Hoechst 33258 induced uncondensed sites in marsupial chromosomes

The fluorochrome Hoechst 33258 induces pronounced uncondensed regions at mitosis at one or more specific sites on the X chromosomes of all eighteen species of marsupials belonging to the family Macropodidae which have been examined. The Y chromosomes of nearly all of these species also show sensitive sites. Autosomal regions which respond to this chemical were observed in only five species and there is evidence of polymorphism for two of these. The regions which respond usually show C-banding, but not all C-banding regions are affected. No specific effect was found in the chromosomes of eleven other species examined which are representative of 5 different Australian marsupial families. The implications of the apparent restriction of sex chromosome sensitive sites to macropods are discussed.     

A specific chromosome element,the telomere of Drosophila polytene chromosomes

The submicroscopic organization of terminal chromosome regions of Drosophila hydei polytene chromosomes is described. A compact region composed of tightly packed fibrils of 100 to 125 Å diameter embedded in an amorphous material is located at each of the chromosome ends of the 5 long chromosome arms. From this compact region, sometimes containing cavities, fibrils extend onto the nearest normal band region. The diameter of the extending fibrils is 100–125 Å, 200–250 Å or 400 Å. Pronase digestion of fixed and squashed chromosomes reduced the electron density of the amorphous matrix in the compact regions but failed to affect the diameter of the fibrils. The extending fibrils, however, showed a decrease in diameter after pronase digestion. The most frequently observed diameter values were 100–125 Å. — The volume of the terminal structures, including the compact region as well as the extending fibrils, is characteristically different for the various elements of the karyotype. Chromosome 2 displays the largest terminal structure, whereas chromosome 4 only occasionally shows the presence of compact regions. — End to end association of the long chromosome arms involves the fusion of the compact terminal structures. The non-random distribution of end to end association seems to be correlated with the volume of the terminal structures. Chromosome 2 which contains the largest compact terminal region is more frequently involved in end to end associations than any other chromosome arm. — The terminal regions show replication of DNA. They belong to the group of regions which display a discontinuous labeling pattern along the chromosomes, representing a late phase of the replication cycle. — The unique structural organization of the terminal chromosome regions, which is never observed at any other location of the genome supports the idea that they are morphological manifestations of the postulated telomeres.     

Comparative analysis of micro and macro B chromosomes in the Korean field mouse Apodemus peninsulae (Rodentia, Murinae) performed by chromosome microdissection and FISH

Comparative analysis of micro B and macro B chromosomes of the Korean field mouse Apodemus peninsulae, collected in populations from Siberia and the Russian Far East, was performed with Giemsa, DAPI, Ag-NOR staining and chromosome painting with whole and partial chromosome probes generated by microdissection and DOP-PCR. DNA composition of micro B chromosomes was different from that of macro B chromosomes. All analyzed micro B chromosomes contained clusters of DNA repeats associated with regions characterized by an uncondensed state in mitosis. Giemsa and DAPI staining did not reveal these regions. Their presence in micro B chromosomes led to their special morphology and underestimation in size. DNA repeat clusters homologous to DNA of micro B chromosome arms were also revealed in telomeric regions of some macro B chromosomes of specimens captured in Siberian regions. Neither active NORs nor clusters of ribosomal DNA were found in the uncondensed regions of micro B chromosomes. Possible evolutionary pathways for the origin of macro and micro B chromosomes are discussed.     

The ultrastructure of human mitotic chromosomes and interphase nuclei treated by Giemsa banding techniques

Total preparations of mitotic chromosomes and interphase nuclei prepared as for Giemsa banding techniques were investigated by standard transmission electron microscopy and by a method of a three dimensional representation. Chromosomes as well as interphase nuclei appear to be composed of irregularely folded fibrils of at least 300 Å thickness. In the G-band regions the chromosomes are thicker containing more foldings of fibrils. Also the fibrils are darker stained in the G-band regions. Loops of fibrils stick out from chromosomes as well as from interphase nuclei. When chromosomes or interphase nuclei come to lie close enough, such loops may stick together and form fibrillar bridges between them. These as well as interchromatid bridges are considered to be artefacts. The fibrils seem to be built up either of one or of several finer fibrils. No further conclusions regarding the fine structure of the fibrils can be drawn.     

Characteristics of the ultrastructural organization of metaphase chromosomes in mouse embryos in the early cleavage stages

The structural organization of the mouse metaphase chromosomes in the early embryonic development (I-IV cleavages) was studied using serial ultrathin section. It was shown that in the first cleavage the metaphase chromosomes consist of DNP fibrils 20-25 nm in diameter, which are distributed nonuniformly along the chromosomes. It was suggested that parts of chromosomal arms formed by tightly packing DNP fibrils may correspond to the G-bands revealed by the routine Giemsa staining. In metaphase chromosomes of 8-16-cell embryos DNP fibrils form chromonema--thick threads about 90 nm in diameter. The chromonemata are evenly organized along chromosomal arms. The centromeric heterochromatin always consists of DNP fibrils tightly arranged in a block having no chromonemal level of organization. In all the cells studied chromosomes form structural contacts (associations) by their centromeric heterochromatin regions.     

Differential decondensation of mitotic chromosomes during hypotonic treatment of living cells as a possible cause of G-banding: an ultrastructural study

The chromosomal ultrastructure of Chinese hamster cells treated with 0.075 M KCl — a solution ordinarily used for making preparations of spread chromosomes — was studied. The hypotonic treatment was shown to result in differential decondensation of chromosomes which consists in the uneven distribution of deoxyribonucleoprotein (DNP) fibrils along chromatids. Fixation of cells with methanol acetic acid causes an abrupt restructuring of chromosomes. However, the DNP preserves its uneven distribution along chromatids. As seen on ultra-thin sections of marker nucleolus organizer chromosomes, the densely packed regions may correspond to G-bands detected in the selfsame chromosomes by standard methods of differential staining. The results suggest that the capacity of chromosomes for differential staining is based on the different resistance of G- and R-bands to the decondensing action of hypotonic solutions on living cells.     

Chromonema and chromomere

A study of ultrathin sections of normal Chinese hamster cells and cells treated with decreasing concentrations of bivalent cations (Ca²⁺ and Mg²⁺) in situ revealed several discrete levels of compaction of DNA-nucleoprotein (DNP) fibrils in mitotic chromosomes and the chromatin of interphase nuclei. At concentrations ranging from 3 mM CaCl₂ and 1 mM MgCl₂ to ten times less, the chromosomes are found to contain fibrous elements (chromonemata) about 100 nm in diameter. As Ca²⁺ concentration is gradually decreased to 0.2–0.1 mM, the chromosomes decondense into a number of discrete chromatin structures, the chromomeres. As decondensation proceeds, these chromomeres acquire a rosettelike structure with DNP fibrils radiating from an electron-dense core. Upon complete decondensation of chromosomes, individual chromomeres persist only in the centromeric regions. The following levels of DNP compaction in mitotic chromosomes are suggested: a 10-nm nucleosomal fibril, a 25-nm nucleomeric fibril, and the chromonema, a fibrous structure, about 100 nm in diameter, composed of chromomeres. Interphase nuclei also contain structures which are morphologically similar to the chromomeres of mitotic chromosomes.     

Ultrastructural composition of nonhistone chromosomal skeleton at different stages of mitosis in situ

In interphase cells of the SPEV culture treated with Triton X-100, 2 M NaCl, and DNAse, in the presence of 2 mM CuCl₂, we clearly revealed a stabilized nuclear protein material (NPM) composed of a peripheral lamina, residual nucleolus, and internal fibrillar network. This network is formed by thin fibrils 10–20 nm in diameter, which are also revealed in the nonhistone matrix of mitotic chromosomes at all stages of mitosis. In mitotic chromosomes, NPM is represented as a network of the 10–20-nm-thick fibrils without any features of the central-axial structures. Beginning from the middle prophase, it is possible to see approached sister chromatids in contact with each other in certain sites, similar to centromeres. At these sites, the thickness of fibrils increases up to 40–50 nm, whereas the fibrils themselves are disposed more tightly; this structure can be seen in the chromosome until telophase. At the end of telophase, the decondensation of chromosomes and formation of two new nuclei whose NPM is analogous to NPM of usual interphase nucleus are observed. Thus, the NPM elements can perform the role of a skeleton in both the interphase nucleus and mitotic chromosomes.     

Differential decondensation of mitotic chromosomes during the hypotonic treatment of cells as a possible cause of G-banding

Chinese hamster metaphase chromosomes were studied after treating the cells with a hypotonic 0.075 M KCl solution, routinely used for differential staining of chromosomes. After the incubation of cells in KCl for 5 seconds-40 minutes and fixation with glutaraldehyde, the chromosomes displayed DNP fibrils about 20 nm in diameter. These fibrils were unevenly packed along the chromosomal arms and formed sites of differential density. In sister chromatids, the even density sites were located symmetrically. The use of serial ultrathin sections of marker chromosomes (e.g., chromosomes with a telomeric disposition of nucleolar organizing regions) made it possible to establish a good correlation between the sites with the light packing of DNP-fibrils and G-bands, identified in the same chromosomes by the standard Giemsa-staining technique. Fixation of the KCl-incubated cells with the methanol: glacial acetic acid (3:1) solution did not change the DNP packing heterogeneity. The chromosomal banding state is reversible, for with the transfer of cells from KCl to the normal cultural medium the chromosomes become homogeneous in length. Thus, the data obtained allow to propose that the capacity of chromosomes for differential staining may be based on an uneven sensitivity of G- and R-bands to the decondensing effect of hypotonic treatment.     

FURTHER ELECTRON MICROSCOPE STUDIES ON FIBRILLAR ORGANIZATION OF THE GROUND CYTOPLASM OF CHAOS CHAOS

Further evidence for fibrillar organization of the ground cytoplasm of Chaos chaos is presented. Fixations with osmium tetroxide at pH 6 or 8 and with glutaraldehyde at pH 6 or 7 were used on two preparations: (a) single actively streaming cells; (b) prechilled cells treated with 0.05% Alcian blue in the cold and returned to room temperature for 5–10 min. In addition, a 50,000 g pellet of homogenized cells was examined after fixation with glutaraldehyde-formaldehyde alone. In sections from actively streaming cells considerable numbers of filaments were observed in the uroid regions after glutaraldehyde fixation, whereas only traces of filaments were seen after osmium tetroxide fixation at either pH 6 or 8. Microtubules were not seen. In sections from dye-treated cells, filaments (4–6 mµ) and fibrils (12–15 mµ) were found with all three fixatives. The 50,000 g pellet was heterogeneous but contained both clumps of fibrils and single thick fibrils like those seen in the cytoplasm of dye-treated cells. Many fibrils of the same dimensions (12–15 mµ wide, 0.5 µ long) were also seen in the supernatant above the pellet. Negative staining showed that some fibrils separated into at least three strands of 4–6 mµ filaments.     

Electron microscopic observations on interband fibrils in Drosophila salivary chromosomes

Acetic alcohol squash, preparations of salivary chromosomes of Drosophila melanogaster stained with uranylacetate during fixation and dehydration were sectioned and examined with the electron microscope. At high magnification the interband regions are seen to be composed of coiled fibrils in the range of 50 Å which often seem to be arranged in pairs. Submicroscopic bodies found along the interband fibrils seem to delimit successive subunits. It is suggested that transverse coiling of the fibrils within the boundaries of each subunit leads to the formation of chromomeres.     

Stepwise Organization of the β-Structure Identifies Key Regions Essential for the Propagation and Cytotoxicity of Insulin Amyloid Fibrils

Amyloid fibrils are supramolecular assemblies, the deposition of which is associated with many serious diseases including Alzheimer, prion, and Huntington diseases. Several smaller aggregates such as oligomers and protofibrils have been proposed to play a role in early stages of the fibrillation process; however, little is known about how these species contribute to the formation of mature amyloid fibrils with a rigid cross-β structure. Here, we identified a new pathway for the formation of insulin amyloid fibrils at a high concentration of salt in which mature fibrils were formed in a stepwise manner via a prefibrillar intermediate: minute prefibrillar species initially accumulated, followed by the subsequent formation of thicker amyloid fibrils. Fourier transform infrared spectra suggested the sequential formation of two types of β-sheets with different strength hydrogen bonds, one of which was developed concomitantly with the mutual assembly of the prefibrillar intermediate to form mature fibrils. Interestingly, fibril propagation and cellular toxicity appeared only after the later step of structural organization, and a comparison of β-sheet regions between the prefibrillar intermediate and mature fibrils using proteolysis led to the proposal of specific regions essential for manifestation of these properties.     

The development of resistance to methotrexate in a mouse melanoma cell line

PG19T3 mouse melanoma cells were selected for resistance to methotrexate. Nine sub-lines that are resistant to concentrations of methotrexate ranging from 1.27×10^–7 M, to 1×10^–4M methotrexate were selected and characterised in terms of their content of dihydrofolate reductase activity and their chromosomes. The intracellular level of dihydrofolate reductase activity increases with increasing resistance such that at the highest level of resistance PG19T3:MTX_R ^{10–4 M} cells contain approximately 1,000 fold more enzyme activity than the parental PG19T3 cells. It is shown that the enhanced activity is due to an increase in the amount of the enzyme rather than any structural change to the enzyme in resistant cellls. Comparisons of pH activity profiles, profiles under different activating conditions and titrations with methotrexate suggest that the sensitive and resistant cells contain identical dihydrofolate reductases. Analysis of the chromosomes of resistant cells shows the presence of up to 5 large marker chromosomes which contain homogeneously staining regions after G-banding. These same regions stain intensely after C-banding and fluoresce brightly after staining with Hoechst 33258. The size of homogeneously staining regions increases throughout the process of selection. For one marker chromosome this increase may have been mediated via a ring chromosome.     

The behavior of nucleolus organizers in structurally changed karyotypes of barley

Two standard karyotype barley lines and 18 lines with karyotypes reconstructed by means of induced reciprocal translocations have been studied with respect to nucleolus formation. The standard karyotype contains two pairs of satellite chromosomes (pairs 6 and 7). Five of the structurally changed karyotypes contain, as a result of reciprocal translocations between the standard satellite chromosomes, only one satellite chromosome pair, each chromosome with two satellites and two nucleolus organizing regions. Under these circumstances, only two of the four NORs are active in nucleolus formation while the other two — probably the transposed ones — remain inactive; hence the maximum number of primary nucleoli per nucleus is two. — When NORs are translocated to chromosomes with no NOR in the standard karyotyp, the normal pattern of nucleolus formation remains unchanged. The same is true after transposition of segments from other chromosomes to the satellites of the standard SAT-chromosome pairs 6 and 7. The results obtained are discussed with respect to effects of translocations on the activity and behaviour of nucleolus organizing regions.     

Analysis of nucleolus organizer regions (NORs) in mitotic and polytene chromosomes ofPhaseolus coccineus by silver staining and Giemsa C-banding

Chromosomes with active nucleolus organizer regions (NORs) were visualized in root tip metaphases ofPhaseolus coccineus using the silver staining technique. A mean number of 5.5 Ag-NORs per cell was observed in 54 cells from eight plants. In the endopolyploid nuclei of the suspensor the silver technique did not demonstrate the reported specificity for nucleolus organizer activity, because there was usually pale staining of nucleoli and preferential staining of heterochromatic regions in the polytene chromosomes including pericentromeric material, telomeres and NORs. The mean number of NORs per nucleolus as detected by this method was 5.8 (28 nucleoli analysed). Using a modified preparation technique, giant chromosomes stained pale, but nucleoli of suspensor cells displayed darkly silver staining internal domains, each of which originating from a nucleolus organizer.—Giemsa C-banding of endopolyploid suspensor nuclei revealed C-positive nucleolus organizers with darkly staining intranucleolar fibrils. The latter were frequently involved in inter-NOR associations. In 34 nucleoli analysed, the mean number of Giemsa C-positive NORs per nucleolus was 6.0.Dedicated to Professor Dr.Lothar Geitler on the occasion of his 80th birthday.     

The salivary gland chromosomes of Dasyneura crataegi (Diptera: Cecidomyiidae)

Four distinctly crossbanded, stout polytene chromosomes are present in the nuclei of both the basal reservoir region and gland proper region of salivary glands of young larvae of the Cecidomyid Dasyneura crataegi. In older larvae, asynchronous progressive splitting of the chromosomes into oligotene fibrils occurs, underlining their truly polytene nature. Three nucleoli are present, located on two of the chromosomes. A series of massive puffs is also organised by one of the nucleolar chromosomes. Three other features of interest shown by the chromosomes of this species are (a) the centromeric association of only two, the nucleolar organising, chromosomes of the four present; (b) the high breakability of the centromeric regions of these two chromosomes; and (c) the marked heterochromatin proliferation which is found at these regions in older larvae. As in most Cecidomyids, the salivary glands are of complex structure with anterior basal reservoir and posterior gland proper zones. Marked differences in the relative and absolute sizes of these two regions are found during the development of the glands, which indicate that their names are inappropriate to their probable functions.     

Elektronenmikroskopische Untersuchungen menschlicher Metaphasen-Chromosomen

Zusammenfassung Chromosomen von Mitosen im Metaphasestadium nach Colchicinbehandlung normaler menschlicher Fibroblastenkulturen wurden mit dem Elektronenmikroskop untersucht.Nach Fixierung in Glutaraldehyd und Einbettung in Epon zeigen Schnittpräparate nach Kontrastierung mit Uranylacetat als feinste erkennbare Elemente etwa 30 Å dicke, schraubig gewundene Fibrillen, die dickere, vielfach und unregelmäßig gefaltete Fibrillen von 100–150 Å Durchmesser aufbauen.Isolierte ganze Chromosomen, die zur Präparation mit hypotoner Salzlösung vorbehandelt, in Alkohol-Essigsäure fixiert und luftgetrocknet wurden, lassen stark gewundene dicke Fibrillen von 200–300 Å durchmesser erkennen, die aus schraubig gewundenen 30 Å dicken Fibrillen bestehen. In Schnittpräparaten von ähnlich vorbehandelten Chromosomen finden sich ebenfalls 200–300 Å dicke Fibrillen, die aus 30 Å dicken feineren Fibrillen in lockerer Anordnung aufgebaut sind. Der größere Durchmesser der dicken Fibrillen in hypoton vorbehandelten Präparaten könnte durch Auflockerung der feinen Fibrillen hervorgerufen sein.In allen Präparaten sind die auch lichtmikroskopisch sichtbaren primären Windungen der Chromatiden angedeutet. Die dickeren Fibrillen lassen sonst keine regelmäßige Anordnung erkennen. Längsunterteilungen im Sinne von Halb- oder Viertelchromatiden sind nicht zu sehen. In Totalpräparaten erscheint die Region des Zentromers weniger dicht, und Kinetochoren sind nicht erkennbar.Es wird die Frage diskutiert, ob nur eine kontinuierliche und vielfach gewundene Fibrille oder mehrere miteinander verflochtene Fibrillen und Stränge ein Chromatid aufbauen.

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Metaphase chromosomes of colchizinized normal human fibroblast cultures were investigated with the electron microscope.Sections of glutaradehyde fixed and epon embedded chromosomes show 30 Å thick coiled fibrils building up folded thicker fibrils of 100–150 Å diameter.Isolated total chromosomes pretreated in hypotonic salt solution, fixed in alcohol-acidic acid and air dried, show also 30 Å thick fibrils coiled into thicker fibrils of 200–300 Å diameter. Sections of similarly treated and epon embedded chromosomes show fibrils of similar dimensions but more loosely coiled than in glutaraldehyde fixed sections.Major coils also seen by light microscopy are noticeable in all preparations. No signs of longitudinal subdivisions of the chromatids are detectable. In whole mount preparations the centromere region appears as less dense and kinetochores cannot be seen.The question is discussed whether one single continuous fibril coiled to a thicker fibril which in turn is irregular folded to a strand laid into the major coils builds up a chromatid, or if many thin fibrils join together to thicker fibrils which again form thicker strands which are finally twisted together to a chromatid.

     

Inter- and intra-genomic transfer of small chromosomal segments in wheat-rye allopolyploids

Newly synthesized wheat-rye allopolyploids, derived from Triticum aestivum Mianyang11 × S. cereale Kustro, were investigated by sequential fluorescent in situ hybridization (FISH) and genomic in situ hybridization (GISH) using rye tandem repeat pSc200 and rye genomic DNA as probes, respectively, over the first, second and third allopolyploid generations. FISH signals of pSc200 could be observed at both telomeres/subtelomeres of all 14 chromosomes of the parental rye. In the first allopolyploid generation, there were ten rye chromosomes bearing FISH signals at both telomeres/subtelomeres and four rye chromosomes bearing FISH signals at only one telomere/subtelomere. However, in the second and the third allopolyploid generations, there were 12 rye chromosomes bearing FISH signals at both telomeres/subtelomeres and 2 rye chromosomes bearing FISH signals at only one telomere/subtelomere. Rye telomeric segments were transferred to the centromeric region of wheat chromosomes in some cells and small segments derived from non-telomeric regions of rye chromosome were transferred to the telomeric region of wheat chromosomes in some other cells. These observations indicated that the rye telomeric/subtelomeric region was unstable in newly synthesized wheat-rye allopolyploids and allopolyploidization was accompanied by rapid inter/intra-genomic exchange. The inter-genomic exchange may have occurred in somatic cells.     

The JIL-1 kinase regulates the structure of Drosophila polytene chromosomes

The JIL-1 kinase localizes to interband regions of Drosophila polytene chromosomes and phosphorylates histone H3 Ser10. Analysis of JIL-1 hypomorphic alleles demonstrated that reduced levels of JIL-1 protein lead to global changes in polytene chromatin structure. Here we have performed a detailed ultrastructural and cytological analysis of the defects in JIL-1 mutant chromosomes. We show that all autosomes and the female X chromosome are similarly affected, whereas the defects in the male X chromosome are qualitatively different. In polytene autosomes, loss of JIL-1 leads to misalignment of interband chromatin fibrils and to increased ectopic contacts between nonhomologous regions. Furthermore, there is an abnormal coiling of the chromosomes with an intermixing of euchromatic regions and the compacted chromatin characteristic of banded regions. In contrast, coiling of the male X polytene chromosome was not observed. Instead, the shortening of the male X chromosome appeared to be caused by increased dispersal of the chromatin into a diffuse network without any discernable banded regions. To account for the observed phenotypes we propose a model in which JIL-1 functions to establish or maintain the parallel alignment of interband chromosome fibrils as well as to repress the formation of contacts and intermingling of nonhomologous chromatid regions. Electronic Supplementary Material Supplementary material is available for this article at and accessible for authorised users     

Transcription of intercalary heterochromatin regions in Drosophila melanogaster cell culture

Labelled RNA preparations (total newly synthesized RNA, as well as stable cytoplasmic RNA) isolated from a cell culture of D. melanogaster were hybridized in situ with polytene chromosomes. Apart from the nucleolus, in all cases the regions adjacent to the chromocentre in the polytene chromosomes and the intercalary heterochromatin regions in the X chromosome and the autosomes are the most intensively labelled. In the case of asynapsis of polytene chromosomes in heterozygotes the label is detected in a number of intercalary heterochromatin sites in one homologue only ("the asymmetrical label"). The same kind of radioactivity distribution in intercalary heterochromatin regions was observed after a hybridization of polytene chromosomes with cloned DNA fragments (Ananiev et al., 1978, 1979) coding for the abundant classes of messenger RNA (Ilyin et al., 1978) in a cultured D. melanogaster cells. In some regions of intercalary heterochromatin which do not contain these fragments the "'asymmetrical" type of label distribution is observed after hybridization with cell RNA. - These results lead one to regard the intercalary heterochromatin regions as "nests" comprising different types of actively transcribable genes, the composition of each nest varying in different stocks of D. melanogaster.