic accumulation of collagen) and cardiovascular disease (which involves cells and macromolecules binding to collagen in the vessel wall). Much is known about the molecular biology of collagens but less is known about collagen fibril structure and how the fibrils are formed (fibrillogenesis). This is explained in part by the fact that the fibrils are non-crystalline, extensively cross-linked, and very large, which makes them refractory to study by conventional biochemical and high-resolution structure-determination techniques. Electron microscopy has become established as the method of choice for studying collagen fibril structure and assembly, and this article describes the electron microscope methods most often used.     

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.     

An electron microscope study of lampbrush chromosomes

Lampbrush chromosomes were isolated from germinal vesicles of oocytes from Necturus maculatus, Triturus viridescens, Pseudotriton montanus and Rana pipiens. After treatment of isolated nuclei with 10 per cent sucrose, chromosomes free of nuclear sap are obtained for examination in either the light microscope or in the electron microscope. For electron microscopy the chromosomes were prepared either by Anderson's critical-point procedure or were embedded in methacrylate and sectioned. The evidence presented in favor of the view that the loops, axis, and the chromomeres of lampbrush chromosomes are formed by two chromonemata is based on the following observations: 1. Treatment of isolated chromosomes with 0.002 M KCN loosens the structure of the loops, and a more or less coiled organization is then observed in most of them with the light microscope. At the electron microscope level, each loop consists of a bundle of microfibrils. The latter are 500 A in diameter, and their complex arrangement within the loops is best studied in stereoscopic preparations. 2. Treatment of chromosomes with 0.002 M KCN also unravels the "chromomeric" regions of the axis. A fibrillar organization then becomes visible in the light microscope. In the electron microscope, wide strands are seen within some chromomeres; their diameter corresponds closely to that of the chromonemata forming the loops associated with the same chromomeres. In thin transverse sections of isolated chromosomes, no special structure is visible in the axial region except random profiles of fibrils similar to those seen in the loops of the same preparations. 3. Two strands sometimes connect adjacent chromomeres. Where gaps exist along the axis, after stretching of the chromosomes, a loop occasionally straddles the break and returns to a chromomere on each side.     

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.