A developmental study of constitutive heterochromatin in Microtus agrestis

In the vole, Microtus agrestis, the constitutive heterochromatin is largely restricted to the giant sex chromosomes but varies in its degree of condensation in various cell types. In the cleavage embryos and fibroblasts it formed one or two long and extended heterochromatic fibers, in hepatocytes it formed two large and diffuse masses and in neurons, spermatogonia and oogonia it formed two large and compact masses. The basic patterns of all differentiated cells were essentially unchanged throughout development.—At all stages of development and in cells of all types, mitotic nuclei displayed two large heteropycnotic chromosomes in prophase and persistent condensation in telophase. Apposition and delayed separation of chromatids of the giant chromosomes was also observed in metaphase and anaphase, respectively. During the first meiotic prophase of spermatocytes and oocytes, the giant chromosomes were also heteropycnotic.—The results strongly suggest that constitutive heterochromatin is localized in the same chromosomes throughout development and represents a specific entity.     

Cytological variations in the constitutive heterochromatin of Microtus agrestis

In the field vole, Microtus agrestis, most of the constitutive heterochromatin is localized in the giant chromosomes. A detailed examination of a large number of adult cell types reveals that this chromatin is actually present as a heterochromatic fiber in all interphase nuclei. Depending upon the cell types, however, the fiber shows varying degrees of condensation and folding ranging from a very long and extended fiber to a large compact chromocenter. The number of cell types with giant chromocenters was less commonly observed than those with extended fibers. This explains why some cells were previously thought to be devoid of heterochromatin.—The results of this investigation strongly indicate that constitutive heterochromatin represents a unique nuclear entity.This investigation was supported by NIH funds (Grant No. HD 1962).     

DNS-Replikation und Morphologie der X-Chromosomen während der Syntheseperiode bei Microtus agrestis

X-chromosome behaviour of female Microtus agrestis in interphase nuclei with and without large chromocenters was examined in cultured epithelial and fibroblast cells. By means of pulse-labeling, the configuration of the X-chromosomes in these nuclei can be illustrated; staining with pararosaniline-methylgreen seems to be most suitable. According to the replication behaviour, three types of chromatin can be discerned in the X-chromosomes: early replicating euchromatin, late replicating sex chromatin, and very late replicating heterochromatin. In fibroblasts only the sex chromatin forms a single, small chromocenter; in epithelial cells both the sex chromatin and the remaining heterochromatin form large chromocenters. Both types of heterochromatin replicate their DNA in the condensed state. It seems likely that the late replicating segments of the X-chromosomes (sex chromatin and remaining heterochromatin) are condensed in every cell, but they may not always be configurated or even visible as typical chromocenters; these segments could be distributed over a wide range of compact to more or less diffuse superstructures.     

Meiotic differences between three triatomine species (Hemiptera,Reduviidae)

We have found the following differences in the male meiosis among three triatomine species: (1) The three largest autosomal bivalents ofTriatoma infestans are heterochromatic.Rhodnius prolixus has two autosomal bivalents with heterochromatic blocks.Triatoma rubrovaria does not show any heteropycnotic autosomes. (2) Sex chromosomes inT. infestans form a chromocenter. At early prophase terminal associations are seen between sex chromosomes inT. rubrovaria, and they maintain a close association until diakinesis. An intimate association between the X and Y chromosomes is observed during early prophase inR. prolixus, but a distant association is maintained by the sex chromosomes at diffuse and diplotene stages in this species. (3) Polyploid nuclei of the nutritive cells are quite distinct. Numerous chromocenters of different shapes and sized are seen in those ofT. infestans. InT. rubrovaria one chromocenter having two positively heteropycnotic elements is observed surrounded by homogeneous chromatin. Only one compact chromocenter is found amongst unevenly distributed chromatin, inR. prolixus.     

Struktur und Position der heterochromatischen Chromosomen in Interphasekernen von Microtus agrestis

Zusammenfassung In Gehirnzellen und Nierenepithelkulturen der Erdmaus, Microtus agrestis, wurden Struktur und Position der Interphasechromozentren untersucht. Die Chromozentren, die von den heterochromatischen Abschnitten der Riesen-Geschlechtschromosomen gebildet werden, erscheinen in Nervenzellen kompakt, in Nierenepithelkulturzellen von unterschiedlichem Kondensationszustand, der vom Zellzyklus unabhängig ist.In Gehirnausstrichen finden sich in 2/3 der Kerne 2 Chromozentren, in 1/3 ein einziges, doppelt großes Chromozentrum, das durch Fusion entstanden ist. Untersuchungen an in Orcein-Essigsäure suspendierten Gehirnzellen zeigen, daß in 88% der Kerne mit fusionierten Chromozentren das Chromozentrum an der Kernmembran, in 12% frei im Kerninneren liegt. In Kernen mit 2 isolierten Chromozentren liegen diese immer an der Kernmembran.Messungen des Abstandes der (in diploiden Kernen) 2 Chromozentren ergeben, daß die Chromozentren 2 Positionen bevorzugen: die Fusion zu einem doppelt großen Chromozentrum und die genau gegenüberliegende Position. Die Verteilung der Abstände deutet auf die Möglichkeit hin, daß sich zumindest die heterochromatischen Chromosomen von einem gewissen Schwellenabstand an somatisch paaren.Eine völlige Übereinstimmung der Position der Chromozentren in Schwesterkernen läßt sich sowohl bei einkernigen getrennten Schwesterzellen als auch in zweikernigen Zellen, die aus einer einkernigen Mutterzelle hervorgegangen sind, beobachten.

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Structure and position of heterochromatic chromosomes in interphase nuclei of Microtus agrestis

Summary Structure and position of chromocenters in interphase nuclei of Microtus agrestis (brain tissue and cultured kidney epithelial cells) were investigated. The chromocenters derived from heterochromatic portions of the very large sex chromosomes appear to be compact in nerve cells and variably condensed in cultured kidney epithelial cells. This variation in condensation is independent of the cell cycle.In smear preparations of brain tissue two thirds of the nuclei contain two chromocenters, whereas one third of the nuclei show one double sized chromocenter, presumably a fusion of two. Suspensions of nerve cells in orcein-acetic acid show that the large fused chromocenters are attached to the nuclear membrane in 88% of the nuclei with one chromocenter. If two isolated chromocenters are present they are always localized adjacent to the nuclear membrane.Measurements of the distance between the two chromocenters show a prevalence of two types of positions: a fusion to a double sized chromocenter and a vis-a-vis position. The frequency distribution of the distances suggest a tendency of somatic pairing of the heterochromatic chromosomes if they come close enough together.In mononucleated sister cells and in sister nuclei of binucleated cells derived from a mononucleated mother cell the position of the chromocenters is identical in the two nuclei.

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Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Differential reaction of condensed and diffuse chromatin to polyamines. I. Reaction of interphase nuclei chromatin to putrescine]

Condensation of the interphase nuclei chromatin under putrescine treatment was studied in cultured human fibroblasts 46, XX: 47, XXX: 49, XXXXY, and aneuploid cells of the Chinese hamster. The effect was tested separately for diffuse and condensed chromatin. Putrescine treatment did not affect the percentage of cell nuclei with X-chromatin bodies in the human cell strains while significantly increasing the percentage of nuclei with coarse chromatin network and chromocenters. In cultured Chinese hamster cells, putrescene did not change the percentage of nuclei with identified chromocenters and no significant condensation of diffuse chromatin was observed either.     

Geschlechtschromatin bei Lepidopteren

Occurence of sex chromatin is widespread amongLepidoptera. Somatic interphase nuclei of female larvae and adults contain a distinct heteropycnotic body which is missing in males. Two types of exceptions exist: one type in which heteropycnotic bodies not discernable from the sex chromatin in other species are formed in both sexes while in another type such chromatin bodies are absent in either sex. Absence of sex chromatin is not linked to a certain systematic entity within the Lepidoptera. In species possessing sex chromatin, the structural aspect of the heteropycnotic body varies within certain limits between different species and tissues. Chromosome analysis of 4 species, 3 of which exhibit normal chromatin bodies in females, while the 4th has no chromatin bodies in both sexes, agree with the interpretation of sex chromatin in the heterogametic females ofLepidoptera as a heteropycnotic Y-chromosome. This interpretation does not apply to those exceptional species which contain heteropycnotic bodies in both sexes.     

Cytofluorometric DNA base determination for the investigation of heterochromatin and heterochromatin amplification

The measurement of chromomycin A3/DAPI fluorescence ratios is shown to allow base content determinations in eu- and heterochromatic regions of interphase cell nuclei. The base content values obtained in chromocenters and euchromatin of Scilla sibirica agree with those measured earlier [12] on the band and non-band areas of the chromosomes of this species. In Sinapis alba three different heterochromatin types, with regard to base content, can be discerned. The heterochromatin amplification observed in the polyploid nuclei of Sinapis roots and hypocotyl involves either all three heterochromatin types, with little resulting change of the total nuclear base content, or only one or the other of them, with a measurable shift of the base content.     

An early stage of ZW/ZZ sex chromosome differentiation in Poecilia sphenops var. melanistica (Poeciliidae,Cyprinodontiformes)

The mitotic and meiotic chromosomes of the American cyprinodont fish Poecilia sphenops var. melanistica were analysed. All 46 chromosomes are telocentric. By specific staining of the constitutive heterochromatin with C-banding and various AT-specific fluorochromes, the homomorphic chromosome pair 1 could be identified as sex chromosomes of the ZW/ZZ type. All female animals exhibit a W chromosome with a large region of telomeric heterochromatin that is not present in the Z chromosome. These sex chromosomes cannot be distinguished by conventional staining; they represent the first demonstration of sex chromosomes in fishes in an early stage of morphological differentiation. The W heterochromatin and the telomeric heterochromatin in the two autosomes 18 show a very bright fluorescence when stained with AT-specific fluorochromes. This allows the direct identification of the chromosomal sex by examining the interphase nuclei: females exhibit three, males only two brightly fluorescent heterochromatic chromocenters in their nuclei. The significance of these ZW/ ZZ sex chromosomes and their specific DNA sequences, the dose compensation of the Z-linked genes, and the experimental possibilities using sex-reversed ZW males are discussed.     

Chromosomal constitution of nucleolus-associated chromatin in man

Summary Use of specific stains permits analysis of the frequency of nucleolus-associated heterochromatin in chromosomes 1 and 9 from human fibroblasts. In 81% of interphase nuclei the heterochromatic segment of both No. 1 chromosomes is associated with the nucleolus, while in 19% only one heterochromatic segment shows such an association with the other occupying a random position in the nucleoplasm. The nucleolar association of chromosome 9 heterochromatin is less constant: in 42.3% of the nuclei both segments are associated with the nucleolus, in 39% of the nuclei only one heterochromatic segment presents such an association, and in 18.7% neither of the two heterochromatic segments is in nucleolar association. In 6% of the cells, one or two chromosome 9 heterochromatic segments are in contact with the nuclear membrane.In situ hybridization using tritium-labeled 28S and 18S RNA shows that in the interphase nucleus the acrocentric short arms, carriers of ribosomal cistrons, are associated with the nucleolus.These observations demonstrate the complexity of the nucleolus-associated chromatin which, in addition to segments of chromosomes 1, 9, 13, 14, 15, 21 and 22, may include the Y chromosome. They also confirm that the nucleolus constitutes one of the orientation points determining the relative localization of chromosomes in the interphase nucleus.     

The replication of human heterochromatin in serial culture

The human C group chromosomes late to start replication in asynchronous and in FUdR synchronized cell lines are X chromosomes. These same chromosomes are also heterochromatic during interphase. During metaphase these allocyclic Xs cannot be identified simply by metaphase position or morphology and show a wide range of measurements for arm ratio, centromere index and total length. Replication starts in the short arm and extends over the entire chromosome during the 2nd and 3rd hr of S until by the 4th hr distinction from other C group chromosomes cannot be made by means of the labeling pattern. When the allocyclic X chromosomes start replication the pattern of H³TdR label over interphase sex chromatin and non-specific heterochromatin shifts from unlabeled to labeled in FUdR synchronized human cell lines. The overall time required for replication of the allocyclic X is less than that for the other chromosomes in both asynchronous and FUdR treated cells. A hypothesis is presented for a direct relation between the delay of onset of replication in heterochromatin and its degree of interphase condensation.The present study was supported by research grants: No. HD-00777 from the National Institutes of Health and No. E-487 from the American Cancer Society, Inc.     

Chromosome banding in Amphibia

The mitotic and meiotic chromosomes and interphase nuclei of the South American tree-frog Centrolenella antisthenesi were studied with various banding techniques. The karyotype is distinguished by a new category of heteromorphic XY/XX sex chromosomes in an initial stage of differentiation. In diakinesis of male meiosis the XY chromosomes exhibit the same pairing configuration as the autosomal bivalents. Analysis of the chromosomes with DNA base pair-specific fluorochromes revealed that unusual large amounts of brightly labeled AT-rich constitutive heterochromatin are located in the centromeric and pericentromeric regions of all autosomes and in the X chromosome. In most types of interphase cell nuclei the brightly fluorescent heterochromatic regions fuse to very large chromocenters.     

Sex chromatin and nucleolar analyses inRumex acetosa L.

Summary Rumex acetosa (sorrel) is a dioecious plant with a XX/XY₁Y₂ sex chromosome system. Both the Y chromosomes are nearly entirely heterochromatic and it has been hypothesised that they can persist as chromocenters in male interphase nuclei. Using specific antibodies against 5-methylcytosine and histone H4 acetylated at terminal lysine 5, global levels of DNA methylation and histone acetylation were studied on the sex chromosomes and autosomes of both sexes. The heterochromatic Y chromosomes did not display a higher methylation level compared to the autosomes. The only prominent hypermethylation signals were found at two nucleolar organising regions located on the autosome pair V, as confirmed by in situ hybridisation with 25S rDNA probe and staining. Immunoanalysis of DNA methylation on female and male interphase nuclei neither revealed any sex-specific differences. Two active (silverpositive) nucleoli and two likely inactive nucleolar organising regions (displaying prominent methylation signals) were found in both sexes. In a fraction of nuclei isolated from leaf cells, two peripheral bodies strongly positive for 4,6-diamidino-2-phenylindole were observed only in males, never in females. These heterochromatin regions were depleted in histone H4 acetylation at terminal lysine 5 and corresponded, according to in situ hybridisation with a Y-chromosome-specific repetitive probe, to the two Y chromosomes. We conclude that the peripheral condensed bodies observed exclusively in male nuclei represent the constitutive heterochromatin of the Y chromosomes which is characterised by a substantial histone H4 underacetylation.     

An electron microscopic study of mitosis in mouse duodenal crypt cells confirms that the prophasic condensation of chromatin begins during the DNA-synthesizing (S) stage of the cycle

The phases of mitosis were examined in the columnar cells at the base of duodenal crypts in adult male mice given an intravenous injection of 3H-thymidine and sacrificed 20 min later. The duodenum was fixed by immersion into glutaraldehyde-formaldehyde, and the cells were examined in the electron microscope, with or without processing for radioautography. Interphase nuclei are characterized by the distribution of chromatin; aside from the cortical chromatin spread along nuclear envelope and nucleolus, there are chromatin accumulations that belong mainly in two different classes: 1) numerous chromatin "specks" ranging in size from about 5 to 70 nm and averaging 47 nm; 2) a few roughly circular or elongated chromatin "packets" measuring from 70 to 230 nm. Early prophase nuclei differ mainly by a large increase in the number of chromatin packets to 20-30 or more per nuclear profile; their average diameter is 128 nm. During mid-prophase, the chromatin packets enlarge gradually to an average 221 nm diameter. Between mid- and late prophase, there is a further increase in diameter to 679 nm. At metaphase, the packets take on the appearance of mature chromosomes, and their diameter increases to 767 nm. At anaphase, daughter chromosomes migrate to each pole, where they fuse into a compact chromatin mass. At telophase, nucleoplasmic areas progressively enlarge within the chromatin mass and separate strands of chromatin, which gradually become segmented into chromatin clumps. Counts of mitotic cells show a high proportion of prophase and telophase nuclei. Calculation from the counts yields the duration of the phases, that is, 5.6, 0.2, 0.1, and 1.6 hr, respectively, for pro-, meta-, ana-, and telophase. Finally, radioautography 20 min after 3H-thymidine injection shows labeling in 54% of the interphase nuclei, 85% of early prophase nuclei, and 73% of mid-prophase nuclei, while there is no label in late prophase, metaphase, anaphase and telophase nuclei. In confirmation of previous light microscopic work, the S stage of the cycle begins when a cell is in interphase and continues through the early prophase and part of mid-prophase. Moreover, the main sites of DNA synthesis are the chromatin specks during interphase and the cortical chromatin during early and mid-prophase. The chromosome condensation taking place in the meantime may be separated into two main steps: 1) a slow, moderate condensation of the chromatin packets during early and mid-prophase and 2) a rapid, pronounced one during late prophase and prometaphase when the packets become chromosomes.     

Quantitative investigation of reproduction of condensed chromatin of sex chromosomes during trophoblast cell polyploidization and endoreduplication in the East European field vole Microtus rossiaemeridionalis

Simultaneous measurement of DNA content in cell nuclei and condensed chromatin bodies formed by heterochromatized regions of sex chromosomes (gonosomal chromatin bodies, GCB) has been performed in two trophoblast cell populations of the East-european field vole Microtus rossiaemeridionalis, namely in the proliferative population of trophoblast cells of the junctional zone of placenta and in the secondary giant trophoblast cells. One or two gonosomal chromatin bodies have been observed in trophoblast cell nuclei of all embryos studied (perhaps both male and female), In the proliferative trophoblast cell population, characterized by low ploidy levels (2c-16c), and in the highly polyploid population of secondary giant trophoblast cells (16c-256c), the total DNA content in GCB increased proportionally to the ploidy level. In separate bodies, the DNA content rose also in direct proportion with the ploidy level seen in the nuclei with both one and two GCBs in the two trophoblast cell populations. A certain increase in percentage of the nuclei with 2-3 GCBs was shown in the nuclei of the junctional zone of placenta; this may be accounted for by genome multiplication via uncompleted mitoses. In the secondary giant trophoblast cell nuclei (16c-256c), the number of GCBs did not exceed 2, and the share of nuclei with two GCBs did not increase, thus suggesting the polytene nature of sex chromosome in these cells. At different poloidy levels, the ratio of DNA content in the nucleus to the total DNA content in GCB did not change significantly giving evidence of a regular replication of sex chromosomes in each cycle of genome reproduction. In all classes of ploidy, the mean total DNA content in trophoblast cell nuclei with single heterochromatic body was less than in the nuclei with two and more GCBs. This may indicate that a single GCB in many cases does not derive from the fusion of two GCBs. To put it another way, in the nuclei with one GCB and in those with two or more GCBs, different chromosome regions may undergo heterochromatization. The regularities observed here are, most probably, associated with the peculiarities in the structure of X- and Y-chromosomes in a range of species of Microtus (M. agrestis, M. rossiaemeridionalis, M. transcaspicus). As a result, gonosomal chromatin bodies may include large blocks of both constitutive heterochromatin of X- and Y-chromosomes (in male and female embryos) and inactivated euchromatin of "lyonized" X-chromosome in female embryos. Therefore the presence of two or more GCBs in trophoblast cells of M. rossiaemeridionalis may be accounted for by both polyploidy and functional state of the nucleus, in which gonosomal constitutive heterochromatin and inactivated euchromatin form two large chromocenters rather than one. The differences in DNA content in GCBs in the nuclei with one and two GCBs seem to be an indirect indication that the two chromocenters may be formed by two different gonosomes, with the extent of their heterochromatization being higher than that in the nuclei with one GCB. GCBs in the trophoblast cells of M. rossiaemeridionalis are observed not only at the early developmental stages, as it was observed in rat at the first half of pregnancy (Zybina and Mosjan, 1967), but also at the later stages, up to the 17th day of gestation. At these stages, the nuclei with non-classical polytene chromosomes rearrange to those with a great number of endochromosomes, probably because of disintegration of chromosomes into oligotene fibrils. However, it does not seem unlikely that this process may involve heterochromatized gonosomal bodies, since only one or two large GCBs can be seen in the nuclei as before. The presence of prominent blocks of constitutive heterochromatin seems to favor a closer association of sister chromatids in polytene chromosomes, which prevents their dissociation into endochromosomes with the result that polyteny of sex chromosomes in the field vole trophoblast is probably retained during a longer period of embryonic development.     

Heterochromatin and chromosomal aberration in microtus agrestis: role of chromosomal association

Mitomycin C (MC) -induced chromatid aberrations among the chromosomes of Microtus agrestis are preferentially localized in the constitutive heterochromatic regions, i.e., major part of the sex chromosomes and the centromeric regions of the autosomes. In the sex chromosomes, intrachanges predominate, while interchanges between the two X chromosomes are very rare. This pattern of distribution of different types of aberrations is interpreted as due to the individual chromocentres that are formed by the two X chromosomes in the interphase.     

Epigenetic characterization of chromatin in cycling cells of pedunculate oak, Quercus robur L.

Cycling cells of Quercus robur have a simple nuclear organization where most of the heterochromatin is visible as DAPI-positive chromocenters, which correspond to DAPI bands at the (peri)centromeric region of each of the 24 chromosomes of the oak complement. Immunofluorescence using 5-mC revealed dispersed distribution of the signal throughout the interphase nucleus/chromosomes without enrichment within DAPI-positive chromocenters/bands, suggesting that DNA methylation was not restricted to constitutive heterochromatin, but was associated with both euchromatic and heterochromatic domains. While H3K9ac exhibited typical euchromatin-specific distribution, the distributional pattern of histone methylation marks H3K9me1, H3K27me2, and H3K4me3 showed some specificity. The H3K9me1 and H3K27me2, both heterochromatin-associated marks, were not restricted to chromocenters, but showed additional dispersed distribution within euchromatin, while H3K27me2 mark also clustered in foci that did not co-localize with chromocenters. Surprisingly, even though H3K4me3 was distributed in the entire chromatin, many chromocenters were enriched with this euchromatin-specific modification. We discuss the distribution of the epigenetic marks in the context of the genome composition and lifestyle of Q. robur.     

Rumex acetosa Y chromosomes: constitutive or facultative heterochromatin?

Condensed Y chromosomes in Rumex acetosa L. root-tip nuclei were studied using 5-azaC treatment and immunohistochemical detection of methylated histones. Although Y chromosomes were decondensed within root meristem in vivo, they became condensed and heteropycnotic in roots cultured in vitro. 5-azacytidine (5-azaC) treatment of cultured roots caused transitional dispersion of their Y chromosome bodies, but 7 days after removal of the drug from the culture medium, Y heterochromatin recondensed and again became visible. The response of Rumex sex chromatin to 5-azaC was compared with that of condensed segments of pericentromeric heterochromatin in Rhoeo spathacea (Sw.) Steam roots. It was shown that Rhoeo chromocentres, composed of AT-rich constitutive heterochromatin, did not undergo decondensation after 5-azaC treatment. The Y-bodies observed within male nuclei of R. acetosa were globally enriched with H3 histone, demethylated at lysine 4 and methylated at lysine 9. This is the first report of histone tail-modification in condensed sex chromatin in plants. Our results suggest that the interphase condensation of Y chromosomes in Rumex is facultative rather than constitutive. Furthermore, the observed response of Y-bodies to 5-azaC may result indirectly from demethylation and the subsequent altered expression of unknown genes controlling tissue-specific Y-inactivation as opposed to the global demethylation of Y-chromosome DNA.     

Heterochromatin,repetitive DNS und Nukleolen in Leberzellen von Microtus agrestis

Zusammenfassung Die Zellstruktur von Leberzellen der Erdmaus, Microtus agrestis, wurde nach Giemsafärbung, Feulgenbehandlung, Behandlung mit Ribonuklease und nach Färbung des konstitutiven Heterochromatins untersucht. Das konstitutive Heterochromatin ist in Leberzellen nicht heteropyknotisch, das fakultative Heterochromatin ist im weiblichen Geschlecht als Sexchromatinkörperchen sichtbar. Bestimmungen des relativen DNS-Gehalts ergaben, daß die Zahl der Sexchromatinkörperchen der Ploidie der Zellkerne proportional ist. Die Nukleolen liegen in Hepatozyten oft randständig; in 59% der diploiden Zellkerne sind 2 Nukleolen enthalten. Nach Anfärbung der repetitiven DNS werden oft auch die Nukleolen gefärbt, nach Ribonukleasebehandlung tritt dieser Effekt nicht auf. Das konstitutive Heterochromatin wird in Form von 2 langen fädigen Strukturen sichtbar.

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Heterochromatin, repetitive DNA and nucleoli in liver cells of Microtus agrestis

Summary The nuclear structure of parenchymal liver cells of embryo and adult Microtus agrestis was studied in smear and section preparations after staining with Giemsa solution and treatment according to Feulgen, after treatment with ribonuclease and after specific staining of constitutive heterochromatin. In liver cell nuclei only the facultative heterochromatin is heteropycnotic, a sex chromatin body is observable in female but not in male animals. Constitutive heterochromatin is not heteropycnotic in liver cells. Measurements of the relative DNA content showed that nuclei with one sex chromatin body are diploid; tetraploid nuclei possess two and octoploid nuclei four sex chromatin bodies. Solely in the diploid cell nuclei of the intrahepatic gall ducts two large chromocenters are found. The nucleoli in hepatocytes often lie at the perimeter of the nucleus. 17% of the diploid nuclei contain one nucleolus, 59% two nucleoli, 23% three and 1% four. After staining of repetitive DNA, the nucleoli often become stained as well; after treatment with ribonuclease this effect does not occur. The constitutive heterochromatin becomes visible in form of two long, threadlike structures. After longer periods of dissociation the sex chromatin body ceases to be visible. Sex chromatin and constitutive heterochromatin are contiguous to the nucleoli.

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Mit dankenswerter Unterstützung durch das Bundesministerium für Bildung und Wissenschaft der Bundesrepublik Deutschland.     

Demonstration of a reaggregation inhibitor in sea urchin embryos

We used a new method based on the study of nuclear areas above certain density thresholds to estimate changes in the condensation of chromatin of a cell. Allium cepa L. root meristematic cells were “labelled” as binucleate by a 1 h treatment with 0.1 % caffeine and were fixed at the middle of each interphase period. The distribution of chromatin densities of Feulgen-stained cells in G1, S and G2 phases was so different that by simply estimating chromatin patterns it would be possible to identify which period of the interphase any cell has reached. G2 nuclei have an increased number of chromatin-dense areas compared with G1 or S nuclei. We postulate that the estimation of chromatin condensation may be useful for the evaluation of intranuclear differentiation at the level of the intact cell.