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.     

Core-Strukturen in den meiotischen und post-meiotischen Kernen der Spermatogenese von Gryllus domesticus

Summary electron microscope study of spermatogenesis and spermiogenesis in Gryllus domesticus has revealed the existence of peculiar lamellate bodies which occur both in spermatocyte and spermatid nuclei. These bodies must be considered as multiple complexes of the axial core structures which are regularly found in paired pachytene chromosomes. Their shape is irregular, and their constituent structural elements, although having dimensions and a fine structure identical to those of regular axial complexes, may assemble in sheets rather than ribbons, often in a concentric rather than planparallel multiple layer system.Spcrmatocyte nuclei may either contain just one or two large bodies of this type, often but not always in close association with the nucleolus and/or the X chromosome, or they may show several such structures of smaller dimensions which have some connection to chromosome fibrils. None of the two types of nuclei simultaneously contains regular axial core complexes. In spermatid nuclei one or two such multiple structures are usually found, again often in association to the X and/or (in O-spermatids) to what appears to be a nucleolus.It is considered likely that the multiple core complexes are due to the self-assembly of those protein molecules which typically assemble only under the control of and in close association with the pachytene chromosomes to form ordered axial complexes. Their occurrence in spermatids shows that the constituent molecular material may not be decomposed during the meiotic divisions after it is dissociated from the chromosomes.

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Mit Unterstützung durch die Göttinger Akademie der Wissenschaften.

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Herrn Prof. Dr. H. Bauer zu seinem 60. Geburtstag gewidmet.     

The CHD remodeling factor Hrp1 stimulates CENP-A loading to centromeres

Centromeres of fission yeast are arranged with a central core DNA sequence flanked by repeated sequences. The centromere-associated histone H3 variant Cnp1 (SpCENP-A) binds exclusively to central core DNA, while the heterochromatin proteins and cohesins bind the surrounding outer repeats. CHD (chromo-helicase/ATPase DNA binding) chromatin remodeling factors were recently shown to affect chromatin assembly in vitro. Here, we report that the CHD protein Hrp1 plays a key role at fission yeast centromeres. The hrp1Δ mutant disrupts silencing of the outer repeats and central core regions of the centromere and displays chromosome segregation defects characteristic for dysfunction of both regions. Importantly, Hrp1 is required to maintain high levels of Cnp1 and low levels of histone H3 and H4 acetylation at the central core region. Hrp1 interacts directly with the centromere in early S-phase when centromeres are replicated, suggesting that Hrp1 plays a direct role in chromatin assembly during DNA replication.     

Visualization of early chromosome condensation: a hierarchical folding, axial glue model of chromosome structure

Current models of mitotic chromosome structure are based largely on the examination of maximally condensed metaphase chromosomes. Here, we test these models by correlating the distribution of two scaffold components with the appearance of prophase chromosome folding intermediates. We confirm an axial distribution of topoisomerase IIalpha and the condensin subunit, structural maintenance of chromosomes 2 (SMC2), in unextracted metaphase chromosomes, with SMC2 localizing to a 150-200-nm-diameter central core. In contrast to predictions of radial loop/scaffold models, this axial distribution does not appear until late prophase, after formation of uniformly condensed middle prophase chromosomes. Instead, SMC2 associates throughout early and middle prophase chromatids, frequently forming foci over the chromosome exterior. Early prophase condensation occurs through folding of large-scale chromatin fibers into condensed masses. These resolve into linear, 200-300-nm-diameter middle prophase chromatids that double in diameter by late prophase. We propose a unified model of chromosome structure in which hierarchical levels of chromatin folding are stabilized late in mitosis by an axial "glue."     

Overall DNA methylation and chromatin structure of normal and abnormal X chromosomes

DNA methylation patterns were studied at the chromosome level in normal and abnormal X chromosomes using an anti-5-methylcytosine antibody. In man, except for the late-replicating X of female cells, the labeled chromosome structures correspond to R- and T-bands and heterochromatin. Depending on the cell type, the species, and cell culture conditions, the late-replicating X in female cells appears to be more or less undermethylated. Under normal conditions, the only structures that remain methylated on the X chromosomes correspond to pseudoautosomal regions, which harbor active genes. Thus, active genes are usually hypomethylated but are located in methylated chromatin. Structural rearrangements of the X chromosome, such as t(X;X)(pter;pter), induce a Turner syndrome-like phenotype that is inconsistent with the resulting triple-X constitution. This suggests a position effect controlling gene inactivation. The derivative chromosomes are always late replicating, and their duplicated short arms, which harbor pseudoautosomal regions, replicate later than the normal late-replicating X chromosomes. The compaction or condensation of this segment is unusual, with a halo of chromatin surrounding a hypocondensed chromosome core. The chromosome core is hypomethylated, but the surrounding chromatin is slightly labeled. Thus, unusual DNA methylation and chromatin condensation are associated with the observed position effect. This strengthens the hypothesis that DNA methylation at the chromosome level is associated with both chromatin structure and gene expression.     

Ultrastructural investigation on the innervation of the Herbst corpuscle

Summary Nerve fibres, running longitudinally as well as circularly between the core lamellae in the Herbst corpuscle are described.These fibres are morphologically different from the central afferent axon. They are most frequently observed in the outer part of the core, and contain inter alia numerous agranular vesicles measuring approx. 450 Å in diameter, dense core vesicles with a diameter approx. 800 Å and microtubuli (250 Å). Occasional specialized junctions are seen between the nerves and the neighbouring lamellae.This study was supported by the Norwegian Council of Agricultural Research.     

Cell cycle control of higher-order chromatin assembly around naked DNA in vitro.

We have developed an in vitro system in which higher-order chromatin structures are assembled around naked DNAs in a cell cycle-dependent manner. Membrane-free soluble extracts specific to interphase and mitotic states were prepared from Xenopus eggs. When high molecular weight DNA is incubated with interphase extracts, fluffy chromatin-like structures are assembled. In contrast, mitotic extracts produce highly condensed chromosome-like structures. Immunofluorescence studies show that a monoclonal antibody MPM-2, which recognizes a class of mitosis-specific phosphoproteins, stains the "core" or "axis" of condensed mitotic chromatin but not interphase chromatin. By adding mitotic extracts, interphase chromatin structures are synchronously converted into the condensed state. The increasingly condensed state of chromatin correlates with the appearance and structural rearrangements of the MPM-2-stained structures. These results suggest that mitosis-specific phosphoproteins recognized by MPM-2 may be directly involved in the assembly of the chromosome scaffold-like structures and chromatin condensation. Although both extracts promote nucleosome assembly at the same rate, topoisomerase II (topo II) activity is four to five times higher in mitotic extracts compared with interphase extracts. The addition of a topo II inhibitor VM-26 into mitotic assembly mixtures disturbs the organization of the MPM-2-stained structures and affects the final stage of chromatin condensation. This in vitro system should be useful for identifying cis- and trans-acting elements responsible for higher-order chromatin assembly and its structural changes in the cell cycle.     

The fine structure of the kinetochore of a mammalian cell in vitro

The chromosomes of Chinese hamster cells were examined with the electron microscope and the following observations were made concerning the structure and organization of the kinetochore. — The kinetochore consists of a dense core 200–300 Å in diameter surrounded hy a less dense zone 200–600 Å wide. The dense core consists of a pair of axial fibrils 50–80 Å in diameter which may be coiled together in a cohelical manner. The less dense zone about the axial elements is composed of numerous microfibrils which loop out at right angles to the axial fibrils. Together the structures comprise a lampbrush-like filament which extends along the surface of each chromatid. Some sections suggested that two such filaments may be present on each chromatid. The fine structure of kinetochores associated with spindle filaments was essentially the same as those free of filaments. The structure and organization of the kinetochore of these mammalian cells was compared to that of lampbrush chromosomes of certain amphibian oöcytes, dipteran polytene chromosome puffs, and the synaptinemal complex seen during meiotic prophase.The authors also wish to thank Dr. Arthur Cole of the Department of Physics for the use of his electron microscope facilities and for his helpful criticism.     

The ultrastructure of G- and C-banded chromosomes

A method of visualizing chromosome bands by electron microscopy has been used to investigate the fine structural organization of G- and C-banded chromosomes. The following information has been obtained:

1. 1. G-bands, produced by trypsinization, were electron dense regions of highly packed chromatin fibres separated by regions in which the chromatin fibres were much less densely packed (interbands).

2. 2. Several degrees of chromatin dispersion were apparent in trypsinized chromosomes. Such dispersion was not a prerequisite for the initial visualization of G-bands, however the progressive pattern of dispersion indicated that the bands were relatively more resistant to dispersion than the interbands.

3. 3. After fixation and trypsinization, individual chromatin fibres measured 250 Å in diameter and appeared morphologically similar to control chromatin fibres seen by whole mount electron microscopy.

4. 4. In trypsinized chromosome complements, the chromosomes often appeared to be interconnected to one another by chromatin fibres. The evidence indicates that these interchromosomal fibres are artefacts produced by the overlapping of dispersed chromatin fibres.

5. 5. When the same metaphase chromosome was observed by both light and electron microscopy, some of the light microscopic G-bands were represented by two or more ultrastructural bands. The number of bands seen in metaphase chromosomes by electron microscopy appears to approach the increased number of bands generally seen in prometaphase chromosomes by light microscopy.

6. 6. C-banding methods (NaOH treatment or overtrypsinization) resulted in the extraction of variable amounts of chromatin from the non C-band regions of the chromosomes, however the constitutive heterochromatin remained highly condensed and resistant to extraction. This result supports the hypothesis that the mechanism of C-banding involves the selective loss of non C-band chromatin.

     

Reorganization and condensation of chromatin in mitotic prophase nuclei of Allium cepa

This paper studies the process and features of chromosome construction in mitotic prophase cells of Allium cepa. The results showed that a prominent reorganization of chromatin occurred during G₂-early prophase. The 250–400 nm thick compact chromatin threads in G₂ nuclei began to disorganize into about 30, 100 and 220 nm chromatin fibres which constituted the loosely organized chromosome outlines in early prophase before chromosome condensation. In middle prophase, chromosome condensation was characterized by the formation of many condensed regions (aggregates of chromatin), which increased in size (1–1.5 m) when prophase proceeded. Meanwhile, the chromatin threads that constituted and connected the condensed regions became increasingly thicker (120–250 nm). In late prophase adjacent condensed regions fused to form cylinder-shaped chromosomes. Based on these observations, we come to the conclusion that the construction of prophase chromosomes is a two-step process, that is, the reorganization and condensation of chromatin. In addition, we report the study of silver-stained, DNA- and histone-depleted prophase chromosomes, describe morphological features of the non-histone protein (NHP) residue in early, middle and late prophase chromosomes, and discuss the roles of NHPs in chromosome construction.     

Meiotic pairing constraints and the activity of sex chromosomes

The state of activity and condensation of the sex chromosomes in gametocytes is frequently different from that found in somatic cells. For example, whereas the X chromosomes of XY males are euchromatic and active in somatic cells, they are usually condensed and inactive at the onset of meiosis; in the somatic cells of female mammals, one X chromosome is heterochromatic and inactive, but both X chromosomes are euchromatic and active early in meiosis. In species in which the female is the heterogametic sex (ZZ males and ZW females), the W chromosome, which is often seen as a condensed chromatin body in somatic cells, becomes euchromatic in early oocytes. We describe an hypothesis which can explain these changes in the activity and condensation of sex chromosomes in gametocytes. It is based on the fact that normal chromosome pairing seems to be essential for the survival of sex cells; chromosomal anomalies resulting in incomplete pairing during meiosis usually result in gametogenic loss. We argue that the changes seen in the sex chromosomes reflect the need to avoid pairing failure during meiosis. Pairing normally requires structural and conformational homology of the two chromosomes, but when the regions is avoided when these regions become heterochromatinized. This hypothesis provides an explanation for the changes found in gametocytes both in species with male heterogamety and those with female heterogamety. It also suggests possible reasons for the frequent origin of large supernumerary chromosomes from sex chromosomes, and for the reported lack of dosage compensation in species with female heterogamety.     

Relationship between relative dry mass and average band width in regions of polytene chromosomes of Drosophila

Whole-mounted polytene chromosomes from Drosophila melanogaster were prepared for high-voltage electron microscopy. Relative dry mass of chromosome regions was estimated by densitometry of electron microscopic negatives. Comparison of dry mass of regions of the male X chromosome with that of regions of associated autosomes established that dry mass values are proportional to DNA content. Relative dry mass values of regions of polytene chromosomes from salivary glands, fat body, and malpighian tubules were correlated with the average diameter of bands in these regions: as mass doubled, band width increased by a factor of approximately 2. To provide a standard for estimating absolute levels of polyteny, band widths were measured for chromosomes representing one major polytene class, 256n. These chromosomes were observed to have an average band width of 0.9 m — These observations provide limits to models of chromatin organization in bands. For each chromatid, this area can accommodate up to five chromatin fibers of 250 Å diameter. This value may represent the extent of folding of a chromatin fiber in an average band. Alternatively, a chromatin fiber of higher-order structure could have a maximum diameter of 560 Å in an average band.     

Human mitotic chromosomes consist predominantly of irregularly folded nucleosome fibres without a 30-nm chromatin structure

How a long strand of genomic DNA is compacted into a mitotic chromosome remains one of the basic questions in biology. The nucleosome fibre, in which DNA is wrapped around core histones, has long been assumed to be folded into a 30-nm chromatin fibre and further hierarchical regular structures to form mitotic chromosomes, although the actual existence of these regular structures is controversial. Here, we show that human mitotic HeLa chromosomes are mainly composed of irregularly folded nucleosome fibres rather than 30-nm chromatin fibres. Our comprehensive and quantitative study using cryo-electron microscopy and synchrotron X-ray scattering resolved the long-standing contradictions regarding the existence of 30-nm chromatin structures and detected no regular structure >11 nm. Our finding suggests that the mitotic chromosome consists of irregularly arranged nucleosome fibres, with a fractal nature, which permits a more dynamic and flexible genome organization than would be allowed by static regular structures.     

Chromatin elimination in the hypotrichous ciliate Stylonychia mytilus

Chromatin elimination in the hypotrichous ciliate Stylonychia mytilus was studied by means of electron microscopy using a microspreading procedure. In the polytene chromosomes of the macronuclear anlagen three organization patterns are observed: Bands of various size composed of 300 Å chromatin fibers, large blocks of 300 Å nucleofilaments which probably represent the heterochromatic regions of the chromosome and axial 120 Å filaments. Those DNA sequences which become eliminated belong to the 300 Å fiber type. The eliminated chromatin occurs in the form of rings of variable size corresponding to a DNA content between 18 and 160 Kb while the axial 120 Å filaments appear to be preserved.     

A neurofibrillar body in the perikaryon of nervus terminalis ganglion cells in teleosts

Summary In three species of Teleosts (Tinea tinea L., Leuciscus cephalus cabeda Risso, Epinephelus guaza L.) a round strongly argentophilic body of considerable size occurs in the cytoplasm of the nervus terminalis ganglion cells. In Tinea, surgical interruption of functional connections of the ganglion cells does not produce any apparent change either in the number or in the size of these cytoplasmic bodies.Electron microscopical observations show that the neurofibrillar body is made up of densely packed and irregularly arranged bundles. In cross section, each of these bundles appears to be composed of neurofilaments (100 Å in thickness) and neurotubules (diameter: 300 Å). Each tubule is surrounded by 9–10 filaments equi-distant from one another, and at a distance of 30–40 Å from the central tubule.The authors are indebted to Prof. G. Palladini for helpful histochemical advice, to Prof. B. Bertolini for electron micrographs and to Mr. D. Scorsini for skilful technical assistance.     

Characterization of chromosome replication during S-phase with bromodeoxyuridine labelling in Chinese hamster ovary and HeLa cells

The same C-banded human polymorphic chromosomes were observed in the light microscope (LM) and then in the scanning electron microscope (SEM) to investigate the structural changes produced by the C-banding technique. C-banded regions, which stained positively in LM, were highly condensed with tightly packed chromatin fibres, resembling non-banded chromosomes. In striking contrast, adjacent non-C-banded regions were represented by loosely arranged fibres, resembling G-banded chromosomes. The significance of these observations in relation to current theories on the effects of C-banding on chromosome structure is discussed.     

Characterization of different types of condensed chromatin inCostus (Zingiberaceae)

The chromatin structure of six diploids species ofCostus was analysed using conventional Giemsa staining, C-banding and DAPI/CMA fluorochromes. The interphase nuclei in all the species show an areticulate structure and the prophase chromosomes show large blocks of proximal condensed chromatin. After banding procedures, each chromosome exhibits only centromeric dot-like DAPI⁺/CMA^– C-bands whereas the satellites (one pair at each karyotype) are weakly stained after C-banding and show a DAPI^–/CMA⁺ fluorescence. Two chromocentres show bright fluorescence with CMA and weak staining after C-banding whereas the others chromocentres show only a small fraction of DAPI⁺ heterochromatin. These results were interpreted to mean that the greater part of the condensed chromatin has an euchromatic nature whereas two types of well localized heterochromatin occur in a small proportion. The Z-stage analysis suggests that heterochromatin and condensed euchromatin decondense at different times. The chromosome number and morphology of all species are given and the implications of the condensed euchromatin are discussed.Dedicated to Prof.Elisabeth Tschermak-Woess on the occasion of her 70th birthday.     

Cylindrical structures in the chloroplasts of Sirogonium melanosporum

Summary Cylindrical structures with outside diameters of 390 to 410 Å have been observed in the chloroplasts of mature vegetative cells of the filamentous, green alga Sirogonium melanosporum. These cylindrical structures are either parallel to one another or randomly oriented in the stroma matrix of the chloroplast. Some of the outer cylindrical structures of the complex appear to be continuous with thylakoid membranes, suggesting a relationship between these two structures.This work was supported by an Institutional Grant from the American Cancer Society to the University of Arizona and by grant GB2440 from the National Science Foundation to R. W. Hoshaw. The authors thank Dr. Wayneferris for the use of the electron microscope supported by NSF grant GB3330.     

The neurohypophysis of the crested newt

Summary In the median eminence of the newt a medial region and two lateral regions are described.In cross section, the medial region appears to be made up of 1) an outer or glandular zone (Zone I) containing aldehyde-thionine-positive and negative nerve fibres and blood capillaries. Nerve fibres appear aligned in palisade array along the capillaries. 2) An inner zone (Zone II) made up of a) a layer of aldehyde-thionine-positive nerve fibres (fibrous layer) belonging to the preoptic hypophyseal tract and b) a layer of ependymal cells lining the infundibular lumen and reaching the blood vessels with their long processes.The lateral regions display a less pronounced stratification and aldehyde-thionine positive nerve fibres are nearly absent.A slender lamina (ependymal border) containing mainly aldehyde-thionine-positive nerve fibres and ependymal cells connects the median eminence to the pars nervosa.At the ultrastructural level, in the outer zone of the medial region at least 4 types of nerve fibres and nerve endings are identified:Type I nerve fibres containing granular vesicles of 700–1000 Å and clear vesicles (250–400 Å).Type II nerve fibres containing granular vesicles and polymorphous granules of 900–1300 Å and clear vesicles (250–400 Å).Type III nerve fibres containing dense granules of 1200–2000 Å and clear vesicles of 250–400 Å.Type IV nerve fibres containing only clear vesicles of 250–400 Å. In the inner zone too, all these nerve fiber types are found among ependymal cells, while the fibrous layer consists of nerve fibres containing granules of 1200–2000 Å in diameter.In the lateral regions Type I, Type II and Type IV nerve fibres and their respective perivascular terminals are found; axons containing dense granules (1200–2000 Å) are scanty. In these regions typical synapses between Type I nerve fibres and processes rich in microtubules are visible.The classification and functional significance of nerve fibres in the median eminence are still unsolved, but it may be assumed that nerve fibres of the medial region belong to both the preoptic hypophyseal and tubero hypophyseal tract, while the lateral regions are characterized by nerve fibres of the tubero hypophyseal tract. Peculiar specializations of the ependymal cells in the median eminence of the newt are also discussed.Work supported by a grant from the Consiglio Nazionale delle Ricerche.The authors are indebted to Mr. G. Gendusa and P. Balbi for technical assistance.     

Meiotic prophase in anthers of asynaptic wheat

The light microscope showed that zygotene and pachytene were completely suppressed in pollen mother cells of an asynaptic mutant of Triticum durum; the chromosomes passed through a normal chromomeral leptotene condition and remained unpaired throughout prophase. The electron microscope confirmed the absence of synaptinemal complexes, as would be expected with no pairing. Prominent opaque axial cores were present in the chromatin from the onset of leptotene up to an indeterminate stage during prophase condensation. At an early time during condensation 150 Å particles appeared between chromatin masses. Coincident to the disappearance of axial cores from the chromatin, polycomplexes consisting of linearly associated core fragments arrayed in single layer sheets appeared between chromatin masses. The aligned fragments were invariably spaced about 625 Å from centre to centre; this is approximately half the distance between centres of the lateral elements (axial cores) of the synaptinemal complex of pachytene of synaptic sister seedlings. There was no central element between the associated fragments. The significance of these observations is discussed, as is also the essential difference between asynapsis and desynapsia.